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Parental Cells (parental + cell)
Terms modified by Parental Cells Selected AbstractsA case of structure determination using pseudosymmetryACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009Sergei Radaev Here, a case is presented of an unusual structure determination which was facilitated by the use of pseudosymmetry. Group A streptococcus uses cysteine protease Mac-1 (also known as IdeS) to evade the host immune system. Native Mac-1 was crystallized in the orthorhombic space group P21212. Surprisingly, crystals of the inactive C94A mutant of Mac-1 displayed monoclinic symmetry with space group P21, despite the use of native orthorhombic Mac-1 microcrystals for seeding. Attempts to solve the structure of the C94A mutant by MAD phasing in the monoclinic space group did not produce an interpretable map. The native Patterson map of the C94A mutant showed two strong peaks along the (1 0 1) diagonal, indicating possible translational pseudosymmetry in space group P21. Interestingly, one-third of the monoclinic reflections obeyed pseudo-orthorhombic P21212 symmetry similar to that of the wild-type crystals and could be indexed and processed in this space group. The pseudo-orthorhombic and monoclinic unit cells were related by the following vector operations: am = bo,co, bm = ao and cm = ,2co,bo. The pseudo-orthorhombic subset of data produced good SAD phases, leading to structure determination with one monomer in the asymmetric unit. Subsequently, the structure of the Mac-1 mutant in the monoclinic form was determined by molecular replacement, which showed six molecules forming three translationally related dimers aligned along the (1 0 1) diagonal. Knowing the geometric relationship between the pseudo-orthorhombic and the monoclinic unit cells, all six molecules can be generated in the monoclinic unit cell directly without the use of molecular replacement. The current case provides a successful example of the use of pseudosymmetry as a powerful phase-averaging method for structure determination by anomalous diffraction techniques. In particular, a structure can be solved in a higher pseudosymmetry subcell in which an NCS operator becomes a crystallographic operator. The geometrical relationships between the subcell and parental cell can be used to generate a complete molecular representation of the parental asymmetric unit for refinement. [source] Acceleration of granulocyte colony-stimulating factor-induced neutrophilic nuclear lobulation by overexpression of Lyn tyrosine kinaseFEBS JOURNAL, Issue 1 2002Tomomi Omura Stimulation with granulocyte colony-stimulating factor (G-CSF) induces myeloid precursor cells to differentiate into neutrophils, and tyrosine phosphorylation of certain cellular proteins is crucial to this process. However, the signaling pathways for neutrophil differentiation are still obscure. As the Src-like tyrosine kinase, Lyn, has been reported to play a role in G-CSF-induced proliferation in avian lymphoid cells, we examined its involvement in G-CSF-induced signal transduction in mammalian cells. Expression plasmids for wild-type Lyn (Lyn) and kinase-negative Lyn (LynKN) were introduced into a murine granulocyte precursor cell line, GM-I62M, that can respond to G-CSF with neutrophil differentiation, and cell lines that overexpressed these molecules (GM-Lyn, GM-LynKN) were established. Upon G-CSF stimulation, both the GM-Lyn and GM-LynKN cells began to differentiate into neutrophils, showing early morphological changes within a few days, much more rapidly than did the parental cells, which started to exhibit nuclear lobulation about 10 days after the cells were transferred to G-CSF-containing medium. However, the time course of expression of the myeloperoxidase gene, another neutrophil differentiation marker, was not affected by the overexpression of Lyn or LynKN. Therefore, in normal cells, protein interactions with Lyn, but not its kinase activity, are important for the induction of G-CSF-induced neutrophilic nuclear lobulation in mammalian granulopoiesis. [source] Role of DNA polymerase , in tolerance of endogenous and exogenous DNA damage in mouse B cellsGENES TO CELLS, Issue 2 2006Akiko Ukai DNA polymerase , (Pol,) is a family A polymerase that contains an intrinsic helicase domain. To investigate the function of Pol, in mammalian cells, we have inactivated its polymerase activity in CH12 mouse B lymphoma cells by targeted deletion of the polymerase core domain that contains the catalytic aspartic acid residue. Compared to parental CH12 cells, mutant cells devoid of Pol, polymerase activity exhibited a slightly reduced growth rate, accompanied by increased spontaneous cell death. In addition, mutant cells showed elevated sensitivity to mitomycin C, cisplatin, etoposide, ,-irradiation and ultraviolet (UV) radiation. Interestingly, mutant cells were more sensitive to the alkylating agent methyl methanesulfonate (MMS) than parental cells. This elevated MMS sensitivity relative to WT cells persisted in the presence of methoxyamine, an inhibitor of the major base excision repair (BER) pathway, suggesting that Pol, is involved in tolerance of MMS through a mechanism that appears to be different from BER. These results reveal an important role for Pol, in preventing spontaneous cell death and in tolerance of not only DNA interstrand cross-links and double strand breaks but also UV adducts and alkylation damage in mammalian lymphocytes. [source] Increased genomic instability and altered chromosomal protein phosphorylation timing in HRAS -transformed mouse fibroblastsGENES, CHROMOSOMES AND CANCER, Issue 5 2009Katherine L. Dunn The RAS-mitogen-activated protein kinase signaling pathway is often deregulated in cancer cells. In metastatic HRAS -transformed mouse fibroblasts (Ciras-3), the RAS-MAPK pathway is constitutively activated. We show here that Ciras-3 cells exhibit a higher incidence of chromosomal instability than 10T1/2 cells, including higher levels of clonal and nonclonal chromosomal aberrations. Stimulation of serum starved 10T1/2 and Ciras-3 cells with phorbol esters (TPA) results in the phosphorylation of histone H3 at serine 10 and serine 28. Regardless of the increased genomic instability in Ciras-3 cells, TPA-induced H3 phosphorylated at serine 10 and H3 phosphorylated at serine 28 partitioned into distinct nuclear subdomains as they did in the parental cells. However, the timing of the response of the H3 phosphorylation event to TPA induction was delayed in Ciras-3 cells. Further Ciras-3 cells, which have a more open chromatin structure, had increased steady state levels of phosphorylated H3 and HMGN1 relative to parental 10T1/2 cells. TPA-induced H3 phosphorylated at serine 10 and 28 were colocalized with the transcriptionally initiated form of RNA polymerase II in 10T1/2 and Ciras-3 cells. Chromatin immunoprecipitation assays demonstrated that TPA-induced H3 phosphorylation at serine 28 was associated with the immediate early JUN promoter, providing direct evidence that this histone post-translational modification is associated with transcriptionally active genes. Together our results demonstrate the increased genomic instability and alterations in the epigenetic program in HRAS -transformed cells. © 2009 Wiley-Liss, Inc. [source] Decreased pyruvate kinase M2 activity linked to cisplatin resistance in human gastric carcinoma cell linesINTERNATIONAL JOURNAL OF CANCER, Issue 4 2004Byong Chul Yoo Abstract Resistance to anticancer drugs is a major obstacle preventing effective treatment of disseminated cancers. Understanding the molecular basis to chemoresistance is likely to provide better treatment. Cell lines resistant to cisplatin or 5-fluorouracil (5-FU) were established from human gastric carcinoma cell lines SNU-638 and SNU-620. Comparative proteomics involving 2-dimensional gel electrophoresis (2-DE) and matrix-associated laser desorption ionization-mass spectroscopy (MALDI-MS) was performed on protein extracts from these parental and drug-resistant derivative lines to screen drug resistance-related proteins. Pyruvate kinase M2 (PK-M2) was identified as a protein showing lower expression in cisplatin-resistant cells compared to parental cells. Consistent with this finding, PK-M2 activity was also lower in cisplatin-resistant cells. Suppression of PK-M2 expression by antisense oligonucleotide resulted in acquired cisplatin resistance in SNU-638 cells. Furthermore, PK-M2 activity in 11 individual human gastric carcinoma cell lines positively correlated with cisplatin sensitivity. Taken together, PK-M2 protein and activity levels were lower in cisplatin-resistant human gastric carcinoma cell lines compared to their parental cell lines. Furthermore, suppression of PK-M2 expression using antisense oligonucleotides increased cisplatin resistance. These data clearly link PK-M2 and cisplatin resistance mechanisms. © 2003 Wiley-Liss, Inc. [source] The role of ZIP8 down-regulation in cadmium-resistant metallothionein-null cellsJOURNAL OF APPLIED TOXICOLOGY, Issue 5 2009Hitomi Fujishiro Abstract The mechanisms of cellular cadmium uptake in mammalian cells remain obscure. To solve this problem, we established cadmium-resistant cells (A7 and B5) from metallothionein-null mouse cells, and found that cadmium accumulation was markedly suppressed in these cells. DNA microarray and real-time PCR analyses revealed that expressions of ZIP (Zrt-, Irt-related protein) 8 and ZIP14 were down-regulated in A7 and B5 cells. In particular, both mRNA and protein levels of ZIP8 were markedly suppressed in A7 and B5 cells. Introduction of short hairpin RNA (shRNA) of ZIP8 into parental cells reduced the accumulation of cadmium to about 35% of that of mock-transfected cells, whereas the introduction of shRNA of divalent metal transporter 1 hardly changed cadmium accumulation. Thus, the cadmium resistance in A7 and B5 cells may be conferred primarily by the down-regulation of ZIP8. In mouse tissues, high expression of ZIP8 was noted in the liver, kidney, lung and testis. These data suggest that ZIP8 plays an important role in cellular uptake of cadmium. Copyright © 2009 John Wiley & Sons, Ltd. [source] Phenotypic comparison of periodontal ligament cells in vivo and in vitroJOURNAL OF PERIODONTAL RESEARCH, Issue 2 2001P. Lekic The mammalian periodontal ligament contains heterogeneous populations of connective tissue cells, the precise function of which is poorly understood. Despite close proximity to bone and the application of high amplitude physical forces, cells in the periodontal ligament (PL) are capable of expressing regulatory factors that maintain PL width during adult life. The study of PL homeostasis and PL cell differentiation requires culture and phenotypic methods for precise characterization of PL cell populations, in particular those cells with an inherently osteogenic program. Currently it is unknown if cells cultured from the PL are phenotypically similar to the parental cells that are present in the tissues. We have compared the phenotype of cells in vivo with cells derived from the PL and expanded in vitro to assess the general validity of in vitro models for the study of phenotypic regulation in vivo. Rat PL cells were isolated by either scraping the root of the extracted first mandibular molars (Group A), or by scraping the alveolar socket following extraction of first mandibular molars (Group B), or by obtaining a mixture of cells after disaggregating a block of tissue consisting of first mandibular molar, PL and the surrounding alveolar bone (Group C). Cultured cells at confluence were fixed and immunostained for ,-smooth muscle actin (,-SMA), osteopontin (OPN), alkaline phosphatase (AP), or bone sialoprotein (BSP). For in vivo assessments, frontal sections of rat first mandibular molar were immunostained for ,-SMA, OPN, AP and BSP. We examined osteogenic differentiation of cultured PL cell cultures by bone nodule-forming assays. In vivo and at all examined sites, >68% of PL cells were immunostained for AP; ,50% and ,51% for OPN and ,-SMA (p=0.3), respectively, while only ,8% were positively stained for BSP (p<0.01). Analysis of cultured PL cells in Groups A, B and C showed 54%, 53% and 56% positive staining for ,-SMA respectively; 51%, 56%, 54% for OPN; 66%, 70%, 69% for AP and 2.2%, 1.4% and 2.8% for BSP. The mean percentage of PL cells in situ stained for the different markers was similar to that of cultured PL cells (Group A,Group B,Group C in situ for p>0.2) except for BSP which was 3 to 4 fold higher in vivo(p<0.01). PL cell cultures treated with dexamethasone showed mineralized tissue formation for all groups (A, B, C), but no mineralized tissue formation was detected in the absence of dexamethasone. As PL cells express quantitatively similar phenotypes in vitro and in vivo, we conclude that the in vitro models used here for assessment of PL cell differentiation appear to be appropriate and are independent of the cell sampling method. Further, dexamethasone-dependent progenitors are present both on the root and bone-related sides of the PL. [source] KAI1 gene suppresses invasion and metastasis of hepatocellular carcinoma MHCC97-H cells in vitro and in animal modelsLIVER INTERNATIONAL, Issue 1 2008Jian-min Yang Abstract Background: Downregulation of KAI1 gene expression has been found in many types of cancer cells and is closely related to cancer invasion and metastasis. This study was aimed at investigating the effects and possible underlying mechanisms of KAI1 gene on invasion and metastasis of human hepatocellular carcinoma (HCC). Methods: The invasive ability, visco-elastic properties and cell adhesion forces were analysed in different HCC cells originating from the MHCC97-H cell line transfected with either the sense or the antisense KAI1 expression plasmid. Tumuorigenicity, metastatic abilities, extracellular matrix (ECM) and intercellular adhesion molecule-1 (ICAM-1) expression were also evaluated in the nude mouse models of the xenografted and orthotopic liver cancer cells. Results: Compared with their parental cells, in the HCC cells transfected with the sense KAI1 gene, the invasive ability in vitro was significantly decreased (P<0.01); the cellular elastic coefficients K1, K2 and , were significantly higher (P<0.05); the cells adhesion forces to fibronectin were significantly lower (P<0.01). The sense KAI1 gene transfection into the cancer cells also inhibited their invasion and lung metastasis in the orthotopic liver cancer nude mice. However, the opposite changes were observed in the HCC cells transfected with the antisense KAI1 gene. KAI1 gene transfection also affected ECM and ICAM-1 expression in the transplanted liver cancer. Conclusion: The KAI1 gene plays an important role in the invasion and metastasis of human HCC and its upregulation in HCC cells suppresses their invasive and metastatic abilities. KAI1 gene functioned as a metastasis inhibitor by regulating the HCC cell biophysical behaviours including aggregation, adhesion, motility and visco-elastic properties. [source] Prostate-specific antitumor activity by probasin promoter-directed p202 expression,MOLECULAR CARCINOGENESIS, Issue 3 2003Yong Wen Abstract p202, an interferon (IFN) inducible protein, arrests cell cycle at G1 phase leading to cell growth retardation. We previously showed that ectopic expression of p202 in human prostate cancer cells renders growth inhibition and suppression of transformation phenotype in vitro. In this report, we showed that prostate cancer cells with stable expression of p202 were less tumorigenic than the parental cells. The antitumor activity of p202 was further demonstrated by an ex vivo treatment of prostate cancer cells with p202 expression vector that showed significant tumor suppression in mouse xenograft model. Importantly, to achieve a prostate-specific antitumor effect by p202, we employed a prostate-specific probasin (ARR2PB) gene promoter to direct p202 expression (ARR2PB-p202) in an androgen receptor (AR),positive manner. The ARR2PB-p202/liposome complex was systemically administered into mice bearing orthotopic AR-positive prostate tumors. We showed that parenteral administration of an ARR2PB-p202/liposome preparation led to prostate-specific p202 expression and tumor suppression in orthotopic prostate cancer xenograft model. Furthermore, with DNA array technique, we showed that the expression of p202 was accompanied by downregulation of G2/M phase cell-cycle regulators, cyclin B, and p55cdc. Together, our results suggest that p202 suppresses prostate tumor growth, and that a prostate-specific antitumor effect can be achieved by systemic administration of liposome-mediated delivery of ARR2PB-p202. © 2003 Wiley-Liss, Inc. [source] ORIGINAL ARTICLE: Leukocyte Activation and Circulating Leukocyte-Derived Microparticles in PreeclampsiaAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 5 2009Christianne A.R. Lok Problem, Preeclampsia shows characteristics of an inflammatory disease including leukocyte activation. Analyses of leukocyte-derived microparticles (MP) and mRNA expression of inflammation-related genes in leukocytes may establish which subgroups of leukocytes contribute to the development of preeclampsia. Method of Study, Blood samples were obtained from preeclamptic patients, normotensive pregnant and non-pregnant controls. sL-selectin and elastase were measured by ELISA. mRNA was isolated from leukocytes and gene expression was determined by multiplex ligation-dependent probe amplification (MLPA). MP were characterized by flow cytometry. Results, Altered concentrations of sL-selectin and elastase confirmed leukocyte activation in preeclampsia. These leukocytes showed up-regulation of Nuclear Factor of Kappa light chain gene enhancer in B Cells inhibitor (NF,B-1A) and cyclin-dependent kinase inhibitor (CDKN)-1A compared with normotensive pregnant women. interleukin-1 Receptor Antagonist (IL-1RA) and tumor necrosis factor (TNF)-R1 were increased compared with those in non-pregnant controls. Monocyte-derived MP were elevated in preeclamptic patients compared with pregnant women. The numbers of cytotoxic T-cell-derived and granulocyte-derived MP were elevated compared with those of non-pregnant women. Conclusion, Leukocytes are activated in preeclampsia. A pro-inflammatory gene expression profile is not prominent, although differences in mRNA expression can be detected. Increased levels of particular subsets of leukocyte-derived MP reflect activation of their parental cells in preeclampsia. [source] Phenotypic variation during cloning procedures: Analysis of the growth behavior of clonal cell linesBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2006Louise M. Barnes Abstract The production of recombinant protein from mammalian cells is a key feature of the biotechnology industry. However, the generation of recombinant mammalian cell lines is still largely performed on an empirical basis and there are many potential areas for enhancement. We have shown previously that despite two rounds of limiting dilution cloning (LDC) of recombinant cell lines, there remained a high degree of heterogeneity in the resulting cell lines. We suggested that a rapid phenotypic drift occurred with these cells. It was unclear if this was a consequence of the added burden of production of a recombinant protein, the selection procedures, or merely an inherent feature of cell growth in culture. To address this, we have subjected untransfected (parental) cells to three successive rounds of LDC and monitored the growth properties of the resultant cells. The results show that despite repeated rounds of cloning, it was not possible to obtain phenotypically similar cell lines. We also demonstated that this phenotypic drift is not due to gross changes in the protein p27, a key regulators of the cell cycle. Although cells with a range of growth properties were observed even after three rounds of cloning, the variation in growth patterns between cell lines decreased after cloning. Hence, we suggest that by cloning it may be possible to generate untransfected cells, which have particular growth properties. Starting with a well-defined population of parental cells may aid in the subsequent generation of tranfectants with desired growth properties. © 2006 Wiley Periodicals, Inc. [source] Patterns of spread in an orthotopic mouse model of bladder cancerBJU INTERNATIONAL, Issue 2006J.P. BRENNAN Purpose:, To develop an orthotopic model of muscle-invasive transitional cell carcinoma (TCC) of the bladder which models primary tumour growth and metastasis. Methodology:, Cell lines were derived from the TCC cell line T24 (Tsu-Pr1) using in vivo selection for metastatic ability (Chaffer et al. Clin Exp Metastasis 2005; 22(2): 115,25). Each of these cell lines (Tsu-Pr1 and sub-lines, B1 and B2) was then injected intramurally into the mouse bladder wall (n = 25 × 3). The cell lines were also injected intravesically and intraperitoneally (n = 15 × 3 in each group). Results:, There were no differences between the three sub-lines in primary tumour formation, presence of macroscopic metastases and survival. This model produced more macroscopic and lymph node metastases in comparison with other orthotopic models reported in the literature. After intraperitoneal injection, the B2 cell line produced a higher number of discrete intra-abdominal masses in comparison with the parental line. This is likely to be related to the phenotype of the cells with parental cells being more mesenchymal, versus the B2 sub-line, which has more epithelial characteristics. Conclusion:, The TSU-Pr1 series is a useful, clinically relevant model of muscle-invasive TCC. In addition, this model may also provide insights into the role of mesenchymal-epithelial transition in the metastatic process. [source] Role of CD21 antigen in diffuse large B-cell lymphoma and its clinical significanceBRITISH JOURNAL OF HAEMATOLOGY, Issue 4 2004Masaki Otsuka Summary Recent advances in immunological and molecular technology have prompted proposals to change tumour classification and treatment strategies. Cell surface antigens are now easy to access, and tumour origins and clinical characteristics are now readily identifiable. However, in diffuse large B-cell lymphoma (DLBCL), one of the heterogeneous forms of haematological malignancy, the clinical significance of tumour surface antigens has not been well documented. We analysed the tumour surface antigens of 50 tumours from newly diagnosed DLBCL patients by flow cytometry in accordance with their clinical characteristics and followed the patients for a median 3·7 years. Statistical analysis showed that CD21 expression was significantly negatively associated with mortality in DLBCL (CD21 negative versus positive; relative risk = 2·36, P < 0·05). As a result of these clinical observations, we generated CD21-overexpressed (CD21+) lymphoma cell lines after gene transfection and analysed tumour cell growth in vivo in immunocompromised mice. Mice challenged with vector-only transfectants and parental cells as controls died within 50 d. In contrast, mice injected with CD21+ transfectants exhibited significantly reduced tumour growth and 83% survived long term (versus control groups; P < 0·05). Interestingly, all established CD21+ transfectants (six clones from different bulks) showed homotypic aggregation during in vitro cell culture, and anti-CD21 antibodies did not block this aggregation. Expression of CD21 is strongly associated with increased survival in DLBCL in vivo. CD21 expression may be indirectly concerned with the expression of additional cell adhesion molecules. [source] Mammary serine protease inhibitor inhibits epithelial growth factor-induced epithelial-mesenchymal transition of esophageal carcinoma cellsCANCER, Issue 1 2009Zhen Cai PhD Abstract BACKGROUND: By using proteomic technology, the authors previously observed the substantial down-regulation of mammary serine protease inhibitor (maspin) in esophageal squamous cell carcinoma and metastases. In the current study, they examined the effects of maspin re-expression in a maspin-null esophageal cancer cell line EC109 and also investigated the underlying mechanism. METHODS: A cell line with stable maspin expression was established. An epithelial growth factor (EGF)-induced epithelial-mesenchymal transition (EMT) model was used to mimic some aspects of the metastatic process in vitro. The effects of maspin reintroduction on EGF-induced EMT and cell growth characteristics were evaluated. Comparative proteomic analysis of transfected cells versus parental cells was then performed to explore the potential mechanism. RESULTS: The introduction of maspin into EC109 cells was able to inhibit EGF-induced EMT and altered cell growth characteristics, including the serum dependence, proliferative response to EGF stimulation, and colony formation ability in soft agar, indicating a conversion from a malignant phenotype to a benign phenotype. Proteomic analysis revealed a significant down-regulation of a group of glycolytic enzymes in maspin-transfected cells. In addition, maspin-transfected cells expressed much lower levels of hypoxia-inducible factor 1, than parental cells or empty vector transfected cells. CONCLUSIONS: Maspin exhibited a metastasis-suppressive effect, which may be a consequence of the reversal of the malignant phenotype of EC109 cells. The switch of cellular metabolic phenotype to low glycolysis by the gain of maspin function may play a key role in the process. This finding provides additional evidence of the tumor metastasis-suppressive activity of maspin and may indicate a new direction for future studies of the mechanism of maspin. Cancer 2009. © 2008 American Cancer Society. [source] Expression of cyclic guanosine monophosphate-dependent protein kinase in metastatic colon carcinoma cells blocks tumor angiogenesisCANCER, Issue 7 2008In-Kiu Kwon PhD Abstract BACKGROUND Type 1 cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) reportedly has exhibited antitumor properties, and its expression is down-regulated in many tumors. METHODS The authors recently demonstrated that PKG re-expression in metastatic colon carcinoma cells results in decreased tumorigenesis: In the current study, they addressed that mechanism. RESULTS Over-expression of PKG in SW620 cells produced smaller, more apoptotic subcutaneous tumors in athymic mice, but the observed effect of PKG expression on growth and apoptosis in vitro was minimal. Closer examination of the subcutaneous xenografts revealed highly vascular tumors produced by the parental SW620 cells, which contrasted greatly with the PKG-expressing tumors, in which cell growth was limited to "islands" surrounding CD31-positive cells. The idea that PKG expression was associated with reduced tumor angiogenesis was supported by decreased levels of vascular endothelial growth factor in these tumors compared with tumors that were derived from parental SW620 cells. Investigation of potential mechanisms revealed that PKG expression was associated with reduced levels of ,-catenin compared with parental cells. Moreover, this effect of exogenous PKG on ,-catenin expression in SW620 cells also occurred in vitro, where the decrease was associated with reduced T-cell factor-dependent transcription. CONCLUSIONS Together the findings indicated that PKG down-regulation in colon cancer cells is important for optimal tumor angiogenesis and that regulation of ,-catenin expression may be important to this process. Cancer 2008. © 2008 American Cancer Society. [source] The role of various Bcl-2 domains in the anti-proliferative effect and modulation of cellular glutathione levels: a prominent role for the BH4 domainCELL PROLIFERATION, Issue 1 2003R. W. M. Hoetelmans Reduced cell proliferation and increased levels of cellular glutathione (GSH) are characteristic for cells that overexpress the anti-apoptotic Bcl-2 protein. We investigated the influence of various Bcl-2 domains on both these characteristics. Rat CC531 colorectal cancer cells were stably transfected with the human bcl- 2 gene (CCbcl2 cells) or with bcl- 2 gene constructs missing a coding sequence for a func-tional domain, BH1 (CC,BH1 cells), BH3 (CC,BH3 cells), BH4 (CC,BH4 cells) or the transmembrane region (CC,TM cells). We measured GSH levels in exponentially and confluent growing bcl- 2-transfected cell populations. The fraction of S-phase cells during exponential growth was significantly reduced in CCbcl2, CC,BH1, CC,BH3, and CC,TM cells compared with parental CC531, neo-transfected CC531 and CC,BH4 cells. GSH levels in these bcl -2 transfectants were significantly higher than in the parental line measured at 50% confluence; at 100% confluence they reached a similar level as found in parental cells. Independently from the presence of BH1, BH3 or TM domains, overexpression of Bcl-2 reduces cellular proliferation under conditions of increased GSH levels. This apparent link is lost in CC,BH4 cells; these cells are not reduced in cellular proliferation and harbour significantly higher GSH levels than found in the other transfectants. Studies on the subcellular localization revealed an extremely low expression of the Bcl-2 protein lacking the N-terminal BH4 domain in nuclear fractions. Nuclear translocation of Bcl-2 requires the presence of the BH4 domain and seems prominent in reducing cellular proliferation. [source] The diphthamide modification on elongation factor-2 renders mammalian cells resistant to ricinCELLULAR MICROBIOLOGY, Issue 8 2008Pradeep K. Gupta Summary Diphthamide is a post-translational derivative of histidine in protein synthesis elongation factor-2 (eEF-2) that is present in all eukaryotes with no known normal physiological role. Five proteins Dph1,Dph5 are required for the biosynthesis of diphthamide. Chinese hamster ovary (CHO) cells mutated in the biosynthetic genes lack diphthamide and are resistant to bacterial toxins such as diphtheria toxin. We found that diphthamide-deficient cultured cells were threefold more sensitive than their parental cells towards ricin, a ribosome- inactivating protein (RIP). RIPs bind to ribosomes at the same site as eEF-2 and cleave the large ribosomal RNA, inhibiting translation and causing cell death. We hypothesized that one role of diphthamide may be to protect ribosomes, and therefore all eukaryotic life forms, from RIPs, which are widely distributed in nature. A protective role of diphthamide against ricin was further demonstrated by complementation where dph mutant CHO cells transfected with the corresponding DPH gene acquired increased resistance to ricin in comparison with the control-transfected cells, and resembled the parental CHO cells in their response to the toxin. These data show that the presence of diphthamide in eEF-2 provides protection against ricin and suggest the hypothesis that diphthamide may have evolved to provide protection against RIPs. [source] Thyroid cancer immuno-therapy with retroviral and adenoviral vectors expressing granulocyte macrophage colony stimulating factor and interleukin-12 in a rat modelCLINICAL ENDOCRINOLOGY, Issue 6 2003Kunihiko Tanaka Summary background, Introduction of genes encoding immuno-stimulatory cytokine(s) into cancer cells is well known to enhance anti-tumour immunity. aim, The present studies were designed to evaluate the therapeutic efficacy of retroviral- and adenoviral-mediated delivery of IL-12 and/or granulocyte macrophage colony-stimulating factor (GM,CSF) genes for thyroid cancer in an immuno-competent rat model. methods, A rat thyroid cancer cell line FRTL-Tc syngeneic to Fisher rat was used. results, Expression of these exogenous cytokines did not affect in vitro cell growth. Subcutaneous injection of FRTL-Tc cells retrovirally transduced with IL-12 or GM,CSF genes formed significantly smaller tumours than that of the parental cells, but had little effect on growth of distant tumours, suggesting no vaccine effect. Similarly, injection of the cells infected with adenovirus expressing IL-12 or GM,CSF (AdIL-12 or AdGM,CSF) almost completely abolished tumourigenicity and injection of AdGM,CSF into pre-established tumours significantly inhibited growth of the tumours injected; neither, however, showed a systemic vaccine affect. On the other hand, injection of AdIL-12 into the pre-established tumours significantly inhibited growth of not only the tumours injected but also distant tumours, indicating induction of systemic anti-tumour immunity. Serum IL-12 was detectable only in this approach. There was neither a synergistic or additive effect of these two cytokines. conclusions, Our data demonstrate in a rat thyroid cancer model that only injection of AdIL-12 into the pre-established tumours elicited systemic anti-tumour immunity, but injection of AdGM,CSF or injection of the cells expressing IL-12 or GM,CSF elicited only local effect, indicating that in situ delivery of IL-12 gene with adenovirus appears most efficacious but may still require adjuvant modalities to enhance the anti-tumour effect. [source] | ||||||||||||||||