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Germinal Centres (germinal + centre)
Terms modified by Germinal Centres Selected AbstractsGenetic and phenotypic analysis of B-cell post-transplant lymphoproliferative disorders provides insights into disease biologyHEMATOLOGICAL ONCOLOGY, Issue 4 2008Efsevia Vakiani Abstract B-cell post-transplant lymphoproliferative disorders (PTLD) are classified as early lesions, polymorphic lymphomas (P-PTLD) and monomorphic lymphomas (M-PTLD). These morphologic categories are thought to reflect a biologic continuum, although supporting genetic data are lacking. To gain better insights into PTLD pathogenesis, we characterized the phenotypes, immunoglobulin (Ig) gene alterations and non-Ig gene (BCL6, RhoH/TTF, c-MYC, PAX5, CIITA, BCL7A, PIM1) mutations of 21 PTLD, including an IM-like lesion, 8 P-PTLD and 12 M-PTLD. Gene expression profile analysis was also performed in 12 cases. All PTLD with clonal Ig rearrangements showed evidence of germinal centre (GC) transit based on the analysis of Ig and BCL6 gene mutations, and 74% had a non-GC phenotype (BCL6,±,MUM1+). Although surface Ig abnormalities were seen in 6/19 (32%) PTLD, only three showed ,crippling' Ig mutations indicating other etiologies for loss of the B-cell receptor. Aberrant somatic hypermutation (ASHM) was almost exclusively observed in M-PTLD (8/12 vs. 1/8 P-PTLD) and all three recurrent cases analysed showed additional mutations in genes targeted by ASHM. Gene expression analysis showed distinct clustering of PTLD compared to B-cell non-Hodgkin lymphomas (B-NHL) without segregation of P-PTLD from non-GC M-PTLD or EBV+ from EBV, PTLD. The gene expression pattern of PTLD appeared more related to that of memory and activated B-cells. Together, our results suggest that PTLD represent a distinct type of B-NHL deriving from an antigen experienced B-cell, whose evolution is associated with accrual of genetic lesions. Copyright © 2008 John Wiley & Sons, Ltd. [source] Immunophenotypic analysis of the Kaposi sarcoma herpesvirus (KSHV; HHV-8)-infected B cells in HIV+ multicentric Castleman disease (MCD)HISTOPATHOLOGY, Issue 5 2008A Chadburn Aims:, Kaposi sarcoma herpesvirus (KSHV) is aetiologically related to Kaposi sarcoma, classical and extracavitary primary effusion lymphoma (PEL; EC-PEL) and multicentric Castleman disease (MCD), entities preferentially occurring in HIV-infected individuals. Characterization of HIV-associated PELs/EC-PELs suggests that the KSHV-infected malignant cells originate from a pre-terminal stage of B-cell differentiation. However, only limited phenotypic studies have been performed on HIV+ MCD, including for PR domain containing 1 with zinc finger domain/B lymphocyte-induced maturation protein 1 (PRDM1/BLIMP1), a key regulator of terminal B-cell differentiation. The aim was to characterize KSHV-infected cells in 17 cases of HIV+ MCD. Methods and results:, Double immunohistochemistry and immunohistochemistry,in situ hybridization were used to characterize the KSHV-infected cells in MCD; the results were compared with the phenotypic profiles of 39 PELs/EC-PELs and seven PEL cell lines. Whereas the immunophenotype of KSHV-infected cells in MCD and malignant KSHV+ PEL cells was similar (PAX5, Bcl-6,; PRDM1/BLIMP1, IRF4/MUM1+; Ki67+), the MCD KSHV-infected cells differed, as they expressed OCT2, cytoplasmic , immunoglobulin; variably expressed CD27; lacked CD138; and were Epstein,Barr virus negative. Conclusions:, Although both PEL and MCD originate from KSHV-infected pre-terminally differentiated B cells, these findings, with previously reported genetic studies, indicate HIV+ MCD may arise from extrafollicular B cells, whereas PELs may originate from cells that have traversed the germinal centre. [source] Resolution of skeletal muscle inflammation in mdx dystrophic mouse is accompanied by increased immunoglobulin and interferon-, productionINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 3 2002Jussara Lagrota-Candido Summary. Mdx mouse, the animal model of Duchenne muscular dystrophy, develops an X-linked recessive inflammatory myopathy with an apparent sustained capacity for muscle regeneration. We analysed whether changes in the skeletal muscle during myonecrosis and regeneration would correlate with functional alterations in peripheral lymphoid tissues. Here we show that during the height of myonecrosis, mdx mice display marked atrophy of peripheral lymph nodes and extensive muscle inflammation. In contrast, enlargement of draining lymph nodes with accumulation of CD4+ CD44+, CD4+ CD25+, CD8+ CD44+ T lymphocytes and type-2 B cells was consistently observed during amelioration of the muscle lesion. In addition, regeneration of the muscular tissue was accompanied by concomitant increase of immunoglobulin-secreting cells in regional lymph nodes and bone marrow. Double immunolabelling analysis revealed intense B cell proliferation and formation of germinal centre in the follicles of dystrophic regional lymph nodes. Furthermore, lymph node cells produced large amounts of IFN-, but not IL-4, IL-6 or IL-10 after in vitro mitogen stimulation with Concanavalin A. As these alterations occurred mainly during the recovery period, we suggested that local activation of the immune system could be an influence which mitigates the myonecrosis of muscular tissue in the mdx dystrophic mouse. [source] Follicular dendritic cells confirm lymphoid organization in the minor salivary glands of primary Sjögren's syndromeJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 9 2008Malin V. Jonsson Background:, Sjögren's syndrome (SS) is an autoimmune chronic inflammatory disorder affecting the salivary and lacrimal glands. The aim of this study was to explore immunophenotypic features of chronic inflammatory reactions in the minor salivary glands in patients with primary SS (pSS). Methods:, Formalin-fixed, paraffin-embedded labial minor salivary gland tissue sections from randomly selected patients with pSS (n = 60) were investigated for the expression of CD21, CD23, CD35 and IgD by immunohistochemistry. Results:, Based on the distribution and staining pattern of CD21, CD23, CD35 and IgD in lymphoid aggregates, several stages of chronic inflammatory reactions were observed. In 12/60 (20%) patients, lymphoid infiltrates with germinal centre (GC)-like features such as extensive networks of CD21-, CD23- and CD35-positive cells were observed in the minor salivary gland tissue. Smaller networks and,/or focal infiltrates with scattered CD21+, CD23+ and CD35+ cells were observed in the remaining 48/60 (80,%) cases. When dividing patients according to the presence (GC+) or the absence (GC,) of GC in the minor salivary glands, the mean focus score was significantly higher in the GC+ patients (P < 0.05). Double staining of the minor salivary glands revealed focal infiltrates with follicular dentritic cell networks and B cells resembling normal GCs in tonsillar tissue. Conclusion:, A particular cellular profile was demonstrated in a sub-group of patients with pSS and could be linked to serological aberrations. These findings warrant further prospective studies. [source] Identification of CXCL13 as a new marker for follicular dendritic cell sarcoma,THE JOURNAL OF PATHOLOGY, Issue 3 2008W Vermi Abstract The homeostatic chemokine CXCL13 is preferentially produced in B-follicles and is crucial in the lymphoid organ development by attracting B-lymphocytes that express its selective receptor CXCR5. Follicular dendritic cells (FDCs) have been identified as the main cellular source of this chemokine in lymphoid organs. Recently, genome-wide approaches have suggested follicular CD4 T-helper cells (THF) as additional CXCL13 producers in the germinal centre and the neoplastic counterpart of THF (CD4+ tumour T-cells in angioimmunoblastic T-cell lymphoma) retains the capability of producing this chemokine. In contrast, no data are available on CXCL13 expression on FDC sarcoma (FDC-S) cells. By using multiple approaches, we investigated the expression of CXCL13 at mRNA and protein level in reactive and neoplastic FDCs. In reactive lymph nodes and tonsils, CXCL13 protein is mainly expressed by a subset of FDCs in B-cell follicles. CXCL13 is maintained during FDC transformation, since both dysplastic FDCs from 13 cases of Castleman's disease and neoplastic FDCs from ten cases of FDC-S strongly and diffusely express this chemokine. This observation was confirmed at mRNA level by using RT-PCR and in situ hybridization. Of note, no CXCL13 reactivity was observed in a cohort of epithelial and mesenchymal neoplasms potentially mimicking FDC-S. FDC-S are commonly associated with a dense intratumoural inflammatory infiltrate and immunohistochemistry showed that these lymphocytes express the CXCL13 receptor CXCR5 and are mainly of mantle zone B-cell derivation (IgD+ and TCL1+). In conclusion, this study demonstrates that CXCL13 is produced by dysplastic and neoplastic FDCs and can be instrumental in recruiting intratumoural CXCR5+ lymphocytes. In addition to the potential biological relevance of this expression, the use of reagents directed against CXCL13 can be useful to properly identify the origin of spindle cell and epithelioid neoplasms. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Down-regulation of ATM protein in HRS cells of nodular sclerosis Hodgkin's lymphoma in children occurs in the absence of ATM gene inactivation,THE JOURNAL OF PATHOLOGY, Issue 3 2007S Bose Abstract The tumour component of classical Hodgkin's lymphoma (cHL), Hodgkin Reed,Sternberg (HRS) cells, are believed to be derived from germinal centre (GC) B cells but intriguingly display a characteristic loss of B cell receptor (BCR) expression. The precise mechanisms by which BCR-negative HRS cell progenitors survive negative selection during the GC reaction remain obscure. Individuals with ataxia telangiectasia, caused by biallelic inactivation of the DNA damage response gene, ataxia telangiectasia mutated (ATM), have a higher risk of cHL development. Here we show that, in contrast to normal GC B cells that expressed low but detectable ATM protein, ATM protein was not detected in HRS cells of 17/18 cases of paediatric cHL, all but one with nodular sclerosis (NS) subtype. A comprehensive analysis of the ATM gene in microdissected HRS cells of nine representative tumours showed no evidence of either loss of heterozygosity or consistent pathogenic mutations. Furthermore, bisulphite sequencing of the ATM promoter from HRS cells of five tumours also revealed the absence of hypermethylation. Since our microarray data suggested significantly reduced ATM transcription in HRS cells compared to GC B cells, we conclude that loss of ATM expression could be the result of alterations in upstream regulators of ATM transcription. Importantly, ATM loss in paediatric cHLs has clinical implications and could be potentially exploited to guide future, less toxic, tumour-specific treatments. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Abstracts of the 24th Meeting of the European Study Group for Cell Proliferation (ESGCP)CELL PROLIFERATION, Issue 3 2001Epidemiology (IMISE), Germany, Leipzig, Organized by the Institute for Medical Informatics, Statistics, University of Leipzig A computer exploration of the rules governing stem cell self-renewal and differentiation Z. Agur1,3, S. J. Fleishman1 and Y. Danie2 1 Institute for Medical BioMathematics, PO Box 282, 6,0991 Bene-Ataroth, Israel; 2 Department of Mathematics, University of Haifa, Mount Carmel, 31905 Haifa, Israel; 3 Correspondence: agur@imbm.org The universal properties of stem cells (SC) have been characterized mathematically, showing that extrinsic regulation is sufficient for maintaining tissue homeostasis (Agur et al., unpublished manuscript). We studied a simple hemopoiesis computer model having similar properties. Our model showed extreme robustness, blood counts locally fluctuating around a constant average, and rapidly returning to this exact average following a severe bone marrow (BM) depletion. A very weak negative feed-back on SC proliferation ascertains haemopoietic homeostasis by generating large variation in the SC compartment passage times. The resulting constant desynchronization in the microenvironment guarantees stable blood production, whereas shifting the balance from self-renewal to differentiation yields oscillatory blood production. Bone marrow cells in mouse and human liver can develop into polyploid and binucleate hepatocytes M. Alison, S. Forbes, K. Hodivala-Dilke, R. Jeffery, R. Poulsom and N. Wright Department of Histopathology, Imperial College School of Medicine, Hammersmith Hospital, London, UK Using a technique to detect the Y-chromosome in human liver cells we have previously demonstrated that hepatocytes can be derived from bone marrow cells (Alison et al. 2000, Nature 406, 257). Bone marrow cells may be useful for providing support for the failing liver and could be used as vehicles for delivering therapeutic genes to the liver. Thus, it becomes important to explore the functional capabilities of these cells. The technique of Y chromosome detection also allows one to examine the ploidy status of these hepatocytes, a factor of considerable relevance, since polyploidization is an integral feature of hepatocyte differentiation (Gupta 2000, Seminars in Cancer Biology 10, 161). We have identified both diploid and polyploid hepatocytes of haematopoietic origin in female mice that have been given a male bone marrow transplant after whole body lethal irradiation. We have also identified Y chromosome-positive hepatocytes of both diploid and polyploid class within liver biopsies both from a female who has received a bone marrow transplant from a male donor, and from a male patient that had received a female orthotopic liver transplant. Moreover, the Y-positive hepatocytes were invariably present in fractal clones further suggestive of intrahepatic division after engraftment. These observations suggest that hepatocytes derived from bone marrow cells have the ability to undergo polyploidization, further indicating that they have the potential to function as normal hepatocytes and contribute towards liver regeneration. Aberrant mitotic mechanisms in Alzheimer's disease T. Arendt, U. Ueberham, M. Holzer, U. Gärtner and M. K. Brückner Department of Neuroanatomy, Paul Flechsig Institute of Brain Research, University of Leipzig, Germany Neurodegeneration in Alzheimer's disease (AD) is accompanied by plastic reorganization of neurons that is aberrant with respect to its localization, morphological appearance and composition of cytoskeletal components. These changes, most likely reflecting an aborted attempt of neuronal repair, are associated with a condition of neuronal de-differentiation characterized by an expression of developmentally regulated genes, post-translational modifications and an accumulation of gene products to an extent which goes beyond those observed during regeneration. This process of de-differentiation is probably triggered by a mitogenic over-stimulation of neurons and mediated through activation of the p21ras -dependent mitogen activated protein kinase (MAPK) cascade which contains a number of proto-oncogenes (trk, p21ras, raf). The MAPK cascade is also involved in the regulation of expression and post-translational modification of the amyloid precursor protein and tau protein as well as in the activation of cell cycle mechanisms. Consequently, neurofibrillary degeneration is closely associated with the neuronal expression of markers of cell cycle activation, indicating that re-entry into the cell cycle might be a critical event in a mechanism that eventually results in cell death. The present paper summarizes recent evidence on an aberrant neuronal expression of cell cycle markers in AD. Moreover, data will be presented on the potentially neuroprotective effects of transfecting neurons with human p16INK4a, a critical inhibitor of G0 -G1 transition, which might be a promising strategy for a neuroprotective gentherapy in AD and related conditions. Up-regulation of c-Myc and Ki-67 proteins is correlated with level of apoptosis in tumour cells after cytokine treatment H. Baisch Institute for Biophysics and Radiobiology, University of Hamburg, Germany Human tumour cells Me-180, MCF-7, and TCC-Sup were treated with tumour necrosis factor , (TNF), interferon , (IFN), and the combination of both (Ti). After 1,3 days of treatment, increasing numbers of cells were released from the culture flask bottom, in particular after treatment with both cytokines (Ti). These floating cells turned out to be apoptotic, as shown by several apoptosis assays [Annexin-V-Fitc, mitochondria membrane depolarization (Mito-Track red), pre-G1 peak (PI), chromatin condensation (Acridin-Orange after acid denaturation), PARP degradation]. The cell lines Me-180 and MCF-7 were sensitive to apoptosis induction whereas TCC-Sup was more resistant. At the same time the proliferation of the cells slowed down and eventually ceased. However, since cytokines may also transmit proliferation promoting signals, I measured the proliferation-associated proteins c-Myc and Ki-67. The results show that both c-Myc and Ki-67 expression is considerably elevated after TNF treatment, only slightly or not after IFN treatment, and the strongest increase was observed after Ti treatment. In a plot of the level of increase of Ki-67 vs. % apoptotic cells a linear relationship was revealed for the sensitive as well as the resistant lines. A similar correlation was observed for c-Myc. These findings support the hypothesis that cells are doomed to apoptosis when they receive conflicting signals. The proliferation promoting proteins Ki-67 and c-Myc are up-regulated, and at the same time the cells stop growing by an as yet unknown signal transmitted by the cytokines. These contradictory stop and go signals lead to apoptosis. Relation between cell proliferation markers and apoptosis in colo-rectal cancer A. Becciolini, M. Balzi, P. Faraoni, J. W. Wilson, C. Gai, S. Ferullo, F. Cianchi, E. Ravagni and C. S. Potten Radiation Biology Laboratory, Clinical Physiopathology Department, University of Florence, Italy The aim of the study was to compare different proliferation markers as factors discriminating the outcome for patients with colo-rectal cancer. Moreover, we analysed the immuno-expression of p53 and Bcl-2 and related them to the proliferative status of the tumour. The analysis examined 63 cases treated with surgery as the first therapeutic approach and affected by colo-rectal cancer without distant metastases. We determined three different proliferation indices: TLI, SPF and Ki67-LI, none of which showed a significant relation with the clinical-histopathological variables. The linear regression analysis demonstrated that there is a good relation between TLI and SPF (r = 0.80), whereas the relation is less good for Ki67-LI and, respectively, TLI (r = 0.72) and SPF (r = 0.77). P53 immunoreactivity was present in 71.4% of the tumours, whereas 39.3% of cases were Bcl-2 positive. The proportion of tumour tissue positive for the two proteins showed an opposite trend in relation to the clinical-histopathological variables. The preliminary evaluation of the prognostic role of the biological parameters indicated that only TLI appears able to discriminate two classes of patients with significantly different DFS. The simultaneous analysis of TLI and p53 or Bcl-2 status identified a group of patients (high proliferative activity and p53-negative or Bcl-2-positive) without relapse or distant metastases. Post-irradiational small intestinal crypt survival can be explained by a pedigree free model H. Berger, D. Drasdo, F. A. Meineke and M. Loeffler Institute for Medical Informatics, Statistics and Epidemiology (IMISE), University of Leipzig, Germany Previous models of the cell kinetics in intestinal crypt assumed the existence of stem cells located in the crypt base. Although no stem cell marker has been found so far the stem cells are believed to have clearly distinguishable properties compared to non-stem cells. The number of stem cells is assumed to be nearly constant and has been estimated from crypt survival curves in X-ray radiation experiments. By asymmetric cell divisions stem cells divide into stem and transient cells. The transient cells are able to perform additional cell divisions until they become mature cells. The mature cells migrate towards the orifice of the crypt and are released into the intestinal lumen from the top of the villi. The hierarchy of cell types ranging from stem cells to mature cells defines a pedigree. We here present a novel model that does not need the assumption of a pedigree to explain the shape of crypt survival curves in radiation experiments with X-rays. The basic units of the model are the individual cells arranged in a cylindrical tissue sheet that represent a single crypt. Hence the model allows consideration of the temporal and the spatial dynamics in the crypt. Our model assumes that the stem cell property is completely determined by the spatial position of a cell. Cell proliferation is controlled by a morphogene secreted at the crypt base that spreads over the crypt and determines in which crypt region cells are able to divide. Interchanging a cell at a stem cell position with any other cell would not change the cell kinetics. Hence, within this concept, ,stemness' is determined extrinsically instead of intrinsically. The intestinal epithelial stem cell lineage and its regulation M. Bjerknes and H. Cheng Departments of Anatomy and Cell Biology and Medical Biophysics, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada M5S 1A8 The presentation describes our investigations into the characterization of intestinal epithelial progenitor populations, their interrelationships, and their regulation. It begins with a brief review of intestinal structure and organization. It then illustrates the information that can be extracted by studying the dynamics of clones derived from genetically labelled progenitor cells. This work leads to the conclusion that the epithelium contains a variety of short- and long-lived progenitor types variously committed to producing single or multiple mature cell types. Furthermore, the lineage relationships amongst the various progenitor types can be established. This forms the basis for Specific Progenitor Assay by Somatic Mutation (SPASM) experiments aimed at determining the sensitivity of progenitors to various factors, and such experiments are outlined. The presentation concludes by describing our observations of clonal interactions in adenomas. Proliferative activity of leukemic blasts and cytosine arabinoside pharmacodynamics are associated with cytogenetically defined prognostic subgroups of acute myeloid leukaemia J. Braess, G. Jahns-Streubel, C. Schoch, D. Haase, T. Haferlach, M. Fiegel, S. Voss, W. Kern and W. Hiddemann Department of Medicine III, Klinikum Großhadern, 81377 Munich, Germany Purpose. The biological mechanisms responsible for the association of specific karyotypes with prognosis in acute myeloid leukemia are at present largely unclear. A prospective study was performed to evaluate the extent to which cytogenetically defined prognostic subgroups of AML differ in proliferative activity as a potential mechanism for differential sensitivities to s-phase specific induction chemotherapy comprising cytosine arabinoside. Patients and methods. 187 patients with de novo AML were included in the study, comprising 25 patients with a favourable karyotype [inv(16), t(8; 21), t(15; 17)], 99 with a normal karyotype, 29 with an unfavourable karyotype (,5, 5q-, ,7, 7q-, complex aberrations) and 34 with cytogenetic aberrations of unknown prognostic significance (all others). Ex vivo proliferative activity (PA) of AML blasts and sensitivity of blasts to cytokine priming (GM-CSF, CG-CSF), as measured by thymidine incorporation, endogenous production of stimulatory cytokines (IL3, GM-CSF, G-CSF) by leukemic blasts, incorporation of the active moiety of cytosine arabinoside , araC-triphosphate , into the DNA and response to induction treatment were determined and related to cytogenetic subgroup. Results. The ,favourable' group demonstrated the highest PA (3.41 pmol/105 cells), significantly (P = 0.02) exceeding the ,unfavourable' group with the lowest PA (0.72) and the group with a normal karyotype (1.06) or with karyotype of unknown significance (1.05) with an intermediate PA. Samples with a high PA (> median of the whole group) were more likely to produce IL3, GM-CSF, G-CSF (56%, 43%, 50%) than cells with a low PA (33%, 36%, 36%; n.s.). The effect of priming by exogenous gm-csf or g-csf was significantly more pronounced in samples with a low PA than in rapidly proliferating samples (P < 0.01). For the whole group, a high PA was closely associated with an increased incorporation of AraCTP into the DNA (P < 0.0001). Clinically a high PA was associated with a better CR rate in the normal group (95% vs. 62%) and in the unfavourable group (75% vs. 33%). Conclusions. The significant differences in proliferative activity between cytogenetic subgroups of AML are associated with increased cytosine arabinoside pharmacodynamics and constitute one potential mechanism for the different response of cytogenetic subgroups to araC-based induction therapy. Cultivation of hematopoietic cells in a 3D bioreactor perfusion system S. W. Braun1, I. J. Koerner2, J. Gerlach1 and P. Neuhaus1 1 Department of Surgery and 2 Department of Hematology, Oncology and Tumor Immunology, Charité, Humboldt-University of Berlin, Germany Purpose. Physiological conditions found in the human bone marrow for maintenance and specific expansion of pluripotent hematopoietic stem cells cannot satisfactorily be imitated in in vitro cultures. Therefore we are developing a bioreactor perfusion system to optimize a long-term ex vivo expansion of hematopoietic cells under well-defined conditions. Materials and methods. The bioreactor incorporates three independent but interwoven capillary membrane systems for decentralized nutrient supply and oxygenation in a cell compartment of 10 ml. Studies were performed with the human myeloid leukemia cell line hl-60 (n = 9) and with bone marrow mononuclear cells (BM-MNC: n = 7). The following culture conditions were investigated in the bioreactor system: (a) membrane materials, (b) perfusion modes and rates, (c) medium supply, (d) influence of agitation and (e) cell harvesting conditions. The expansion and viability of hematopoietic cells during cultivation were characterized by growth kinetics, metabolism parameters and morphological appearance. Results. The best results were obtained using polyethersulphone membranes with unidirectional flow across the cell compartment at a perfusion rate of 10 ml/min and a continuous medium supply rate of 5 ml/h. Mild agitation improved the results. A perfusion rate of 15 ml/min (total volume: 300 ml) using perfusion mode combinations was necessary for optimal cell harvesting (> 95%). Under these conditions, expansion rates up to 194% per day, viability rates up to 89% and stable glucose/lactate metabolism were achieved for Hl-60. For growth optimization of BM-MNC in the bioreactor, the addition of cytokines as well as co-culture with stroma cells was favourable. Conclusion. The bioreactor perfusion system proved to be suitable for the expansion of non-adherent hematopoietic cells. However, to achieve a higher viability of the expanded cells, further studies are required to optimize the system for clinical application. P2 receptor-evoked proliferation of Müller glial cells: dependence on calcium and on receptor tyrosine kinases A. Bringmann, I. Milenkovic, V. Moll, H. Kodal and A. Reichenbach Paul Flechsig Institute of Brain Research, Department of Neurophysiology, University of Leipzig, D-04109 Leipzig, Germany Massive gliosis is accompanied by proliferation of macroglial cells. Extracellular ATP has been suggested as a candidate molecule which may induce gliosis. In the sensory retina, the Müller cell is the principal macroglial cell. During proliferative gliosis of the retina, P2 receptor-mediated responses in Müller cells have been shown to be up-regulated. The intracellular signalling pathways that were activated by extracellular ATP and that result in an enhanced proliferation rate were investigated in cultured Müller cells of the guinea pig, using an assay of the DNA synthesis rate. We found that the ATP-evoked proliferation of Müller cells is dependent on Ca2+ and on transactivation of growth factor receptor tyrosine kinases (RTKs). Inhibition of the Ca2+ influx, of the activity of Ca2+ -dependent K+ channels, or of the protein kinase C blocked the mitogenic effect of ATP. Using tyrphostins it was revealed that P2 receptor activation resulted in a transactivation of at least two different RTKs: of the platelet-derived growth factor RTK and, apparently subsequently, of the epidermal growth factor RTK. It is concluded that extracellular ATP activates multiple signalling pathways in cultured Müller cells, to exert its proliferation-enhancing effect. Asynchronous induction of DNA synthesis in G0 nuclei by Xenopus egg extracts R. F. Brooks and W. Sun Guy's, King's and St Thomas' School of Biomedical Sciences, King's College London, Guy's Campus, London SE1 1UL, UK Cell-free extracts of Xenopus eggs support the complete replication of chromosomal DNA in vitro, under normal cell cycle controls. This replication requires the assembly of a prereplication complex (pre-RC) at origins of replication, involving the sequential binding of the six-subunit origin recognition complex (ORC), the Cdc6 and Cdt1 proteins and a complex of six MCM proteins (MCM2-7). With intact mammalian nuclei as template, replication is dependent on the retention of pre-RCs assembled in vivo prior to nuclear isolation, as some component required for their assembly is unable to cross an intact nuclear envelope in egg extracts. Early G0 nuclei from 3T3 cells show significant levels of replication but with prolonged quiescence the pre-RCs are gradually dismantled, as judged by a progressive loss of the capacity to replicate in egg extracts and a decline in chromatin-bound MCMs. The early G0 nuclei that initiate DNA synthesis do so asynchronously, despite exposure to the same concentration of replication factors present in the extracts. This asynchrony is not due to variation in the level of MCM proteins retained by individual nuclei. Nor can it be attributed to differences in the rate of nuclear import, which shows that the initiation of DNA synthesis is not triggered merely by the accumulation of replication factors to some critical threshold. However, when replication begins, it does so at many hundred independent foci scattered throughout the nucleus. The signal for the initiation of DNA synthesis must therefore be a global event distributed throughout the nucleus, its timing varying from one nucleus to another. The nature of this signal remains unknown, but could involve an abrupt increase in cyclin-dependent kinase (Cdk) activity analogous to that which triggers the onset of mitosis. Estrogen-induced protection against apoptosis is in the human breast cancer cell line MCF-7 mediated by procathepsin-D P. Budtz, J. Hofgaard and M. Riis University of Copenhagen, August Krogh Institute, 13 Universityitetsparken, DK 2100 Copenhagen, Denmark The mechanism by which estrogen (E2) stimulates growth of breast cancer cells may be direct through gene activation and/or indirect through the stimulation of autocrine growth factors. It has been suggested that estrogen binding to receptors induces synthesis of specific mRNAs encoding proteins, which are secreted from the cells and via binding to receptors functioning as growth factors (the autocrine hypothesis)1. Procathepsin D (PcD) is normally processed intracellularly into the mature lysosomal protease cathepsin D, but is in response to E2 in the breast cancer cell line MCF-7 secreted in its unprocessed 52-kDa form, which has been suggested to act as such an autocrine growth factor2,3. In a previous study, where E2 stimulated MCF-7 cells were incubated in the presence of a PcD antibody, we showed a significantly reduced growth rate and, at day 8, a cell pool size of about 60%, compared to controls without anti-PcD added. Since the proliferation did not differ significantly, the reduced (net) growth rate following inactivation of E2 -induced PcD was assumed to be due to an increased apoptotic rate4. We have now confirmed this assumption in that the apoptotic index, whether measured following TUNEL staining, or immunostaining of caspase-cleaved PARP or cytokeratin 18, was strongly increased in anti-PcD treated E2 -stimulated cells, but with different expression patterns with time. We conclude that estrogen-induced protection against apoptosis in the human breast cancer cell line MCF-7 is (at least partly) mediated by procathepsin-D. Apoptosis manifests itself differently following induction by antiestrogens (tamoxifen, ici 182. 780), topoisomerase II-inhibitor (etoposide), and DNA intrastrand crosslinking (cisplatin) P. Budtz and K. Møller University of Copenhagen, August Krogh Institute, 13 Universityitetsparken, DK 2100-Copenhagen, Denmark Previous controversies concerning the ability of the MCF-7 cell line to undergo apoptosis (see Budtz 1999, Cell Prolif 32, 289) may have many reasons: methods used, MCF-7 subline, growth conditions, period of time in which the cells have been exposed to the agents. In order to rule out in some detail the manifestation of apoptosis in MCF-7, we have compared cell cultures under growth-promoting conditions (E2) or under growth-inhibiting conditions (antiestrogens: TAM and ICI 182.780; topoisomerase II inhibitor: etoposide; alkylating: cisplatin; unknown mechanism: BSA), studying as a function of time (6 h, 3 and 7 days) the expression of apoptosis in the early (Annexin V), intermediate (caspase-cleaved PARP and cytokeratin 18) and late phase of apoptosis (TUNEL). The effect of these drugs on expression patterns of apoptosis varied. The protective role of E2 was confirmed in that the apoptotic index (AI) was reduced to under 50% of controls for up to 7 days with cytokeratin 18-staining and for up to 3 days with TUNEL and PARP. Of the proapoptotic drugs, all showed increasing AI with time but to a different degree. Thus, compared to controls, the AI at day 7 was increased 25-fold by the antiestrogens, 20-fold by etoposid, 15-fold by cisplatin, 10-fold by BSA, for TAM and etoposid as revealed by TUNEL, for ICI 182.780, cisplatin and BSA as revealed by PARP. Apoptotic induction as revealed by caspase-cleaved cytokeratin 18 was far less pronounced (2,4-fold at day 7, maximal for ICI 182.780). The results show that AI obtained by different staining methods for apoptosis is not directly comparable, and further that caspase 6 and/or 7 (which cleave cytokeratin 18) appear to be of minor importance for execution of apoptosis in MCF-7. Gene expression in hematopoietic tissues: a comparative in vivo study of progenitor cell proliferation both in fetal and adult mice K. T. Chang, O. P,enák, V. Skora, L. ,efc and E. Neýas Department of Pathophysiology, 1st Medical Faculty, Charles University, Prague, Czech Republic Introduction. We have studied the expression of growth factors involved in the hematopoietic microenvironment during embryonic expansion of hematopoiesis in the fetal liver and during hematopoietic regeneration after damage induced by cyclophosphamide (CY). Total RNA was obtained from fetal liver, and from adult liver, bone marrow and spleen. This RNA was transcribed to cDNA that served as a template for real-time PCR. Using specific pairs of primers, mRNAs for the following hematopoietic growth factors/inhibitors were investigated: stem cell factor (SCF), flt3-ligand (flt3L), macrophage inflammatory protein-1, (MIP-1), stromal derived factor 1 (SDF-1) and its receptor CXCR4. Methods. We set up a semiquantitative real-time (RT) PCR with product detection via FastStart SYBR Green I fluorescent intercalation dye. The housekeeping gene for ,-actin was used as an internal standard. The level of particular mRNA was measured as a difference in PCR cycles necessary for the beginning of the exponential phase of PCR reaction. Results. The expression of SDF-1 in fetal liver increases as the fetus matures and reaches its maximum in adults. In contrast, its receptor (CXCR4) follows the expression pattern of its ligand in prenatal development, but is suppressed in adult liver. We found a significant increase in mRNA level for SCF in bone marrow on day 2 after CY. This expression pattern of SCF was not present in spleen. Regarding flt3-ligand and MIP-1,, we did not find significant changes in their expression either in bone marrow or in spleen. Expression of SDF-1 was increased on day 2 after CY in bone marrow, whereas on day 5, it was suppressed. There was no significant change in SDF-1 expression in the spleen. The receptor for SDF-1 (CXCR4) did not exert marked changes in bone marrow, whereas in spleen its mRNA level was decreased on day 2. Conclusion. SCF and SDF-1 may account for progenitor cell proliferation and trafficking in regenerating bone marrow and in fetal liver. This gene expression pattern corresponds with the proliferative kinetics of hematopoietic progenitor cells after cyclophosphamide treatment that had been previously studied. Deterministic and stem cell intrinsic regulation of hematopoietic stem cell self-renewal and differentiation R. H. Cho1, L. Karlsson2, M. Thoman1, H. B. Sieburg3 and C. E. Müller-Sieburg1 1 Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121; 2 R. W. Johnson Pharmaceutical Research Institute, San Diego, CA 92121; 3 Department of Mathematics, University of California, San Diego, CA 92093, USA Commitment and self-renewal are essential and defining functions of stem cells. Decisions about proliferation and differentiation are made at the level of a single stem cell and thus require examination at the clonal level. We have developed a simple, but highly effective method to obtain clonal stem cells with extensive in vivo repopulation capacity [2000, Exp Hematol,28, 1080]. This method yielded about 50% clonally engrafted mice. Thus, the method efficiently identified and isolated clonal, repopulating stem cells to a previously unprecedented level. Clonally derived stem cells differed extensively in their kinetics and extent of repopulation, revealing very primitive and more mature types of stem cells. However, daughter stem cells derived from individual stem cells show remarkably similar repopulation and differentiation capacity. This indicates that stem cell proliferation and differentiation are predetermined and non-stochastic. Amongst the clonally repopulated mice, we identified lineage dominant stem cells. Lineage dominant stem cells give rise to all mature hematopoietic cells, but the progeny is markedly skewed towards either the lymphoid or myeloid lineage. No evidence of transformation or hematolymphoid proliferative disorders was found. This lineage dominant pattern of repopulation can be recreated in secondary and tertiary hosts, indicating that these stem cells have extensive self-renewal capacity and that lineage dominance is stable, stem cell intrinsic behaviour. Collectively, the data indicate that lineage commitment decisions are made at the level of the most primitive stem cells. This appears to cause intrinsic, stable changes at the level of the stem cell that result in progeny with reduced fitness in the underrepresented lineage. Survivin expression in human soft tissue sarcomas A. Costa, S. Pilotti, G. Abolafio, R. Motta and M. G. Daidone Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy Survivin is a member of the IAP (inhibition of apoptosis) gene family whose expression appears to be cell-cycle dependent and regulated during ontogeny, with mRNA and protein levels elevated in fetal tissues and down-regulated in adult normal cells. Survivin interacts with microtubules and can be phosphorylated by p34 cdc2, thus being on interface protein between cell cycle control and apoptosis. Survivin expression has been detected in most human cancers, including breast, gastric and colorectal cancers, lymphomas and melanomas, and in some preneoplastic lesions. We investigated by immunohistochemistry survivin expression in histologically heterogeneous tumours such as soft tissue sarcomas (85 cases) and in a series of benign lesions (five cases). Survivin was detected at a cytoplasmic level in all the five benign lipomatous neoplasms and in 79% of the malignant lesions, with a wide range of positive cells (5,100%) and a median value of 70%. In angiosarcoma, desmoid tumours and malignant fibrous histiocytoma, the median value of survivin-positive cells was less than 50% compared to chondrosarcoma, leiomyosarcoma, liposarcoma, rhabdomyosarcoma and schwannoma. Dermatofibrosarcoma protuberans, a generally well-differentiated histotype, rarely showed survivin expression (in 3/12 cases). In contrast, in the other lipomatous sarcomas, mixoid liposarcoma was characterized by a nuclear staining. In summary, survivin is present in most soft tissue sarcomas, with a histology-related variability. The nuclear localization observed in mixoid liposarcoma should indicate a role of survivin in the differentiation of lipomatous lesions. The transient manipulation of transcription factor gene expression in multipotent haemopoietic progenitor cells M. Cross and Z. McIvor Laboratory of Molecular Medicine, IZKF, University of Leipzig, Germany The lineage commitment of haemopoietic progenitor cells is a largely intrinsic process in which transcription factors are likely to play a pivotal role. We have recently reported the use of GFP coexpression vectors to direct the transient expression of regulatory genes in the multipotent murine haemopoietic progenitor cell line FDCPmix (McIvor et al. 2000, BJH 110, 674). This assay allows the rapid and precise evaluation of effects on survival, proliferation, self-renewal and proliferation. Here, we show the consequences of directing the expression of selected haemopoietic transcription factor genes on developmental potential and gene expression in the FDCPmix cells. The transient expression of PU.1 (a transcription factor associated predominantly with myelopoiesis) results in a marked reduction in self-renewal potential as assayed by colon forming efficiency, and a delay in erythropoiesis, with little adverse affect on myelopoiesis in the presence of GM- and G-CSF. Interestingly, PU.1 expression appears simultaneously to cause a strong increase in transcription from cotransfected promoters (with or without PU.1 binding sites). This ability to increase non-specific transcription is shared by GATA-1, a transcription factor involved in erythroid differentiation, but not by the stem cell transcription factor SCL. This suggests that the over expression of certain lineage-restricted transcription factors in multipotent progenitors can influence the balance between self-renewal and lineage commitment, and that commitment under these conditions may be associated with an increase in overall transcriptional activity. Proliferation and apoptosis-related markers: prognosis and predictive role in breast cancer M. G. Daidone Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy In breast cancer, proliferative activity and apoptosis represent two biological processes thoroughly investigated for their association with tumour progression and their involvement in determining the cellular response to clinical treatments. In general, the prognostic relevance of proliferation indices, including the mitotic activity component of all pathologic grading systems, overcomes that of apoptosis-related markers (evaluated as bcl-2 or bax expression, or apoptotic index). Conversely, either proliferative activity or apoptosis proved to be relevant in indicating susceptibility to chemical, hormonal and physical agents in large retrospective studies. Recently, results from treatment protocols prospectively planned to test the clinical utility of proliferation markers as a primary or a secondary objective (which are thus associated with a high level of evidence in grading the clinical utility of the investigated markers) have indicated the fraction of cells in the S-phase, evaluated by flow cytometry or as 3H-thymidine incorporation, as a marker to identify, within node-negative cancer patients, those at a very low risk of relapse (even in the presence of other conventional patho-biologic features) or those who will benefit from regimens including antimetabolites. In addition, the outcome of an ancillary study of a randomized adjuvant chemotherapy trial supported the hypothesis of a cell cycle-related benefit from different drug schedules. Bcl-2 overexpression is associated with a favourable outcome following tamoxifen, possibly as a surrogate marker of differentiation or as an estrogen-regulated protein, but is also indicative of a possible resistance to chemotherapy. Neoadjuvant treatments allow investigation of the relative balance between proliferation and apoptosis following drug treatment in an in vivo system. Effects of keratinocyte growth factor (KGF) on the radiation response of in vitro reconstituted oral mucosa W. Dörr and A. Lacmann Department of Radiotherapy and Radiation Oncology, Medical Faculty Carl Gustav Carus, Technical University Dresden The present study was initiated to quantify the effect of KGF on the radiation response of in vitro reconstituted oral mucosa. For this, keratinocyte cultures were established from mouse tongue mucosal explants, grown on collagen gel contracted by fibroblasts. Differentiation and stratification were induced by lifting the cultures to the air,medium interface. KGF in a concentration of 100 ng/ml was added to the medium either before (days , 3,0) or after (days 0 to + 3) irradiation with single doses of 20 and 30 Gy or 5 fractions of 4 Gy, respectively. Samples for histological processing were taken on days 4 or 11 p. irr. Irradiation results in a substantial decrease in epithelial thickness and cell number. KGF significantly reduced this radiation response in all irradiation protocols. The effect on epithelial thickness was independent of the KGF treatment interval. In contrast, cell numbers were significantly higher after postirradiation treatment compared to preirradiation administration. The latter indicates a reduction in individual cell size of the daughter cells if KGF is applied after irradiation. In conclusion, KGF stimulates proliferation of keratinocytes in in vitro reconstituted oral mucosa, and ameliorates the response of the organotypic coculture to irradiation. Hierarchical and flexible models of proliferative organization: a revisit from the standpoint of hematopoiesis and radiosensitivity J. D. Down BioTransplant Inc., Charlestown Navy Yard, Boston, MA, USA Two decades have passed since Michalowski conceived of the division of tissue systems into two types with respect to their proliferative organization and radiation response (1981, Radiat Environ Biophys 19, 157). Hierarchical (type H) and flexible (type F) models have generally been ascribed to tissue systems of rapid and slow renewal, respectively, and provide a theoretical framework for describing the kinetics of radiation responses. These models may also be associated with sensitivity of the early and late reacting tissue to changes in radiation dose fractionation (high and low ,/, ratios, respectively) whereby late radiation responses have a greater recovery from damage with reduction in dose per fraction (Thames et al. 1982, Int J Radiat Oncol Biol Phys8, 219). The bone marrow is often exemplified as a typical early responding and H-type tissue where acute radiation injury (myelodepression and marrow lethality) conforms to poor recovery during fractionated irradiation and thus leads to high estimates of the ,/, ratio. However, more recent studies utilizing long-term repopulation assays in vitro and in vivo have shown the existence of more primitive stem cells capable of fractionated irradiation dose-sparing and a low ,/, ratio that is more consistent with a late responding F-type tissue. The whole hematopoietic system may therefore be better described as a hybrid H-F type tissue. It is conceivable that a similar phenomenon may also arise in certain leukemias. With the advent of stem cell subset isolation techniques, such a concept may prove valuable in defining the cellular and molecular determinants of two characteristic radiation responses but within the same cell pedigree. Apart from the relevance to establishing long-term hematopoietic cell chimerism after total body irradiation and stem cell transplantation, it remains possible that the H-F hybrid model has broader applications if it can be extended to other rapidly renewing normal tissue systems and their malignant counterparts. Modelling blastulation and gastrulation D. Drasdo1 and G. Forgacs2 1 Institute for Medical Informatics, Statistics and Epidemiology (IMISE), University of Leipzig, Leipzig, Germany (drasdo@imise.uni-Leipzig.de); 2Department of Physics and Biology, University of Missouri, Columbia, Missouri, USA Blastulation is the process leading from the zygote to a hollow sphere of cells (blastula) by successive cell divisions that conserve the total mass of the embryo5,6. In some species, as in synapta digita or sea urchin, the blastula is one cell thick. At a certain blastula size an invagination occurs in a restricted area forming a gastrula. The invaginated part later forms the digestive tract. We here present a single-cell based model of blastulation and gastrulation in which each model parameter can be linked to experimentally accessible kinetic or material parameters. The model describes a two-dimensional circular cut along the perimeter of the blastula7. We find that the expansion of the blastula can be driven by the cell division alone. Until a certain size the polarity and stiffness of the one-cell-thick layer surrounding the cavity is sufficient to stabilize the spherical shape of the blastula. Above this size the spherical shape becomes unstable and a buckling instability occurs which destabilizes the blastula and eventually derails normal development. To ensure a single invagination, gastrulation must occur prior to the appearance of the instability. Within our model we avoid the instability by a sudden change of some model parameters. We interpret these changes as a consequence of specific gene activity. After changing the physical characteristics of some cells, normal development resumes, and gastrulation proceeds. The physical mechanism underlying the buckling instability is generic7 and hence may also be responsible for the unphysiological buckling during the postirradiation recovery in intestinal crypts or during the extra buckling in the oral mucosa observed after administering growth factors. Growth law and spatial fluctuations in growing tumours prior to vascularization D. Drasdo Institute for Medical Informatics, Statistics and Epidemiology (IMISE), University of Leipzig, Germany (drasdo@imise.uni-leipzig.de) Understanding of the principles underlying tumour growth laws is essential in order to predict tumour growth and consider this knowledge in tumour treatment strategies. For this purpose, direct mechanistic approaches seem particularly useful in contrast to heuristic models, since the former are better candidates to allow conclusions from the macroscopic behaviour of a growing tumour to potential underlying microscopic rules. Along this line of thought, we here construct a lattice model of tumour growth, guided by a recently established stochastic single-cell growth model in which all model parameters can be directly linked to physical properties of the single cells. The lattice model allows one to proceed up to very large cell numbers and can thus be compared to experiments of growing tumour spheroid. In contrast to the usual approach to approximate the growth law of spheroid tumours by the Gompertz law, our findings suggest that the growth law is well described by an exponential function for small cell numbers crossing over into a power law for large cell numbers. The observation of power laws supports recent findings, which suggest a power-law growth of breast tumours. The advantage of a stochastic single cell approach becomes even more obvious when processes are studied that affect the spatial organization of a tumour: We model the effect of an antimitotic drug on a tumour by the assumptions that a cell in mitosis dies with a probability Pdeath > 0.5 and are then phagocytosed by macrophages or neighbour cells. We find spatial density fluctuations of the cells that later result in a fragmentation of a tumour. The intensity of the fragmentation depends very much on the growth law prior to the treatment: If the tumour was growing power-law-like before the treatment, the fragmentation is the strongest if Pdeath, 0.5; if it was an exponential, the fragmentation is the strongest for Pdeath= 1. Clearly, the approach may be oversimplified but is useful in identifying the potential origin of effects on tumour treatment. Biological role of p16ink4a deletion in hematologic neoplasias M. H. Dreyling, G. Hutter, G. Ott, C. Schoch and W. Hiddemann Department of Medicine III, University Hospital Grosshadern/LMU, GSF Munich and Department of Pathology, University of Würzburg, Gemany Introduction. Deletion of p16ink4a is one of the most frequent genetic abnormalities in various tumour types. The gene product p16 is an inhibitor of the cyclin-dependent kinase 4 which regulates the transition from G1 to S phase. However, in hematologic malignancies, the deleted tumour suppressor region spans about 120 kb including other candidate genes (p15ink4b, p19arf). Methods. We have analysed 20 cell lines and 122 patient samples with MDS, AML, CML-BC, ALL, diffuse large cell lymphoma (DLCL), follicular lymphoma, and mantle cell lymphoma (MCL to define the extension and frequency of non-homozygous p16ink4a deletions applying two colour FISH and other molecular methods (Southern blot, SSCP). Results. p16ink4a defines the minimal tumour suppressor region and is deleted in 20% of ALL, 38% of DLCL, and 40% of MCL. Interestingly, the p16ink4a deletion was deleted in 6 of 7 cases with secondary DLCL or Richter's syndrome (86%), whereas deletions were infrequent in FL (10%; P < 0.001) or primary DLCL (20%; P < 0.001). In addition, the p16ink4a deletion was one of the most frequent secondary alterations in MCL and closely correlated to cell proliferation (P = 0.01). Conclusions. Similar to p53, alteration of p16ink4a is a genetic marker of the secondary transformation to aggressive lymphoma. In addition, the p16ink4a deletion is frequently non-homozygous and closely correlated to cell cycle activity. Whereas no point mutation could be detected in these cases, methylation of the promotor region is currently being investigated to characterize alternative mechanisms of gene inactivation. A genomic approach to understanding human origins I. Ebersberger, W. Enard, A. von Haeseler, F. Heissig, I. Hellmann, H. Kaessmann, P. Khaitovich, T. Kitano, S. Meyer, C. Schwarz, D. Serre, V. Wiebe, M. Winkler, S. Zöllner and S. Pääbo Max-Planck-Institute for Evolutionary Anthropology, Inselstrasse 22, D-04103 Leipzig, Germany In defining what traits are unique as well as shared between contemporary humans and their closest living and extinct evolutionary relatives, we may be able approach some of the genetic traits that define humans as a species. Our laboratory attempts to achieve this in several different approaches, three of which will be discussed here. We study the intraspecific DNA sequence diversity in humans, the great apes and Neanderthals. The results from sequencing a 10 000-bp region on the X chromosome indicate that humans are unique among the apes in having little diversity as well as a signal of population expansion. The comparison of mitochondrial DNA sequences from three Neanderthal individuals suggests that these may be more similar to modern humans than to the great apes in terms of their within-group diversity. We have sequenced a total of 256 kb of genomic DNA around the genes ZFX and ZFY in chimpanzees. When compared to other genomic sequences from the chimpanzee, the results show that different regions in the human genome differ significantly in their rates and modes of evolution and that the mutational mechanisms underlying transversions and transitions differ. We have furthermore studied the relative levels of expression of 20 000 genes in humans, chimpanzees and macaques. The results suggest that, whereas the rate of change of the transcriptome in the liver and blood has been similar between chimpanzees and humans, the rate of overall transcriptome change in the human brain has accelerated about 3-fold. A number of genes that differ significantly in their expression levels between humans and chimpanzees have been identified. In vivo imaging reveals muscle fibre de-differentiation makes a significant contribution to the regenerating tail blastema K. Echeverri1, J. Clarke2 and E. M. Tanaka1 1 Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany; 2 Department of Anatomy and Developmental Biology, University College London, London, UK During tail regeneration in urodele amphibians such as axolotls, all the tissue types including muscle, dermis, spinal cord and cartilage are regenerated. It is still unknown how this diversity of cell types is reformed with such precision. In particular, the number and variety of mature cell types in the remaining stump that contribute to the blastema is unclear. We have used Nomarski imaging to clearly document the process of regeneration in the larval axolotl tail. We have also used in vivo fluorescent labelling of single muscle fibres to show that mature muscle de-differentiates and contributes significantly to the blastema. Our data shows that muscle de-differentiation occurs by the ,falling apart' of the muscle fibre into mononucleate cells, followed by rapid cell proliferation and an increase in cell-to-cell contact by the extension of processes. We further show that both direct clipping of the muscle fibre and severe tissue damage around the fibre are required to initiate de-differentiation. Our observations also make it possible for the first time to assess how many of the blastema cells arise specifically from muscle de-differentiation. Our calculations indicate that up to one-third of the cells in the blastema come from de-differentiation of mature muscle fibres. A mathematical model of human granulopoiesis to identify optimal G-CSF scheduling during chemotherapy C. Engel, P. Stumpp, H. Franke, S. Schmitz and M. Loeffler Institute for Medical Informatics, Statistics, and Epidemiology (IMISE), University of Leipzig, Leipzig, Germany G-CSF is widely used to improve granulopoietic recovery during myelotoxic chemotherapy regimen. However, optimal leukocyte recovery largely depends on G-CSF administration timing. In order to identify optimal G-CSF scheduling we developed a quantitative mathematical model of human granulopoiesis. The model was applied to simulate the leukocyte population dynamics during 2-weekly CHO[E]P-chemotherapy (CHO[E]P-14) with 10 days (d4-d13) of G-CSF support and 3-weekly CHO[E]P (CHO[E]P-21) without G-CSF. The model describes the population kinetics of hematopoietic stem cells, granuloid precursors and mature blood neutrophils. Major assumptions were that,G-CSF dose-dependently increases granulopoietic amplification, shortens maturation and enhances the release of mature neutrophils to the blood. Chemotherapy is assumed to cause an acute depletion of stem cells and immature precursors. Clinical trial data on the average time course of leukocytes during six cycles of CHO[E]P-21 and CHO[E]P-14 were available from about 1300 patients of a large multicentre trial of the German High-Grade Non-Hodgkin's Lymphoma Study Group. In a first step, the model parameters quantifying the extent of myelosuppression of CHO[E]P were determined by fitting the model to CHO[E]P-21 data. Without changing these parameters, CHO[E]P-14 + G-CSF (d4-d13) could be simulated with good agreement to the clinical data by changing the cycle interval and introducing exogenous G-CSF administration. Simulations of different G-CSF schedulings showed that sufficient recovery of leukocytes should be achieved with G-CSF support from d8-d12 (5 days), which is 5 days less than the schedule currently used. Based on our model results a pilot study has recently been initiated to evaluate the safety and feasibility of the proposed 5-day scheduling. A mathematical model of murine granulo- and erythropoiesis during continuous rhG-CSF stimulation C. Engel, G. de Haan, W. Nijhof and M. Loeffler Institute for Medical Informatics, Statistics, and Epidemiology (IMISE), University of Leipzig, Leipzig, Germany It was our aim to develop a quantitative simulation model to comprehensively explain the experimentally observable phenomena in normal murine hemopoiesis during long-term exogenous G-CSF administration in vivo. We use a mathematical model which describes hemopoiesis by concatenated cell compartments and cell flux rates. Model assumptions about cell kinetic effects of G-CSF are tested by comparing model simulations with a large set of experimental data reported from different authors and laboratories. From these model simulations we conclude that the superposition of the following effects explains the complex behaviour of G-CSF stimulated hemopoiesis: G-CSF (1) enhances mitotic amplification of CFU-GM (mainly in the spleen) and proliferative granuloblasts (mainly in the marrow); (2) accelerates marrow maturation of postmitotic cells; (3) blocks marrow erythropoiesis at the BFU-E stage; (4) induces migration of CFU-S, CFU-GM and BFU-E from marrow to spleen. Furthermore, we conclude a novel mechanism: since G-CSF-induced neutrophilia cannot be explained quantitatively by only assuming enhanced amplification and accelerated differentiation, we hypothesize that (5) G-CSF-mediated prevention of intramedullary apoptosis (,ineffective granulopoiesis') of mature cells is a relevant mechanism to gain more blood neutrophils. (6) Granulopoiesis once shifted to the spleen is more responsive to G-CSF than in the bone marrow. All effects are quantified by dose,response characteristics. The model permits estimation of the contribution of each mentioned G-CSF effect on in vivo hemopoiesis and allows predictions and suggestions to be made for further experimental studies. Cyclosphospamide-induced apoptosis in the experimental model of murine lymphosarcoma O. V. Falameyeva1, S. Y. Djanayeva1, V. I. Kaledin2, O. N. Poteryaeva1 and T. A. Korolenko1 1 Institute of Physiology RAMS, Novosibirsk, Russia; 2 Institute of Cytology and Genetics RAS, Novosibirsk, Russia Introduction. The initiation of apoptosis (programmed cell death) relies on the activation of cysteine and aspartic proteinases; caspase-mediated proteolysis is a critical event in apoptosis. The aim of the study was to investigate the role of cathepsins B, L, D (CB, CL, CD) and the cystatin C (CC) ratio in the apoptosis model of lymphosarcoma (LS) treatment by cyclophospahmide (CPA). Methods. LS tumour cells (1 × 106) were transplanted i.m. into CBA mice. Mice were treated with CPA (Sigma, USA, i.v., 25 mg/kg or 50 mg/kg, single). CB and CL activities were determined against fluorogenic substrates (with specific inhibitor CA 074 for CB), and CD activity against azocasein as a substrate. CC concentration was measured using ELISA kits (Krka, Novo Mesto, Slovenia). Results. LS development was followed by an increase in specific CB and CL activity and a low CC level in tumour tissue. CPA treatment at a dose of 25 mg/kg did not influence tumour weight or CC level. However, CPA treatment at a dose of 50 mg/kg was followed by a significant reduction of tumour weight (180%) and increased CB (4 times), CL (6 times) and CD (1.5 times) activities and a several-fold increase in CC concentration. DNA study ladder structures suggested apoptosis development. Conclusion. Treatment of LS by CPA at the two dose levels had different effects on apoptosis in tumour cells. CB, CL and CD activities and CC level are markers of apoptosis in tumour cells. Lineage interactions during continuous combined EPO and G-CSF administration explained by a comprehensive model of murine erythro- and granulopoiesis C. Gebauer, C. Engel, G. de Haan, W. Nijhof and M. Loeffler Institute for Medical Informatics, Statistics and Epidemiology (IMISE), University of Leipzig, Leipzig, Germany We developed a mathematical model of murine granulo- and erythropoiesis to quantitatively describe the cell kinetic effects of combined G-CSF and EPO treatment with respect to possible synergistic or antagonistic effects between lineages and growth factors. Dose,response data of bone marrow and spleen hematopoiesis were obtained from a full-factorial experiment in which EPO and G-CSF were given continuously for 7 days. The model describes hematopoietic cell stages by concatenated compartments. Assumptions about growth factor effects were tested by comparing model simulations with experimental data. The model simulations suggest that the following assumptions can comprehensively explain our experimental findings. Synergistic effects. An increased splenic contribution of both lineages is explained by migration of early progenitors and stimulation of both lineages by EPO and G-CSF. Antagonistic effects. G-CSF dose-dependently blocks marrow erythropoiesis, which can be compensated by EPO to a certain extent. Bone marrow suppression of erythropoiesis is quantitatively compensated by the splenic increase. The behaviour of granulo- and erythropoiesis in splenectomized mice during combined EPO and G-CSF treatment is also well explained by this model. The suggested model gives basic insight into the quantitative effects of combined growth factor treatment on murine hematopoiesis. Transferred to human hematopoiesis, such models could help to optimize multiple growth factor treatment during chemotherapy and to detect possible hazardous combinations. Links between molecular defects and cell kinetics in CML M. Y. Gordon, S. B. Marley, J. L. Lewis, R. J. Davidson, D. X. Nguyen and J. M. Goldman Department of Haematology, Imperial College School of Medicine, Hammersmith Campus, London, UK Expression of the p210Bcr,Abl tyrosine kinase is responsible for the abnormal proliferation of stem/progenitor cells in CML and results in expansion of the myeloid mass. Much of this expansion may be a consequence of increased proliferation by granulocyte-macrophage colony-forming cells (CFU-GM) which has been demonstrated in vitro by replating primary CFU-GM colonies and observing secondary colony formation. The Abl tyrosine kinase inhibitor STI571 suppresses CML, but not normal, CFU-GM proliferation supporting the view that p210Bcr,Abl is responsible for CML CFU-GM expansion. The PI3-kinase, Ras and Jak/Stat signalling cascades are activated by p210Bcr,Abl and may therefore participate in transducing the signals from p210Bcr,Abl which result in increased proliferation. Inhibitors of PI3-kinase (wortmannin and LY294002), like STI571, preferentially suppressed CML CFU-GM proliferation. PI3-kinase is downstream of Ras and Jak/Stat. Inhibition of Jak2 with tyrphostin AG490, but not of Ras with a farnesyltransferase inhibitor (FTI II , Calbiochem) reproduced the effects of STI571 on CML CFU-GM. The relevant intermediates downstream of PI3-kinase have not yet been identified but signalling may culminate with components of the retinoblastoma protein (pRb) pathway since deletion of the cyclin-dependent kinase inhibitors (CDKIs) p16, p21 and p27 results in hyperproliferation of CFU-GM. Knowledge of signal transduction pathways controlling progenitor cell proliferation should aid the selection of molecular therapies designed to suppress myeloid expansion in CML. A share of cell,cell contact interactions in cell growth regulation in vitro R. Grigorian, N. Sarkissian, A. Sayadian and G. Gasparian Laboratory of Genetics, Institute of Zoology of Armenian NAS, 7, Sevak St., Yerevan 37014, Armenia (E-mail:zool@sci.am) Integration of different intracellular signal transduction pathways is believed to suggest the interchangeability, at least partially, of various signal-inducing factors. Two experimental cell systems were applied to test this idea. The first was a sparse culture of normal fibroblasts where cell,cell touching is an infrequent event. In this system the cell density rise may be mimicked by adding ethanol-fixed homologous cells, and the medium enrichment may be simulated by replacing the medium with the conditioned one. This allows one to identify effects of cell,cell contacts and diffusible factors of the medium separately. The second system consisted of the cells cultured in serum-free medium. Varying the cell population density enables the role of cell,cell contacts to be elucidated. Using the systems described we have been studying the contribution of intercellular contacts in cell regulation. It was shown that they could both inhibit and stimulate cell multiplication. With unavailability of serum factors, a cell survival-promoting influence of intercellular contacts was found. The results suggest that insoluble molecules exposed on the cell surface and soluble serum factors can substitute each other in cell behaviour control. Thus, direct cell,cell interaction may be considered as a kind of instructive event subsequently processed in the integrated system of cell signalling. Isolation and characterization of primitive hematopoietic stem cells (HSC) via Hoechst sorting from various hematopoietic cell sources Y. Guo, M. Follo, M. Lübbert and M. Engelhardt Hematology/Oncology Department, University Medical Center, 79106 Freiburg, Germany Efflux of the vital dye Hoechst (HO) 33342 allows the identification of a small ,side population' (SP) of HOlow cells. Murine SP cells have previously been described as CD34- and highly enriched for cells capable of multilineage repopulation. Human SP cells, however, seem more heterogeneous, express CD34 and to date have been less extensively characterized. Therefore, we analysed the frequency of and potential differences between SP cells from murine BM (n = 10), CB (n = 15), apheresis (AP, n = 23), normal BM (n = 6), the Kasumi-1 cell line and BM from patients with hematological malignancies (n = 9), functionally confirming SP cells with verapamil. Kasumi-1 cells revealed no SP pattern. From all murine and human SP cells, we observed a median frequency of 0.02% (range: 0.001,0.33), a mostly CD34 phenotype, and a CFU and LTC-IC increase of 1- and 49.5-fold, respectively. From murine BM, SP cells were found with highest percentages (median 0.03%) and were increased for CFUs by a median of 127.5-fold (n = 5) as compared to unsorted cells. In humans, the SP frequency was lower (0.02%), with highest numbers in G-CSF- or chemotherapy plus G-CSF-mobilized AP (median: 0.03%) and lower from CB (0.02%) or BM (0.01%) specimens. Human SP cells were mostly CD34 (median 96.3%, range 99.4,50.2%, n = 10). The CFU potential from human SP cells was low, increasing 4.6-fold from CB, but displaying no clonogenic potential from AP and normal BM as compared to unsorted cells. We found a small population by HO 33342 exclusion within BM cells from leukemia patients: the SP frequency was 0.01% and the clonogenic potential did not differ from AP and normal BM. Human SP cells from donor AP, CB, BM and patient AP samples were enriched for LTC-ICs with a median of 138-, 53-, 15- and 10-fold, respectively. Ongoing in vivo repopulating assays in NOD/SCID mice will further characterize the SP population, and detection of the presence of chromosomal abnormalities from patients should allow us to determine their normal or malignant nature. These results demonstrate that the refinement of this sorting strategy may permit to better define primitive HCSs of different ontogeny, and may aid in purification of normal vs. malignant HCSs from patients with hematological malignancies. Expression GM-CSF by mouse erythroid nuclear cells T. V. Injelevskaya, L. V. Eremina, S. V. Sennikov and V. A. Kozlov Institute of Clinical Immunology SB RAMS, 14, Yadrintsevskaya Str., 630099, Novosibirsk, Russia Previous research showed that treatment of erythroid nuclear cells by mitomicin C stimulates exogenous spleen colony formation (8,9 days) in lethally irradiated mice when mixed with bone marrow transplant from intact mice. The aim of our research was to discover possible cytokine mechanisms of this effect. We found that erythroid cells isolated from the spleens of newborn mice expressed mRNA GM-CSF. Gene expression was determined by RT-PCR. Mouse erythroid nuclear cells isolated from spleen with erythroid hyperplasia (after acute hypoxia, phenilhydrazine-induced anemia and after bleeding) expressed mRNA GM-CSF after addition of EPO. Further, we have studied GM-CSF production by erythroid nuclear cells isolated from spleen with erythroid hyperplasia and cultivated for 24 h. Conditional media of erythroid cells (1 × 106 cells/ml) was obtained and GM-CSF concentration measured by the method of electrochemiluminescence using polyclonal and monoclonal antibodies. Measured GM-CSF production of erythroid nuclear cells isolated from spleen with erythroid hyperplasia was 2210.98 pg/ml after acute hypoxia, 4201.84 pg/ml after phenylhydrazine-induced anemia, 6785.48 pg/ml after bleeding and 8875.72 pg/ml from newborn mice which is comparable to the production of thymic cells and splenocytes after mitogenic stimulation. We conclude that promotion of colony formation activity of bone marrow cells could be mediated by production of GM-CSF by erythroid cells. Anti-angiogenic drugs retard progression of two experimental leukemias P. O. Iversen, D. R. Sørensen and H. B. Benestad Institute Basic Medical Sciences, University of Oslo, Oslo, Norway Angiogenesis is essential for growth, invasion and metastasis of solid tumours. Angiogenesis inhibitors, like endostatin (ES) and phosphomannopentaose sulphate (PI-88), have retarded and in some cases stopped growth in various models of cancer. A role for angiogenesis in the pathophysiology of hematological malignancies has also been suggested. We have tested this suggestion in two leukemia models, i.e. juvenile myelomonocytic leukemia (JMML) developing in severe combined immunodeficient, non-obese diabetic (SCID/NOD) mice, and a promyelocytic leukemia transplantable to the Brown Norwegian rat (BNML). Flow cytometry was used to monitor the growth of transplanted cells in the bone marrow of the mouse and rat recipients. Human-specific fluorescent antibodies to CD45 were employed to mark JMML and normal human bone marrow cells; the RM-124 antibody to tag the BNML cells. Immunohistochemistry with antivon Willebrand factor was used to assess the density of microvessels in bone marrow sections. Eight weeks after leukemic cell transplantation, with daily injections of the drugs for the last 4 weeks, the leukemic cell burden had been reduced from almost 90% of all mouse marrow cells retrieved to about 15% with ES, to about 35% with PI-88, and to about 10% with ES + PI-88. Normal human bone marrow cells transplanted as a control constituted 55,60% of the mouse marrow cells. This ratio was not detectably changed by drug treatments. In SCID/NOD mice, microvessel density after JMML transplantation was high, almost 10 times higher than in the other groups. However, PI-88 only reduced the vessel density to twice control value. JMML colony numbers in semisolid cultures were not significantly altered by ES or PI-88. Very similar findings were made with the BNML model, confirming the synergism between ES and PI-88 concerning both leukemic cell growth and reduction of microvascularity. Again, neither leukemic nor normal cell growth in cell suspension cultures (3H-thymidine incorporation) was significantly affected by ES or PI-88. In conclusion, antiangiogenic therapy may be a valuable adjunct to cytotoxic or other modes of leukemia therapy. The behaviour of hematopoietic stem cells is autonomously regulated L. M. Kamminga, I. Akkerman, E. Weersing, A. Ausema, B. Dontje, G. Van Zant and G. de Haan Department of Stem Cell Biology, University of Groningen, Groningen, the Netherlands Mechanisms that affect the function of primitive hematopoietic stem cells with long-term proliferative potential remain largely unknown. We have previously shown that DBA (D2) stem cells are more numerous, cycle more actively and are more easily mobilized to the peripheral blood compared to C57BL/6 (B6). To assess whether these properties of stem cells are cell-extrinsically or cell-autonomously regulated, we developed a model in which D2 and B6 stem cells coexist in a single animal. Chimeric mice were produced by transplanting lethally irradiated B6D2F1 (BDF1) recipients with mixtures of D2 and B6 day-14 fetal liver cells to prevent ,graft vs. graft' disease. Donor leukocyte contribution was assessed using anti-H2Kb/d antibodies. We determined a series of stem cell characteristics of D2 and B6 cells in animals with chimeric hematopoiesis. After pegylated G-CSF administration peripheral blood D2 CFU-GM were 4,8 fold more numerous than B6 progenitors. In addition, D2 and B6 progenitors maintained their genotype-specific cycling activity in BDF1 recipients. Chimeric marrow was harvested and D2 and B6 cell populations were separated by flow cytometry. Parallel cobblestone area-forming cell (CAFC) analysis on both samples showed that the number of late appearing CAFC subsets within the D2 cell population was ~3 fold higher than within the B6 fraction. Finally, a secondary transplantation was performed using unfractionated chimeric marrow, administered to lethally irradiated BDF1 recipients. Comparison of the proportion of animals possessing D2 and/or B6 leukocytes 5 months after transplant revealed that the frequency of D2 LTRA was ~10 fold higher than B6 LTRA numbers. These data demonstrate that genetically distinct stem cell populations, coexisting in individual animals, independently maintain their parental phenotypes. This indicates that stem cell properties are predominantly regulated cell-autonomously. Involvement of laminins and Cdx genes in intestinal development and homeostasis M. Kedinger, C. Domon-Dell, O. Lefebvre, I. Duluc, J. N. Freund and P. Simon-Assmann INSERM U.381 ,Ontogenesis and Pathology of the Gut', 67200 Strasbourg, France The importance of epithelial,mesenchymal cell interactions during intestinal morphogenesis and differentiation, but also in the homeostasis of the adult epithelium, has been largely described in our laboratory. Two families of molecules have retained our attention, the laminins and the transcription factors Cdx. We show their involvement in the cellular cross-talk taking place in the developmental phase but also in the development of tumours when their expression is altered. Laminins are major constituents of the subepithelial basement membrane (BM). Various laminin isoforms are expressed in the intestinal tissue, and their expression pattern varies as a function of the developmental stage and along the crypt,villus axis. They are produced in a complementary manner by both epithelial and mesenchymal cells. The role of two of them has been analysed by using various experimental procedures. Laminin-1, localized early in the fetal gut and restricted to the crypt BM in the adult tissue, is required for cell proliferation and the onset of cell differentiation; its expression can be modulated by glucocorticoids. Laminin-5, localized in the adult villus BM, is up-regulated in intestinal epithelial cells by the hepatocyte growth factor produced by mesenchymal cells, and may be involved in cell migration. On the pathological side, the expression of laminin-1 is correlated to the growth of tumours, their infiltration by stromal cells and angiogenesis and to the expression of the integrin ,6,4. Cdx1 and Cdx2 genes are the mammalian homologues of the drosophila caudal homeobox gene. They are expressed specifically in the intestinal epithelium from fetal stages to the adult stage where they are restricted, respectively, to the crypt cells, and to the upper crypt and villus cells. It has also been demonstrated that Cdx1 and Cdx2 expression is altered in colorectal cancers. We showed that both genes are modulated by the nature of the mesenchymal cells and that they are additionally regulated by complementary signalling pathways: the expression of Cdx1 has been shown to be stimulated by the Wnt/, catenin pathway in embryonic intestinal endodermal cells as well as in colonic tumour cell lines, and the expression of Cdx2 is stimulated (i) by laminin-1; the various effects of laminin-1 (cell differentiation and changes in cell,cell and cell,matrix adhesion properties) may be secondary to this prime effect of laminin on Cdx2, and (ii) by the tumour suppressor gene PTEN which antagonizes the PI3K pathways. Taken together, these data indicate that intestinal development and homeostasis depend on epithelial,mesenchymal interactions that involve various laminin isoforms and the Cdx homeobox genes. Interestingly, deregulation of the expression of both laminins and Cdx genes are also involved in colon tumorigenesis. Potential toxicity of sodium selenite to human hematopoiesis Z. Kheder1, B. Millo2, J. Zawierta3 and B. Machalinski1 1 Department of General Pathology, 2 Department of Clinical Biochemistry and Laboratory Diagnostic and 3 Department of Biochemistry and Chemistry, Pomeranian Medical Academy, Szczecin, Poland The influence of the selenium on human hematopoiesis has not been addressed sufficiently in the literature. However, the environmental selenium content has already been connected with increasing morbidity due to the hematological diseases. Knowledge of the linkage between selenium compounds and human hematopoiesis would be of interest for several reasons. Insight into the potential toxicity of selenium to normal hematopoietic cell growth, viability, differentiation and apoptosis might be gained. With these issues in mind we sought to characterize the influence of sodium selenite on human hematopoiesis using a variety of techniques. We also attempt to discern the influence of Na2SeO3 on the morphology of the different organs by employing a murine model. Accordingly, we exposed human bone marrow (BM) and cord blood (CB) hematopoietic cells ex vivo to different doses of Na2SeO3 and subsequently looked for the effect of the exposure on the clonogenecity of human CFU-GM (colony forming unit of granulocyte-macrophages) and BFU-E (burst forming unit of erythocytes). We found that relatively low concentrations of sodium selenite (100 and 1000 g/l) were toxic to marrow myeloid (CFU-GM) and erythroid (BFU-E) progenitors. The inhibition of colony growth was more evident in the BM and after incubation at 37 °C compared to 4 °C. A similar effect was also observed when viability of the cells was examined using the trypan blue exclusion test. Moreover, we employed two different assays evaluating the early phase (Annexin V -test using FACS technique) as well as the executive phase of apoptosis (ELISA test) in BM and CB hematopoietic cells. Both phases of apoptosis were detectable in BM and CB hematopoietic cells exposed to different doses of sodium selenite. Finally, we injected normal mice with increasing doses of Na2SeO3 and subsequently evaluated them for the presence of any morphological changes in the livers, spleens, kidneys, lungs and bones. We found that sodium selenite caused morphological changes in the spleens and lungs. In contrast, livers, kidneys and bones appeared to be relatively resistant to this substance. This study shows that sodium selenite could potentially damage human cells involved in hematopoiesis. Effect of sodium selenite on bone marrow transplant engraftment Z. Kheder1, A. Jarema2 and B. Machalinski1 1 Department of General Pathology and 2 Clinic of Radiotherapy, Pomeranian Medical Academy, Szczecin, Poland Our previous studies suggested that Na2SeO3 might be toxic to early human bone marrow and cord blood hematopoietic cells. Expanding our preliminary report, we performed an additional experiment designed to evaluate whether hematopoietic cells, treated with sodium selenite in ex vivo conditions, were able to engraft into marrow cavities of lethally irradiated mice and successfully reconstitute hematopoiesis. Accordingly, we aspirated bone marrow from freshly decapitated Balb C mice. Next we isolated the adherent depleted mononuclear cell (A,MNC) fraction, and subsequently treated it with different doses of sodium selenite for 12 h at 37 °C. Meanwhile, other groups of mice were exposed to 800 cGy from a gamma irradiation source 24 h prior to transplantation. Cells exposed to Na2SeO3 in ex vivo conditions were then evaluated for their ability to engraft in the irradiated mice. We report here that untreated cells as well as the cells exposed to a low (100 µg/l) dose of Na2SeO3 were able to establish significant numbers of new hematopoietic islets on the surface of spleens. In contrast, much fewer hematopoietic islets were found in the cells exposed to higher doses of sodium selenite (1000 and 10000 µg/l). The morphological parameter values of peripheral blood derived from the examined mice confirmed this relation. Based on the results we conclude that selenium may significantly influence hematopoiesis. 3D cellular automata modelling of granulopoiesis: feedback regulation and stem cell dynamics T. Komatsuzaki1, Y. Saikawa2, Y. Iwata1, S. Koizumi2 and H. Sato1 1 Department of Human & Mechanical Systems Engineering, Faculty of Engineering, and 2 Department of Pediatrics, Faculty of Medicine, Kanazawa University, Kanazawa, Japan The two specific questions at issue in hematopoiesis are (1) how the self-maintenance and differentiation commitment of the hematopoietic stem cells (HSC) are achieved and (2) how the peripheral control of hematopoiesis is regulated. Several mathematical models that incorporate a negative feedback circuit have been proposed to explore the regulatory dynamics of hematopoiesis and are still unsatisfactory. In the present study, we have applied a 3D cellular automaton (CA) model to granlopoiesis. CA is a discrete dynamical system for modelling complex processes and will allow us to analyse the theoretical relationship between the microscopic mechanisms and the macroscopic behaviour of hematopoietic processes. The modelling parameters include the number of mitotic event of the cells in a proliferating phase, the transit time of each of 15 differential stages from CFU-G to the mature cells, and the neighbourhood rules for HSC proliferation. Consistent with clinical observations, the HSC population was maintained at a frequency of 0.2% in a typical set of simulation models. Importantly, stabilization of the cell production was induced in a negative feedback manner following external perturbation of the peripheral granulocyte numbers. Kinetic analyses of the HSC responding to this peripheral perturbation demonstrated the regulatory relationship between self-renewal and lineage-commitment processes. Since this model is capable of marking the HSC and subsequently tracing their fate, CA modelling will provide a framework to analyse the behaviour of HSC and to understand abnormal kinetics of the diseases with periodic hematopoiesis. The role of cystatin C and Stefin A as markers of tumour growth and efficacy of antitumour therapy T. A. Korolenko1, O. V. Falameyeva, O. N. Poteryaeva, O. A. Levina, I. G., Svechnikova1, V. I. Kaledin2 and J. Nowicky3 1 Laboratory of Cellular Biochemistry, Institute of Physiology RAMS; 2 Institute of Cytology and Genetics RAS, Novosibirsk, Russia; 3 Nowicky Pharma, Austria, Vienna Aim. To study the effectiveness of the macrophage stimulators carboxymethylated glucan (CMG), Ukraine (U) treatment in murine LS lymphosarcoma and Lewis lung adenocarcinoma with special attention to cysteine proteinases (CP) and CP inhibitors cystatin c and Stefin a as markers of tumour growth and metastasis. Methods. CBAC57BL/6 mice were used. Animals with LS lymphosarcoma or Lewis lung adenocarcinoma were treated with cyclophosphane (CPA, 150 mg/kg, i.p., single) and/or CMG (Institute of Chemistry SAS, Slovakia, 25 mg/kg, i.p., single) and/or U (Nowicky Pharma, Austria, 0.5 mg per mouse i.p., single) as macrophage stimulators. The activity of cysteine proteinases (cathepsins B and L) was measured against fluorogenic substrates with specific inhibitor (CA-074 for cathepsin B); concentrations of CP inhibitors cystatin c and Stefin a were determined using ELISA kits (Krka, Slovenia). Results. Significant antitumour effects were found in both types of tumour with the help of a combination of CPA and macrophage stimulator CMG or U (reproducible and significant retardation of tumour growth and as a consequence prolongation of life-span). In Lewis lung adenocarcinoma, treatment with CPA + CMG significantly decreased not only tumour growth but also the metastasizing to the lung. In untreated groups, CP activity in tumour tissue was increased and a low concentration of CP inhibitors was noted. CPA or CPA + U treatment of LS lymphosarcoma increased extracellular CP inhibitor cystatin c by 7 times in tumour tissue (to a lower degree in spleen and serum) as compared to untreated mice. Intracellular inhibitor of CP Stefin a concentration was low in the tumours of untreated and treated mice. CPA + U or U treatment of Ls lymphosarcoma increased cystatin c concentration in tumour tissue and liver. Conclusion. In the tumour models studied, macrophage stimulation in combined treatment was the most effective (especially treatment with CPA + CMG in Lewis lung adenocarcinoma). The concentration of the extracellular CP inhibitor cystatin c is an important marker of malignization and antitumour treatment efficacy as compared to the level of Stefin a, an intracellular CP inhibitor. P53 pathway is not involved in apoptosis induction by iron deprivation J. Kováø, J. Truksa and T. Valenta Cell Growth Control Laboratory, Institute of Molecular Genetics, Prague, Czech Republic Iron deprivation induces apoptosis in some cultured tumour cells (human Raji, mouse 38C13) while other cells (human HeLa, mouse EL4) are resistant. In order to obtain a better understanding of mechanisms involved in apoptosis induction by iron deprivation we studied the expression of genes involved in the p53 pathway of apoptosis induction. We compared the p53 level in sensitive vs. resistant cells under control and iron-depriving conditions. Western blot analysis and flow cytometry showed that the p53 level was not changed by iron deprivation in both sensitive and resistant cells. However, some decrease in the level of p53 mRNA due to iron deprivation was seen. Interestingly, the p21CIP1/WAF1 level in sensitive 38C13 cells increased after iron deprivation. In contrast, the p21CIP1/WAF1 level in sensitive Raji cells seemed to decrease. The level of p21CIP1/WAF in resistant cell lines did not change. The level of p21CIP1/WAF1 mRNA did not seem to change significantly in resistant as well as in sensitive cells. Similarly, the expression of another p53-controlled gene mdm2 was not significantly affected by iron deprivation in resistant as well as in sensitive cells. In either cell type iron deprivation did not lead to any significant change in the level or expression of antiapoptotic Bcl-2. The level of proapoptotic Bax protein was somewhat increased due to iron deprivation, however, in both sensitive and resistant cells. The level of bax mRNA did not change in either cell type. Taken together, these results suggest that the classical p53 pathway of apoptosis induction is not involved in apoptosis induction by iron deprivation. A novel model of hematopoietic and epithelial stem cells M. Loeffler1, I. Roeder1, F. A. Meineke1 and C. S. Potten2 1 Institute for Medical Informatics and Statistics, University of Leipzig, Germany; 2 Epigraft, Manchester, UK Highly proliferating tissues like hematopoiesis, epidermal and intestinal epithelia are regenerated from tissue-specific stem cells. The definition of stem cells implies that they are relatively undifferentiated cells capable of proliferation, self-maintenance, the production of large numbers of differentiated functional progeny, the regeneration of the tissue after injury and a flexibility in the use of these options8. Despite many efforts to identify individual stem cells using markers, functional assays have remained the diagnostic gold standard. They typically investigate the growth potential in vitro (e.g. colony formation) or in vivo (e.g. colony formation in reconstituting tissues, clonal competition assays). A variety of theoretical models has been suggested to provide a consistent explanation of somatic stem cell functioning. Classical models are based on the idea that a stem cell has a particular ,stemness' identity discriminating it from the descendent transit cells. In order to maintain these distinctions, mechanisms like asymmetric cell divisions have been postulated for stem cells and restricted proliferative pedigrees for the transit cell populations. In this presentation we critically review these approaches and their conceptual limitations. To overcome these limitations we propose a novel class of models based on a system theoretic approach. We postulate that each cell in a proliferating tissue at any time has several options for future development (potentials). Such options are, e.g., going into or leaving the cell cycle, expressing a particular property or not, choosing between two differentiation pathways. There are now two levels of regulation. One determines the set of options (potentials) a particular cell has. The second determines which of these options are selected (activated) in the particular setting (propensities). Both levels of regulation are assumed to depend on the general status of the tissue, the previous history of the individual cell and on the local growth environment in which it is placed. This concept makes no a priori distinction between stem cells and other cells but generates cells that gain and loose appropriate properties and hence behave accordingly in functional settings. This concept has been implemented as computer models for the hematopoietic tissue and for the small intestinal crypt. Simulations demonstrate that this class of model is powerful, removes many conceptual limitations in previous models and provides a comprehensive understanding of in vivo data on hematopoietic cell kinetics, colony formation, clone competition, retroviral marking experiments and recovery from injury. The model also leads to a reinterpretation of the in vitro functional hematopoietic colony assays. An analogous model for the spatial organization of the crypt epithelium provides explanations for clonogenicity data after injury and for data on conversion of single crypts to monoclonicity after mutation experiments. Differential expression of EGR-1 gene within gpi-deficient myeloid cells from patients with paroxysmal nocturnal hemoglobinuria A. Lyakisheva, R. E. Schmidt and J. Schubert Department of Clinical Immunology, Hannover Medical School, Germany Paroxysmal nocturnal hemoglobinuria (PNH) has been characterized by the appearance of peripheral blood cells deficient in GPI-anchored surface proteins. This defect could be attributed to mutations within the PIG-A gene encoding for a protein involved in GPI-anchor biosynthesis. Since GPI-deficient cells do not expand by themselves, the mechanism enabling the PNH stem cell to expand within the bone marrow has not yet been clarified. Therefore, a second event leading to clonal expansion needs to be assumed. According to our hypothesis this could be due to an intrinsic growth advantage because of additional mutations within their cellular activating repertoire. In order to identify genes differentially expressed in PNH cells in comparison to normal cells, we performed PCR-based RNA fingerprinting. As a model for GPI-deficient clone we used cultured normal and GPI-deficient T-cell lines from one PNH patient. Twelve candidate genes with a differential expression profile were isolated. Furthermore, testing of gene expression in GPI-deficient granulocytes by Northern blot analysis has confirmed EGR-1 as a candidate gene that is highly up-regulated in all analysed PNH cases in comparison to normal cells. In order to address external influences such as cytokines leading to a differential expression of EGR-1 in PNH granulocytes we tested expression in patients receiving high doses of G-CSF for mobilization of CD34+ cells. Application of exogenous G-CSF did not lead to an overexpression of EGR-1 in the patient's granulocytes. In addition, incubation of normal granulocytes in patients' plasma did not induce an increase in the EGR-1 mRNA level. Further analysis of granulocytes obtained from patients with other hematological diseases revealed an overexpression of this gene only in some cases of myeloproliferative and myelodysplastic diseases. We conclude from our data that clonal expansion of GPI-deficient myeloid cells from PNH patients could be due to the overexpression of transcription factors such as EGR-1. Further analysis will address the molecular events leading to the gene overexpression within the deficient cells. Introduction of plasmid DNA and oligonucleotides (ODN) into human normal and myelogenous leukemia derived cells: comparison study and clinical implications for gene therapy W. Marlicz, A. Machalinska and B. Machalinski Department of General Pathology, Pomeranian Academy of Medicine, Szczecin, Poland The efficient introduction of foreign DNA into human normal and neoplastic hematopoietic cells is still the limiting step of gene therapy. With this in mind we sought to determine the efficacy of several nonviral transfection techniques [cationic liposomes , lipofectamine and cholesterol conjugated liposomes (DC-Chol liposomes), electroporation and streptolysin O] in efficient delivery of plasmid DNA and oligonucleotides (ODN) into normal human progenitor cells as well as human chronic myelogenous leukemia derived cells. For this purpose, human normal progenitor A,T, MNC, K-562 cells and leukemic cells obtained from CML patients in blast crisis were transfected using non-viral techniques with oligodeoxynucleotides (antic-myb) and reporter plasmids containing genes encoding chloramphenicol acetyl transferase (CAT plasmid) and human placental alkaline phosphatase (PAP plasmid). The efficacy of plasmid transfections were evaluated within 48 h using CAT and PAP assays. The efficacy of ODN transfections was evaluated 2 and 5 h after transfection by dot-blot. Additionally, the intracellular localization of biotin-labelled oligomers in transfected cells was visualized by electron microscopy. We found that none of the methods examined allowed delivery of plasmid DNA into normal human progenitor cells as well as cells obtained from CML patients. Only cationic liposomes enabled efficient transfection of plasmid DNA into K-562 cells. Additionally, cationic liposomes allowed for efficient transfection of normal progenitors (~9%) and leukemic cells (~16%) with ODN. Moreover, we found that labelled oligomers were localized in significant amounts in the cell nuclei shortly after transfection. Of note, ultrastructural analysis of electroporated and streptolysin O treated cells revealed profound damage of cell membranes and presence of ODN mostly in vesicular structures. These observations support our previous conclusions that cationic liposomes could be useful in at least some forms of gene therapy and could be efficiently employed as adjuvant compounds for bone marrow purging with oligodeoxynucleotides. A functional analysis of receptors with intrinsic tyrosine kinase activity on human AML and CML derived cells W. Marlicz1, A. Machalinska1, M. Masiuk1, A. Karbicka1, M. Kawa1, B. Kolodziej2, W. Domagala2 and B. Machalinski1 1 Department of General Pathology; 2 Department of Pathomorphology, Pomeranian Academy of Medicine, Szczecin, Poland A number of hematopoietic cytokines are bound by receptors with intrinsic tyrosine kinase activity (TKR). Examples include C-KIT, INS-R, and C-MET. Knowledge of the role of these receptors in regulating the biology of human leukemic cells would be of interest for several reasons. For example, insight into the importance of a given receptor/ligand pair in regulating leukemic cell growth might contribute to better prognosis and/or response to the chemotherapy. With these issues in mind, we sought to characterize the expression of several receptors with intrinsic tyrosine kinase activity [C-KIT, IGF-1R, INS (insulin) R, C-MET, CSF-1R, STK-1R, PDGF-AA and C-TRK] on human leukemic blasts, as well as expression of ligands binding to receptors with intrinsic tyrosine kinase activity (KL, IGF-1, INS, HGF, CSF-1, FLT-3, PDGF and NGF) in the same cells. We also attempted to discern the importance of these growth factors in regulating proliferation and inhibiting apoptosis of human leukemic blasts. In our studies, the expression of receptors with intrinsic tyrosine kinase activity and their ligands was evaluated both at the mRNA level by RT-PCR and at the protein level by employing FACS analysis in AML derived (n = 30) and CML (n = 10) cells. RT-PCR analysis of total cellular RNA revealed mRNAs for all of these receptors, except for the NGF receptor TRK-A in blasts derived from AML and CML patients. Furthermore, the FACS analysis confirmed the presence of INS-R, C-KIT-R, CSF-1-R, IGF-1-R and C-MET-R on these cells. Of note, we observed that the level of receptors with intrinsic tyrosine kinase activity expression was higher on CML derived blasts as compared to AML cells. To our surprise, in repeated experiments, none of the exogenously added recombinant ligand binding to studied receptors (except for KL and bFGF) either costimulated growth of leukemic CFU-GM colonies in methylcellulose cultures or affected survival of leukemic blasts in the serum-free liquid cultures. Finally, since mRNAs for several TKR ligands were found to be expressed by AML and CML derived blasts (KL, INS, IGF-1, HGF), the potential role of intrinsic tyrosine kinase receptor/ligand axes in autocrine regulation of leukemic cell growth is currently being investigated in our laboratory. Radiation-induced effects on cell proliferation T. J. McMillan Department of Biological Sciences, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster, UK It has been recognized for many years that radiation can stop the cell cycle on a temporary and permanent basis as well as cause cell death. A revolution in our understanding of intracellular cell signalling systems means that we now have a very good idea of how many of these effects are mediated. There are, however, some important questions that remain to be answered. The genetic and biochemical characterization of the G1/S and the G2/M damage-responsive cell cycle checkpoints has given us important insights into the significance of these checkpoints in the overall cellular response to radiation. In particular, the intimate interplay between the checkpoints and DNA repair functions has been shown to be critical and linked to this is the key role for radiation-induced apoptosis in some tissues. It is not yet clear, however, which forms of radiation-induced damage are critical for these signalling pathways. Most emphasis has been placed on the radiation-induced double-strand break in radiobiology but, since other lesions can trigger the signalling pathways, does this increase the importance of these so-called ,lesser lesions'? In addition, a key step in the biochemical pathways that is still blank is the damage detection step. Protein complexes including the DNA-dependent protein kinase can bind to sites of DNA damage but whether this is truly the main damage detection step is still not clear. Despite the gaps, this ,new knowledge' has opened up some intriguing possibilities for the use of radiation in a therapeutic context and as the practicalities of gene therapy approaches are improved we must hope that they can be applied with success to radiation therapy in the clinic. Fidelity and infidelity in hematopoietic development F. Melchers Basel Institute for Immunology, Basel, Switzerland Transplantation of DHJH -rearranged surrogate light (SL) chain-expressing wild-type preB-I cells into RAG-deficient mice leads to long-term reconstitution of some of the mature B-cell compartments, to the establishment of normal levels of serum IgM, a third of the normal serum IgA levels, but not of detectable IgG levels. Neither T lineage nor myeloid cells of donor origin can be detected. The transplanted wild-type preB-I cells do not home back to the bone marrow. B lymphocyte development in PAX-5-deficient mice becomes arrested at the transition of preB-I to preB-II cells, i.e. at the stage where VH to DHJH rearrangements occur and where the preB cell receptor with µH chains and SL chains is normally formed. T-lineage and myeloid cell development is normal. PreB-I cells of PAX-5-deficient mice have a wild-type preB-I cell-like phenotype and they proliferate normally in vitro on stromal cells in the presence of IL-7. However, removal of IL-7 from the tissue cultures, unlike with wild-type preB-I cells, does not induce B-cell differentiation to sIgM+ B cells, but induces macrophage differentiation. Addition of GM-CSF to the IL-7-deprived cells induces the differentiation of MHC class II-expressing, antigen-presenting dendritic cells. In vitro differentiation to granulocytes and osteoclasts can also be observed in the presence of the appropriate cytokines. Moreover, transplantation of PAX-5-deficient preB-I cells into RAG-deficient hosts, while not allowing B-cell differentiation, leads to the full reconstitution of the thymus with all stages of CD4, CD8, and CD4+ CD8+ thymocytes, to normal positive and negative selection of thymocytes in the thymus, and to the development of normal, reactive mature CD4+ and CD8+ T-cell compartments in the peripheral lymphoid tissues. When clones of PAX-5-deficient preB-I cells carrying identifiable DHJH -rearranged H chain alleles are used for transplantation, the thymocytes and T cells all carry these DHJH rearrangements. On the other side, Ig,, Ig,, VpreB and ,5 are turned off in the thymocytes, demonstrating that the expression of these genes does not commit cells irreversibly to the B lineage. Furthermore, PAX-5-deficient preB-I cells home back to the bone marrow of the RAG-deficient host, can be reisolated and regrown in tissue culture, and retransplanted into a secondary RAG-deficient host which again develops thymocytes and T cells and allows the homing of the transplanted clonal preB-I cells to the bone marrow. Hence, the PAX-5-deficient preB-I cells are not only multipotent for T-lineage and myeloid cell differentiation but are also long-term reconstituting cells. Dynamical properties of germinal centre reactions M. Meyer-Hermann Institut für Theoretische Physik, TU Dresden, 01062 Dresden, Germany A humoral immunity response has a lot of interesting features. The presentation of a specific antigen leads to the activation of a set of B cells. In an early phase of immune response these may differentiate into antibody-producing cells which bind to the presented antigen epitopes with a certain affinity. Additionally, the activated B cells proliferate at a high rate in the environment of the follicular dendritic cells and develop into germinal centres. Here, the type of antibodies is optimized, i.e. the affinity of the antibodies to the presented antigen is enhanced. A new model is presented which describes the time evolution of the diverse cell populations as well as the optimization process in the germinal centre. The dynamics are represented by an appropriate set of standard differential equations. As all parameters are fixed by experimental data, it becomes possible to analyse essential features and quantities of germinal centre reactions quantitatively. Such quantities are the recycling probability, i.e. the probability that already positively selected centrocytes re-enter the proliferation process. Also, possible time delays for the production of plasma and memory cells from germinal centres are discussed. Furthermore, the question of how the result of a germinal centre may be optimized (concerning the number and quality of resulting antibody-producing cells) is addressed. Chimerism in embryos and adults following the injection of neural and hematopoietic stem cells into murine blastocysts A. M. Müller, S. Kreutzfeldt, S. Petrovic, N. Kirchhof, C. Schmittwolf, B. Mühl, A. Merkel and F. Harder Institut für Medizinische Strahlenkunde und Zellforschung (MSZ), University of Würzburg, Würzburg 97078, Germany Until recently, it was generally believed that the differentiation potential of a mammalian somatic stem cell is restricted to one tissue only. Recent reports, however, show that mammalian somatic stem cells can, in the course of regeneration, repopulate heterologous cell systems and therefore possess an unexpected plasticity. In order to analyse and compare the developmental potential of different somatic stem cell types, we have isolated murine adult bone marrow-derived hematopoietic stem cells (HSCs; LIN,, ckit+, SCA1+ cells) and murine fetal brain-derived neural stem cells (NSCs). NSCs are grown in vitro in media containing EGF and bFGF. Somatic stem cells were derived from transgenic mouse lines (i.e. eGFP, lacZ, bcl2). About 40 somatic stem cells were injected into preimplantation murine blastocysts and the repopulation pattern in developing chimeric animals was analysed. We detect progeny of the stem cells in more than 40% of the developing animals. Analysis of the tissue distribution of the injected donor cells revealed that progeny of the HSCs are preferentially detected in hematopoietic and at a lower frequency in non-hematopoietic tissues. Following the in vitro propagation of NSCs and their injection into blastocysts, NSCs are also capable of generating chimeric embryos and adults. Analysis of the donor contribution revealed NSC-derived hematopoietic repopulation, but overall, progeny of NSCs are mostly detected in neural tissues. Our results indicate that, even if somatic stem cells from different tissue origins are injected into an environment which is permissive for the development of all cell lineages of the developing embryo, they show plasticity but mostly retain their tissue-specific identity. Increased long-term donor cell engraftment in mice treated with growth factors prior to low dose total body irradiation and bone marrow transplantation E. J. K. Noach, A. Dethmers-Ausema, J. H. Dillingh, B. Dontje, E. Weersing, I. Akkerman, E. Vellenga and G. de Haan Department of Stem Cell Biology, University of Groningen, Groningen, the Netherlands Low toxicity conditioning regimens prior to bone marrow transplantation (BMT) are currently widely explored. However, methods to establish stable high levels of long-term donor cell engraftment still need to be improved and mechanisms of engraftment need to be clarified. Our aim is to develop new transplantation protocols using hematopoietic growth factors prior to low dose total body irradiaton (TBI) in recipients of autologous BMT to increase stem cell depletion and to create a high level of long-term donor cell engraftment. We hypothesize that a correlation exists between severeness of stem cell depletion in the host and successful donor cell engraftment after transplantation. Mice (C57BL/6, CD45.2) were treated for 1 or 7 days with different types of growth factors [SCF + IL-11, SCF + Flt3-ligand (FL) or G-CSF] prior to sublethal TBI (4 Gy). Percentage donor cell chimerism was measured by FACS analysis after transplantation of 3 × 106 CD45.1 congenic bone marrow cells into recipients. Although in recipients pretreated for 7 days with SCF + IL-11 or SCF + FL a strongly increased donor cell engraftment compared to controls was found immediately after transplantation, we observed a discrepancy between the engraftment patterns in the SCF + IL-11 and SCF + FL group: whereas engraftment remained very high and stabile in the SCF + FL treated recipients, donor cell contribution in the SCF + IL-11 group dropped towards control values after 24 weeks. In contrast, 1-day pretreatment with SCF + IL-11 and SCF + FL resulted initially in reduced donor cell engraftment, but a continuous increase over time was observed, finally resulting in a highly significantly increased donor cell contribution. G-CSF treatment showed no long-term beneficial effects on engraftment. We performed in vitro stem cell assays before and after TBI to determine the extent of stem cell depletion. A strong correlation (R2 = 0.957) existed between long-term donor cell engraftment and remaining stem cells after TBI, except for the groups treated for 1 day with SCF + IL-11 or SCF + FL. Growth factor-induced differential competitiveness between remaining recipient stem cells and transplanted donor cells may cause skewing of engraftment patterns over prolonged periods of time in these groups. We conclude that long-term donor cell engraftment can be strongly augmented by treatment of recipients with hematopoietic growth factors acting on primitive cells prior to low dose TBI. Potent support of hematopoietic stem cell activity by embryonic stromal cells from the subregions of the mouse AGM region R. Oostendorp, N. Kusadasi, K. Harvey, M. de Bruijn, R. Ploemacher and E. Dzierzak 3rd Medical Clinic, Technical University, Munich, Germany; Departments of Cell Biology and Hematology, Erasmus University, Rotterdam, the Netherlands The first adult repopulating hematopoietic stem cells (HSC) are autonomously generated in the aorta-gonads-mesonephros (AGM) region of murine and human embryonic development in mid-gestation. HSC activity is initially localized to the dorsal aorta, surrounding mesenchyme and other major vascular regions such as the vitelline and umbilical arteries. Thereafter, HSC activity rapidly increases in the urogenital ridges (UG) and the liver. To examine which embryonic microenvironment best supports and/or expands HSCs we established stromal cell lines from AGM subregions and the embryonic liver of mid-gestation mouse embryos. Several embryonic stromal clones were found to maintain adult hematopoietic progenitors and HSCs to a similar degree to, or better than, previously described murine adult and fetal stromal cell lines. Interestingly, the stromal clones most strongly supportive of early progenitors or HSCs are derived from the UG subregion of the AGM. This finding suggests that the AGM provides its own microenvironment for HSC growth and maintenance of the first HSCs during mid-gestation. Currently, we are comparing HSC-supportive with non-supportive stromal cell lines from the same or other embyonic subregions on a molecular level. These studies will be very important to establish which factors are required for generation and maintenance of HSC in the mid-gestation embryo. Simulations of B-cell selection in the germinal centre M. Or-Guil, E. F. Manffra and H. Kantz Max Planck Institute for the Physics of Complex Systems, Noethnitzer Str. 38, 01187 Dresden, Germany During the course of an acute immune reaction, germinal centres are founded with the aim of generating B cells through hypermutation able to produce antibodies with higher affinity towards pathogen epitopes than the B cells of the native repertoire. Although many details of the germinal centre function are well known, the mechanisms governing the dynamics of the B cells and the selection processes within the germinal centre are still unclear. The introduction of mathematical models and computer simulations might shed some light on these questions. In this talk, we approach the question of how a newly generated clone can take over the B-cell population in the germinal centre within a few days. We show that a selection mechanism based upon global communication of centrocytes releasing activation regulating cytokines when activated by antigen complexes can explain the rapid take-over of a clone population. A mathematical model for reconstitution of granulopoiesis after high-dose chemotherapy with autologous stem cell transplantation I. Østby, L. S. Rusten, G. Kvalheim, H. B. Benestad and P. Gøttum Clinical Stem Cell Laboratory at the Norwegian Radium Hospital and the Department of Informatics at the University of Oslo, Norway We have built a mathematical model of human granulopoiesis based on continuous cell maturation. The model utilizes a convection-reaction partial differential equation, with feedback from a cytokine compartment on proliferation, maturation and mobilization of granulocytes from marrow to blood. The cytokine compartment models production and turnover of endogenous G-CSF. Stability analysis indicated that the model was not prone to unphysiological oscillations. Model predictions and observed hematological recovery were compared in 19 patients with relapsed Hodgkin's disease undergoing high-dose chemotherapy employing purified CD34+ cells as stem cell support. G-CSF was not given after the reinfusion. The number of CD34+ and CFU-GM cells reinfused both showed a weak correlation with the measured engraftment time [neutrophils > 0.5 × 109; Spearman r = ,0.13(CD34+), r = ,0.30(CFU-GM)]. The number of CFU-GM cells was used as input to the model, which showed a curvilinear relationship with the estimated engraftment time. The simulations indicated that the long-term granulocyte level was related to the number of earlier progenitors. With a constant maturation rate, the maximal proliferation rate had to be as high as 0.072 h,1 (cycle time 9.6 h) to model the recovery in some patients. By allowing for decreased maturation rates in the mitotic stages, a considerable proliferative reserve is introduced. A left shift of the mobilization curve and the addition of a tissue compartment to be filled up during the initial phase of recovery were necessary to achieve precise modelling of the engraftment. In conclusion, the model captures essential features of regenerating granulopoiesis and could be potentially useful for e.g. simulating different approaches to the use of ex vivo expansion in stem/progenitor cell populations. Angiopoietin-1, angiopoietin-2, and Tie-2 in tumour and non-tumour tissues during growth of experimental melanoma J. Pomyje, J. H. ,ivný, T. Stopka, J. ,imák, H. Va,ková and E. Ne,as Department of Pathophysiology, 1st Medical Faculty, Charles University, Prague, Czech Republic Tumour progression is dependent on the formation of new vessels in tumour tissue. Tumour cells produce a variety of factors which influence vessel growth and maintenance in both tumour and tumour-adjacent tissues. Angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and their tyrosine kinase receptor Tie-2 have been shown to play an important role in the processes of growth and remodelling of normal as well as tumour vessels. We studied gene expression of angiogenic factors Ang-1, Ang-2, and their tyrosine kinase receptor Tie-2, in the tumour and non-tumour tissues of mice bearing the experimental melanoma B16, to search for an effect of tumour on remote tissues. Using semiquantitative RT-PCR and real time PCR we measured Ang-1, Ang-2 and Tie-2 mRNAs levels in the tumour, bone marrow, liver and spleen. Melanoma tissue over-expressed Ang-2 mRNA compared to spleen, liver and bone marrow of normal mice, suggesting its role during melanoma progression. On the other hand, there was a significant decrease of Ang-2 mRNA expression in bone marrow collected at days 5 and 10 of tumour growth compared to the expression of Ang-2 mRNA in bone marrow of normal mice and those at days 15 and 20 of tumour growth. These data demonstrate for the first time an ectopic effect of the tumour on a gene coding an angiogenic factor, and also suggest that tumour growth may influence angiogenesis and/or vasculogenesis in distant organs. A potential novel marker for intestinal epithelial stem cells C. S. Potten1, C. Booth1, G. L. Tudor1, D. Booth2, S.-I. Sakakibara3,4 and H. Okano3,4 1 EpiStem Limited, Incubator Building, Grafton Street, Manchester, UK; 2 Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Manchester, UK; 3 Department of Neuroanatomy, Biomedical Research Center, Osaka University, Osaka, Japan; 4 Core Research for Evolution, Science & Technology (CREST), Japan Science and Technology Corporation, Minoto-Ku, Tokyo, Japan Stem cells are the permanent residents of replacing tissues. They are responsible for cell replacement throughout the lifetime of the animal. However, with the possible exception of some parts of the haemopoietic progenitor cell lineage, there are no effective stem cell markers. This has restricted the study of these cells, since experiments must use functional responses or markers induced by mutational events. Each of these rely on certain assumptions and subjective interpretation. Mushashi-1 (Msi-1) is a neural RNA binding protein originally identified in Drosophila, where it was required for the early asymmetric divisions in sensory organ precursor cells. The mammalian homologue is thought to be involved in the early asymetric divisions that generate neural stem and progenitor cells, with its expression down-regulated in the differentiated progeny. Stem cell numbers, position and hierarchy have been well documented in the small intestine via their functional responses. Asymmetric divisions are thought to regulate stem cell number in development, normal adult steady state, following injury, tissue regeneration and in tumours. We have therefore examined the expression of this protein in the intestine in these various states. Msi-1 is strongly expressed in many cells of the developing crypts of the 2-day-old mouse, but is not present in the more differentiated villus cells. In the adult, expression is restricted to just a few cells towards the crypt base, consistent with expression in the stem cells and possibly their immediate daughter cells. A similar pattern is found in the human small intestine. Following irradiation, when the number of clonogenic stem cells present in the crypt is known to increase, many more cells in the lower crypt express Msi-1. Expression patterns then gradually return to control levels when the crypt is restored. Adenomas are believed to develop from changes in the regulation of the stem cell compartment, i.e changes in stem cell asymmetric division. Min ± mice spontaneously develop intestinal adenomas due to mutation of the APC gene. Msi-1 is dramatically over expressed in these cells (including the very early aberrent crypts), consistent with such a purturbed stem cell state. These observations indicate that Msi-1 is a potential marker for intestinal epithelial stem cells. Further preliminary observations suggest it may be a more universal marker for epithelial stem cells. Network model of cellular ageing C. J. Proctor and T. B. L. Kirkwood Department of Gerontology, University of Newcastle, Westgate Road, Newcastle upon Tyne NE4 6BE, UK Multiple molecular and cellular mechanisms contribute to ageing. Mathematical models are valuable as predictive and analytic tools for understanding this complex process and how the mechanisms interact. One of the major mechanisms of cellular ageing is the accumulation of defective (mutant) mitochondria. Most of the damage arises from small reactive molecules (free radicals) formed during normal cell metabolism. Defective mitochondria produce less cellular energy than healthy mitochondria, and since they accumulate with age there may be serious consequences for the cell. It has also been shown that damage to the nuclear DNA of the cell accumulates with age. Another major factor in the ageing of dividing cells is telomere loss due to the inactivation of the enzyme telomerase in human somatic cells. There is considerable heterogeneity in cell division potential within fibroblast populations, which suggests that stochastic models are appropriate. Furthermore, the mechanisms described above may act synergistically. Few of them are able to explain cellular senescence if considered individually. Therefore, in order to model these mechanisms a network approach is required. We describe a network model that is being developed in close collaboration with experimental research teams in our Department and elsewhere. Our model highlights the importance of interactions between the different mechanisms contributing to cellular ageing. Effect of keratinocyte growth factor (KGF) on proliferation, clonogenic capacity, and radiation response of human epithelial tumour cells in vitro M. Rave-Fränk, A. Hille, C. Damm, M. Jäckel, O. Pradier, W. Dörr and H. Schmidberger Klinik für Strahlentherapie und Radioonkologie, Universityität Göttingen, D-37075 Göttingen, Germany Purpose. To investigate the effect of recombinant human KGF on proliferation, clonogenic capacity, and radiation response of low passage human epithelial tumour cells in vitro. Materials and methods. Five tumour cell cultures derived from head and neck squamous cell carcinomas, three cultures derived from pleural effusions of lung carcinomas and normal nasal epithelial cells were analysed in passage 2,6. Cells were incubated with rhKGF (10,200 ng/ml) immediately after plating for clonal growth in serum-depleted media. To determine cellular radiosensitivity single doses of 1,8 Gy of X-rays were applied. Colony formation as well as the number of cell doublings was determined after 10,14 days of growth in KGF-treated and control cells. Results. Normal epithelial cells showed a 2- to 3-fold increase in the number of cell doublings due to KGF treatment. In contrast, in tumour cell cultures only slight stimulation of proliferation occurred in three out of eight samples. Neither radiation-induced impairment of proliferation nor clonogenic cell survival was influenced by the addition of KGF in the tumour cell cultures analysed. Conclusion. rhKGF has been shown to ameliorate the radiation tolerance of normal epithelia. The minimum in vitro tumour cell response to rhKGF, compared to normal epithelial cells, suggests a potential for selective protection of normal epithelia during radiotherapy. The adult human epidermal keratinoblast: a target for tissue engineering and gene therapy D. Reissig1, S. Zellmer2 and J. Salvetter1 1 Institute of Anatomy and 2 Institute of Biochemistry, Faculty of Medicine, University of Leipzig, Leipzig, Germany Adult human stem cells, especially keratinocytes, have become the focus of increasing interest for tissue engineering and as targets for gene therapy. The epidermis consists mostly of keratinocytes/keratinoblasts, the latter representing a keratinoblast stem cell system localized at the level of the stratum basale. Only the highly proliferative keratinoblasts are suitable for tissue engineering and gene therapy, and only they are able to form a stable artificial epidermis in vitro. Keratinocytes/keratinoblasts were isolated from human epidermis and selectively enriched on the basis of their ability to adhere rapidly to the surface of the plastic tissue culture vessel. After 3 days, colony formation begins. The colonies develop into compact, homogenously structured holoclones of small cells c. 10 µm in diameter. The expression of ,6, ,1, and ,4 intergrins and cytokeratins CK10 and CK19, which are important markers of cell state and ageing, was determined by flow cytometry. The transfection efficiency of the green fluorescent protein (complexes of pEGFP-CMV and Effectene) of the selected keratinocytes/keratinoblasts was greater than 40%. These results indicate that only these precisely characterized highly proliferative stem cells can be used for tissue engineering and gene therapeutic applications. Proliferative senescence of fibroblasts H. P. Rodemann Section of Radiobiology & Molecular Environmental Research, University of Tübingen, Germany A terminal differentiation lineage including a sequence of three potentially mitotic progenitor fibroblasts (MFI , MFII , MFIII) in the precursor compartment and three types of postmitotic fibrocytes (PMFIV , PMFV , PMFVI) in the functional compartment of the fibroblast/fibrocyte cells system has been identified by in vitro and ex vivo/in vitro studies. Consequently, the clonal heterogeneity in fibroblast cultures from donors of all ages has been associated with this differentiation lineage. With respect to the ageing process of the fibroblast/fibrocyte cell system, it can be concluded from these studies that cellular ageing of fibroblasts under normal physiological conditions is not based on the loss of mitotic potential of progenitor fibroblasts, but is characterized by the genetically determined life span of functioning postmitotic fibrocytes. However, premature terminal differentiation of progenitor fibroblasts to postmitotic fibrocytes can be induced by exogenous stress such as ionizing radiation. Radiation exposure in a dose range of 1,10 Gy causes a significant change in the distribution of surviving MF progenitor cells towards later differentiation stages within the precursor compartment as well as the differentiaton-dependent transition of progenitor fibroblasts to postmitotic fibrocytes. In the context of radiation-induced differentiation representing the proliferative senescence of the fibroblast/fibrocyte cell system, the transforming growth factor-,1 (TGF-,1) plays a key regulatory role. By the use of TGF-,1 knock-out cells (TGF-,1,/,) as well as neutralizing antibodies against active TGF-,1 the important function of this cytokine in the terminal differentiation process/proliferative senescence of the fibroblast system could be demonstrated. This work is supported by a grant from the Deutsche Forschungsgemeinschaft (Ro 527/4,2) and the BMBF-/IZKF Program 01KS9602. Application of a new concept of stem cell development to clonal competition processes in chronic myeloid leukaemia I. Roeder, C. Werner and M. Loeffler Institute for Medical Informatics, Statistics, and Epidemiology (IMISE), University of Leipzig, Leipzig, Germany Different clonal growth and competition phenomena, clonal fluctuations, as well as disappearance/reappearance of clones in primitive hematopoiesis have been described for several animal systems. Qualitatively similar patterns can be found in human hematopoietic malignancies, such as chronic myeloid leukaemia (CML), where the clonal competition of a normal and a malignant clone can be followed by cytogenetic and molecular monitoring. Although the regulation mechanisms which underlie these phenomena are widely unknown, there is evidence that dynamical processes of stem cell proliferation and stem cell,stroma interactions are involved. Using the emerging knowledge of molecular characteristics, cell kinetics and cell,stroma effects, we developed a mechanistic disease model for CML, which explains macroscopic observations on a population basis by kinetic processes at a microscopic level. The model is based on a new general concept of stem cell development, which explains the self-renewing capacity of primitive hematopoietic progenitors as a consequence of reversible, environment-dependent changes of actual expressed cell properties (concept of microplasticity). The balanced supply of hematopoietic cells is achieved by the dynamic regulation of cell,stroma attachment/detachment kinetics. Single-cell-based simulations allow the description of clonal fluctuation and competition phenomena both on the level of cell populations and on the level of individual clones. Distributions of cytogenetic responses in populations of CML patients after treatment with hydroxyurea or interferon-, have been used to consolidate the model, which is now applied to other treatment modalities such as STI571 or autologous bone marrow transplantation. Neuronal morphology: shape characteristics and models A. Schierwagen and J. van Pelt Universityität Leipzig, Institut für Informatik, Augustuspl.10, 04109 Leipzig, Germany The development of neuronal morphology and the forming of neural networks are key processes involved in neural tissue formation. This paper presents methods for describing quantitatively neuronal shapes and for modelling neuronal branching patterns. Models of neuronal arborizations may be assorted in reconstruction models and (stochastic or mechanistic) growth models. The growth model approach is discussed in more detail. First, Van Pelt's BESTL model is outlined, together with results of its application to several neuron types. Then mechanistic growth models are presented which describe the neurite growth process on the basis of intra- and extracellular mechanisms. In conclusion, the paper demonstrates that growth models are indispensable computational tools if the development of neuronal morphology is to be understood as a complex process governed by intracellular mechanisms, activity-dependent processes and interaction with the local environment. Activated Notch1 instructively induces multilineage myeloid differentiation of the hematopoietic stem cell line FDCP-mix T. Schroeder and U. Just GSF-Institute of Clinical Molecular Biology and Tumour Genetics, München, Germany Hematopoietic stem cells self-renew and differentiate in response to local environmental signals provided by hematopoietic stromal cells in the bone marrow. Here we present evidence suggesting that Jagged1/Notch signalling is involved in this process by inducing myeloid differentiation of hematopoietic stem cells. Activated Notch1, even when induced transiently, irreversibly reduced the self-renewal capacity of the multipotent hematopoietic stem cell line FDCP-mix accompanied by increased and accelerated differentiation along the granulocyte, macrophage and erythroid lineage. Differentiation was induced by activated Notch1 even under conditions that normally promote self-renewal but required the presence of hematopoietic cytokines for survival and proliferation. Apoptosis of FDCP-mix cells was not influenced by activated Notch1 and proliferation was only slightly inhibited. Similar to activated Notch1, the Notch ligand Jagged1, which is normally expressed on bone marrow stromal cells, induced an increased and accelerated myeloid differentiation of FDCP-mix cells. These results indicate that activation of mNotch1 provides a cytokine-independent instructive signal for multilineage myeloid differentiation of hematopoietic stem cells and reduces the probability of stem cell self-renewal. Effects of keratinocyte growth factor on survival and colony size of irradiated human keratinocytes in vitro D. Slonina, H. Hoinkis and W. Dörr c/o Department of Radiotherapy and Radiation Oncology, Medical Faculty Carl Gustav Carus, Technical University, Dresden, Germany The efficacy of recombinant human keratinocyte growth factor (rhKGF) in ameliorating acute radiation effects in oral mucosa was recently demonstrated. However, the exact mechanisms of these effects of KGF remain unclear. The present study was initiated in order to investigate rhKGF-induced changes in survival and colony size of normal human epidermal keratinocytes in vitro. For this, primary human neonatal keratinocytes (HEKn) were irradiated with single doses of 0 Gy (control) and 2 Gy (200 kV X-rays) and subsequently incubated in the presence or absence of 100 ng/ml rhKGF. Plating efficiency (PE) and surviving fraction (SF) were determined in a standard clonogenic assay. In cell cultures with or without rhKGF the mean PE was 4.6%. Irradiation with 2 Gy without KGF resulted in a SF2 of 51%; with KGF, a comparable SF2 of 54% was observed. This indicates that KGF did not change the survival characteristics of HEKn keratinocytes. The size of the individual colonies, however, was significantly increased in all cultures incubated with rhKGF compared to control cultures without rhKGF. The number of extremely large colonies (, 2 mm) was clearly higher (P < 0.0001) in the rhKGF-treated cultures. In conclusion, rhKGF does not affect keratinocyte survival after irradiation, but effectively stimulates proliferation of surviving cells. Mathematical models of hematopoiesis dynamics in irradiated mammals O. A. Smirnova Research Centre of Spacecraft Radiation Safety, Moscow, Russia Mathematical models are developed which describe the dynamics of bone-marrow hematopoiesis in mammals exposed to acute and chronic irradiation. The blood-forming system is represented here as a combination of major hematopoietic lines: thrombocytopoiesis, lymphopoiesis, erythropoiesis and granulocytopoiesis. The models are the systems of non-linear differential equations whose variables are represented by concentrations of mature blood cells and their bone-marrow precursors capable and incapable of dividing. The models quantitatively describe the ordinary effects of acute and chronic irradiation on the hematopoietic system of mammals (mice and rats). They also reproduce paradoxical effects of low dose rate chronic exposures, namely, the relaxation of lymphopoiesis, granulocytopoiesis and erythropoiesis systems to new equilibrium states characterized by elevated stationary concentrations of cells of some compartments. In addition, the models imitate experimentally observed increase of radioresistance of some hematopoietic lines under certain regimes of preliminary single and chronic exposures to radiation. Proceeding from the results obtained, an explanation of ,an adaptive response' of preirradiated animals on subsequent acute irradiation is suggested. The model can be used for predicting hematopoiesis dynamics in irradiated mammals. Mathematical models of dynamics of small intestine epithelium system in non-irradiated and irradiated mammals O. A. Smirnova Research Centre of Spacecraft Radiation Safety, Moscow, Russia Three mathematical models are developed which describe the dynamics of basic (columnar) cells in the small intestine of mammals (mice) under normal conditions and under acute and chronic irradiation. The models include the chalone mechanism of regulation of the small intestine epithelium system and take account of the principal stages of development of columnar cells. Also, the models make allowance for peculiarities of small intestine epithelium system functioning related to the motion of columnar cells over crypt and villus in the course of their division and maturation. The models are systems of non-linear differential equations whose variables are concentrations of columnar villus cells and their crypt precursors capable and incapable of dividing. The first model predicts dynamical regimes (including limit cycle) which are specific to the small intestinal system in the absence of radiation. The two other models describe quantitatively experimentally observed dynamics of cell concentrations in the crypt-villus system of mice exposed to acute and chronic radiation. The models can be used for simulating the dynamics of small intestinal system in non-irradiated and irradiated mammals. Differing sensitivity of tumour cells to apoptosis induction by iron deprivation H. ,týbrová, J. Truksa, T. Valenta and J. Kováø Cell Growth Control Laboratory, Institute of Molecular Genetics, Prague, Czech Republic We studied the sensitivity of tumour cells to apoptosis induction by iron deprivation. Iron deprivation was achieved by the employment of a defined iron-deficient culture medium. Some cells were found to be highly sensitive to iron deprivation (38C13, Raji). Under iron-depriving conditions, they died within 48 h (38C13) or 96 h (Raji), respectively. On the other hand, some tumour cells lines were completely resistant to the iron deprivation under the same experimental conditions (EL4, HeLa). DNA fragmentation analysis and flow cytometric analysis revealed characteristic apoptotic changes in sensitive cells but not in resistant cells. We compared the effect of iron deprivation with the effect of other apoptotic inducers (rotenone, hydroxyurea, methotrexate and doxorubicin). We found that there was a correlation between sensitivity to iron deprivation and sensitivity to other apoptosis inducers. Cells sensitive to iron deprivation (38C13, Raji) had a greater degree of sensitivity to other apoptosis inducers while cells resistant to iron deprivation (EL4, HeLa) were resistant to other apoptosis inducers in most cases. We conclude that some tumour cells are sensitive to apoptosis induction by iron deprivation while other tumour cells are resistant. Cells sensitive to iron deprivation seem to be of hematopoietic origin. Stimulatory effect of enterocyte-derived factor(s) on proliferative activity of haematopoetic precursors in vitro V. V. Temchura, S. V. Sennikov, T. V. Injelevskaya, V. A. Trufakin and V. A. Kozlov Institute of Clinical Immunology SB RAMS, Novosibirsk, Russia It is known that several haematopoietic cytokines are expressed in the mouse fetal intestine in the same fashion as in the fetal thymus and liver, the latter both known as haematopoietic tissues. We hypothesized that postnatal intestinal epithelial cells (IEC) may act as a cell source of growth factor(s) that act on early haematopoiesic precursors. We found that transplantation of bone marrow cells cultivated in medium containing 20% of serum-free mouse IEC supernatants (SIEC) to lethally irradiated animals led to the stimulation of formation of spleen nodules within 8 days. Quantitatively, the proliferative stimulation reached 17.8 ± 2% as compared to the control group. Then SIEC was separated into three fractions (F): F1 (> 30 kDa), F2 (10,30 kDa) and F3 (< 10 kDa). Stimulation activity of F1 was comparable to native SIEC (17.1 ± 0.3%), F2 had less activity (8.1 ± 0.7%) and F3 had no up-regulation of proliferative activity. Haemopoietic cytokines were measured using the electrochemiluminescence method with polyclonal and monoclonal antibodies. Production of granulocyte-macrophage colony-stimulating factor (GM-CSF) was detected in SIEC (37 241 pg/ml), in F1 (28 305 pg/ml) and in F2 (5490 pg/ml). Thus, these findings suggest that IEC SIEC has a proliferative-stimulating effect on SHC under these conditions. This activity may be mediated by GM-CSF, possibly in cooperation with either known cytokines or unknown specific factors secreted by the gut epithelium. Apoptosis induction in tumour cells by thiol deprivation and thiol excess J. Truksa, H. ,týbrová, K. Spìváková, T. Valenta and J. Kováø Cell Growth Control Laboratory, Institute of Molecular Genetics, Prague, Czech Republic We found that thiol deprivation (2-mercaptoethanol concentrations about 0.3 µm and lower) resulted in apoptotic death in some cultured tumour cells (mouse 38C13) while other cells were resistant (mouse EL4). On the other hand, thiol excess (2-mercaptoethanol concentrations higher than 300 µm) induced apoptosis in both cell lines. Experiments with other thiol compounds, l -cysteine and l -cystine, revealed that a free SH group is required for growth and survival of 38C13. Nonthiol antioxidant ascorbic acid was unable to maintain survival of 38C13 cells. However, it induced apoptosis in both tested cell lines at high concentrations (600 µm and higher). Thus the effect of thiol excess seems to be coupled with the excess of antioxidants while apoptotic induction by thiol deprivation seems to be free SH group specific. Immunofluorescence analysis did not show any significant change in the expression or activation of p53 due to thiol deprivation or thiol excess. The level of antiapoptotic protein Bcl-2 showed no changes in the case of thiol deprivation. However, in the case of thiol excess a significant decrease in the Bcl-2 level in both tested cell lines was detected. On the other hand, proapoptotic protein Bax displayed unchanged levels for thiol deprivation as well as for thiol excess. We conclude that p53 and Bax do not play any substantial role in apoptosis induction by thiol deprivation or by thiol excess while the Bcl-2 protein could be involved in apoptotic death induced by thiol excess. Nickel salts induce a decrease in histone H4 acetylation and down-regulate MDRI gene expression in multidrug-resistant human ovarian carcinoma cells A. Trussardi, S. Yatouji, F. Bontemps, L. Lilette, C. Trentesaux and J. Dufer Unité MéDIAN, CNRS FRE2141, IFR53, Faculté de Pharmacie, Reims, France Human ovarian carcinoma multidrug-resistant OV1-VCR cancer cells displayed, as compared to their parental IGROV1 cell line, nuclear texture changes compatible with a chromatin decondensation, as previously shown by image cytometry. These changes were associated with an increased (about 5-fold) DNase I sensitivity in OV1-VCR nuclei, suggesting an increased chromatin accessibility. It has been shown that high levels of chromatin acetylation across complete chromatin domains induced chromatin changes detected as ,general DNase I sensitivity'. By Western blotting, the level of acetylated histone H4 appears increased in OV1-VCR cells. As nickel salts have been described as inhibitors of histone H4 acetylation9, IGROV1 and OV1-VCR cells were treated with 1 µg/cm2 of nickel chloride or nickel subsulfide for 48 h. This treatment greatly reduces histone H4 acetylation in both cell lines. This decrease can be observed on the four putative acetylation sites of the histone (lys5, 8, 12 and 16). Furthermore, in OV1-VCR cells, nickel treatment significantly down-regulates the mdr1 gene expression. This effect does not appear to be non-specific, as ubiquitously expressed gene ,2m remains unaffected, either in IGROV1 or OV1-VCR cells. This work was supported by the Comité Départemental des Ardennes de la Ligue Française contre le Cancer. The use of mouse chimeras to study genes regulating stem cell function G. Van Zant Departments of Medicine and Physiology, University of Kentucky Medical Center, Lexington, KY 40536-0093, USA Self-renewing tissues rely on stem cell populations to continuously supply those mature cells with inherently short lifespans or those lost through wear and tear, disease or accident. Stem cells must therefore function throughout the lifespan of an animal; failure to do so would result in life-threatening aplasias. Since stem cells are capable of not only supplying differentiated cells to a tissue, but also replicating to resupply their own numbers, it would seem that such populations would be exempt from age-related depletion. An alternative perspective is that they may be exquisitely sensitive to age-related functional decline if their regenerative properties are not fully maintained. Using lympho-hematopoiesis as a model, it is widely recognized that production of most blood cells is maintained at near-normal levels throughout the lifespans of rodents and humans. An exception is the redistribution in lymphoid cell subpopulations during ageing resulting in the contraction of some subsets, and, of course, the involution of the thymus. It is further recognized that the response to hematological and immunological stress is blunted in older animals and humans, despite the fact that in mice the number of stem cells actually increases in the bone marrow during normal ageing. I will discuss our use of mouse chimeras, both cellular and genomic, to study the genetics of stem cell regulation during ageing. Cellular chimeras made by aggregating embryos of different mouse strains is a powerful tool to (a) identify cell autonomous (genetic) stem cell traits and (b) through natural stem cell competition, to uncover even very subtle differences in stem cell regulation over the lifetime of a chimera. Genomic chimeras, recombinant inbred mice and congenic mice, have been used to take a forward genetic approach (phenotype to genotype) to identify and characterize genetic loci responsible for stem cell regulation. My talk will present our latest results using genomic chimeras and will re-examine our earlier results using cellular chimeras. Tumour vascularization and angiogenesis P. Vaupel1 and M. A. Konerding2 1 Institute of Physiology & Pathophysiology; 2 Institute of Anatomy, University of Mainz, 55099 Mainz, Germany During embryogenesis all organs and nearly all tissues develop specific vascular patterns fulfilling the individual specific demands. Tumours, in contrast, induce, after an initial avascular growth phase, a structurally and functionally different vascular bed, whereby both pre-existent host and newly formed vessels are involved in the supply of nutrients and drainage of waste products. Neoangiogenesis is primarily induced by VEGF, which in turn is triggered by HIF-1. Compared to all other kinds of physiological and pathophysiological secondary angiogenesis (such as normal growth, menstrual cycle, wound healing or chronic inflammation), tumour angiogenesis exhibits numerous abnormalities. These include the lack of further differentiation, formation of sinusoidal vessels, endothelial cell and basement membrane discontinuities, all of which can result in high vascular permeability and direct contact of the flowing blood with tumour cells. In addition, there is also no doubt that tumour cells participate in vessel wall lining, a fact which should be considered in antivascular therapy concepts. Microvessel elongations, tortuousity, blind ends, increased interbranching distances as well as low degree of vascular hierarchy contribute to disturbances of the nutritive blood flow and can induce rheological problems. Arterio-venous shunts and vascular networks supplied and drained from the venous side further impair adequate supply and drainage which in turn promote the development of a hostile metabolic environment. These poorly supplied areas are heterogeneously distributed over the tumour mass and can be seen in the immediate vicinity of vascular hot spots. The vessel densities in these hot spots can be even higher than in the surrounding tissue. No flow regulation according to metabolic demands can be expected in tumours due to the absence of physiological/pharmacological receptors and the sinusoidal vessel wall structure which lacks smooth muscle cells. So far, no sufficient lymphatic drainage has been observed in solid tumours. Cells of the side population (SP) contribute to liver regeneration G. G. Wulf, K.-L. Luo, M. K. Brenner and M. A. Goodell Centre for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 11030, USA High efflux capacity for xenobiotics is a distinctive cell function of primitive hemopoietic stem cells. Exploiting this feature by flow cytometry after staining with the fluorescent dye Hoechst 33342, we previously identified highly potent hemopoietic stem cells in the side population (SP) of the murine bone marrow. In the liver, Hoechst 33342 staining delineated a very similar population of small blast-like cells with partial expression of CD45. In orthotopic liver cell transplantations conditioning the recipients with the hepatotoxic agent DDC, hepatic SP cells contributed to the mature liver parenchyma including bile duct epithelium, irrespective of their CD45 status. In animals with stable hemopoietic chimerism 12 month after transplantation with bone marrow SP cells, a part of the hepatic SP cells were bone marrow derived, and were recruitable to liver regeneration after transient DDC treatment. These findings introduce SP cells from different sources as a facultative liver regenerating cell population. Alterations of glycosphingolipid composition in differentiating lymphocytes of intact animals and animals with sarcoma 45 A. Zakaryan, L. Hovsepyan and K. Karagezyan Laboratory of Lipid Biochemistry, Institute of Molecular Biology of Armenian NAS, 7 Hasratyan St., Yerevan, 375014, Republic of Armenia (E-mail:annalg@mb.sci.am) The galactosylceramide (GalCer), cerebroside sulphate (CBS) and ganglioside composition in bone marrow progenitors of lymphocytes, thymocytes and mature lymphocytes of intact rats and rats grafted with sarcoma 45 was studied. Five fractions of GalCer and CBS of the cells from both intact and tumour-bearing animals were obtained by thin-layer chromatography: three fractions of GalCer and two of CBS. The quantity of GalCer and CBS in healthy animals was shown to be rather stable during lymphocyte differentiation. Only in thymocytes the decrease in one fraction of GalCer and two fractions of CBS was observed. In rats grafted with tumour cells a pronounced drop in GalCer and CBS contents took place. It began in the thymic population and was most strongly expressed in mature lymphocytes. In tumour-bearing animals the rate of ganglioside sialylation diminished which was expressed in an increase in mono- and di- and a decrease in tri- and tetrasialoganglioside levels. Thus, tumour-induced alterations in lymphocyte glycosphingolipid composition involve all stages of lymphocyte differentiation. This modification is believed to be a result of inhibition of synthesis of these lipids. The alterations observed may reflect a shift of the normal balance between lymphocyte subpopulations and the suppression of the antineoplastic activity of effector cells. [source] Carcinomas arising in multilocular thymic cysts of the neck: a clinicopathological study of three casesHISTOPATHOLOGY, Issue 1 2004C A Moran Aims :,To report three cases of primary carcinoma of the neck arising in multilocular thymic cysts (MTC). Methods and results :,The patients were three men aged 47, 50 and 52 years who presented with a painless neck mass of several weeks' duration. The patients had no history of previous surgical procedures or of malignancy elsewhere. The tumours in all three patients were located on the right lateral side of the neck; all patients underwent complete surgical resection of the mass. Grossly, the tumours were cystic and measured between 20 and 30 mm in greatest diameter. Histologically, the tumours showed cyst walls lined by squamous epithelium. The cyst walls contained prominent germinal centres with lymphoid hyperplasia, cholesterol cleft granulomas, and scattered keratinized structures reminiscent of Hassall's corpuscles. In addition, a neoplastic cellular proliferation composed of round to oval cells arranged in sheets and originating from the lining of the cystic structures was present. The neoplastic cells showed moderate amounts of eosinophilic cytoplasm, round nuclei, and, in some areas, prominent nucleoli. Mitotic figures were easily found, and cellular pleomorphism was present in several areas. In two cases the tumours showed features of basaloid carcinoma of the thymus, while in one case the pattern was that of squamous cell carcinoma. Immunohistochemical studies for keratin showed a strong positive reaction in the tumour cells, while leucocyte common antigen strongly stained the lymphoid background. Follow-up information obtained in two patients showed them to be alive 6 months after initial diagnosis. One patient was lost to follow-up. Conclusion :,The cases described here represent an unusual variant of carcinoma arising in multilocular thymic cyst in the neck region. [source] Antibody response to influenza infection of mice: different patterns for glycoprotein and nucleocapsid antigensIMMUNOLOGY, Issue 4 2003Robert Sealy Summary Our previous studies of C57BL/6 mice intranasally infected with influenza virus (A/PR8) revealed a spike of virus-specific immunoglobulin A (IgA)-secreting antibody-forming cells (AFC) in the mediastinal lymph node (MLN) 7 days post-infection. Here we show that these AFC are directed only against viral glycoprotein, and not nucleocapsid antigens. The early IgA spike associates with a decline in glycoprotein-specific AFC during week 2 post-infection. In contrast to the glycoprotein-specific AFC, nucleocapsid-specific, IgA-secreting AFC develop gradually in the MLN and persist for more than 3 weeks post-infection. As peripheral lymph node reactions wane, the nucleocapsid-specific AFC appear as long-sustained populations in the bone marrow. Microanatomical examination of the respiratory tract in infected mice shows foci of infection established in the lung 2 days post-infection, from which virus spreads to infect the entire lining of the trachea by day 3. At this time, viral haemagglutinin can be seen within the MLN, probably on projections from infected dendritic cells. This feature disappears within a day, though viral antigen expression continues to spread throughout the respiratory tract. Total IgA- and IgG-secreting AFC appear histologically in large numbers during the first week post-infection, significantly preceding the appearance of germinal centres (revealed by peanut agglutinin staining in week 2). To explain these results, we suggest that the initial immunogenic encounter of B cells with viral antigens occurs about 3 days post-infection in the MLN, with antigens transported by dendritic cells from airway mucosa, the only site of viral replication. Viral glycoproteins expressed as integral membrane components on the surface of infected dendritic cells [probably in the absence of cognate T helper (Th) cells] promote members of expanding relevant B-cell clones to undergo an IgA switch and terminal local plasmacytoid differentiation. Anti-glycoprotein specificities are thus selectively depleted from progeny of activated B-cell clones which are channelled to participate in germinal centre formation (perhaps by cognate T helper cells when they become sufficiently frequent). One product of the germinal centre reaction is the long-sustained, bone marrow-resident population, which is accordingly rich in anti-nucleoprotein, but not anti-glycoprotein specificities. Of note, we find that AFC responses toward influenza virus and Sendai virus differ, even though viral replication is limited to the airway mucosa in each case. The response towards Sendai virus exhibits neither the early appearance of anti-glycoprotein AFC expressing IgA in draining lymph nodes, nor the subsequent relative deficit of this specificity from bone marrow AFC populations. [source] Life and death within germinal centres: a double-edged swordIMMUNOLOGY, Issue 2 2002Liliana Guzman-Rojas Summary Within germinal centres, B lymphocytes are destined to die by apoptosis via Fas signalling, unless they are positively rescued by antigen and by signals initiated by CD40,CD154 interactions. Thus, while the germinal centre microenvironment can become a virtual graveyard for most B lymphocytes that fail to bind antigen with high affinity, it concomitantly provides the necessary stimuli for the survival of cells that successfully accomplish affinity maturation. Such dichotomy in the physiology of germinal centre reaction that results in survival of the functional B-cell repertoire and the elimination of abnormal cells, dictates the fate towards B-cell homeostasis or disease. Consequently, the death and survival-signalling arms within germinal centres predominantly reside on the timely and controlled expression of Fas and its ligand (FasL), and CD40 and CD154, respectively. In keeping with this notion, lymphoproliferation or deficient immunity are documented landmarks of inactivation of either the Fas/FasL or CD40/CD154 signalling pathways. The present review considers two different scenarios in the control of B-cell survival and death within germinal centres. The first is an idealistic scenario, in which a discriminatory and co-ordinate signalling initiated by the CD40/CD154 and Fas/FasL pairs, respectively, leads the rescue of the functional B-cell repertoire and the elimination of the abnormal phenotype. The second is a gloomy scenario in which both the lack and the hyperexpression of either receptor/ligand pairs, are seen as equally deleterious. [source] Presence of Subepithelial Lymphoid Nodules in the Teat of EwesANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 3 2007V. S. Mavrogianni Summary A total of 87 clinically healthy ovine teats were examined bacteriologically (by scraping the mucosa) and histologically. Teats examined were those of lactating mammary glands with no bacteria isolated (n = 23); of mammary glands after cessation of lactation with no bacteria isolated (n = 25); of lactating mammary glands with bacteria isolated (n = 22); and of mammary glands after cessation of lactation with bacteria isolated (n = 17). The salient histological feature was subepithelial leucocytic infiltration. In teat cisterns, lymphocytes were the predominant cell type and in teat ducts, lymphocytes and neutrophils were seen in equal proportions. Subepithelial lymphoid nodules, some with germinal centres, were detected in 43 (49%) teats. The majority of lymphoid nodules was observed at the border between teat duct and teat cistern. Presence of bacteria was significantly associated with the presence of leucocytic activity (P < 0.001) and with the presence of lymphoid nodules (P = 0.032). We conclude that the presence of induced subepithelial lymphoid tissue at the border between teat duct and teat cistern appears to be important in protecting the mammary gland during the early stages of bacterial invasion. The findings call for further investigations into the lymphoid structures of the teat; these should elucidate the role and development of mammary mucosa-associated lymphoid tissues and may lead to strategies for enhancing non-specific defence mechanisms of the mammary gland. [source] The Guinea Fowl Spleen at Embryonic and Post-Hatch PeriodsANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 3 2006B. I. Onyeanusi Summary The spleen of the guinea fowl was bean-shaped but without a dented hilus. It is supplied by three short arteries that came from the ventral surface, two on the cranial end and one at the caudal end of the organ. The whole organ had a thin but tough capsule covering the outer surface except at the point of entry of the blood vessels. By day 18 of incubation, the spleen had a thin but well-defined capsule and internal to this been complete network of sinusoids filled with erythrocytes, lymphocytes and granulocytes. By day 19, dark and light staining zones, which could be termed red and white pulps, had appeared. By day 20, the granulocytes with a lot of granules within their cytoplasm, had become the biggest-sized cells in the spleen. At day 21, arteries and veins were noticed clearly in the spleen and many lymphocytes, few granulocytes and reticular cells surrounded these. Red pulp with its sinusoids was now distinct. A giant cell containing three nuclei was seen within the red pulp. At day 1 post-hatch, the capsule was at its greatest thickness so far and muscle cells were seen at the inner most part of the capsule. Granulocytes that had been a constant feature suddenly disappeared. At day 5, the small lymphocytes had dominated the large and medium-sized ones. By 2 weeks, the red and white pulps were virtually equal in distribution but by 3 weeks, the red pulp was convincingly greater. By 7 weeks, plasma cells had appeared in the peripheral splenic cords. Monocytes were observed in the sinusoids. Two germinal centres were identified for the first time in week 13 post-hatch. [source] Histology, Immunohistochemistry and Ultrastructure of the Tonsil of the Soft Palate of the HorseANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 1 2006P. Kumar Summary The tonsil of the soft palate was an oval, flat structure located centro-rostrally on the oral surface of the soft palate. Its stratified squamous non-keratinized epithelium was perforated by holes or small crypts the deeper parts of which were loosely spongiform inter-digitated with lymphoid tissue. These unusual features have not previously been reported in tonsils of any species. Crypts and reticulated epithelium as found in the lingual and palatine tonsils were not observed. Lectins showed varying affinities for specific layers of the epithelium. M cells were not observed. A few Langerhans cells were distributed among surface epithelial cells. Lymphoid tissue was arranged loosely and in isolated lymphoid follicles in the subepithelial lamina propria mucosae. Although IgA+ cells and macrophages were proportionately more numerous the amount of lymphoid tissue was much less than in the lingual and palatine tonsils. Most of the follicular germinal centres lacked a darkly stained corona. CD4 positive were more numerous than CD8+ lymphocytes and were distributed in the parafollicular and inter-follicular areas. Large clusters of mucus acini positive for glycogen, acidic and neutral mucopolysaccharides separated lymphoid tissue from deeply placed striated muscle. Only a few high endothelial venules were observed in the parafollicular and inter-follicular areas. These had relatively few vesiculo vacuolar or other organelles in their high endothelial cells and few lymphocytes attaching to their walls. [source] Abstracts of the 24th Meeting of the European Study Group for Cell Proliferation (ESGCP)CELL PROLIFERATION, Issue 3 2001Epidemiology (IMISE), Germany, Leipzig, Organized by the Institute for Medical Informatics, Statistics, University of Leipzig A computer exploration of the rules governing stem cell self-renewal and differentiation Z. Agur1,3, S. J. Fleishman1 and Y. Danie2 1 Institute for Medical BioMathematics, PO Box 282, 6,0991 Bene-Ataroth, Israel; 2 Department of Mathematics, University of Haifa, Mount Carmel, 31905 Haifa, Israel; 3 Correspondence: agur@imbm.org The universal properties of stem cells (SC) have been characterized mathematically, showing that extrinsic regulation is sufficient for maintaining tissue homeostasis (Agur et al., unpublished manuscript). We studied a simple hemopoiesis computer model having similar properties. Our model showed extreme robustness, blood counts locally fluctuating around a constant average, and rapidly returning to this exact average following a severe bone marrow (BM) depletion. A very weak negative feed-back on SC proliferation ascertains haemopoietic homeostasis by generating large variation in the SC compartment passage times. The resulting constant desynchronization in the microenvironment guarantees stable blood production, whereas shifting the balance from self-renewal to differentiation yields oscillatory blood production. Bone marrow cells in mouse and human liver can develop into polyploid and binucleate hepatocytes M. Alison, S. Forbes, K. Hodivala-Dilke, R. Jeffery, R. Poulsom and N. Wright Department of Histopathology, Imperial College School of Medicine, Hammersmith Hospital, London, UK Using a technique to detect the Y-chromosome in human liver cells we have previously demonstrated that hepatocytes can be derived from bone marrow cells (Alison et al. 2000, Nature 406, 257). Bone marrow cells may be useful for providing support for the failing liver and could be used as vehicles for delivering therapeutic genes to the liver. Thus, it becomes important to explore the functional capabilities of these cells. The technique of Y chromosome detection also allows one to examine the ploidy status of these hepatocytes, a factor of considerable relevance, since polyploidization is an integral feature of hepatocyte differentiation (Gupta 2000, Seminars in Cancer Biology 10, 161). We have identified both diploid and polyploid hepatocytes of haematopoietic origin in female mice that have been given a male bone marrow transplant after whole body lethal irradiation. We have also identified Y chromosome-positive hepatocytes of both diploid and polyploid class within liver biopsies both from a female who has received a bone marrow transplant from a male donor, and from a male patient that had received a female orthotopic liver transplant. Moreover, the Y-positive hepatocytes were invariably present in fractal clones further suggestive of intrahepatic division after engraftment. These observations suggest that hepatocytes derived from bone marrow cells have the ability to undergo polyploidization, further indicating that they have the potential to function as normal hepatocytes and contribute towards liver regeneration. Aberrant mitotic mechanisms in Alzheimer's disease T. Arendt, U. Ueberham, M. Holzer, U. Gärtner and M. K. Brückner Department of Neuroanatomy, Paul Flechsig Institute of Brain Research, University of Leipzig, Germany Neurodegeneration in Alzheimer's disease (AD) is accompanied by plastic reorganization of neurons that is aberrant with respect to its localization, morphological appearance and composition of cytoskeletal components. These changes, most likely reflecting an aborted attempt of neuronal repair, are associated with a condition of neuronal de-differentiation characterized by an expression of developmentally regulated genes, post-translational modifications and an accumulation of gene products to an extent which goes beyond those observed during regeneration. This process of de-differentiation is probably triggered by a mitogenic over-stimulation of neurons and mediated through activation of the p21ras -dependent mitogen activated protein kinase (MAPK) cascade which contains a number of proto-oncogenes (trk, p21ras, raf). The MAPK cascade is also involved in the regulation of expression and post-translational modification of the amyloid precursor protein and tau protein as well as in the activation of cell cycle mechanisms. Consequently, neurofibrillary degeneration is closely associated with the neuronal expression of markers of cell cycle activation, indicating that re-entry into the cell cycle might be a critical event in a mechanism that eventually results in cell death. The present paper summarizes recent evidence on an aberrant neuronal expression of cell cycle markers in AD. Moreover, data will be presented on the potentially neuroprotective effects of transfecting neurons with human p16INK4a, a critical inhibitor of G0 -G1 transition, which might be a promising strategy for a neuroprotective gentherapy in AD and related conditions. Up-regulation of c-Myc and Ki-67 proteins is correlated with level of apoptosis in tumour cells after cytokine treatment H. Baisch Institute for Biophysics and Radiobiology, University of Hamburg, Germany Human tumour cells Me-180, MCF-7, and TCC-Sup were treated with tumour necrosis factor , (TNF), interferon , (IFN), and the combination of both (Ti). After 1,3 days of treatment, increasing numbers of cells were released from the culture flask bottom, in particular after treatment with both cytokines (Ti). These floating cells turned out to be apoptotic, as shown by several apoptosis assays [Annexin-V-Fitc, mitochondria membrane depolarization (Mito-Track red), pre-G1 peak (PI), chromatin condensation (Acridin-Orange after acid denaturation), PARP degradation]. The cell lines Me-180 and MCF-7 were sensitive to apoptosis induction whereas TCC-Sup was more resistant. At the same time the proliferation of the cells slowed down and eventually ceased. However, since cytokines may also transmit proliferation promoting signals, I measured the proliferation-associated proteins c-Myc and Ki-67. The results show that both c-Myc and Ki-67 expression is considerably elevated after TNF treatment, only slightly or not after IFN treatment, and the strongest increase was observed after Ti treatment. In a plot of the level of increase of Ki-67 vs. % apoptotic cells a linear relationship was revealed for the sensitive as well as the resistant lines. A similar correlation was observed for c-Myc. These findings support the hypothesis that cells are doomed to apoptosis when they receive conflicting signals. The proliferation promoting proteins Ki-67 and c-Myc are up-regulated, and at the same time the cells stop growing by an as yet unknown signal transmitted by the cytokines. These contradictory stop and go signals lead to apoptosis. Relation between cell proliferation markers and apoptosis in colo-rectal cancer A. Becciolini, M. Balzi, P. Faraoni, J. W. Wilson, C. Gai, S. Ferullo, F. Cianchi, E. Ravagni and C. S. Potten Radiation Biology Laboratory, Clinical Physiopathology Department, University of Florence, Italy The aim of the study was to compare different proliferation markers as factors discriminating the outcome for patients with colo-rectal cancer. Moreover, we analysed the immuno-expression of p53 and Bcl-2 and related them to the proliferative status of the tumour. The analysis examined 63 cases treated with surgery as the first therapeutic approach and affected by colo-rectal cancer without distant metastases. We determined three different proliferation indices: TLI, SPF and Ki67-LI, none of which showed a significant relation with the clinical-histopathological variables. The linear regression analysis demonstrated that there is a good relation between TLI and SPF (r = 0.80), whereas the relation is less good for Ki67-LI and, respectively, TLI (r = 0.72) and SPF (r = 0.77). P53 immunoreactivity was present in 71.4% of the tumours, whereas 39.3% of cases were Bcl-2 positive. The proportion of tumour tissue positive for the two proteins showed an opposite trend in relation to the clinical-histopathological variables. The preliminary evaluation of the prognostic role of the biological parameters indicated that only TLI appears able to discriminate two classes of patients with significantly different DFS. The simultaneous analysis of TLI and p53 or Bcl-2 status identified a group of patients (high proliferative activity and p53-negative or Bcl-2-positive) without relapse or distant metastases. Post-irradiational small intestinal crypt survival can be explained by a pedigree free model H. Berger, D. Drasdo, F. A. Meineke and M. Loeffler Institute for Medical Informatics, Statistics and Epidemiology (IMISE), University of Leipzig, Germany Previous models of the cell kinetics in intestinal crypt assumed the existence of stem cells located in the crypt base. Although no stem cell marker has been found so far the stem cells are believed to have clearly distinguishable properties compared to non-stem cells. The number of stem cells is assumed to be nearly constant and has been estimated from crypt survival curves in X-ray radiation experiments. By asymmetric cell divisions stem cells divide into stem and transient cells. The transient cells are able to perform additional cell divisions until they become mature cells. The mature cells migrate towards the orifice of the crypt and are released into the intestinal lumen from the top of the villi. The hierarchy of cell types ranging from stem cells to mature cells defines a pedigree. We here present a novel model that does not need the assumption of a pedigree to explain the shape of crypt survival curves in radiation experiments with X-rays. The basic units of the model are the individual cells arranged in a cylindrical tissue sheet that represent a single crypt. Hence the model allows consideration of the temporal and the spatial dynamics in the crypt. Our model assumes that the stem cell property is completely determined by the spatial position of a cell. Cell proliferation is controlled by a morphogene secreted at the crypt base that spreads over the crypt and determines in which crypt region cells are able to divide. Interchanging a cell at a stem cell position with any other cell would not change the cell kinetics. Hence, within this concept, ,stemness' is determined extrinsically instead of intrinsically. The intestinal epithelial stem cell lineage and its regulation M. Bjerknes and H. Cheng Departments of Anatomy and Cell Biology and Medical Biophysics, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada M5S 1A8 The presentation describes our investigations into the characterization of intestinal epithelial progenitor populations, their interrelationships, and their regulation. It begins with a brief review of intestinal structure and organization. It then illustrates the information that can be extracted by studying the dynamics of clones derived from genetically labelled progenitor cells. This work leads to the conclusion that the epithelium contains a variety of short- and long-lived progenitor types variously committed to producing single or multiple mature cell types. Furthermore, the lineage relationships amongst the various progenitor types can be established. This forms the basis for Specific Progenitor Assay by Somatic Mutation (SPASM) experiments aimed at determining the sensitivity of progenitors to various factors, and such experiments are outlined. The presentation concludes by describing our observations of clonal interactions in adenomas. Proliferative activity of leukemic blasts and cytosine arabinoside pharmacodynamics are associated with cytogenetically defined prognostic subgroups of acute myeloid leukaemia J. Braess, G. Jahns-Streubel, C. Schoch, D. Haase, T. Haferlach, M. Fiegel, S. Voss, W. Kern and W. Hiddemann Department of Medicine III, Klinikum Großhadern, 81377 Munich, Germany Purpose. The biological mechanisms responsible for the association of specific karyotypes with prognosis in acute myeloid leukemia are at present largely unclear. A prospective study was performed to evaluate the extent to which cytogenetically defined prognostic subgroups of AML differ in proliferative activity as a potential mechanism for differential sensitivities to s-phase specific induction chemotherapy comprising cytosine arabinoside. Patients and methods. 187 patients with de novo AML were included in the study, comprising 25 patients with a favourable karyotype [inv(16), t(8; 21), t(15; 17)], 99 with a normal karyotype, 29 with an unfavourable karyotype (,5, 5q-, ,7, 7q-, complex aberrations) and 34 with cytogenetic aberrations of unknown prognostic significance (all others). Ex vivo proliferative activity (PA) of AML blasts and sensitivity of blasts to cytokine priming (GM-CSF, CG-CSF), as measured by thymidine incorporation, endogenous production of stimulatory cytokines (IL3, GM-CSF, G-CSF) by leukemic blasts, incorporation of the active moiety of cytosine arabinoside , araC-triphosphate , into the DNA and response to induction treatment were determined and related to cytogenetic subgroup. Results. The ,favourable' group demonstrated the highest PA (3.41 pmol/105 cells), significantly (P = 0.02) exceeding the ,unfavourable' group with the lowest PA (0.72) and the group with a normal karyotype (1.06) or with karyotype of unknown significance (1.05) with an intermediate PA. Samples with a high PA (> median of the whole group) were more likely to produce IL3, GM-CSF, G-CSF (56%, 43%, 50%) than cells with a low PA (33%, 36%, 36%; n.s.). The effect of priming by exogenous gm-csf or g-csf was significantly more pronounced in samples with a low PA than in rapidly proliferating samples (P < 0.01). For the whole group, a high PA was closely associated with an increased incorporation of AraCTP into the DNA (P < 0.0001). Clinically a high PA was associated with a better CR rate in the normal group (95% vs. 62%) and in the unfavourable group (75% vs. 33%). Conclusions. The significant differences in proliferative activity between cytogenetic subgroups of AML are associated with increased cytosine arabinoside pharmacodynamics and constitute one potential mechanism for the different response of cytogenetic subgroups to araC-based induction therapy. Cultivation of hematopoietic cells in a 3D bioreactor perfusion system S. W. Braun1, I. J. Koerner2, J. Gerlach1 and P. Neuhaus1 1 Department of Surgery and 2 Department of Hematology, Oncology and Tumor Immunology, Charité, Humboldt-University of Berlin, Germany Purpose. Physiological conditions found in the human bone marrow for maintenance and specific expansion of pluripotent hematopoietic stem cells cannot satisfactorily be imitated in in vitro cultures. Therefore we are developing a bioreactor perfusion system to optimize a long-term ex vivo expansion of hematopoietic cells under well-defined conditions. Materials and methods. The bioreactor incorporates three independent but interwoven capillary membrane systems for decentralized nutrient supply and oxygenation in a cell compartment of 10 ml. Studies were performed with the human myeloid leukemia cell line hl-60 (n = 9) and with bone marrow mononuclear cells (BM-MNC: n = 7). The following culture conditions were investigated in the bioreactor system: (a) membrane materials, (b) perfusion modes and rates, (c) medium supply, (d) influence of agitation and (e) cell harvesting conditions. The expansion and viability of hematopoietic cells during cultivation were characterized by growth kinetics, metabolism parameters and morphological appearance. Results. The best results were obtained using polyethersulphone membranes with unidirectional flow across the cell compartment at a perfusion rate of 10 ml/min and a continuous medium supply rate of 5 ml/h. Mild agitation improved the results. A perfusion rate of 15 ml/min (total volume: 300 ml) using perfusion mode combinations was necessary for optimal cell harvesting (> 95%). Under these conditions, expansion rates up to 194% per day, viability rates up to 89% and stable glucose/lactate metabolism were achieved for Hl-60. For growth optimization of BM-MNC in the bioreactor, the addition of cytokines as well as co-culture with stroma cells was favourable. Conclusion. The bioreactor perfusion system proved to be suitable for the expansion of non-adherent hematopoietic cells. However, to achieve a higher viability of the expanded cells, further studies are required to optimize the system for clinical application. P2 receptor-evoked proliferation of Müller glial cells: dependence on calcium and on receptor tyrosine kinases A. Bringmann, I. Milenkovic, V. Moll, H. Kodal and A. Reichenbach Paul Flechsig Institute of Brain Research, Department of Neurophysiology, University of Leipzig, D-04109 Leipzig, Germany Massive gliosis is accompanied by proliferation of macroglial cells. Extracellular ATP has been suggested as a candidate molecule which may induce gliosis. In the sensory retina, the Müller cell is the principal macroglial cell. During proliferative gliosis of the retina, P2 receptor-mediated responses in Müller cells have been shown to be up-regulated. The intracellular signalling pathways that were activated by extracellular ATP and that result in an enhanced proliferation rate were investigated in cultured Müller cells of the guinea pig, using an assay of the DNA synthesis rate. We found that the ATP-evoked proliferation of Müller cells is dependent on Ca2+ and on transactivation of growth factor receptor tyrosine kinases (RTKs). Inhibition of the Ca2+ influx, of the activity of Ca2+ -dependent K+ channels, or of the protein kinase C blocked the mitogenic effect of ATP. Using tyrphostins it was revealed that P2 receptor activation resulted in a transactivation of at least two different RTKs: of the platelet-derived growth factor RTK and, apparently subsequently, of the epidermal growth factor RTK. It is concluded that extracellular ATP activates multiple signalling pathways in cultured Müller cells, to exert its proliferation-enhancing effect. Asynchronous induction of DNA synthesis in G0 nuclei by Xenopus egg extracts R. F. Brooks and W. Sun Guy's, King's and St Thomas' School of Biomedical Sciences, King's College London, Guy's Campus, London SE1 1UL, UK Cell-free extracts of Xenopus eggs support the complete replication of chromosomal DNA in vitro, under normal cell cycle controls. This replication requires the assembly of a prereplication complex (pre-RC) at origins of replication, involving the sequential binding of the six-subunit origin recognition complex (ORC), the Cdc6 and Cdt1 proteins and a complex of six MCM proteins (MCM2-7). With intact mammalian nuclei as template, replication is dependent on the retention of pre-RCs assembled in vivo prior to nuclear isolation, as some component required for their assembly is unable to cross an intact nuclear envelope in egg extracts. Early G0 nuclei from 3T3 cells show significant levels of replication but with prolonged quiescence the pre-RCs are gradually dismantled, as judged by a progressive loss of the capacity to replicate in egg extracts and a decline in chromatin-bound MCMs. The early G0 nuclei that initiate DNA synthesis do so asynchronously, despite exposure to the same concentration of replication factors present in the extracts. This asynchrony is not due to variation in the level of MCM proteins retained by individual nuclei. Nor can it be attributed to differences in the rate of nuclear import, which shows that the initiation of DNA synthesis is not triggered merely by the accumulation of replication factors to some critical threshold. However, when replication begins, it does so at many hundred independent foci scattered throughout the nucleus. The signal for the initiation of DNA synthesis must therefore be a global event distributed throughout the nucleus, its timing varying from one nucleus to another. The nature of this signal remains unknown, but could involve an abrupt increase in cyclin-dependent kinase (Cdk) activity analogous to that which triggers the onset of mitosis. Estrogen-induced protection against apoptosis is in the human breast cancer cell line MCF-7 mediated by procathepsin-D P. Budtz, J. Hofgaard and M. Riis University of Copenhagen, August Krogh Institute, 13 Universityitetsparken, DK 2100 Copenhagen, Denmark The mechanism by which estrogen (E2) stimulates growth of breast cancer cells may be direct through gene activation and/or indirect through the stimulation of autocrine growth factors. It has been suggested that estrogen binding to receptors induces synthesis of specific mRNAs encoding proteins, which are secreted from the cells and via binding to receptors functioning as growth factors (the autocrine hypothesis)1. Procathepsin D (PcD) is normally processed intracellularly into the mature lysosomal protease cathepsin D, but is in response to E2 in the breast cancer cell line MCF-7 secreted in its unprocessed 52-kDa form, which has been suggested to act as such an autocrine growth factor2,3. In a previous study, where E2 stimulated MCF-7 cells were incubated in the presence of a PcD antibody, we showed a significantly reduced growth rate and, at day 8, a cell pool size of about 60%, compared to controls without anti-PcD added. Since the proliferation did not differ significantly, the reduced (net) growth rate following inactivation of E2 -induced PcD was assumed to be due to an increased apoptotic rate4. We have now confirmed this assumption in that the apoptotic index, whether measured following TUNEL staining, or immunostaining of caspase-cleaved PARP or cytokeratin 18, was strongly increased in anti-PcD treated E2 -stimulated cells, but with different expression patterns with time. We conclude that estrogen-induced protection against apoptosis in the human breast cancer cell line MCF-7 is (at least partly) mediated by procathepsin-D. Apoptosis manifests itself differently following induction by antiestrogens (tamoxifen, ici 182. 780), topoisomerase II-inhibitor (etoposide), and DNA intrastrand crosslinking (cisplatin) P. Budtz and K. Møller University of Copenhagen, August Krogh Institute, 13 Universityitetsparken, DK 2100-Copenhagen, Denmark Previous controversies concerning the ability of the MCF-7 cell line to undergo apoptosis (see Budtz 1999, Cell Prolif 32, 289) may have many reasons: methods used, MCF-7 subline, growth conditions, period of time in which the cells have been exposed to the agents. In order to rule out in some detail the manifestation of apoptosis in MCF-7, we have compared cell cultures under growth-promoting conditions (E2) or under growth-inhibiting conditions (antiestrogens: TAM and ICI 182.780; topoisomerase II inhibitor: etoposide; alkylating: cisplatin; unknown mechanism: BSA), studying as a function of time (6 h, 3 and 7 days) the expression of apoptosis in the early (Annexin V), intermediate (caspase-cleaved PARP and cytokeratin 18) and late phase of apoptosis (TUNEL). The effect of these drugs on expression patterns of apoptosis varied. The protective role of E2 was confirmed in that the apoptotic index (AI) was reduced to under 50% of controls for up to 7 days with cytokeratin 18-staining and for up to 3 days with TUNEL and PARP. Of the proapoptotic drugs, all showed increasing AI with time but to a different degree. Thus, compared to controls, the AI at day 7 was increased 25-fold by the antiestrogens, 20-fold by etoposid, 15-fold by cisplatin, 10-fold by BSA, for TAM and etoposid as revealed by TUNEL, for ICI 182.780, cisplatin and BSA as revealed by PARP. Apoptotic induction as revealed by caspase-cleaved cytokeratin 18 was far less pronounced (2,4-fold at day 7, maximal for ICI 182.780). The results show that AI obtained by different staining methods for apoptosis is not directly comparable, and further that caspase 6 and/or 7 (which cleave cytokeratin 18) appear to be of minor importance for execution of apoptosis in MCF-7. Gene expression in hematopoietic tissues: a comparative in vivo study of progenitor cell proliferation both in fetal and adult mice K. T. Chang, O. P,enák, V. Skora, L. ,efc and E. Neýas Department of Pathophysiology, 1st Medical Faculty, Charles University, Prague, Czech Republic Introduction. We have studied the expression of growth factors involved in the hematopoietic microenvironment during embryonic expansion of hematopoiesis in the fetal liver and during hematopoietic regeneration after damage induced by cyclophosphamide (CY). Total RNA was obtained from fetal liver, and from adult liver, bone marrow and spleen. This RNA was transcribed to cDNA that served as a template for real-time PCR. Using specific pairs of primers, mRNAs for the following hematopoietic growth factors/inhibitors were investigated: stem cell factor (SCF), flt3-ligand (flt3L), macrophage inflammatory protein-1, (MIP-1), stromal derived factor 1 (SDF-1) and its receptor CXCR4. Methods. We set up a semiquantitative real-time (RT) PCR with product detection via FastStart SYBR Green I fluorescent intercalation dye. The housekeeping gene for ,-actin was used as an internal standard. The level of particular mRNA was measured as a difference in PCR cycles necessary for the beginning of the exponential phase of PCR reaction. Results. The expression of SDF-1 in fetal liver increases as the fetus matures and reaches its maximum in adults. In contrast, its receptor (CXCR4) follows the expression pattern of its ligand in prenatal development, but is suppressed in adult liver. We found a significant increase in mRNA level for SCF in bone marrow on day 2 after CY. This expression pattern of SCF was not present in spleen. Regarding flt3-ligand and MIP-1,, we did not find significant changes in their expression either in bone marrow or in spleen. Expression of SDF-1 was increased on day 2 after CY in bone marrow, whereas on day 5, it was suppressed. There was no significant change in SDF-1 expression in the spleen. The receptor for SDF-1 (CXCR4) did not exert marked changes in bone marrow, whereas in spleen its mRNA level was decreased on day 2. Conclusion. SCF and SDF-1 may account for progenitor cell proliferation and trafficking in regenerating bone marrow and in fetal liver. This gene expression pattern corresponds with the proliferative kinetics of hematopoietic progenitor cells after cyclophosphamide treatment that had been previously studied. Deterministic and stem cell intrinsic regulation of hematopoietic stem cell self-renewal and differentiation R. H. Cho1, L. Karlsson2, M. Thoman1, H. B. Sieburg3 and C. E. Müller-Sieburg1 1 Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121; 2 R. W. Johnson Pharmaceutical Research Institute, San Diego, CA 92121; 3 Department of Mathematics, University of California, San Diego, CA 92093, USA Commitment and self-renewal are essential and defining functions of stem cells. Decisions about proliferation and differentiation are made at the level of a single stem cell and thus require examination at the clonal level. We have developed a simple, but highly effective method to obtain clonal stem cells with extensive in vivo repopulation capacity [2000, Exp Hematol,28, 1080]. This method yielded about 50% clonally engrafted mice. Thus, the method efficiently identified and isolated clonal, repopulating stem cells to a previously unprecedented level. Clonally derived stem cells differed extensively in their kinetics and extent of repopulation, revealing very primitive and more mature types of stem cells. However, daughter stem cells derived from individual stem cells show remarkably similar repopulation and differentiation capacity. This indicates that stem cell proliferation and differentiation are predetermined and non-stochastic. Amongst the clonally repopulated mice, we identified lineage dominant stem cells. Lineage dominant stem cells give rise to all mature hematopoietic cells, but the progeny is markedly skewed towards either the lymphoid or myeloid lineage. No evidence of transformation or hematolymphoid proliferative disorders was found. This lineage dominant pattern of repopulation can be recreated in secondary and tertiary hosts, indicating that these stem cells have extensive self-renewal capacity and that lineage dominance is stable, stem cell intrinsic behaviour. Collectively, the data indicate that lineage commitment decisions are made at the level of the most primitive stem cells. This appears to cause intrinsic, stable changes at the level of the stem cell that result in progeny with reduced fitness in the underrepresented lineage. Survivin expression in human soft tissue sarcomas A. Costa, S. Pilotti, G. Abolafio, R. Motta and M. G. Daidone Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy Survivin is a member of the IAP (inhibition of apoptosis) gene family whose expression appears to be cell-cycle dependent and regulated during ontogeny, with mRNA and protein levels elevated in fetal tissues and down-regulated in adult normal cells. Survivin interacts with microtubules and can be phosphorylated by p34 cdc2, thus being on interface protein between cell cycle control and apoptosis. Survivin expression has been detected in most human cancers, including breast, gastric and colorectal cancers, lymphomas and melanomas, and in some preneoplastic lesions. We investigated by immunohistochemistry survivin expression in histologically heterogeneous tumours such as soft tissue sarcomas (85 cases) and in a series of benign lesions (five cases). Survivin was detected at a cytoplasmic level in all the five benign lipomatous neoplasms and in 79% of the malignant lesions, with a wide range of positive cells (5,100%) and a median value of 70%. In angiosarcoma, desmoid tumours and malignant fibrous histiocytoma, the median value of survivin-positive cells was less than 50% compared to chondrosarcoma, leiomyosarcoma, liposarcoma, rhabdomyosarcoma and schwannoma. Dermatofibrosarcoma protuberans, a generally well-differentiated histotype, rarely showed survivin expression (in 3/12 cases). In contrast, in the other lipomatous sarcomas, mixoid liposarcoma was characterized by a nuclear staining. In summary, survivin is present in most soft tissue sarcomas, with a histology-related variability. The nuclear localization observed in mixoid liposarcoma should indicate a role of survivin in the differentiation of lipomatous lesions. The transient manipulation of transcription factor gene expression in multipotent haemopoietic progenitor cells M. Cross and Z. McIvor Laboratory of Molecular Medicine, IZKF, University of Leipzig, Germany The lineage commitment of haemopoietic progenitor cells is a largely intrinsic process in which transcription factors are likely to play a pivotal role. We have recently reported the use of GFP coexpression vectors to direct the transient expression of regulatory genes in the multipotent murine haemopoietic progenitor cell line FDCPmix (McIvor et al. 2000, BJH 110, 674). This assay allows the rapid and precise evaluation of effects on survival, proliferation, self-renewal and proliferation. Here, we show the consequences of directing the expression of selected haemopoietic transcription factor genes on developmental potential and gene expression in the FDCPmix cells. The transient expression of PU.1 (a transcription factor associated predominantly with myelopoiesis) results in a marked reduction in self-renewal potential as assayed by colon forming efficiency, and a delay in erythropoiesis, with little adverse affect on myelopoiesis in the presence of GM- and G-CSF. Interestingly, PU.1 expression appears simultaneously to cause a strong increase in transcription from cotransfected promoters (with or without PU.1 binding sites). This ability to increase non-specific transcription is shared by GATA-1, a transcription factor involved in erythroid differentiation, but not by the stem cell transcription factor SCL. This suggests that the over expression of certain lineage-restricted transcription factors in multipotent progenitors can influence the balance between self-renewal and lineage commitment, and that commitment under these conditions may be associated with an increase in overall transcriptional activity. Proliferation and apoptosis-related markers: prognosis and predictive role in breast cancer M. G. Daidone Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy In breast cancer, proliferative activity and apoptosis represent two biological processes thoroughly investigated for their association with tumour progression and their involvement in determining the cellular response to clinical treatments. In general, the prognostic relevance of proliferation indices, including the mitotic activity component of all pathologic grading systems, overcomes that of apoptosis-related markers (evaluated as bcl-2 or bax expression, or apoptotic index). Conversely, either proliferative activity or apoptosis proved to be relevant in indicating susceptibility to chemical, hormonal and physical agents in large retrospective studies. Recently, results from treatment protocols prospectively planned to test the clinical utility of proliferation markers as a primary or a secondary objective (which are thus associated with a high level of evidence in grading the clinical utility of the investigated markers) have indicated the fraction of cells in the S-phase, evaluated by flow cytometry or as 3H-thymidine incorporation, as a marker to identify, within node-negative cancer patients, those at a very low risk of relapse (even in the presence of other conventional patho-biologic features) or those who will benefit from regimens including antimetabolites. In addition, the outcome of an ancillary study of a randomized adjuvant chemotherapy trial supported the hypothesis of a cell cycle-related benefit from different drug schedules. Bcl-2 overexpression is associated with a favourable outcome following tamoxifen, possibly as a surrogate marker of differentiation or as an estrogen-regulated protein, but is also indicative of a possible resistance to chemotherapy. Neoadjuvant treatments allow investigation of the relative balance between proliferation and apoptosis following drug treatment in an in vivo system. Effects of keratinocyte growth factor (KGF) on the radiation response of in vitro reconstituted oral mucosa W. Dörr and A. Lacmann Department of Radiotherapy and Radiation Oncology, Medical Faculty Carl Gustav Carus, Technical University Dresden The present study was initiated to quantify the effect of KGF on the radiation response of in vitro reconstituted oral mucosa. For this, keratinocyte cultures were established from mouse tongue mucosal explants, grown on collagen gel contracted by fibroblasts. Differentiation and stratification were induced by lifting the cultures to the air,medium interface. KGF in a concentration of 100 ng/ml was added to the medium either before (days , 3,0) or after (days 0 to + 3) irradiation with single doses of 20 and 30 Gy or 5 fractions of 4 Gy, respectively. Samples for histological processing were taken on days 4 or 11 p. irr. Irradiation results in a substantial decrease in epithelial thickness and cell number. KGF significantly reduced this radiation response in all irradiation protocols. The effect on epithelial thickness was independent of the KGF treatment interval. In contrast, cell numbers were significantly higher after postirradiation treatment compared to preirradiation administration. The latter indicates a reduction in individual cell size of the daughter cells if KGF is applied after irradiation. In conclusion, KGF stimulates proliferation of keratinocytes in in vitro reconstituted oral mucosa, and ameliorates the response of the organotypic coculture to irradiation. Hierarchical and flexible models of proliferative organization: a revisit from the standpoint of hematopoiesis and radiosensitivity J. D. Down BioTransplant Inc., Charlestown Navy Yard, Boston, MA, USA Two decades have passed since Michalowski conceived of the division of tissue systems into two types with respect to their proliferative organization and radiation response (1981, Radiat Environ Biophys 19, 157). Hierarchical (type H) and flexible (type F) models have generally been ascribed to tissue systems of rapid and slow renewal, respectively, and provide a theoretical framework for describing the kinetics of radiation responses. These models may also be associated with sensitivity of the early and late reacting tissue to changes in radiation dose fractionation (high and low ,/, ratios, respectively) whereby late radiation responses have a greater recovery from damage with reduction in dose per fraction (Thames et al. 1982, Int J Radiat Oncol Biol Phys8, 219). The bone marrow is often exemplified as a typical early responding and H-type tissue where acute radiation injury (myelodepression and marrow lethality) conforms to poor recovery during fractionated irradiation and thus leads to high estimates of the ,/, ratio. However, more recent studies utilizing long-term repopulation assays in vitro and in vivo have shown the existence of more primitive stem cells capable of fractionated irradiation dose-sparing and a low ,/, ratio that is more consistent with a late responding F-type tissue. The whole hematopoietic system may therefore be better described as a hybrid H-F type tissue. It is conceivable that a similar phenomenon may also arise in certain leukemias. With the advent of stem cell subset isolation techniques, such a concept may prove valuable in defining the cellular and molecular determinants of two characteristic radiation responses but within the same cell pedigree. Apart from the relevance to establishing long-term hematopoietic cell chimerism after total body irradiation and stem cell transplantation, it remains possible that the H-F hybrid model has broader applications if it can be extended to other rapidly renewing normal tissue systems and their malignant counterparts. Modelling blastulation and gastrulation D. Drasdo1 and G. Forgacs2 1 Institute for Medical Informatics, Statistics and Epidemiology (IMISE), University of Leipzig, Leipzig, Germany (drasdo@imise.uni-Leipzig.de); 2Department of Physics and Biology, University of Missouri, Columbia, Missouri, USA Blastulation is the process leading from the zygote to a hollow sphere of cells (blastula) by successive cell divisions that conserve the total mass of the embryo5,6. In some species, as in synapta digita or sea urchin, the blastula is one cell thick. At a certain blastula size an invagination occurs in a restricted area forming a gastrula. The invaginated part later forms the digestive tract. We here present a single-cell based model of blastulation and gastrulation in which each model parameter can be linked to experimentally accessible kinetic or material parameters. The model describes a two-dimensional circular cut along the perimeter of the blastula7. We find that the expansion of the blastula can be driven by the cell division alone. Until a certain size the polarity and stiffness of the one-cell-thick layer surrounding the cavity is sufficient to stabilize the spherical shape of the blastula. Above this size the spherical shape becomes unstable and a buckling instability occurs which destabilizes the blastula and eventually derails normal development. To ensure a single invagination, gastrulation must occur prior to the appearance of the instability. Within our model we avoid the instability by a sudden change of some model parameters. We interpret these changes as a consequence of specific gene activity. After changing the physical characteristics of some cells, normal development resumes, and gastrulation proceeds. The physical mechanism underlying the buckling instability is generic7 and hence may also be responsible for the unphysiological buckling during the postirradiation recovery in intestinal crypts or during the extra buckling in the oral mucosa observed after administering growth factors. Growth law and spatial fluctuations in growing tumours prior to vascularization D. Drasdo Institute for Medical Informatics, Statistics and Epidemiology (IMISE), University of Leipzig, Germany (drasdo@imise.uni-leipzig.de) Understanding of the principles underlying tumour growth laws is essential in order to predict tumour growth and consider this knowledge in tumour treatment strategies. For this purpose, direct mechanistic approaches seem particularly useful in contrast to heuristic models, since the former are better candidates to allow conclusions from the macroscopic behaviour of a growing tumour to potential underlying microscopic rules. Along this line of thought, we here construct a lattice model of tumour growth, guided by a recently established stochastic single-cell growth model in which all model parameters can be directly linked to physical properties of the single cells. The lattice model allows one to proceed up to very large cell numbers and can thus be compared to experiments of growing tumour spheroid. In contrast to the usual approach to approximate the growth law of spheroid tumours by the Gompertz law, our findings suggest that the growth law is well described by an exponential function for small cell numbers crossing over into a power law for large cell numbers. The observation of power laws supports recent findings, which suggest a power-law growth of breast tumours. The advantage of a stochastic single cell approach becomes even more obvious when processes are studied that affect the spatial organization of a tumour: We model the effect of an antimitotic drug on a tumour by the assumptions that a cell in mitosis dies with a probability Pdeath > 0.5 and are then phagocytosed by macrophages or neighbour cells. We find spatial density fluctuations of the cells that later result in a fragmentation of a tumour. The intensity of the fragmentation depends very much on the growth law prior to the treatment: If the tumour was growing power-law-like before the treatment, the fragmentation is the strongest if Pdeath, 0.5; if it was an exponential, the fragmentation is the strongest for Pdeath= 1. Clearly, the approach may be oversimplified but is useful in identifying the potential origin of effects on tumour treatment. Biological role of p16ink4a deletion in hematologic neoplasias M. H. Dreyling, G. Hutter, G. Ott, C. Schoch and W. Hiddemann Department of Medicine III, University Hospital Grosshadern/LMU, GSF Munich and Department of Pathology, University of Würzburg, Gemany Introduction. Deletion of p16ink4a is one of the most frequent genetic abnormalities in various tumour types. The gene product p16 is an inhibitor of the cyclin-dependent kinase 4 which regulates the transition from G1 to S phase. However, in hematologic malignancies, the deleted tumour suppressor region spans about 120 kb including other candidate genes (p15ink4b, p19arf). Methods. We have analysed 20 cell lines and 122 patient samples with MDS, AML, CML-BC, ALL, diffuse large cell lymphoma (DLCL), follicular lymphoma, and mantle cell lymphoma (MCL to define the extension and frequency of non-homozygous p16ink4a deletions applying two colour FISH and other molecular methods (Southern blot, SSCP). Results. p16ink4a defines the minimal tumour suppressor region and is deleted in 20% of ALL, 38% of DLCL, and 40% of MCL. Interestingly, the p16ink4a deletion was deleted in 6 of 7 cases with secondary DLCL or Richter's syndrome (86%), whereas deletions were infrequent in FL (10%; P < 0.001) or primary DLCL (20%; P < 0.001). In addition, the p16ink4a deletion was one of the most frequent secondary alterations in MCL and closely correlated to cell proliferation (P = 0.01). Conclusions. Similar to p53, alteration of p16ink4a is a genetic marker of the secondary transformation to aggressive lymphoma. In addition, the p16ink4a deletion is frequently non-homozygous and closely correlated to cell cycle activity. Whereas no point mutation could be detected in these cases, methylation of the promotor region is currently being investigated to characterize alternative mechanisms of gene inactivation. A genomic approach to understanding human origins I. Ebersberger, W. Enard, A. von Haeseler, F. Heissig, I. Hellmann, H. Kaessmann, P. Khaitovich, T. Kitano, S. Meyer, C. Schwarz, D. Serre, V. Wiebe, M. Winkler, S. Zöllner and S. Pääbo Max-Planck-Institute for Evolutionary Anthropology, Inselstrasse 22, D-04103 Leipzig, Germany In defining what traits are unique as well as shared between contemporary humans and their closest living and extinct evolutionary relatives, we may be able approach some of the genetic traits that define humans as a species. Our laboratory attempts to achieve this in several different approaches, three of which will be discussed here. We study the intraspecific DNA sequence diversity in humans, the great apes and Neanderthals. The results from sequencing a 10 000-bp region on the X chromosome indicate that humans are unique among the apes in having little diversity as well as a signal of population expansion. The comparison of mitochondrial DNA sequences from three Neanderthal individuals suggests that these may be more similar to modern humans than to the great apes in terms of their within-group diversity. We have sequenced a total of 256 kb of genomic DNA around the genes ZFX and ZFY in chimpanzees. When compared to other genomic sequences from the chimpanzee, the results show that different regions in the human genome differ significantly in their rates and modes of evolution and that the mutational mechanisms underlying transversions and transitions differ. We have furthermore studied the relative levels of expression of 20 000 genes in humans, chimpanzees and macaques. The results suggest that, whereas the rate of change of the transcriptome in the liver and blood has been similar between chimpanzees and humans, the rate of overall transcriptome change in the human brain has accelerated about 3-fold. A number of genes that differ significantly in their expression levels between humans and chimpanzees have been identified. In vivo imaging reveals muscle fibre de-differentiation makes a significant contribution to the regenerating tail blastema K. Echeverri1, J. Clarke2 and E. M. Tanaka1 1 Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany; 2 Department of Anatomy and Developmental Biology, University College London, London, UK During tail regeneration in urodele amphibians such as axolotls, all the tissue types including muscle, dermis, spinal cord and cartilage are regenerated. It is still unknown how this diversity of cell types is reformed with such precision. In particular, the number and variety of mature cell types in the remaining stump that contribute to the blastema is unclear. We have used Nomarski imaging to clearly document the process of regeneration in the larval axolotl tail. We have also used in vivo fluorescent labelling of single muscle fibres to show that mature muscle de-differentiates and contributes significantly to the blastema. Our data shows that muscle de-differentiation occurs by the ,falling apart' of the muscle fibre into mononucleate cells, followed by rapid cell proliferation and an increase in cell-to-cell contact by the extension of processes. We further show that both direct clipping of the muscle fibre and severe tissue damage around the fibre are required to initiate de-differentiation. Our observations also make it possible for the first time to assess how many of the blastema cells arise specifically from muscle de-differentiation. Our calculations indicate that up to one-third of the cells in the blastema come from de-differentiation of mature muscle fibres. A mathematical model of human granulopoiesis to identify optimal G-CSF scheduling during chemotherapy C. Engel, P. Stumpp, H. Franke, S. Schmitz and M. Loeffler Institute for Medical Informatics, Statistics, and Epidemiology (IMISE), University of Leipzig, Leipzig, Germany G-CSF is widely used to improve granulopoietic recovery during myelotoxic chemotherapy regimen. However, optimal leukocyte recovery largely depends on G-CSF administration timing. In order to identify optimal G-CSF scheduling we developed a quantitative mathematical model of human granulopoiesis. The model was applied to simulate the leukocyte population dynamics during 2-weekly CHO[E]P-chemotherapy (CHO[E]P-14) with 10 days (d4-d13) of G-CSF support and 3-weekly CHO[E]P (CHO[E]P-21) without G-CSF. The model describes the population kinetics of hematopoietic stem cells, granuloid precursors and mature blood neutrophils. Major assumptions were that,G-CSF dose-dependently increases granulopoietic amplification, shortens maturation and enhances the release of mature neutrophils to the blood. Chemotherapy is assumed to cause an acute depletion of stem cells and immature precursors. Clinical trial data on the average time course of leukocytes during six cycles of CHO[E]P-21 and CHO[E]P-14 were available from about 1300 patients of a large multicentre trial of the German High-Grade Non-Hodgkin's Lymphoma Study Group. In a first step, the model parameters quantifying the extent of myelosuppression of CHO[E]P were determined by fitting the model to CHO[E]P-21 data. Without changing these parameters, CHO[E]P-14 + G-CSF (d4-d13) could be simulated with good agreement to the clinical data by changing the cycle interval and introducing exogenous G-CSF administration. Simulations of different G-CSF schedulings showed that sufficient recovery of leukocytes should be achieved with G-CSF support from d8-d12 (5 days), which is 5 days less than the schedule currently used. Based on our model results a pilot study has recently been initiated to evaluate the safety and feasibility of the proposed 5-day scheduling. A mathematical model of murine granulo- and erythropoiesis during continuous rhG-CSF stimulation C. Engel, G. de Haan, W. Nijhof and M. Loeffler Institute for Medical Informatics, Statistics, and Epidemiology (IMISE), University of Leipzig, Leipzig, Germany It was our aim to develop a quantitative simulation model to comprehensively explain the experimentally observable phenomena in normal murine hemopoiesis during long-term exogenous G-CSF administration in vivo. We use a mathematical model which describes hemopoiesis by concatenated cell compartments and cell flux rates. Model assumptions about cell kinetic effects of G-CSF are tested by comparing model simulations with a large set of experimental data reported from different authors and laboratories. From these model simulations we conclude that the superposition of the following effects explains the complex behaviour of G-CSF stimulated hemopoiesis: G-CSF (1) enhances mitotic amplification of CFU-GM (mainly in the spleen) and proliferative granuloblasts (mainly in the marrow); (2) accelerates marrow maturation of postmitotic cells; (3) blocks marrow erythropoiesis at the BFU-E stage; (4) induces migration of CFU-S, CFU-GM and BFU-E from marrow to spleen. Furthermore, we conclude a novel mechanism: since G-CSF-induced neutrophilia cannot be explained quantitatively by only assuming enhanced amplification and accelerated differentiation, we hypothesize that (5) G-CSF-mediated prevention of intramedullary apoptosis (,ineffective granulopoiesis') of mature cells is a relevant mechanism to gain more blood neutrophils. (6) Granulopoiesis once shifted to the spleen is more responsive to G-CSF than in the bone marrow. All effects are quantified by dose,response characteristics. The model permits estimation of the contribution of each mentioned G-CSF effect on in vivo hemopoiesis and allows predictions and suggestions to be made for further experimental studies. Cyclosphospamide-induced apoptosis in the experimental model of murine lymphosarcoma O. V. Falameyeva1, S. Y. Djanayeva1, V. I. Kaledin2, O. N. Poteryaeva1 and T. A. Korolenko1 1 Institute of Physiology RAMS, Novosibirsk, Russia; 2 Institute of Cytology and Genetics RAS, Novosibirsk, Russia Introduction. The initiation of apoptosis (programmed cell death) relies on the activation of cysteine and aspartic proteinases; caspase-mediated proteolysis is a critical event in apoptosis. The aim of the study was to investigate the role of cathepsins B, L, D (CB, CL, CD) and the cystatin C (CC) ratio in the apoptosis model of lymphosarcoma (LS) treatment by cyclophospahmide (CPA). Methods. LS tumour cells (1 × 106) were transplanted i.m. into CBA mice. Mice were treated with CPA (Sigma, USA, i.v., 25 mg/kg or 50 mg/kg, single). CB and CL activities were determined against fluorogenic substrates (with specific inhibitor CA 074 for CB), and CD activity against azocasein as a substrate. CC concentration was measured using ELISA kits (Krka, Novo Mesto, Slovenia). Results. LS development was followed by an increase in specific CB and CL activity and a low CC level in tumour tissue. CPA treatment at a dose of 25 mg/kg did not influence tumour weight or CC level. However, CPA treatment at a dose of 50 mg/kg was followed by a significant reduction of tumour weight (180%) and increased CB (4 times), CL (6 times) and CD (1.5 times) activities and a several-fold increase in CC concentration. DNA study ladder structures suggested apoptosis development. Conclusion. Treatment of LS by CPA at the two dose levels had different effects on apoptosis in tumour cells. CB, CL and CD activities and CC level are markers of apoptosis in tumour cells. Lineage interactions during continuous combined EPO and G-CSF administration explained by a comprehensive model of murine erythro- and granulopoiesis C. Gebauer, C. Engel, G. de Haan, W. Nijhof and M. Loeffler Institute for Medical Informatics, Statistics and Epidemiology (IMISE), University of Leipzig, Leipzig, Germany We developed a mathematical model of murine granulo- and erythropoiesis to quantitatively describe the cell kinetic effects of combined G-CSF and EPO treatment with respect to possible synergistic or antagonistic effects between lineages and growth factors. Dose,response data of bone marrow and spleen hematopoiesis were obtained from a full-factorial experiment in which EPO and G-CSF were given continuously for 7 days. The model describes hematopoietic cell stages by concatenated compartments. Assumptions about growth factor effects were tested by comparing model simulations with experimental data. The model simulations suggest that the following assumptions can comprehensively explain our experimental findings. Synergistic effects. An increased splenic contribution of both lineages is explained by migration of early progenitors and stimulation of both lineages by EPO and G-CSF. Antagonistic effects. G-CSF dose-dependently blocks marrow erythropoiesis, which can be compensated by EPO to a certain extent. Bone marrow suppression of erythropoiesis is quantitatively compensated by the splenic increase. The behaviour of granulo- and erythropoiesis in splenectomized mice during combined EPO and G-CSF treatment is also well explained by this model. The suggested model gives basic insight into the quantitative effects of combined growth factor treatment on murine hematopoiesis. Transferred to human hematopoiesis, such models could help to optimize multiple growth factor treatment during chemotherapy and to detect possible hazardous combinations. Links between molecular defects and cell kinetics in CML M. Y. Gordon, S. B. Marley, J. L. Lewis, R. J. Davidson, D. X. Nguyen and J. M. Goldman Department of Haematology, Imperial College School of Medicine, Hammersmith Campus, London, UK Expression of the p210Bcr,Abl tyrosine kinase is responsible for the abnormal proliferation of stem/progenitor cells in CML and results in expansion of the myeloid mass. Much of this expansion may be a consequence of increased proliferation by granulocyte-macrophage colony-forming cells (CFU-GM) which has been demonstrated in vitro by replating primary CFU-GM colonies and observing secondary colony formation. The Abl tyrosine kinase inhibitor STI571 suppresses CML, but not normal, CFU-GM proliferation supporting the view that p210Bcr,Abl is responsible for CML CFU-GM expansion. The PI3-kinase, Ras and Jak/Stat signalling cascades are activated by p210Bcr,Abl and may therefore participate in transducing the signals from p210Bcr,Abl which result in increased proliferation. Inhibitors of PI3-kinase (wortmannin and LY294002), like STI571, preferentially suppressed CML CFU-GM proliferation. PI3-kinase is downstream of Ras and Jak/Stat. Inhibition of Jak2 with tyrphostin AG490, but not of Ras with a farnesyltransferase inhibitor (FTI II , Calbiochem) reproduced the effects of STI571 on CML CFU-GM. The relevant intermediates downstream of PI3-kinase have not yet been identified but signalling may culminate with components of the retinoblastoma protein (pRb) pathway since deletion of the cyclin-dependent kinase inhibitors (CDKIs) p16, p21 and p27 results in hyperproliferation of CFU-GM. Knowledge of signal transduction pathways controlling progenitor cell proliferation should aid the selection of molecular therapies designed to suppress myeloid expansion in CML. A share of cell,cell contact interactions in cell growth regulation in vitro R. Grigorian, N. Sarkissian, A. Sayadian and G. Gasparian Laboratory of Genetics, Institute of Zoology of Armenian NAS, 7, Sevak St., Yerevan 37014, Armenia (E-mail:zool@sci.am) Integration of different intracellular signal transduction pathways is believed to suggest the interchangeability, at least partially, of various signal-inducing factors. Two experimental cell systems were applied to test this idea. The first was a sparse culture of normal fibroblasts where cell,cell touching is an infrequent event. In this system the cell density rise may be mimicked by adding ethanol-fixed homologous cells, and the medium enrichment may be simulated by replacing the medium with the conditioned one. This allows one to identify effects of cell,cell contacts and diffusible factors of the medium separately. The second system consisted of the cells cultured in serum-free medium. Varying the cell population density enables the role of cell,cell contacts to be elucidated. Using the systems described we have been studying the contribution of intercellular contacts in cell regulation. It was shown that they could both inhibit and stimulate cell multiplication. With unavailability of serum factors, a cell survival-promoting influence of intercellular contacts was found. The results suggest that insoluble molecules exposed on the cell surface and soluble serum factors can substitute each other in cell behaviour control. Thus, direct cell,cell interaction may be considered as a kind of instructive event subsequently processed in the integrated system of cell signalling. Isolation and characterization of primitive hematopoietic stem cells (HSC) via Hoechst sorting from various hematopoietic cell sources Y. Guo, M. Follo, M. Lübbert and M. Engelhardt Hematology/Oncology Department, University Medical Center, 79106 Freiburg, Germany Efflux of the vital dye Hoechst (HO) 33342 allows the identification of a small ,side population' (SP) of HOlow cells. Murine SP cells have previously been described as CD34- and highly enriched for cells capable of multilineage repopulation. Human SP cells, however, seem more heterogeneous, express CD34 and to date have been less extensively characterized. Therefore, we analysed the frequency of and potential differences between SP cells from murine BM (n = 10), CB (n = 15), apheresis (AP, n = 23), normal BM (n = 6), the Kasumi-1 cell line and BM from patients with hematological malignancies (n = 9), functionally confirming SP cells with verapamil. Kasumi-1 cells revealed no SP pattern. From all murine and human SP cells, we observed a median frequency of 0.02% (range: 0.001,0.33), a mostly CD34 phenotype, and a CFU and LTC-IC increase of 1- and 49.5-fold, respectively. From murine BM, SP cells were found with highest percentages (median 0.03%) and were increased for CFUs by a median of 127.5-fold (n = 5) as compared to unsorted cells. In humans, the SP frequency was lower (0.02%), with highest numbers in G-CSF- or chemotherapy plus G-CSF-mobilized AP (median: 0.03%) and lower from CB (0.02%) or BM (0.01%) specimens. Human SP cells were mostly CD34 (median 96.3%, range 99.4,50.2%, n = 10). The CFU potential from human SP cells was low, increasing 4.6-fold from CB, but displaying no clonogenic potential from AP and normal BM as compared to unsorted cells. We found a small population by HO 33342 exclusion within BM cells from leukemia patients: the SP frequency was 0.01% and the clonogenic potential did not differ from AP and normal BM. Human SP cells from donor AP, CB, BM and patient AP samples were enriched for LTC-ICs with a median of 138-, 53-, 15- and 10-fold, respectively. Ongoing in vivo repopulating assays in NOD/SCID mice will further characterize the SP population, and detection of the presence of chromosomal abnormalities from patients should allow us to determine their normal or malignant nature. These results demonstrate that the refinement of this sorting strategy may permit to better define primitive HCSs of different ontogeny, and may aid in purification of normal vs. malignant HCSs from patients with hematological malignancies. Expression GM-CSF by mouse erythroid nuclear cells T. V. Injelevskaya, L. V. Eremina, S. V. Sennikov and V. A. Kozlov Institute of Clinical Immunology SB RAMS, 14, Yadrintsevskaya Str., 630099, Novosibirsk, Russia Previous research showed that treatment of erythroid nuclear cells by mitomicin C stimulates exogenous spleen colony formation (8,9 days) in lethally irradiated mice when mixed with bone marrow transplant from intact mice. The aim of our research was to discover possible cytokine mechanisms of this effect. We found that erythroid cells isolated from the spleens of newborn mice expressed mRNA GM-CSF. Gene expression was determined by RT-PCR. Mouse erythroid nuclear cells isolated from spleen with erythroid hyperplasia (after acute hypoxia, phenilhydrazine-induced anemia and after bleeding) expressed mRNA GM-CSF after addition of EPO. Further, we have studied GM-CSF production by erythroid nuclear cells isolated from spleen with erythroid hyperplasia and cultivated for 24 h. Conditional media of erythroid cells (1 × 106 cells/ml) was obtained and GM-CSF concentration measured by the method of electrochemiluminescence using polyclonal and monoclonal antibodies. Measured GM-CSF production of erythroid nuclear cells isolated from spleen with erythroid hyperplasia was 2210.98 pg/ml after acute hypoxia, 4201.84 pg/ml after phenylhydrazine-induced anemia, 6785.48 pg/ml after bleeding and 8875.72 pg/ml from newborn mice which is comparable to the production of thymic cells and splenocytes after mitogenic stimulation. We conclude that promotion of colony formation activity of bone marrow cells could be mediated by production of GM-CSF by erythroid cells. Anti-angiogenic drugs retard progression of two experimental leukemias P. O. Iversen, D. R. Sørensen and H. B. Benestad Institute Basic Medical Sciences, University of Oslo, Oslo, Norway Angiogenesis is essential for growth, invasion and metastasis of solid tumours. Angiogenesis inhibitors, like endostatin (ES) and phosphomannopentaose sulphate (PI-88), have retarded and in some cases stopped growth in various models of cancer. A role for angiogenesis in the pathophysiology of hematological malignancies has also been suggested. We have tested this suggestion in two leukemia models, i.e. juvenile myelomonocytic leukemia (JMML) developing in severe combined immunodeficient, non-obese diabetic (SCID/NOD) mice, and a promyelocytic leukemia transplantable to the Brown Norwegian rat (BNML). Flow cytometry was used to monitor the growth of transplanted cells in the bone marrow of the mouse and rat recipients. Human-specific fluorescent antibodies to CD45 were employed to mark JMML and normal human bone marrow cells; the RM-124 antibody to tag the BNML cells. Immunohistochemistry with antivon Willebrand factor was used to assess the density of microvessels in bone marrow sections. Eight weeks after leukemic cell transplantation, with daily injections of the drugs for the last 4 weeks, the leukemic cell burden had been reduced from almost 90% of all mouse marrow cells retrieved to about 15% with ES, to about 35% with PI-88, and to about 10% with ES + PI-88. Normal human bone marrow cells transplanted as a control constituted 55,60% of the mouse marrow cells. This ratio was not detectably changed by drug treatments. In SCID/NOD mice, microvessel density after JMML transplantation was high, almost 10 times higher than in the other groups. However, PI-88 only reduced the vessel density to twice control value. JMML colony numbers in semisolid cultures were not significantly altered by ES or PI-88. Very similar findings were made with the BNML model, confirming the synergism between ES and PI-88 concerning both leukemic cell growth and reduction of microvascularity. Again, neither leukemic nor normal cell growth in cell suspension cultures (3H-thymidine incorporation) was significantly affected by ES or PI-88. In conclusion, antiangiogenic therapy may be a valuable adjunct to cytotoxic or other modes of leukemia therapy. The behaviour of hematopoietic stem cells is autonomously regulated L. M. Kamminga, I. Akkerman, E. Weersing, A. Ausema, B. Dontje, G. Van Zant and G. de Haan Department of Stem Cell Biology, University of Groningen, Groningen, the Netherlands Mechanisms that affect the function of primitive hematopoietic stem cells with long-term proliferative potential remain largely unknown. We have previously shown that DBA (D2) stem cells are more numerous, cycle more actively and are more easily mobilized to the peripheral blood compared to C57BL/6 (B6). To assess whether these properties of stem cells are cell-extrinsically or cell-autonomously regulated, we developed a model in which D2 and B6 stem cells coexist in a single animal. Chimeric mice were produced by transplanting lethally irradiated B6D2F1 (BDF1) recipients with mixtures of D2 and B6 day-14 fetal liver cells to prevent ,graft vs. graft' disease. Donor leukocyte contribution was assessed using anti-H2Kb/d antibodies. We determined a series of stem cell characteristics of D2 and B6 cells in animals with chimeric hematopoiesis. After pegylated G-CSF administration peripheral blood D2 CFU-GM were 4,8 fold more numerous than B6 progenitors. In addition, D2 and B6 progenitors maintained their genotype-specific cycling activity in BDF1 recipients. Chimeric marrow was harvested and D2 and B6 cell populations were separated by flow cytometry. Parallel cobblestone area-forming cell (CAFC) analysis on both samples showed that the number of late appearing CAFC subsets within the D2 cell population was ~3 fold higher than within the B6 fraction. Finally, a secondary transplantation was performed using unfractionated chimeric marrow, administered to lethally irradiated BDF1 recipients. Comparison of the proportion of animals possessing D2 and/or B6 leukocytes 5 months after transplant revealed that the frequency of D2 LTRA was ~10 fold higher than B6 LTRA numbers. These data demonstrate that genetically distinct stem cell populations, coexisting in individual animals, independently maintain their parental phenotypes. This indicates that stem cell properties are predominantly regulated cell-autonomously. Involvement of laminins and Cdx genes in intestinal development and homeostasis M. Kedinger, C. Domon-Dell, O. Lefebvre, I. Duluc, J. N. Freund and P. Simon-Assmann INSERM U.381 ,Ontogenesis and Pathology of the Gut', 67200 Strasbourg, France The importance of epithelial,mesenchymal cell interactions during intestinal morphogenesis and differentiation, but also in the homeostasis of the adult epithelium, has been largely described in our laboratory. Two families of molecules have retained our attention, the laminins and the transcription factors Cdx. We show their involvement in the cellular cross-talk taking place in the developmental phase but also in the development of tumours when their expression is altered. Laminins are major constituents of the subepithelial basement membrane (BM). Various laminin isoforms are expressed in the intestinal tissue, and their expression pattern varies as a function of the developmental stage and along the crypt,villus axis. They are produced in a complementary manner by both epithelial and mesenchymal cells. The role of two of them has been analysed by using various experimental procedures. Laminin-1, localized early in the fetal gut and restricted to the crypt BM in the adult tissue, is required for cell proliferation and the onset of cell differentiation; its expression can be modulated by glucocorticoids. Laminin-5, localized in the adult villus BM, is up-regulated in intestinal epithelial cells by the hepatocyte growth factor produced by mesenchymal cells, and may be involved in cell migration. On the pathological side, the expression of laminin-1 is correlated to the growth of tumours, their infiltration by stromal cells and angiogenesis and to the expression of the integrin ,6,4. Cdx1 and Cdx2 genes are the mammalian homologues of the drosophila caudal homeobox gene. They are expressed specifically in the intestinal epithelium from fetal stages to the adult stage where they are restricted, respectively, to the crypt cells, and to the upper crypt and villus cells. It has also been demonstrated that Cdx1 and Cdx2 expression is altered in colorectal cancers. We showed that both genes are modulated by the nature of the mesenchymal cells and that they are additionally regulated by complementary signalling pathways: the expression of Cdx1 has been shown to be stimulated by the Wnt/, catenin pathway in embryonic intestinal endodermal cells as well as in colonic tumour cell lines, and the expression of Cdx2 is stimulated (i) by laminin-1; the various effects of laminin-1 (cell differentiation and changes in cell,cell and cell,matrix adhesion properties) may be secondary to this prime effect of laminin on Cdx2, and (ii) by the tumour suppressor gene PTEN which antagonizes the PI3K pathways. Taken together, these data indicate that intestinal development and homeostasis depend on epithelial,mesenchymal interactions that involve various laminin isoforms and the Cdx homeobox genes. Interestingly, deregulation of the expression of both laminins and Cdx genes are also involved in colon tumorigenesis. Potential toxicity of sodium selenite to human hematopoiesis Z. Kheder1, B. Millo2, J. Zawierta3 and B. Machalinski1 1 Department of General Pathology, 2 Department of Clinical Biochemistry and Laboratory Diagnostic and 3 Department of Biochemistry and Chemistry, Pomeranian Medical Academy, Szczecin, Poland The influence of the selenium on human hematopoiesis has not been addressed sufficiently in the literature. However, the environmental selenium content has already been connected with increasing morbidity due to the hematological diseases. Knowledge of the linkage between selenium compounds and human hematopoiesis would be of interest for several reasons. Insight into the potential toxicity of selenium to normal hematopoietic cell growth, viability, differentiation and apoptosis might be gained. With these issues in mind we sought to characterize the influence of sodium selenite on human hematopoiesis using a variety of techniques. We also attempt to discern the influence of Na2SeO3 on the morphology of the different organs by employing a murine model. Accordingly, we exposed human bone marrow (BM) and cord blood (CB) hematopoietic cells ex vivo to different doses of Na2SeO3 and subsequently looked for the effect of the exposure on the clonogenecity of human CFU-GM (colony forming unit of granulocyte-macrophages) and BFU-E (burst forming unit of erythocytes). We found that relatively low concentrations of sodium selenite (100 and 1000 g/l) were toxic to marrow myeloid (CFU-GM) and erythroid (BFU-E) progenitors. The inhibition of colony growth was more evident in the BM and after incubation at 37 °C compared to 4 °C. A similar effect was also observed when viability of the cells was examined using the trypan blue exclusion test. Moreover, we employed two different assays evaluating the early phase (Annexin V -test using FACS technique) as well as the executive phase of apoptosis (ELISA test) in BM and CB hematopoietic cells. Both phases of apoptosis were detectable in BM and CB hematopoietic cells exposed to different doses of sodium selenite. Finally, we injected normal mice with increasing doses of Na2SeO3 and subsequently evaluated them for the presence of any morphological changes in the livers, spleens, kidneys, lungs and bones. We found that sodium selenite caused morphological changes in the spleens and lungs. In contrast, livers, kidneys and bones appeared to be relatively resistant to this substance. This study shows that sodium selenite could potentially damage human cells involved in hematopoiesis. Effect of sodium selenite on bone marrow transplant engraftment Z. Kheder1, A. Jarema2 and B. Machalinski1 1 Department of General Pathology and 2 Clinic of Radiotherapy, Pomeranian Medical Academy, Szczecin, Poland Our previous studies suggested that Na2SeO3 might be toxic to early human bone marrow and cord blood hematopoietic cells. Expanding our preliminary report, we performed an additional experiment designed to evaluate whether hematopoietic cells, treated with sodium selenite in ex vivo conditions, were able to engraft into marrow cavities of lethally irradiated mice and successfully reconstitute hematopoiesis. Accordingly, we aspirated bone marrow from freshly decapitated Balb C mice. Next we isolated the adherent depleted mononuclear cell (A,MNC) fraction, and subsequently treated it with different doses of sodium selenite for 12 h at 37 °C. Meanwhile, other groups of mice were exposed to 800 cGy from a gamma irradiation source 24 h prior to transplantation. Cells exposed to Na2SeO3 in ex vivo conditions were then evaluated for their ability to engraft in the irradiated mice. We report here that untreated cells as well as the cells exposed to a low (100 µg/l) dose of Na2SeO3 were able to establish significant numbers of new hematopoietic islets on the surface of spleens. In contrast, much fewer hematopoietic islets were found in the cells exposed to higher doses of sodium selenite (1000 and 10000 µg/l). The morphological parameter values of peripheral blood derived from the examined mice confirmed this relation. Based on the results we conclude that selenium may significantly influence hematopoiesis. 3D cellular automata modelling of granulopoiesis: feedback regulation and stem cell dynamics T. Komatsuzaki1, Y. Saikawa2, Y. Iwata1, S. Koizumi2 and H. Sato1 1 Department of Human & Mechanical Systems Engineering, Faculty of Engineering, and 2 Department of Pediatrics, Faculty of Medicine, Kanazawa University, Kanazawa, Japan The two specific questions at issue in hematopoiesis are (1) how the self-maintenance and differentiation commitment of the hematopoietic stem cells (HSC) are achieved and (2) how the peripheral control of hematopoiesis is regulated. Several mathematical models that incorporate a negative feedback circuit have been proposed to explore the regulatory dynamics of hematopoiesis and are still unsatisfactory. In the present study, we have applied a 3D cellular automaton (CA) model to granlopoiesis. CA is a discrete dynamical system for modelling complex processes and will allow us to analyse the theoretical relationship between the microscopic mechanisms and the macroscopic behaviour of hematopoietic processes. The modelling parameters include the number of mitotic event of the cells in a proliferating phase, the transit time of each of 15 differential stages from CFU-G to the mature cells, and the neighbourhood rules for HSC proliferation. Consistent with clinical observations, the HSC population was maintained at a frequency of 0.2% in a typical set of simulation models. Importantly, stabilization of the cell production was induced in a negative feedback manner following external perturbation of the peripheral granulocyte numbers. Kinetic analyses of the HSC responding to this peripheral perturbation demonstrated the regulatory relationship between self-renewal and lineage-commitment processes. Since this model is capable of marking the HSC and subsequently tracing their fate, CA modelling will provide a framework to analyse the behaviour of HSC and to understand abnormal kinetics of the diseases with periodic hematopoiesis. The role of cystatin C and Stefin A as markers of tumour growth and efficacy of antitumour therapy T. A. Korolenko1, O. V. Falameyeva, O. N. Poteryaeva, O. A. Levina, I. G., Svechnikova1, V. I. Kaledin2 and J. Nowicky3 1 Laboratory of Cellular Biochemistry, Institute of Physiology RAMS; 2 Institute of Cytology and Genetics RAS, Novosibirsk, Russia; 3 Nowicky Pharma, Austria, Vienna Aim. To study the effectiveness of the macrophage stimulators carboxymethylated glucan (CMG), Ukraine (U) treatment in murine LS lymphosarcoma and Lewis lung adenocarcinoma with special attention to cysteine proteinases (CP) and CP inhibitors cystatin c and Stefin a as markers of tumour growth and metastasis. Methods. CBAC57BL/6 mice were used. Animals with LS lymphosarcoma or Lewis lung adenocarcinoma were treated with cyclophosphane (CPA, 150 mg/kg, i.p., single) and/or CMG (Institute of Chemistry SAS, Slovakia, 25 mg/kg, i.p., single) and/or U (Nowicky Pharma, Austria, 0.5 mg per mouse i.p., single) as macrophage stimulators. The activity of cysteine proteinases (cathepsins B and L) was measured against fluorogenic substrates with specific inhibitor (CA-074 for cathepsin B); concentrations of CP inhibitors cystatin c and Stefin a were determined using ELISA kits (Krka, Slovenia). Results. Significant antitumour effects were found in both types of tumour with the help of a combination of CPA and macrophage stimulator CMG or U (reproducible and significant retardation of tumour growth and as a consequence prolongation of life-span). In Lewis lung adenocarcinoma, treatment with CPA + CMG significantly decreased not only tumour growth but also the metastasizing to the lung. In untreated groups, CP activity in tumour tissue was increased and a low concentration of CP inhibitors was noted. CPA or CPA + U treatment of LS lymphosarcoma increased extracellular CP inhibitor cystatin c by 7 times in tumour tissue (to a lower degree in spleen and serum) as compared to untreated mice. Intracellular inhibitor of CP Stefin a concentration was low in the tumours of untreated and treated mice. CPA + U or U treatment of Ls lymphosarcoma increased cystatin c concentration in tumour tissue and liver. Conclusion. In the tumour models studied, macrophage stimulation in combined treatment was the most effective (especially treatment with CPA + CMG in Lewis lung adenocarcinoma). The concentration of the extracellular CP inhibitor cystatin c is an important marker of malignization and antitumour treatment efficacy as compared to the level of Stefin a, an intracellular CP inhibitor. P53 pathway is not involved in apoptosis induction by iron deprivation J. Kováø, J. Truksa and T. Valenta Cell Growth Control Laboratory, Institute of Molecular Genetics, Prague, Czech Republic Iron deprivation induces apoptosis in some cultured tumour cells (human Raji, mouse 38C13) while other cells (human HeLa, mouse EL4) are resistant. In order to obtain a better understanding of mechanisms involved in apoptosis induction by iron deprivation we studied the expression of genes involved in the p53 pathway of apoptosis induction. We compared the p53 level in sensitive vs. resistant cells under control and iron-depriving conditions. Western blot analysis and flow cytometry showed that the p53 level was not changed by iron deprivation in both sensitive and resistant cells. However, some decrease in the level of p53 mRNA due to iron deprivation was seen. Interestingly, the p21CIP1/WAF1 level in sensitive 38C13 cells increased after iron deprivation. In contrast, the p21CIP1/WAF1 level in sensitive Raji cells seemed to decrease. The level of p21CIP1/WAF in resistant cell lines did not change. The level of p21CIP1/WAF1 mRNA did not seem to change significantly in resistant as well as in sensitive cells. Similarly, the expression of another p53-controlled gene mdm2 was not significantly affected by iron deprivation in resistant as well as in sensitive cells. In either cell type iron deprivation did not lead to any significant change in the level or expression of antiapoptotic Bcl-2. The level of proapoptotic Bax protein was somewhat increased due to iron deprivation, however, in both sensitive and resistant cells. The level of bax mRNA did not change in either cell type. Taken together, these results suggest that the classical p53 pathway of apoptosis induction is not involved in apoptosis induction by iron deprivation. A novel model of hematopoietic and epithelial stem cells M. Loeffler1, I. Roeder1, F. A. Meineke1 and C. S. Potten2 1 Institute for Medical Informatics and Statistics, University of Leipzig, Germany; 2 Epigraft, Manchester, UK Highly proliferating tissues like hematopoiesis, epidermal and intestinal epithelia are regenerated from tissue-specific stem cells. The definition of stem cells implies that they are relatively undifferentiated cells capable of proliferation, self-maintenance, the production of large numbers of differentiated functional progeny, the regeneration of the tissue after injury and a flexibility in the use of these options8. Despite many efforts to identify individual stem cells using markers, functional assays have remained the diagnostic gold standard. They typically investigate the growth potential in vitro (e.g. colony formation) or in vivo (e.g. colony formation in reconstituting tissues, clonal competition assays). A variety of theoretical models has been suggested to provide a consistent explanation of somatic stem cell functioning. Classical models are based on the idea that a stem cell has a particular ,stemness' identity discriminating it from the descendent transit cells. In order to maintain these distinctions, mechanisms like asymmetric cell divisions have been postulated for stem cells and restricted proliferative pedigrees for the transit cell populations. In this presentation we critically review these approaches and their conceptual limitations. To overcome these limitations we propose a novel class of models based on a system theoretic approach. We postulate that each cell in a proliferating tissue at any time has several options for future development (potentials). Such options are, e.g., going into or leaving the cell cycle, expressing a particular property or not, choosing between two differentiation pathways. There are now two levels of regulation. One determines the set of options (potentials) a particular cell has. The second determines which of these options are selected (activated) in the particular setting (propensities). Both levels of regulation are assumed to depend on the general status of the tissue, the previous history of the individual cell and on the local growth environment in which it is placed. This concept makes no a priori distinction between stem cells and other cells but generates cells that gain and loose appropriate properties and hence behave accordingly in functional settings. This concept has been implemented as computer models for the hematopoietic tissue and for the small intestinal crypt. Simulations demonstrate that this class of model is powerful, removes many conceptual limitations in previous models and provides a comprehensive understanding of in vivo data on hematopoietic cell kinetics, colony formation, clone competition, retroviral marking experiments and recovery from injury. The model also leads to a reinterpretation of the in vitro functional hematopoietic colony assays. An analogous model for the spatial organization of the crypt epithelium provides explanations for clonogenicity data after injury and for data on conversion of single crypts to monoclonicity after mutation experiments. Differential expression of EGR-1 gene within gpi-deficient myeloid cells from patients with paroxysmal nocturnal hemoglobinuria A. Lyakisheva, R. E. Schmidt and J. Schubert Department of Clinical Immunology, Hannover Medical School, Germany Paroxysmal nocturnal hemoglobinuria (PNH) has been characterized by the appearance of peripheral blood cells deficient in GPI-anchored surface proteins. This defect could be attributed to mutations within the PIG-A gene encoding for a protein involved in GPI-anchor biosynthesis. Since GPI-deficient cells do not expand by themselves, the mechanism enabling the PNH stem cell to expand within the bone marrow has not yet been clarified. Therefore, a second event leading to clonal expansion needs to be assumed. According to our hypothesis this could be due to an intrinsic growth advantage because of additional mutations within their cellular activating repertoire. In order to identify genes differentially expressed in PNH cells in comparison to normal cells, we performed PCR-based RNA fingerprinting. As a model for GPI-deficient clone we used cultured normal and GPI-deficient T-cell lines from one PNH patient. Twelve candidate genes with a differential expression profile were isolated. Furthermore, testing of gene expression in GPI-deficient granulocytes by Northern blot analysis has confirmed EGR-1 as a candidate gene that is highly up-regulated in all analysed PNH cases in comparison to normal cells. In order to address external influences such as cytokines leading to a differential expression of EGR-1 in PNH granulocytes we tested expression in patients receiving high doses of G-CSF for mobilization of CD34+ cells. Application of exogenous G-CSF did not lead to an overexpression of EGR-1 in the patient's granulocytes. In addition, incubation of normal granulocytes in patients' plasma did not induce an increase in the EGR-1 mRNA level. Further analysis of granulocytes obtained from patients with other hematological diseases revealed an overexpression of this gene only in some cases of myeloproliferative and myelodysplastic diseases. We conclude from our data that clonal expansion of GPI-deficient myeloid cells from PNH patients could be due to the overexpression of transcription factors such as EGR-1. Further analysis will address the molecular events leading to the gene overexpression within the deficient cells. Introduction of plasmid DNA and oligonucleotides (ODN) into human normal and myelogenous leukemia derived cells: comparison study and clinical implications for gene therapy W. Marlicz, A. Machalinska and B. Machalinski Department of General Pathology, Pomeranian Academy of Medicine, Szczecin, Poland The efficient introduction of foreign DNA into human normal and neoplastic hematopoietic cells is still the limiting step of gene therapy. With this in mind we sought to determine the efficacy of several nonviral transfection techniques [cationic liposomes , lipofectamine and cholesterol conjugated liposomes (DC-Chol liposomes), electroporation and streptolysin O] in efficient delivery of plasmid DNA and oligonucleotides (ODN) into normal human progenitor cells as well as human chronic myelogenous leukemia derived cells. For this purpose, human normal progenitor A,T, MNC, K-562 cells and leukemic cells obtained from CML patients in blast crisis were transfected using non-viral techniques with oligodeoxynucleotides (antic-myb) and reporter plasmids containing genes encoding chloramphenicol acetyl transferase (CAT plasmid) and human placental alkaline phosphatase (PAP plasmid). The efficacy of plasmid transfections were evaluated within 48 h using CAT and PAP assays. The efficacy of ODN transfections was evaluated 2 and 5 h after transfection by dot-blot. Additionally, the intracellular localization of biotin-labelled oligomers in transfected cells was visualized by electron microscopy. We found that none of the methods examined allowed delivery of plasmid DNA into normal human progenitor cells as well as cells obtained from CML patients. Only cationic liposomes enabled efficient transfection of plasmid DNA into K-562 cells. Additionally, cationic liposomes allowed for efficient transfection of normal progenitors (~9%) and leukemic cells (~16%) with ODN. Moreover, we found that labelled oligomers were localized in significant amounts in the cell nuclei shortly after transfection. Of note, ultrastructural analysis of electroporated and streptolysin O treated cells revealed profound damage of cell membranes and presence of ODN mostly in vesicular structures. These observations support our previous conclusions that cationic liposomes could be useful in at least some forms of gene therapy and could be efficiently employed as adjuvant compounds for bone marrow purging with oligodeoxynucleotides. A functional analysis of receptors with intrinsic tyrosine kinase activity on human AML and CML derived cells W. Marlicz1, A. Machalinska1, M. Masiuk1, A. Karbicka1, M. Kawa1, B. Kolodziej2, W. Domagala2 and B. Machalinski1 1 Department of General Pathology; 2 Department of Pathomorphology, Pomeranian Academy of Medicine, Szczecin, Poland A number of hematopoietic cytokines are bound by receptors with intrinsic tyrosine kinase activity (TKR). Examples include C-KIT, INS-R, and C-MET. Knowledge of the role of these receptors in regulating the biology of human leukemic cells would be of interest for several reasons. For example, insight into the importance of a given receptor/ligand pair in regulating leukemic cell growth might contribute to better prognosis and/or response to the chemotherapy. With these issues in mind, we sought to characterize the expression of several receptors with intrinsic tyrosine kinase activity [C-KIT, IGF-1R, INS (insulin) R, C-MET, CSF-1R, STK-1R, PDGF-AA and C-TRK] on human leukemic blasts, as well as expression of ligands binding to receptors with intrinsic tyrosine kinase activity (KL, IGF-1, INS, HGF, CSF-1, FLT-3, PDGF and NGF) in the same cells. We also attempted to discern the importance of these growth factors in regulating proliferation and inhibiting apoptosis of human leukemic blasts. In our studies, the expression of receptors with intrinsic tyrosine kinase activity and their ligands was evaluated both at the mRNA level by RT-PCR and at the protein level by employing FACS analysis in AML derived (n = 30) and CML (n = 10) cells. RT-PCR analysis of total cellular RNA revealed mRNAs for all of these receptors, except for the NGF receptor TRK-A in blasts derived from AML and CML patients. Furthermore, the FACS analysis confirmed the presence of INS-R, C-KIT-R, CSF-1-R, IGF-1-R and C-MET-R on these cells. Of note, we observed that the level of receptors with intrinsic tyrosine kinase activity expression was higher on CML derived blasts as compared to AML cells. To our surprise, in repeated experiments, none of the exogenously added recombinant ligand binding to studied receptors (except for KL and bFGF) either costimulated growth of leukemic CFU-GM colonies in methylcellulose cultures or affected survival of leukemic blasts in the serum-free liquid cultures. Finally, since mRNAs for several TKR ligands were found to be expressed by AML and CML derived blasts (KL, INS, IGF-1, HGF), the potential role of intrinsic tyrosine kinase receptor/ligand axes in autocrine regulation of leukemic cell growth is currently being investigated in our laboratory. Radiation-induced effects on cell proliferation T. J. McMillan Department of Biological Sciences, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster, UK It has been recognized for many years that radiation can stop the cell cycle on a temporary and permanent basis as well as cause cell death. A revolution in our understanding of intracellular cell signalling systems means that we now have a very good idea of how many of these effects are mediated. There are, however, some important questions that remain to be answered. The genetic and biochemical characterization of the G1/S and the G2/M damage-responsive cell cycle checkpoints has given us important insights into the significance of these checkpoints in the overall cellular response to radiation. In particular, the intimate interplay between the checkpoints and DNA repair functions has been shown to be critical and linked to this is the key role for radiation-induced apoptosis in some tissues. It is not yet clear, however, which forms of radiation-induced damage are critical for these signalling pathways. Most emphasis has been placed on the radiation-induced double-strand break in radiobiology but, since other lesions can trigger the signalling pathways, does this increase the importance of these so-called ,lesser lesions'? In addition, a key step in the biochemical pathways that is still blank is the damage detection step. Protein complexes including the DNA-dependent protein kinase can bind to sites of DNA damage but whether this is truly the main damage detection step is still not clear. Despite the gaps, this ,new knowledge' has opened up some intriguing possibilities for the use of radiation in a therapeutic context and as the practicalities of gene therapy approaches are improved we must hope that they can be applied with success to radiation therapy in the clinic. Fidelity and infidelity in hematopoietic development F. Melchers Basel Institute for Immunology, Basel, Switzerland Transplantation of DHJH -rearranged surrogate light (SL) chain-expressing wild-type preB-I cells into RAG-deficient mice leads to long-term reconstitution of some of the mature B-cell compartments, to the establishment of normal levels of serum IgM, a third of the normal serum IgA levels, but not of detectable IgG levels. Neither T lineage nor myeloid cells of donor origin can be detected. The transplanted wild-type preB-I cells do not home back to the bone marrow. B lymphocyte development in PAX-5-deficient mice becomes arrested at the transition of preB-I to preB-II cells, i.e. at the stage where VH to DHJH rearrangements occur and where the preB cell receptor with µH chains and SL chains is normally formed. T-lineage and myeloid cell development is normal. PreB-I cells of PAX-5-deficient mice have a wild-type preB-I cell-like phenotype and they proliferate normally in vitro on stromal cells in the presence of IL-7. However, removal of IL-7 from the tissue cultures, unlike with wild-type preB-I cells, does not induce B-cell differentiation to sIgM+ B cells, but induces macrophage differentiation. Addition of GM-CSF to the IL-7-deprived cells induces the differentiation of MHC class II-expressing, antigen-presenting dendritic cells. In vitro differentiation to granulocytes and osteoclasts can also be observed in the presence of the appropriate cytokines. Moreover, transplantation of PAX-5-deficient preB-I cells into RAG-deficient hosts, while not allowing B-cell differentiation, leads to the full reconstitution of the thymus with all stages of CD4, CD8, and CD4+ CD8+ thymocytes, to normal positive and negative selection of thymocytes in the thymus, and to the development of normal, reactive mature CD4+ and CD8+ T-cell compartments in the peripheral lymphoid tissues. When clones of PAX-5-deficient preB-I cells carrying identifiable DHJH -rearranged H chain alleles are used for transplantation, the thymocytes and T cells all carry these DHJH rearrangements. On the other side, Ig,, Ig,, VpreB and ,5 are turned off in the thymocytes, demonstrating that the expression of these genes does not commit cells irreversibly to the B lineage. Furthermore, PAX-5-deficient preB-I cells home back to the bone marrow of the RAG-deficient host, can be reisolated and regrown in tissue culture, and retransplanted into a secondary RAG-deficient host which again develops thymocytes and T cells and allows the homing of the transplanted clonal preB-I cells to the bone marrow. Hence, the PAX-5-deficient preB-I cells are not only multipotent for T-lineage and myeloid cell differentiation but are also long-term reconstituting cells. Dynamical properties of germinal centre reactions M. Meyer-Hermann Institut für Theoretische Physik, TU Dresden, 01062 Dresden, Germany A humoral immunity response has a lot of interesting features. The presentation of a specific antigen leads to the activation of a set of B cells. In an early phase of immune response these may differentiate into antibody-producing cells which bind to the presented antigen epitopes with a certain affinity. Additionally, the activated B cells proliferate at a high rate in the environment of the follicular dendritic cells and develop into germinal centres. Here, the type of antibodies is optimized, i.e. the affinity of the antibodies to the presented antigen is enhanced. A new model is presented which describes the time evolution of the diverse cell populations as well as the optimization process in the germinal centre. The dynamics are represented by an appropriate set of standard differential equations. As all parameters are fixed by experimental data, it becomes possible to analyse essential features and quantities of germinal centre reactions quantitatively. Such quantities are the recycling probability, i.e. the probability that already positively selected centrocytes re-enter the proliferation process. Also, possible time delays for the production of plasma and memory cells from germinal centres are discussed. Furthermore, the question of how the result of a germinal centre may be optimized (concerning the number and quality of resulting antibody-producing cells) is addressed. Chimerism in embryos and adults following the injection of neural and hematopoietic stem cells into murine blastocysts A. M. Müller, S. Kreutzfeldt, S. Petrovic, N. Kirchhof, C. Schmittwolf, B. Mühl, A. Merkel and F. Harder Institut für Medizinische Strahlenkunde und Zellforschung (MSZ), University of Würzburg, Würzburg 97078, Germany Until recently, it was generally believed that the differentiation potential of a mammalian somatic stem cell is restricted to one tissue only. Recent reports, however, show that mammalian somatic stem cells can, in the course of regeneration, repopulate heterologous cell systems and therefore possess an unexpected plasticity. In order to analyse and compare the developmental potential of different somatic stem cell types, we have isolated murine adult bone marrow-derived hematopoietic stem cells (HSCs; LIN,, ckit+, SCA1+ cells) and murine fetal brain-derived neural stem cells (NSCs). NSCs are grown in vitro in media containing EGF and bFGF. Somatic stem cells were derived from transgenic mouse lines (i.e. eGFP, lacZ, bcl2). About 40 somatic stem cells were injected into preimplantation murine blastocysts and the repopulation pattern in developing chimeric animals was analysed. We detect progeny of the stem cells in more than 40% of the developing animals. Analysis of the tissue distribution of the injected donor cells revealed that progeny of the HSCs are preferentially detected in hematopoietic and at a lower frequency in non-hematopoietic tissues. Following the in vitro propagation of NSCs and their injection into blastocysts, NSCs are also capable of generating chimeric embryos and adults. Analysis of the donor contribution revealed NSC-derived hematopoietic repopulation, but overall, progeny of NSCs are mostly detected in neural tissues. Our results indicate that, even if somatic stem cells from different tissue origins are injected into an environment which is permissive for the development of all cell lineages of the developing embryo, they show plasticity but mostly retain their tissue-specific identity. Increased long-term donor cell engraftment in mice treated with growth factors prior to low dose total body irradiation and bone marrow transplantation E. J. K. Noach, A. Dethmers-Ausema, J. H. Dillingh, B. Dontje, E. Weersing, I. Akkerman, E. Vellenga and G. de Haan Department of Stem Cell Biology, University of Groningen, Groningen, the Netherlands Low toxicity conditioning regimens prior to bone marrow transplantation (BMT) are currently widely explored. However, methods to establish stable high levels of long-term donor cell engraftment still need to be improved and mechanisms of engraftment need to be clarified. Our aim is to develop new transplantation protocols using hematopoietic growth factors prior to low dose total body irradiaton (TBI) in recipients of autologous BMT to increase stem cell depletion and to create a high level of long-term donor cell engraftment. We hypothesize that a correlation exists between severeness of stem cell depletion in the host and successful donor cell engraftment after transplantation. Mice (C57BL/6, CD45.2) were treated for 1 or 7 days with different types of growth factors [SCF + IL-11, SCF + Flt3-ligand (FL) or G-CSF] prior to sublethal TBI (4 Gy). Percentage donor cell chimerism was measured by FACS analysis after transplantation of 3 × 106 CD45.1 congenic bone marrow cells into recipients. Although in recipients pretreated for 7 days with SCF + IL-11 or SCF + FL a strongly increased donor cell engraftment compared to controls was found immediately after transplantation, we observed a discrepancy between the engraftment patterns in the SCF + IL-11 and SCF + FL group: whereas engraftment remained very high and stabile in the SCF + FL treated recipients, donor cell contribution in the SCF + IL-11 group dropped towards control values after 24 weeks. In contrast, 1-day pretreatment with SCF + IL-11 and SCF + FL resulted initially in reduced donor cell engraftment, but a continuous increase over time was observed, finally resulting in a highly significantly increased donor cell contribution. G-CSF treatment showed no long-term beneficial effects on engraftment. We performed in vitro stem cell assays before and after TBI to determine the extent of stem cell depletion. A strong correlation (R2 = 0.957) existed between long-term donor cell engraftment and remaining stem cells after TBI, except for the groups treated for 1 day with SCF + IL-11 or SCF + FL. Growth factor-induced differential competitiveness between remaining recipient stem cells and transplanted donor cells may cause skewing of engraftment patterns over prolonged periods of time in these groups. We conclude that long-term donor cell engraftment can be strongly augmented by treatment of recipients with hematopoietic growth factors acting on primitive cells prior to low dose TBI. Potent support of hematopoietic stem cell activity by embryonic stromal cells from the subregions of the mouse AGM region R. Oostendorp, N. Kusadasi, K. Harvey, M. de Bruijn, R. Ploemacher and E. Dzierzak 3rd Medical Clinic, Technical University, Munich, Germany; Departments of Cell Biology and Hematology, Erasmus University, Rotterdam, the Netherlands The first adult repopulating hematopoietic stem cells (HSC) are autonomously generated in the aorta-gonads-mesonephros (AGM) region of murine and human embryonic development in mid-gestation. HSC activity is initially localized to the dorsal aorta, surrounding mesenchyme and other major vascular regions such as the vitelline and umbilical arteries. Thereafter, HSC activity rapidly increases in the urogenital ridges (UG) and the liver. To examine which embryonic microenvironment best supports and/or expands HSCs we established stromal cell lines from AGM subregions and the embryonic liver of mid-gestation mouse embryos. Several embryonic stromal clones were found to maintain adult hematopoietic progenitors and HSCs to a similar degree to, or better than, previously described murine adult and fetal stromal cell lines. Interestingly, the stromal clones most strongly supportive of early progenitors or HSCs are derived from the UG subregion of the AGM. This finding suggests that the AGM provides its own microenvironment for HSC growth and maintenance of the first HSCs during mid-gestation. Currently, we are comparing HSC-supportive with non-supportive stromal cell lines from the same or other embyonic subregions on a molecular level. These studies will be very important to establish which factors are required for generation and maintenance of HSC in the mid-gestation embryo. Simulations of B-cell selection in the germinal centre M. Or-Guil, E. F. Manffra and H. Kantz Max Planck Institute for the Physics of Complex Systems, Noethnitzer Str. 38, 01187 Dresden, Germany During the course of an acute immune reaction, germinal centres are founded with the aim of generating B cells through hypermutation able to produce antibodies with higher affinity towards pathogen epitopes than the B cells of the native repertoire. Although many details of the germinal centre function are well known, the mechanisms governing the dynamics of the B cells and the selection processes within the germinal centre are still unclear. The introduction of mathematical models and computer simulations might shed some light on these questions. In this talk, we approach the question of how a newly generated clone can take over the B-cell population in the germinal centre within a few days. We show that a selection mechanism based upon global communication of centrocytes releasing activation regulating cytokines when activated by antigen complexes can explain the rapid take-over of a clone population. A mathematical model for reconstitution of granulopoiesis after high-dose chemotherapy with autologous stem cell transplantation I. Østby, L. S. Rusten, G. Kvalheim, H. B. Benestad and P. Gøttum Clinical Stem Cell Laboratory at the Norwegian Radium Hospital and the Department of Informatics at the University of Oslo, Norway We have built a mathematical model of human granulopoiesis based on continuous cell maturation. The model utilizes a convection-reaction partial differential equation, with feedback from a cytokine compartment on proliferation, maturation and mobilization of granulocytes from marrow to blood. The cytokine compartment models production and turnover of endogenous G-CSF. Stability analysis indicated that the model was not prone to unphysiological oscillations. Model predictions and observed hematological recovery were compared in 19 patients with relapsed Hodgkin's disease undergoing high-dose chemotherapy employing purified CD34+ cells as stem cell support. G-CSF was not given after the reinfusion. The number of CD34+ and CFU-GM cells reinfused both showed a weak correlation with the measured engraftment time [neutrophils > 0.5 × 109; Spearman r = ,0.13(CD34+), r = ,0.30(CFU-GM)]. The number of CFU-GM cells was used as input to the model, which showed a curvilinear relationship with the estimated engraftment time. The simulations indicated that the long-term granulocyte level was related to the number of earlier progenitors. With a constant maturation rate, the maximal proliferation rate had to be as high as 0.072 h,1 (cycle time 9.6 h) to model the recovery in some patients. By allowing for decreased maturation rates in the mitotic stages, a considerable proliferative reserve is introduced. A left shift of the mobilization curve and the addition of a tissue compartment to be filled up during the initial phase of recovery were necessary to achieve precise modelling of the engraftment. In conclusion, the model captures essential features of regenerating granulopoiesis and could be potentially useful for e.g. simulating different approaches to the use of ex vivo expansion in stem/progenitor cell populations. Angiopoietin-1, angiopoietin-2, and Tie-2 in tumour and non-tumour tissues during growth of experimental melanoma J. Pomyje, J. H. ,ivný, T. Stopka, J. ,imák, H. Va,ková and E. Ne,as Department of Pathophysiology, 1st Medical Faculty, Charles University, Prague, Czech Republic Tumour progression is dependent on the formation of new vessels in tumour tissue. Tumour cells produce a variety of factors which influence vessel growth and maintenance in both tumour and tumour-adjacent tissues. Angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and their tyrosine kinase receptor Tie-2 have been shown to play an important role in the processes of growth and remodelling of normal as well as tumour vessels. We studied gene expression of angiogenic factors Ang-1, Ang-2, and their tyrosine kinase receptor Tie-2, in the tumour and non-tumour tissues of mice bearing the experimental melanoma B16, to search for an effect of tumour on remote tissues. Using semiquantitative RT-PCR and real time PCR we measured Ang-1, Ang-2 and Tie-2 mRNAs levels in the tumour, bone marrow, liver and spleen. Melanoma tissue over-expressed Ang-2 mRNA compared to spleen, liver and bone marrow of normal mice, suggesting its role during melanoma progression. On the other hand, there was a significant decrease of Ang-2 mRNA expression in bone marrow collected at days 5 and 10 of tumour growth compared to the expression of Ang-2 mRNA in bone marrow of normal mice and those at days 15 and 20 of tumour growth. These data demonstrate for the first time an ectopic effect of the tumour on a gene coding an angiogenic factor, and also suggest that tumour growth may influence angiogenesis and/or vasculogenesis in distant organs. A potential novel marker for intestinal epithelial stem cells C. S. Potten1, C. Booth1, G. L. Tudor1, D. Booth2, S.-I. Sakakibara3,4 and H. Okano3,4 1 EpiStem Limited, Incubator Building, Grafton Street, Manchester, UK; 2 Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Manchester, UK; 3 Department of Neuroanatomy, Biomedical Research Center, Osaka University, Osaka, Japan; 4 Core Research for Evolution, Science & Technology (CREST), Japan Science and Technology Corporation, Minoto-Ku, Tokyo, Japan Stem cells are the permanent residents of replacing tissues. They are responsible for cell replacement throughout the lifetime of the animal. However, with the possible exception of some parts of the haemopoietic progenitor cell lineage, there are no effective stem cell markers. This has restricted the study of these cells, since experiments must use functional responses or markers induced by mutational events. Each of these rely on certain assumptions and subjective interpretation. Mushashi-1 (Msi-1) is a neural RNA binding protein originally identified in Drosophila, where it was required for the early asymmetric divisions in sensory organ precursor cells. The mammalian homologue is thought to be involved in the early asymetric divisions that generate neural stem and progenitor cells, with its expression down-regulated in the differentiated progeny. Stem cell numbers, position and hierarchy have been well documented in the small intestine via their functional responses. Asymmetric divisions are thought to regulate stem cell number in development, normal adult steady state, following injury, tissue regeneration and in tumours. We have therefore examined the expression of this protein in the intestine in these various states. Msi-1 is strongly expressed in many cells of the developing crypts of the 2-day-old mouse, but is not present in the more differentiated villus cells. In the adult, expression is restricted to just a few cells towards the crypt base, consistent with expression in the stem cells and possibly their immediate daughter cells. A similar pattern is found in the human small intestine. Following irradiation, when the number of clonogenic stem cells present in the crypt is known to increase, many more cells in the lower crypt express Msi-1. Expression patterns then gradually return to control levels when the crypt is restored. Adenomas are believed to develop from changes in the regulation of the stem cell compartment, i.e changes in stem cell asymmetric division. Min ± mice spontaneously develop intestinal adenomas due to mutation of the APC gene. Msi-1 is dramatically over expressed in these cells (including the very early aberrent crypts), consistent with such a purturbed stem cell state. These observations indicate that Msi-1 is a potential marker for intestinal epithelial stem cells. Further preliminary observations suggest it may be a more universal marker for epithelial stem cells. Network model of cellular ageing C. J. Proctor and T. B. L. Kirkwood Department of Gerontology, University of Newcastle, Westgate Road, Newcastle upon Tyne NE4 6BE, UK Multiple molecular and cellular mechanisms contribute to ageing. Mathematical models are valuable as predictive and analytic tools for understanding this complex process and how the mechanisms interact. One of the major mechanisms of cellular ageing is the accumulation of defective (mutant) mitochondria. Most of the damage arises from small reactive molecules (free radicals) formed during normal cell metabolism. Defective mitochondria produce less cellular energy than healthy mitochondria, and since they accumulate with age there may be serious consequences for the cell. It has also been shown that damage to the nuclear DNA of the cell accumulates with age. Another major factor in the ageing of dividing cells is telomere loss due to the inactivation of the enzyme telomerase in human somatic cells. There is considerable heterogeneity in cell division potential within fibroblast populations, which suggests that stochastic models are appropriate. Furthermore, the mechanisms described above may act synergistically. Few of them are able to explain cellular senescence if considered individually. Therefore, in order to model these mechanisms a network approach is required. We describe a network model that is being developed in close collaboration with experimental research teams in our Department and elsewhere. Our model highlights the importance of interactions between the different mechanisms contributing to cellular ageing. Effect of keratinocyte growth factor (KGF) on proliferation, clonogenic capacity, and radiation response of human epithelial tumour cells in vitro M. Rave-Fränk, A. Hille, C. Damm, M. Jäckel, O. Pradier, W. Dörr and H. Schmidberger Klinik für Strahlentherapie und Radioonkologie, Universityität Göttingen, D-37075 Göttingen, Germany Purpose. To investigate the effect of recombinant human KGF on proliferation, clonogenic capacity, and radiation response of low passage human epithelial tumour cells in vitro. Materials and methods. Five tumour cell cultures derived from head and neck squamous cell carcinomas, three cultures derived from pleural effusions of lung carcinomas and normal nasal epithelial cells were analysed in passage 2,6. Cells were incubated with rhKGF (10,200 ng/ml) immediately after plating for clonal growth in serum-depleted media. To determine cellular radiosensitivity single doses of 1,8 Gy of X-rays were applied. Colony formation as well as the number of cell doublings was determined after 10,14 days of growth in KGF-treated and control cells. Results. Normal epithelial cells showed a 2- to 3-fold increase in the number of cell doublings due to KGF treatment. In contrast, in tumour cell cultures only slight stimulation of proliferation occurred in three out of eight samples. Neither radiation-induced impairment of proliferation nor clonogenic cell survival was influenced by the addition of KGF in the tumour cell cultures analysed. Conclusion. rhKGF has been shown to ameliorate the radiation tolerance of normal epithelia. The minimum in vitro tumour cell response to rhKGF, compared to normal epithelial cells, suggests a potential for selective protection of normal epithelia during radiotherapy. The adult human epidermal keratinoblast: a target for tissue engineering and gene therapy D. Reissig1, S. Zellmer2 and J. Salvetter1 1 Institute of Anatomy and 2 Institute of Biochemistry, Faculty of Medicine, University of Leipzig, Leipzig, Germany Adult human stem cells, especially keratinocytes, have become the focus of increasing interest for tissue engineering and as targets for gene therapy. The epidermis consists mostly of keratinocytes/keratinoblasts, the latter representing a keratinoblast stem cell system localized at the level of the stratum basale. Only the highly proliferative keratinoblasts are suitable for tissue engineering and gene therapy, and only they are able to form a stable artificial epidermis in vitro. Keratinocytes/keratinoblasts were isolated from human epidermis and selectively enriched on the basis of their ability to adhere rapidly to the surface of the plastic tissue culture vessel. After 3 days, colony formation begins. The colonies develop into compact, homogenously structured holoclones of small cells c. 10 µm in diameter. The expression of ,6, ,1, and ,4 intergrins and cytokeratins CK10 and CK19, which are important markers of cell state and ageing, was determined by flow cytometry. The transfection efficiency of the green fluorescent protein (complexes of pEGFP-CMV and Effectene) of the selected keratinocytes/keratinoblasts was greater than 40%. These results indicate that only these precisely characterized highly proliferative stem cells can be used for tissue engineering and gene therapeutic applications. Proliferative senescence of fibroblasts H. P. Rodemann Section of Radiobiology & Molecular Environmental Research, University of Tübingen, Germany A terminal differentiation lineage including a sequence of three potentially mitotic progenitor fibroblasts (MFI , MFII , MFIII) in the precursor compartment and three types of postmitotic fibrocytes (PMFIV , PMFV , PMFVI) in the functional compartment of the fibroblast/fibrocyte cells system has been identified by in vitro and ex vivo/in vitro studies. Consequently, the clonal heterogeneity in fibroblast cultures from donors of all ages has been associated with this differentiation lineage. With respect to the ageing process of the fibroblast/fibrocyte cell system, it can be concluded from these studies that cellular ageing of fibroblasts under normal physiological conditions is not based on the loss of mitotic potential of progenitor fibroblasts, but is characterized by the genetically determined life span of functioning postmitotic fibrocytes. However, premature terminal differentiation of progenitor fibroblasts to postmitotic fibrocytes can be induced by exogenous stress such as ionizing radiation. Radiation exposure in a dose range of 1,10 Gy causes a significant change in the distribution of surviving MF progenitor cells towards later differentiation stages within the precursor compartment as well as the differentiaton-dependent transition of progenitor fibroblasts to postmitotic fibrocytes. In the context of radiation-induced differentiation representing the proliferative senescence of the fibroblast/fibrocyte cell system, the transforming growth factor-,1 (TGF-,1) plays a key regulatory role. By the use of TGF-,1 knock-out cells (TGF-,1,/,) as well as neutralizing antibodies against active TGF-,1 the important function of this cytokine in the terminal differentiation process/proliferative senescence of the fibroblast system could be demonstrated. This work is supported by a grant from the Deutsche Forschungsgemeinschaft (Ro 527/4,2) and the BMBF-/IZKF Program 01KS9602. Application of a new concept of stem cell development to clonal competition processes in chronic myeloid leukaemia I. Roeder, C. Werner and M. Loeffler Institute for Medical Informatics, Statistics, and Epidemiology (IMISE), University of Leipzig, Leipzig, Germany Different clonal growth and competition phenomena, clonal fluctuations, as well as disappearance/reappearance of clones in primitive hematopoiesis have been described for several animal systems. Qualitatively similar patterns can be found in human hematopoietic malignancies, such as chronic myeloid leukaemia (CML), where the clonal competition of a normal and a malignant clone can be followed by cytogenetic and molecular monitoring. Although the regulation mechanisms which underlie these phenomena are widely unknown, there is evidence that dynamical processes of stem cell proliferation and stem cell,stroma interactions are involved. Using the emerging knowledge of molecular characteristics, cell kinetics and cell,stroma effects, we developed a mechanistic disease model for CML, which explains macroscopic observations on a population basis by kinetic processes at a microscopic level. The model is based on a new general concept of stem cell development, which explains the self-renewing capacity of primitive hematopoietic progenitors as a consequence of reversible, environment-dependent changes of actual expressed cell properties (concept of microplasticity). The balanced supply of hematopoietic cells is achieved by the dynamic regulation of cell,stroma attachment/detachment kinetics. Single-cell-based simulations allow the description of clonal fluctuation and competition phenomena both on the level of cell populations and on the level of individual clones. Distributions of cytogenetic responses in populations of CML patients after treatment with hydroxyurea or interferon-, have been used to consolidate the model, which is now applied to other treatment modalities such as STI571 or autologous bone marrow transplantation. Neuronal morphology: shape characteristics and models A. Schierwagen and J. van Pelt Universityität Leipzig, Institut für Informatik, Augustuspl.10, 04109 Leipzig, Germany The development of neuronal morphology and the forming of neural networks are key processes involved in neural tissue formation. This paper presents methods for describing quantitatively neuronal shapes and for modelling neuronal branching patterns. Models of neuronal arborizations may be assorted in reconstruction models and (stochastic or mechanistic) growth models. The growth model approach is discussed in more detail. First, Van Pelt's BESTL model is outlined, together with results of its application to several neuron types. Then mechanistic growth models are presented which describe the neurite growth process on the basis of intra- and extracellular mechanisms. In conclusion, the paper demonstrates that growth models are indispensable computational tools if the development of neuronal morphology is to be understood as a complex process governed by intracellular mechanisms, activity-dependent processes and interaction with the local environment. Activated Notch1 instructively induces multilineage myeloid differentiation of the hematopoietic stem cell line FDCP-mix T. Schroeder and U. Just GSF-Institute of Clinical Molecular Biology and Tumour Genetics, München, Germany Hematopoietic stem cells self-renew and differentiate in response to local environmental signals provided by hematopoietic stromal cells in the bone marrow. Here we present evidence suggesting that Jagged1/Notch signalling is involved in this process by inducing myeloid differentiation of hematopoietic stem cells. Activated Notch1, even when induced transiently, irreversibly reduced the self-renewal capacity of the multipotent hematopoietic stem cell line FDCP-mix accompanied by increased and accelerated differentiation along the granulocyte, macrophage and erythroid lineage. Differentiation was induced by activated Notch1 even under conditions that normally promote self-renewal but required the presence of hematopoietic cytokines for survival and proliferation. Apoptosis of FDCP-mix cells was not influenced by activated Notch1 and proliferation was only slightly inhibited. Similar to activated Notch1, the Notch ligand Jagged1, which is normally expressed on bone marrow stromal cells, induced an increased and accelerated myeloid differentiation of FDCP-mix cells. These results indicate that activation of mNotch1 provides a cytokine-independent instructive signal for multilineage myeloid differentiation of hematopoietic stem cells and reduces the probability of stem cell self-renewal. Effects of keratinocyte growth factor on survival and colony size of irradiated human keratinocytes in vitro D. Slonina, H. Hoinkis and W. Dörr c/o Department of Radiotherapy and Radiation Oncology, Medical Faculty Carl Gustav Carus, Technical University, Dresden, Germany The efficacy of recombinant human keratinocyte growth factor (rhKGF) in ameliorating acute radiation effects in oral mucosa was recently demonstrated. However, the exact mechanisms of these effects of KGF remain unclear. The present study was initiated in order to investigate rhKGF-induced changes in survival and colony size of normal human epidermal keratinocytes in vitro. For this, primary human neonatal keratinocytes (HEKn) were irradiated with single doses of 0 Gy (control) and 2 Gy (200 kV X-rays) and subsequently incubated in the presence or absence of 100 ng/ml rhKGF. Plating efficiency (PE) and surviving fraction (SF) were determined in a standard clonogenic assay. In cell cultures with or without rhKGF the mean PE was 4.6%. Irradiation with 2 Gy without KGF resulted in a SF2 of 51%; with KGF, a comparable SF2 of 54% was observed. This indicates that KGF did not change the survival characteristics of HEKn keratinocytes. The size of the individual colonies, however, was significantly increased in all cultures incubated with rhKGF compared to control cultures without rhKGF. The number of extremely large colonies (, 2 mm) was clearly higher (P < 0.0001) in the rhKGF-treated cultures. In conclusion, rhKGF does not affect keratinocyte survival after irradiation, but effectively stimulates proliferation of surviving cells. Mathematical models of hematopoiesis dynamics in irradiated mammals O. A. Smirnova Research Centre of Spacecraft Radiation Safety, Moscow, Russia Mathematical models are developed which describe the dynamics of bone-marrow hematopoiesis in mammals exposed to acute and chronic irradiation. The blood-forming system is represented here as a combination of major hematopoietic lines: thrombocytopoiesis, lymphopoiesis, erythropoiesis and granulocytopoiesis. The models are the systems of non-linear differential equations whose variables are represented by concentrations of mature blood cells and their bone-marrow precursors capable and incapable of dividing. The models quantitatively describe the ordinary effects of acute and chronic irradiation on the hematopoietic system of mammals (mice and rats). They also reproduce paradoxical effects of low dose rate chronic exposures, namely, the relaxation of lymphopoiesis, granulocytopoiesis and erythropoiesis systems to new equilibrium states characterized by elevated stationary concentrations of cells of some compartments. In addition, the models imitate experimentally observed increase of radioresistance of some hematopoietic lines under certain regimes of preliminary single and chronic exposures to radiation. Proceeding from the results obtained, an explanation of ,an adaptive response' of preirradiated animals on subsequent acute irradiation is suggested. The model can be used for predicting hematopoiesis dynamics in irradiated mammals. Mathematical models of dynamics of small intestine epithelium system in non-irradiated and irradiated mammals O. A. Smirnova Research Centre of Spacecraft Radiation Safety, Moscow, Russia Three mathematical models are developed which describe the dynamics of basic (columnar) cells in the small intestine of mammals (mice) under normal conditions and under acute and chronic irradiation. The models include the chalone mechanism of regulation of the small intestine epithelium system and take account of the principal stages of development of columnar cells. Also, the models make allowance for peculiarities of small intestine epithelium system functioning related to the motion of columnar cells over crypt and villus in the course of their division and maturation. The models are systems of non-linear differential equations whose variables are concentrations of columnar villus cells and their crypt precursors capable and incapable of dividing. The first model predicts dynamical regimes (including limit cycle) which are specific to the small intestinal system in the absence of radiation. The two other models describe quantitatively experimentally observed dynamics of cell concentrations in the crypt-villus system of mice exposed to acute and chronic radiation. The models can be used for simulating the dynamics of small intestinal system in non-irradiated and irradiated mammals. Differing sensitivity of tumour cells to apoptosis induction by iron deprivation H. ,týbrová, J. Truksa, T. Valenta and J. Kováø Cell Growth Control Laboratory, Institute of Molecular Genetics, Prague, Czech Republic We studied the sensitivity of tumour cells to apoptosis induction by iron deprivation. Iron deprivation was achieved by the employment of a defined iron-deficient culture medium. Some cells were found to be highly sensitive to iron deprivation (38C13, Raji). Under iron-depriving conditions, they died within 48 h (38C13) or 96 h (Raji), respectively. On the other hand, some tumour cells lines were completely resistant to the iron deprivation under the same experimental conditions (EL4, HeLa). DNA fragmentation analysis and flow cytometric analysis revealed characteristic apoptotic changes in sensitive cells but not in resistant cells. We compared the effect of iron deprivation with the effect of other apoptotic inducers (rotenone, hydroxyurea, methotrexate and doxorubicin). We found that there was a correlation between sensitivity to iron deprivation and sensitivity to other apoptosis inducers. Cells sensitive to iron deprivation (38C13, Raji) had a greater degree of sensitivity to other apoptosis inducers while cells resistant to iron deprivation (EL4, HeLa) were resistant to other apoptosis inducers in most cases. We conclude that some tumour cells are sensitive to apoptosis induction by iron deprivation while other tumour cells are resistant. Cells sensitive to iron deprivation seem to be of hematopoietic origin. Stimulatory effect of enterocyte-derived factor(s) on proliferative activity of haematopoetic precursors in vitro V. V. Temchura, S. V. Sennikov, T. V. Injelevskaya, V. A. Trufakin and V. A. Kozlov Institute of Clinical Immunology SB RAMS, Novosibirsk, Russia It is known that several haematopoietic cytokines are expressed in the mouse fetal intestine in the same fashion as in the fetal thymus and liver, the latter both known as haematopoietic tissues. We hypothesized that postnatal intestinal epithelial cells (IEC) may act as a cell source of growth factor(s) that act on early haematopoiesic precursors. We found that transplantation of bone marrow cells cultivated in medium containing 20% of serum-free mouse IEC supernatants (SIEC) to lethally irradiated animals led to the stimulation of formation of spleen nodules within 8 days. Quantitatively, the proliferative stimulation reached 17.8 ± 2% as compared to the control group. Then SIEC was separated into three fractions (F): F1 (> 30 kDa), F2 (10,30 kDa) and F3 (< 10 kDa). Stimulation activity of F1 was comparable to native SIEC (17.1 ± 0.3%), F2 had less activity (8.1 ± 0.7%) and F3 had no up-regulation of proliferative activity. Haemopoietic cytokines were measured using the electrochemiluminescence method with polyclonal and monoclonal antibodies. Production of granulocyte-macrophage colony-stimulating factor (GM-CSF) was detected in SIEC (37 241 pg/ml), in F1 (28 305 pg/ml) and in F2 (5490 pg/ml). Thus, these findings suggest that IEC SIEC has a proliferative-stimulating effect on SHC under these conditions. This activity may be mediated by GM-CSF, possibly in cooperation with either known cytokines or unknown specific factors secreted by the gut epithelium. Apoptosis induction in tumour cells by thiol deprivation and thiol excess J. Truksa, H. ,týbrová, K. Spìváková, T. Valenta and J. Kováø Cell Growth Control Laboratory, Institute of Molecular Genetics, Prague, Czech Republic We found that thiol deprivation (2-mercaptoethanol concentrations about 0.3 µm and lower) resulted in apoptotic death in some cultured tumour cells (mouse 38C13) while other cells were resistant (mouse EL4). On the other hand, thiol excess (2-mercaptoethanol concentrations higher than 300 µm) induced apoptosis in both cell lines. Experiments with other thiol compounds, l -cysteine and l -cystine, revealed that a free SH group is required for growth and survival of 38C13. Nonthiol antioxidant ascorbic acid was unable to maintain survival of 38C13 cells. However, it induced apoptosis in both tested cell lines at high concentrations (600 µm and higher). Thus the effect of thiol excess seems to be coupled with the excess of antioxidants while apoptotic induction by thiol deprivation seems to be free SH group specific. Immunofluorescence analysis did not show any significant change in the expression or activation of p53 due to thiol deprivation or thiol excess. The level of antiapoptotic protein Bcl-2 showed no changes in the case of thiol deprivation. However, in the case of thiol excess a significant decrease in the Bcl-2 level in both tested cell lines was detected. On the other hand, proapoptotic protein Bax displayed unchanged levels for thiol deprivation as well as for thiol excess. We conclude that p53 and Bax do not play any substantial role in apoptosis induction by thiol deprivation or by thiol excess while the Bcl-2 protein could be involved in apoptotic death induced by thiol excess. Nickel salts induce a decrease in histone H4 acetylation and down-regulate MDRI gene expression in multidrug-resistant human ovarian carcinoma cells A. Trussardi, S. Yatouji, F. Bontemps, L. Lilette, C. Trentesaux and J. Dufer Unité MéDIAN, CNRS FRE2141, IFR53, Faculté de Pharmacie, Reims, France Human ovarian carcinoma multidrug-resistant OV1-VCR cancer cells displayed, as compared to their parental IGROV1 cell line, nuclear texture changes compatible with a chromatin decondensation, as previously shown by image cytometry. These changes were associated with an increased (about 5-fold) DNase I sensitivity in OV1-VCR nuclei, suggesting an increased chromatin accessibility. It has been shown that high levels of chromatin acetylation across complete chromatin domains induced chromatin changes detected as ,general DNase I sensitivity'. By Western blotting, the level of acetylated histone H4 appears increased in OV1-VCR cells. As nickel salts have been described as inhibitors of histone H4 acetylation9, IGROV1 and OV1-VCR cells were treated with 1 µg/cm2 of nickel chloride or nickel subsulfide for 48 h. This treatment greatly reduces histone H4 acetylation in both cell lines. This decrease can be observed on the four putative acetylation sites of the histone (lys5, 8, 12 and 16). Furthermore, in OV1-VCR cells, nickel treatment significantly down-regulates the mdr1 gene expression. This effect does not appear to be non-specific, as ubiquitously expressed gene ,2m remains unaffected, either in IGROV1 or OV1-VCR cells. This work was supported by the Comité Départemental des Ardennes de la Ligue Française contre le Cancer. The use of mouse chimeras to study genes regulating stem cell function G. Van Zant Departments of Medicine and Physiology, University of Kentucky Medical Center, Lexington, KY 40536-0093, USA Self-renewing tissues rely on stem cell populations to continuously supply those mature cells with inherently short lifespans or those lost through wear and tear, disease or accident. Stem cells must therefore function throughout the lifespan of an animal; failure to do so would result in life-threatening aplasias. Since stem cells are capable of not only supplying differentiated cells to a tissue, but also replicating to resupply their own numbers, it would seem that such populations would be exempt from age-related depletion. An alternative perspective is that they may be exquisitely sensitive to age-related functional decline if their regenerative properties are not fully maintained. Using lympho-hematopoiesis as a model, it is widely recognized that production of most blood cells is maintained at near-normal levels throughout the lifespans of rodents and humans. An exception is the redistribution in lymphoid cell subpopulations during ageing resulting in the contraction of some subsets, and, of course, the involution of the thymus. It is further recognized that the response to hematological and immunological stress is blunted in older animals and humans, despite the fact that in mice the number of stem cells actually increases in the bone marrow during normal ageing. I will discuss our use of mouse chimeras, both cellular and genomic, to study the genetics of stem cell regulation during ageing. Cellular chimeras made by aggregating embryos of different mouse strains is a powerful tool to (a) identify cell autonomous (genetic) stem cell traits and (b) through natural stem cell competition, to uncover even very subtle differences in stem cell regulation over the lifetime of a chimera. Genomic chimeras, recombinant inbred mice and congenic mice, have been used to take a forward genetic approach (phenotype to genotype) to identify and characterize genetic loci responsible for stem cell regulation. My talk will present our latest results using genomic chimeras and will re-examine our earlier results using cellular chimeras. Tumour vascularization and angiogenesis P. Vaupel1 and M. A. Konerding2 1 Institute of Physiology & Pathophysiology; 2 Institute of Anatomy, University of Mainz, 55099 Mainz, Germany During embryogenesis all organs and nearly all tissues develop specific vascular patterns fulfilling the individual specific demands. Tumours, in contrast, induce, after an initial avascular growth phase, a structurally and functionally different vascular bed, whereby both pre-existent host and newly formed vessels are involved in the supply of nutrients and drainage of waste products. Neoangiogenesis is primarily induced by VEGF, which in turn is triggered by HIF-1. Compared to all other kinds of physiological and pathophysiological secondary angiogenesis (such as normal growth, menstrual cycle, wound healing or chronic inflammation), tumour angiogenesis exhibits numerous abnormalities. These include the lack of further differentiation, formation of sinusoidal vessels, endothelial cell and basement membrane discontinuities, all of which can result in high vascular permeability and direct contact of the flowing blood with tumour cells. In addition, there is also no doubt that tumour cells participate in vessel wall lining, a fact which should be considered in antivascular therapy concepts. Microvessel elongations, tortuousity, blind ends, increased interbranching distances as well as low degree of vascular hierarchy contribute to disturbances of the nutritive blood flow and can induce rheological problems. Arterio-venous shunts and vascular networks supplied and drained from the venous side further impair adequate supply and drainage which in turn promote the development of a hostile metabolic environment. These poorly supplied areas are heterogeneously distributed over the tumour mass and can be seen in the immediate vicinity of vascular hot spots. The vessel densities in these hot spots can be even higher than in the surrounding tissue. No flow regulation according to metabolic demands can be expected in tumours due to the absence of physiological/pharmacological receptors and the sinusoidal vessel wall structure which lacks smooth muscle cells. So far, no sufficient lymphatic drainage has been observed in solid tumours. Cells of the side population (SP) contribute to liver regeneration G. G. Wulf, K.-L. Luo, M. K. Brenner and M. A. Goodell Centre for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 11030, USA High efflux capacity for xenobiotics is a distinctive cell function of primitive hemopoietic stem cells. Exploiting this feature by flow cytometry after staining with the fluorescent dye Hoechst 33342, we previously identified highly potent hemopoietic stem cells in the side population (SP) of the murine bone marrow. In the liver, Hoechst 33342 staining delineated a very similar population of small blast-like cells with partial expression of CD45. In orthotopic liver cell transplantations conditioning the recipients with the hepatotoxic agent DDC, hepatic SP cells contributed to the mature liver parenchyma including bile duct epithelium, irrespective of their CD45 status. In animals with stable hemopoietic chimerism 12 month after transplantation with bone marrow SP cells, a part of the hepatic SP cells were bone marrow derived, and were recruitable to liver regeneration after transient DDC treatment. These findings introduce SP cells from different sources as a facultative liver regenerating cell population. Alterations of glycosphingolipid composition in differentiating lymphocytes of intact animals and animals with sarcoma 45 A. Zakaryan, L. Hovsepyan and K. Karagezyan Laboratory of Lipid Biochemistry, Institute of Molecular Biology of Armenian NAS, 7 Hasratyan St., Yerevan, 375014, Republic of Armenia (E-mail:annalg@mb.sci.am) The galactosylceramide (GalCer), cerebroside sulphate (CBS) and ganglioside composition in bone marrow progenitors of lymphocytes, thymocytes and mature lymphocytes of intact rats and rats grafted with sarcoma 45 was studied. Five fractions of GalCer and CBS of the cells from both intact and tumour-bearing animals were obtained by thin-layer chromatography: three fractions of GalCer and two of CBS. The quantity of GalCer and CBS in healthy animals was shown to be rather stable during lymphocyte differentiation. Only in thymocytes the decrease in one fraction of GalCer and two fractions of CBS was observed. In rats grafted with tumour cells a pronounced drop in GalCer and CBS contents took place. It began in the thymic population and was most strongly expressed in mature lymphocytes. In tumour-bearing animals the rate of ganglioside sialylation diminished which was expressed in an increase in mono- and di- and a decrease in tri- and tetrasialoganglioside levels. Thus, tumour-induced alterations in lymphocyte glycosphingolipid composition involve all stages of lymphocyte differentiation. This modification is believed to be a result of inhibition of synthesis of these lipids. The alterations observed may reflect a shift of the normal balance between lymphocyte subpopulations and the suppression of the antineoplastic activity of effector cells. [source] BAFF: a local and systemic target in autoimmune diseasesCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 2 2009I. Moisini Summary BAFF (B lymphocyte activating factor of the tumour necrosis factor family) is a vital homeostatic cytokine for B cells that helps regulate both innate and adaptive immune responses. Increased serum levels of BAFF are found in a number of different autoimmune diseases, and BAFF is found in inflammatory sites in which there is lymphoid neogenesis. BAFF antagonism has been used in several autoimmune disease models, resulting in B cell depletion, decreased activation of T cells and dendritic cells (DC) and a reduction in the overall inflammatory burden. BAFF, through its interaction with BAFF-R, is required for survival of late transitional, marginal zone and mature naive B cells, all of which are depleted by BAFF blockade. Through their interactions with TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) and BCMA (B cell maturation protein), BAFF and its homologue APRIL (a proliferation-inducing ligand), support the survival of at least some subsets of plasma cells; blockade of both cytokines results in a decrease in serum levels of immunoglobulin (Ig)G. In contrast, neither BAFF nor APRIL is required for the survival or reactivation of memory B cells or B1 cells. BAFF also helps DC maturation and interleukin (IL)-6 release and is required for proper formation of a follicular dendritic cell (FDC) network within germinal centres, although not for B cell affinity maturation. The clinical efficacy of BAFF blockade in animal models of autoimmunity may be caused both by the decline in the number of inflammatory cells and by the inhibition of DC maturation within target organs. Blockade of BAFF and its homologue APRIL are being explored for human use; several Phase I and II clinical trials of BAFF inhibitors for autoimmunity have been completed and Phase III trials are in progress. [source] Immunomodulatory activity of a methionine aminopeptidase-2 inhibitor on B cell differentiationCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 3 2009R. C. Priest Summary Methionine aminopeptidase-2 (MetAP-2) inhibitors have potent anti-angiogenesis activity and are being developed for the treatment of solid tumours. The recently observed specific expression of MetAP-2 in germinal centre B cells suggests that it has a role in regulating B cell function. We have demonstrated a potent MetAP-2-dependent inhibitory effect on the antibody secretion from B cell receptor and CD40 co-stimulated primary human B cells in the presence of interleukin-21. The effect of MetAP-2 inhibition on antibody secretion was due to a block in differentiation of B cells into plasma cells. Immunohistochemical analysis of germinal centres from human, mouse and marmoset spleen showed a similar expression pattern of MetAP-2 in the marmoset and man, whereas mouse spleen showed no detectable expression. In a marmoset, T dependent immunization model, the MetAP-2 inhibitor suppressed an antigen-specific antibody response. Furthermore, histological analysis showed loss of B cells in the spleen and disrupted germinal centre formation. These results provide experimental evidence to support a novel role for MetAP-2 in immunomodulation. These effects of MetAP-2 are mediated by disruption of the germinal centre reaction and a block in the differentiation of B cells into plasma cells. [source] | |||||||||||||||||||||||||