CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 2 2009
Jian Zhong
SUMMARY 1Cardiac ryanodine RyR2 receptors regulate Ca2+ release from the sarcoplasmic reticulum (SR). FK506 binding protein (FKBP) 12.6 prevents aberrant SR Ca2+ leakage during diastole, thereby maintaining the integrity of RyR2 function. Previous studies have focused mainly on FKBP12.6 deficiency and so the pathophysiological consequences of FKBP12.6 overexpression remain unclear. Herein, we investigate the effect of FKBP12.6 overexpression on cardiac hypertrophic and apoptotic signalling. 2Human FKBP12.6 cDNA was cloned into pAdTrack-CMV and the resulting plasmid, along with a control empty plasmid, were transfected into bacteria. The resulting virus, namely Ad-FKBP12.6 containing green fluorescent protein, was propagated and purified. Neonatal rat cardiomyocytes were infected with this virus. Protein and DNA synthesis were measured by [3H]-leucine and [3H]-thymidine incorporation, respectively. Expression of p38 mitogen-activated protein kinase (MAPK), phosphorylated extracellular signal-regulated kinase 1 or 2 (p-ERK1/2) and Bax were examined by western blotting. 3Compared with control cells, cardiomyocytes that overexpressed FKBP12.6 became hypertrophic and hyperplastic, with increased levels of both p38 MAPK and p-ERK1/2. At the same time, overexpression of FKBP12.6 induced apoptosis of cardiomyocytes, as determined by both Bax protein expression and DNA fragmentation. Rapamycin treatment downregulated the expression of p-ERK1/2, p38 MAPK and Bax in stimulated cardiomyocytes, with or without FKBP12.6 overexpression, and enhanced protein synthesis, but had no effect on DNA synthesis in cardiomyocytes. 4In conclusion, FKBP12.6 overexpression may participate in pathophysiological processes through both hypertrophic and apoptotic signalling pathways, leading to cardiomyocyte damage and death. [source]

Hypocretin/orexin in fish physiology with emphasis on zebrafish

ACTA PHYSIOLOGICA, Issue 3 2010
P. Panula
Abstract One hypocretin/orexin (hcrt) gene has been identified in several fish species. The first pufferfish gene was identified in 2002 and the zebrafish gene was cloned in 2004. Its structure is very similar to that of mammals, and it encodes for two active peptides with C-termini similar to those of mammals. The gene is expressed in the brain in only one hypothalamic nucleus, which sends projections to the telencephalon, diencephalon, mesencephalon and rhombencephalon. The terminal fibres are found in close contact with many aminergic cell groups, including those of raphe serotonergic, locus coeruleus noradrenergic, several dopaminergic cell groups and the sole histaminergic hypothalamic cluster. One receptor corresponding to mammalian hcrt 2 receptor has been identified in fish. Overexpression of hcrt in zebrafish has been reported to consolidate wakefulness and inhibit rest. On the other hand, fish lacking the hcrt receptor show short and fragmented sleep instead of sleepiness and cataplexy. Food deprivation increases hcrt mRNA expression in zebrafish brain, and intracerebroventricular hcrt peptides stimulate food consumption and feeding behaviour in goldfish. Hcrt peptides thus have important roles in fish physiology. Many genetic and functional methods available render fish, especially zebrafish, a suitable organism to study new aspects of hcrt physiology in vertebrates. [source]

Thyroid hormone receptor , can control action potential duration in mouse ventricular myocytes through the KCNE1 ion channel subunit

ACTA PHYSIOLOGICA, Issue 2 2010
A. Mansén
Abstract Aims:, The reduced heart rate and prolonged QTend duration in mice deficient in thyroid hormone receptor (TR) ,1 may involve aberrant expression of the K+ channel ,-subunit KCNQ1 and its regulatory ,-subunit KCNE1. Here we focus on KCNE1 and study whether increased KCNE1 expression can explain changes in cardiac function observed in TR,1-deficient mice. Methods:, TR-deficient, KCNE1-overexpressing and their respective wildtype (wt) mice were used. mRNA and protein expression were assessed with Northern and Western blot respectively. Telemetry was used to record electrocardiogram and temperature in freely moving mice. Patch-clamp was used to measure action potentials (APs) in isolated cardiomyocytes and ion currents in Chinese hamster ovary (CHO) cells. Results:, KCNE1 was four to 10-fold overexpressed in mice deficient in TR,1. Overexpression of KCNE1 with a heart-specific promoter in transgenic mice resulted in a cardiac phenotype similar to that in TR,1-deficient mice, including a lower heart rate and prolonged QTend time. Cardiomyocytes from KCNE1-overexpressing mice displayed increased AP duration. CHO cells transfected with expression plasmids for KCNQ1 and KCNE1 showed an outward rectifying current that was maximal at equimolar plasmids for KCNQ1-KCNE1 and decreased at higher KCNE1 levels. Conclusion:, The bradycardia and prolonged QTend time in hypothyroid states can be explained by altered K+ channel function due to decreased TR,1-dependent repression of KCNE1 expression. [source]

Overexpression of CD7 in classical Hodgkin lymphoma-infiltrating T lymphocytes,

CYTOMETRY, Issue 3 2009
Adam C. Seegmiller
Abstract Background: Diagnosis of Hodgkin lymphoma (HL) is sometimes complicated by the scarcity of neoplastic cells in a reactive inflammatory background. Immunophenotyping by flow cytometry (FC) has not played a significant role in HL diagnosis because of its consistent failure to identify these neoplastic cells. However, HL-infiltrating T cells have been shown to play a role in HL pathogenesis. This study characterizes the FC immunophenotype of these T lymphocytes to determine whether they can be used to assist in the diagnosis of HL. Methods: Cell suspensions from 76 lymph nodes involved by HL and 156 lymph nodes with reactive lymphadenopathy (LAD) were analyzed by flow cytometry to assess the expression of T-cell antigens. Results: The CD4:CD8 ratio and CD7 expression in both CD4(+) and CD8(+) T cells are increased in HL compared with reactive lymph nodes and there are significant differences between these features in different subtypes of HL. However, only the expression of CD7 in CD4(+) T cells distinguishes between HL and reactive LAD. This is especially true for classical HL in younger patients. Using a CD7 mean fluorescence intensity (MFI) cutoff value generated by this data, 37/47 FNA specimens were correctly diagnosed. Conclusions: There are significant differences in the immunophenotypes of HL-infiltrating T cells. Of these, the CD7 expression in CD4(+) T cells discriminates between HL and reactive LAD, suggesting that this could be a useful and practical adjunctive tool in the diagnosis of HL. It may also further our understanding of the pathophysiology of this disease. © 2008 Clinical Cytometry Society [source]

Overexpression of CD49f in precursor B-cell acute lymphoblastic leukemia: Potential usefulness in minimal residual disease detection

CYTOMETRY, Issue 2 2009
Joseph A. DiGiuseppe
Abstract Background: The persistence of minimal residual disease (MRD) following therapy is an established prognostic factor in precursor B-cell acute lymphoblastic leukemia (pB-ALL). Detection of MRD in pB-ALL by flow cytometric immunophenotyping requires demonstration of abnormal antigen expression in leukemic B-cell precursors relative to that of normal B-cell precursors. The gene encoding CD49f (integrin ,-6) is one of several whose overexpression in pB-ALL at diagnosis has been associated with the subsequent detection of MRD. However, whether CD49f might be a useful reagent in the immunophenotypic detection of MRD in pB-ALL has not been evaluated. Methods: We evaluated CD49f expression by 4-color flow cytometry in normal B-cell precursors, and in a series of cases of pB-ALL, both at diagnosis and at intervals following the initiation of therapy. Results: In 10 control marrow samples, CD49f was undetectable or extremely dim in all but a minor subset of normal CD19+ B-lineage cells, whereas in 11 of 15 cases (73%) of pB-ALL, CD49f was moderate or bright at diagnosis, and persisted or became brighter after initiation of therapy. MRD detected using CD49f corresponded precisely with that obtained using a standard panel of antibodies, and permitted the detection of leukemic populations comprising as little as 0.02% of cells. Of the four pB-ALL cases in which CD49f was undetectable or dim at diagnosis, MRD was detected in two; in one of these, CD49f expression was substantially increased in the leukemic cells that persisted following initiation of therapy. Conclusions: CD49f is commonly overexpressed in p-B-ALL, and represents a potentially useful marker for the immunophenotypic detection of MRD. © 2008 Clinical Cytometry Society How to cite this article: DiGiuseppe JA, Fuller SG, Borowitz MJ. Overexpression of CD49f in precursor B-cell acute lymphoblastic leukemia: potential usefulness in minimal residual disease detection. Cytometry Part B 2008. [source]

Overexpression of profilin reduces the migration of invasive breast cancer cells

CYTOSKELETON, Issue 2 2004
Partha Roy
Abstract The exact role profilin plays in cell migration is not clear. In this study, we have evaluated the effect of overexpression of profilin on the migration of breast cancer cells. Overexpression was carried out by stably expressing GFP-profilin in BT474 cells. It was observed that even a moderate level of overexpression of profilin significantly impaired the ability of BT474 cells to spread on fibronectin-coated substrate and migrate in response to EGF. GFP-profilin expressing cells also showed increased resistance to detachment in response to trypsin and increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin compared to the parental and GFP-expressing (control) cell lines. These results suggest that perturbation of profilin levels may offer a good strategy for controlling the metastatic potential of breast cancer cells. Cell Motil. Cytoskeleton 57:84,95, 2004. © 2004 Wiley-Liss, Inc. [source]

Expression of constructs of the neuronal isoform of myosin-Va interferes with the distribution of melanosomes and other vesicles in melanoma cells

CYTOSKELETON, Issue 2 2002
João Carlos da Silva Bizario
Abstract Myosin-Va has been implicated in melanosome translocation, but the exact molecular mechanisms underlying this function are not known. In the dilute, S91 melanoma cells, melanosomes move to the cell periphery but do not accumulate in the tips of dendrites as occurs in wild-type B16 melanocytes; rather, they return and accumulate primarily at the pericentrosomal region in a microtubule-dependent manner. Expression of the full-length neuronal isoform of myosin-Va in S91 cells causes melanosomes to disperse, occupying a cellular area approximately twice that observed in non-transfected cells, suggesting a partial rescue of the dilute phenotype. Overexpression of the full tail domain in S91 cells is not sufficient to induce melanosome dispersion, rather it causes melanosomal clumping. Overexpression of the head and head-neck domains of myosin-Va in B16 cells does not alter the melanosome distribution. However, overexpression of the full tail domain in these cells induces melanosome aggregation and the appearance of tail-associated, aggregated particles or vesicular structures that exhibit variable degrees of staining for melanosomal and Golgi ,-COP markers, as well as colocalization with the endogenous myosin-Va. Altogether, the present data suggest that myosin-Va plays a role in regulating the direction of microtubule-dependent melanosome translocation, in addition to promoting the capture of melanosomes at the cell periphery as suggested by previous studies. These studies also reinforce the notion that myosin-V has a broader function in melanocytes by acting on vesicular targeting or intracellular protein trafficking. Cell Motil. Cytoskeleton 51:57,75, 2002. © 2002 Wiley-Liss, Inc. [source]

Human Papillomavirus and Overexpression of P16INK4a in Nonmelanoma Skin Cancer

DERMATOLOGIC SURGERY, Issue 3 2004
Ingo Nindl PhD
Background. P16INK4a overexpression has been identified as a specific biomarker in high-risk human papillomavirus (HPV),infected cervical (pre)cancer lesions. Objective. To evaluate the overexpression of this cyclin-dependent kinase inhibitor in skin tumors depending on HPV infections, we analyzed normal skin, benign skin disease, and skin cancer specimens. Methods. Biopsies of 23 patients with normal histology (3), psoriasis (2), verrucae vulgaris (2), actinic keratoses (5), squamous cell carcinoma (SCC) in situ (3), Bowen's carcinoma (1), and SCC (7) were analyzed. Specimens of 23 patients were immunostained using the monoclonal antibody E6H4 specific for p16INK4a. HPV status was assessed by a polymerase chain reaction (PCR) system to detect all currently known HPV types. MY (MY09/MY11 and MYN9/MYN10)-, CP (CP65/CP70 and CP66/CP69)-nested PCR, and three single PCR methods CN1, CN3, and CN4 were used in a first step, and HPV typing was performed by restriction fragment length polymorphism analysis. Only ,-globin,positive patients were included in this study. Results. HPV DNA was detected in all actinic keratoses, SCC in situ, Bowen's carcinoma, and SCC, in 50% (one of two) of verrucae vulgaris, in 66% (two of three) of normal skin, and in none of two psoriasis. P16INK4a expression was not detected in normal skin, psoriasis, and verrucae vulgares. Overexpression of p16INK4a was detected in a subset of dysplastic cells (10% to 80%) of all skin (pre)cancer lesions such as actinic keratoses, SCC in situ, Bowen's carcinoma, and SCC infected with HPV independent of sun exposure. Conclusion. P16INK4a appears to be overexpressed in a portion of dysplastic cells from actinic keratoses and SCC. Further studies to examine the association of HPV infection and the overexpression of p16INK4a are warranted. [source]

Identification and characterization of Xenopus OMP25

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2004
Masafumi Inui
This study describes the isolation of mitochondrial outer membrane protein 25 (OMP25) from Xenopus laevis and an analysis of its role in early development. X. laevis OMP25 (xOMP25) is a transmembrane protein of the mitochondrial outer membrane with a PDZ domain in the cytoplasmic tail, and an approximate molecular size of 25 kDa. We isolated xOMP25 from a cDNA library of X. laevis tailbud embryos. Amino acid sequence analysis of xOMP25 showed 57% identity to mouse OMP25, with 73% identity in the PDZ domains. XOMP25 mRNA is expressed maternally, and at a constant level throughout early development. The transcript is localized to eye, otic vesicle, branchial arch and neural tube. Mitochondrial targeting of an EGFP-fusion protein of xOMP25 was visualized using a mitochondria-specific fluorescent dye. Overexpression of xOMP25 in embryos caused curved axes, small eyes and disorganized head structures. Knockdown of xOMP25 protein using antisense morpholino oligonucleotides resulted in slightly shortened axes and decreased neural tissue. Although the mechanism remains unclear, our results implicate xOMP25 protein in the formation of the intact neural tube. [source]

Overexpression of the transcription factor Msx1 is insufficient to drive complete regeneration of refractory stage Xenopus laevis hindlimbs

DEVELOPMENTAL DYNAMICS, Issue 6 2009
Donna M. Barker
Abstract Xenopus laevis tadpoles are capable of hindlimb regeneration, although this ability declines with age. Bmp signaling is one pathway known to be necessary for successful regeneration to occur. Using an inducible transgenic line containing an activated version of the Bmp target Msx1, we assessed the ability of this transcription factor to enhance regeneration in older limbs. Despite considerable evidence correlating msx1 expression with regenerative success in vertebrate regeneration models, we show that induction of msx1 during hindlimb regeneration fails to induce complete regeneration. However, we did observe some improvement in regenerative outcome, linked to morphological changes in the early wound epithelium and a corresponding increase in proliferation in the underlying distal mesenchyme, neither of which are maintained later. Additionally, we show that Msx1 is not able to rescue limb regeneration in a Bmp signalling-deficient background, indicating that additional Bmp targets are required for regeneration in anuran limbs. Developmental Dynamics 238:1366,1378, 2009. © 2009 Wiley-Liss, Inc. [source]

Xenopus aristaless-related homeobox (xARX) gene product functions as both a transcriptional activator and repressor in forebrain development

DEVELOPMENTAL DYNAMICS, Issue 2 2005
Daniel W. Seufert
Abstract Mutations in the aristaless-related homeobox (ARX) gene have been found in patients with a variety of X-linked mental retardation syndromes with forebrain abnormalities, including lissencephaly. Arx is expressed in the developing mouse, Xenopus, and zebrafish forebrain. We have used whole-mount in situ hybridization, overexpression, and loss-of-function studies to investigate the involvement of xArx in Xenopus brain development. We verified that xArx is expressed in the prospective diencephalon, as the forebrain is patterned and specified during neural plate stages. Expression spreads into the ventral and medial telencephalon as development proceeds through neural tube and tadpole stages. Overexpression of xArx resulted in morphological abnormalities in forebrain development, including loss of rostral midline structures, syn- or anophthalmia, dorsal displacement of the nasal organ, and ventral neural tube hyperplasia. Additionally, there is a delay in expression of many molecular markers of brain and retinal development. However, expression of some markers, dlx5 and wnt8b, was enhanced in xArx -injected embryos. Loss-of-function experiments indicated that xArx was necessary for normal forebrain development. Expansion of wnt8b expression depended on xArx function as a transcriptional repressor, whereas ectopic expression of dlx5, accompanied by development of ectopic otic structures, depended on function of Arx as a transcriptional activator. These results suggest that Arx acts as a bifunctional transcriptional regulator in brain development. Developmental Dynamics 232:313,324, 2005. © 2004 Wiley-Liss, Inc. [source]

Local activation of protein kinase A inhibits morphogenetic movements during Xenopus gastrulation

DEVELOPMENTAL DYNAMICS, Issue 1 2003
Byung-Ho Song
Abstract cAMP-dependent protein kinase (PKA) has various biological roles in many organisms. However, little is known about its role in the developmental processes of vertebrates. In this study, we describe the functional analysis of PKA during gastrulation movements in Xenopus laevis. Overexpression of constitutively active PKA (cPKA) in the dorsal equatorial region of the embryo affects morphogenetic movement during gastrulation. We also show that intrinsic differences of PKA activities along the dorsoventral axis are set up and the level of PKA activity on the dorsal region is lower than that on the ventral region from late blastula to gastrula stages. In addition, PKA activation in animal explants inhibits activin-induced elongation. In cPKA-injected embryos, there were no changes in the expressions of markers involved in mesoderm specification, although the correct expression domains of these genes were altered. The effects of PKA activation can be restored by coexpression of PKI, a pseudosubstrate of PKA. We further analyzed the effects of PKA activation on the behavior of migratory gastrulating cells in vitro. Expression of cPKA in head mesoderm cells causes less polarized and/or randomized migration as demonstrated by a directional cell migration assay. Finally, we show that RhoA GTPase lies downstream of PKA, affecting activin-induced convergent extension movements. Taken together, these results suggest that overexpressed PKA can modulate a pathway responsible for morphogenetic movements during Xenopus gastrulation. Developmental Dynamics 227:91,103, 2003. © 2003 Wiley-Liss, Inc. [source]

Retinal patterning by Pax6-dependent cell adhesion molecules

DEVELOPMENTAL NEUROBIOLOGY, Issue 11 2010
Elisabeth Rungger-Brändle
Abstract Long-standing evidence gained from Pax6 mutant embryos pointed to an involvement of Pax6-dependent cell adhesion molecules in patterning the central nervous system and, in particular, the retina. However, direct evidence for such pathways remained elusive. We here present direct evidence that knockdown of Pax6 expression by morpholino antisense molecules in Xenopus embryos and knockdown of maternal N-cadherin (mNcad), N-cadherin (Ncad) and neural cell adhesion molecule (NCAM) produce similar phenotypes. Eye formation is reduced and retinal lamination is heavily disorganized. In Pax6 knockdown embryos, the levels of mRNAs coding for these cell adhesion molecules are markedly reduced. Overexpression of Pax6 efficiently rescues the phenotype of Pax6 knockdown embryos and restores expression of these putative target genes. Rescue of Pax6-deficiency by the putative target gene mNcad moderately rescues eye formation. The promoters of the genes coding for cell adhesion molecules contain several putative Pax6 binding sites, as determined by computer analysis. Chromatin immunoprecipitation shows that, in embryonic heads, Pax6 binds to promoter regions containing such predicted binding sites. Thus, several cell adhesion molecules are direct target genes of Pax6 and cooperate in retinal patterning. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 764,780, 2010 [source]

Overexpression of cyclooxygenase-2 is associated with chemoradiotherapy resistance and prognosis in esophageal squamous cell carcinoma patients

DISEASES OF THE ESOPHAGUS, Issue 8 2008
W.-Z. Huang
SUMMARY Our objective was to investigate whether cyclooxygenase-2 (COX-2) expression can predict the patient's response to chemoradiotherapy (CRT) and ensuing prognosis in esophageal squamous cell carcinoma (ESCC). The clinicopathological and follow-up data of 112 patients with ESCC who underwent CRT from January 2001 to June 2006 were analyzed retrospectively. The immunohistochemical expression level of COX-2 was examined for all biopsy specimens of primary tumors, and the correlation of COX-2 expression with the patient's response to CRT and prognosis was examined. COX-2 positive immunostaining was detected in 111 (99.1%) of the patients, including overexpression in 54 (48.2%) patients and low expression in 58 (51.8%) of the patients. The response of tumors with a low level expression of COX-2 (70.7%, 41/58) was significantly higher than that of tumors with COX-2 overexpression (42.6%, 23/54; P = 0.003). Patients with a low level of COX-2 expression had a higher downstaged rate than those with a high level of COX-2 expression (9/13 vs 2/8), but the difference was not statistically significant (P = 0.08). In the definitive CRT group (91 cases), COX-2 overexpression was significantly associated with poor 3-year overall survival (P = 0.028). Multivariate analysis showed that only metastatic stage (nonregional node metastasis) was an independent prognosis factor. The assessment of COX-2 status may provide additional information to identify ESCC patients with poor chances of response to CRT and potential candidates for more individualized treatment. [source]

The PPAR, agonist GW501516 suppresses interleukin-6-mediated hepatocyte acute phase reaction via STAT3 inhibition

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 5 2007
T. Kino
Abstract Background, Interleukin-6 and downstream liver effectors acute phase reactants are implicated in the systemic inflammatory reaction. Peroxisome proliferator-activated receptor , (PPAR,), which binds to and is activated by a variety of fatty acids, was recently shown to have anti-inflammatory actions. Materials and methods, We examined the ability of the synthetic PPAR, agonist GW501516 to suppress interleukin-6-induced expression of acute phase proteins in human hepatoma HepG2 cells and rat primary hepatocytes. Results, GW501516 dose-dependently suppressed interleukin-6-induced mRNA expression of the acute phase protein ,1-antichymotrypsin in HepG2 cells. The compound also suppressed interleukin-6-induced mRNA expression of ,2-acid glycoprotein, ,-fibrinogen and ,2-macroglobulin in and the secretion of C-reactive protein by rat primary hepatocytes. Depletion of the PPAR, receptor, but not of PPAR, or ,, attenuated the suppressive effect of GW501516 on interleukin-6-induced ,1-antichymotrypsin mRNA expression, indicating that PPAR, specifically mediated this effect. Since interleukin-6 stimulates the transcriptional activity of the ,1-antichymotrypsin promoter by activating the signal transducer and activator of transcription (STAT) 3, we examined functional interaction of this transcription factor and PPAR, on this promoter. Overexpression of PPAR, enhanced the suppressive effect of GW501516 on STAT3-activated transcriptional activity of the ,1-antichymotrypsin promoter, while GW501516 suppressed interleukin-6-induced binding of this transcription factor to this promoter. Conclusions, These findings indicate that agonist-activated PPAR, interferes with interleukin-6-induced acute phase reaction in the liver by inhibiting the transcriptional activity of STAT3. PPAR, agonists might be useful for the suppression of systemic inflammatory reactions in which IL-6 plays a central role. [source]

Transforming growth factor-beta1 affects interleukin-10 production in the bone marrow of patients with chronic idiopathic neutropenia

EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 6 2007
Katerina Pyrovolaki
Abstract Background:, Chronic idiopathic neutropenia (CIN) is a bone marrow (BM) failure syndrome characterized by accelerated apoptosis of myeloid progenitor cells because of a local imbalance between pro-inflammatory and anti-inflammatory cytokines. In this study, we investigated the interplay among transforming growth factor-beta1 (TGF-,1), interleukin-10 (IL-10), and soluble flt-3 ligand (sFL) within the BM of CIN patients and probed the role of these cytokines in the pathophysiology of CIN. Design:, We used long-term BM cultures (LTBMC) to evaluate TGF-,1, IL-10, and sFL levels in CIN patients (n = 70) and healthy subjects (n = 35). Cytokine levels in LTBMC supernatants were correlated with the number of circulating neutrophils and the proportion of BM CD34+/CD33+ myeloid progenitor cells. Results:, CIN patients had increased TGF-,1 and sFL levels in LTBMCs compared with controls and individual cytokine values were found to be correlated inversely with the number of neutrophils and the proportion of CD34+/CD33+ cells. Patients displayed low supernatant IL-10 levels compared with controls and cytokine values were found to be correlated positively with the number of neutrophils and the proportion of CD34+/CD33+ cells. The levels of TGF-,1 were found to be inversely correlated with IL-10 and positively with sFL values in LTBMC, supernatants suggesting a possible interplay among these cytokines in CIN BM. Neutralization of TGF-,1 in LTBMCs increased IL-10 levels significantly in patients but not in controls, while neutralization had no effect on sFL levels. Conclusion:, Excessive production of TGF-,1 within the BM microenvironment of CIN patients results in downregulation of IL-10 and reduction of myeloid progenitor cells. Overexpression of sFL probably represents a compensatory mechanism to the low myeloid progenitor cells. [source]

Constitutive expression of the FK506 binding protein 51 (FKBP51) in bone marrow cells and megakaryocytes derived from idiopathic myelofibrosis and non-neoplastic haematopoiesis

EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 4 2004
Oliver Bock
Abstract: Objectives:, Overexpression of FK506 binding protein 51 (FKBP51) in megakaryocytic progenitor cells generated from purified CD34+ cells in patients with idiopathic myelofibrosis (IMF) has been demonstrated. It has been suggested that FKBP51 is involved in the dysregulation of the apoptotic programme with consecutive prolongation of cell survival. The knowledge of FKBP51 and its expression in bone marrow cells and mature megakaryocytes in non-neoplastic haematopoiesis and IMF is sparse. Methods:, To evaluate a potential overexpression of FKBP51 in patients with IMF (n = 37) compared with non-neoplastic haematopoiesis (n = 31), total bone marrow cells as well as single megakaryocytes, isolated by laser microdissection, were quantitatively analysed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). By applying immunohistochemistry, FKBP51 gene expression was correlated with staining pattern and cellular localisation of the corresponding FKBP51 protein. Results:, We demonstrated that FKBP51 is constitutively expressed in non-neoplastic haematopoiesis. FKBP51 gene expression by total bone marrow cells as well as megakaryocytes was not significantly different in IMF. FKBP51 protein expression could be localised to myeloid progenitor cells as well as megakaryocytes. In particular, megakaryocytes were stained almost exclusively nuclear for FKBP51. No differences in expression patterns between both IMF and control cases could be demonstrated. Conclusions:, For the first time, FKBP51 expression, in particular gene expression and subcellular localization was described in bone marrow cells of non-neoplastic and neoplastic haematopoiesis grown in vivo. We conclude that FKBP51 could be temporarily overexpressed in megakaryocytic progenitors rather than contribute to the accumulation of mature megakaryocytes in IMF. [source]

Protection of hematopoietic cells from O6 -alkylation damage by O6 -methylguanine DNA methyltransferase gene transfer: studies with different O6 -alkylating agents and retroviral backbones

EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2001
Michael Jansen
Abstract: Overexpression of O6 -methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O6 -alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O6 -alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus-based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) was demonstrated for all four drugs. In comparison to mock-transduced controls, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on average 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT-cDNA from a murine embryonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8,12 µg/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer. [source]

Overexpression of GAP-43 modifies the distribution of the receptors for myelin-associated growth-inhibitory proteins in injured Purkinje axons

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2009
Simona Foscarin
Abstract Neurons with enhanced intrinsic growth capabilities can elongate their axons into non-permissive territories, but the mechanisms that enable the outgrowing processes to overcome environmental inhibition are largely unknown. To address this issue, we examined adult mouse Purkinje cells that overexpress the axonal growth-associated protein GAP-43. After injury, these neurons exhibit sprouting along the intracortical neuritic course and at the severed stump in the white matter. To determine whether GAP-43-overexpressing Purkinje cells are responsive to extrinsic inhibitory cues, we investigated the content and subcellular localization of major receptors for myelin-associated inhibitory proteins, PlexinB1 and the Nogo receptor (NgR) with the related co-receptors LINGO-1 and p75. Expression of these molecules, estimated by measuring perikaryal immunostaining intensity and Western blot, was not different in wild-type or transgenic mice, and it was not overtly modified after axotomy. Following injury, however, the content of PlexinB1 was significantly reduced in GAP-43-overexpressing neurites. Furthermore, in the same axons the distribution of both PlexinB1 and NgR was altered, being inverse to that of GAP-43. Labelling for the two receptors was conspicuously reduced on the axonal surface and it was almost undetectable in the outgrowing sprouts, which showed strong GAP-43 immunoreactivity. These observations indicate that although GAP-43 overexpression does not modify the expression of receptors for myelin-associated inhibitory factors, it interferes with their subcellular localization and exposure on the neuritic membrane. Therefore, GAP-43 promotes axon growth by multiple synergistic mechanisms that potentiate the intrinsic motility of the elongating processes, while reducing their sensitivity to environmental inhibition. [source]

Overexpression of APP provides neuroprotection in the absence of functional benefit following middle cerebral artery occlusion in rats

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2007
Jared Clarke
Abstract Cerebral ischaemia leads to a transient accumulation of ,-amyloid precursor protein (APP) and ,-amyloid (A,) peptides adjacent to the ischaemic lesion. There is conflicting evidence that APP/A, fragments may either enhance neuronal plasticity or be neurotoxic. The aim of the current study was to assess the effect of overexpression of human APP in rats on functional recovery following cerebral ischaemia. Adult APP-overexpressing (hAPP695 Tg) rats subjected to transient middle cerebral artery occlusion (MCAO) had significantly smaller infarct volumes than non-transgenic littermates, yet did not perform better on a series of sensorimotor or learning tests during a 6-month follow-up period. In fact, transgenic animals were found to be significantly more impaired in both the beam-walking and Morris water maze tests following MCAO. Immunohistochemistry showed human A,-positive staining in the cortex and hippocampus of APP transgenic rats. The present data suggest that while overexpression of APP in rats may provide some histological neuroprotection in the event of cerebral ischaemia, this does not translate into significant functional recovery. [source]

Axonal morphogenesis controlled by antagonistic roles of two CRMP subtypes in microtubule organization

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2003
Junichi Yuasa-Kawada
Abstract During development, cells undergo dynamic morphological changes by rearrangements of the cytoskeleton including microtubules. However, molecular mechanisms underlying the microtubule remodeling between orientated and disoriented formations are almost unknown. Here we found that novel subtypes of collapsin response mediator proteins (CRMP-As) and the originals (CRMP-Bs), which occur from the alternative usage of different first coding exons, are involved in this conversion of microtubule patterns. Overexpression of CRMP2A and CRMP2B in chick embryonic fibroblasts induced orientated and disoriented patterns of microtubules, respectively. Moreover, sequential overexpression of another subtype overcame the effect of the former expression of the countersubtype. Overexpression experiments in cultured chick retinae showed that CRMP2B promoted axon branching and suppressed axon elongation of ganglion cells, while CRMP2A blocked these effects when co-overexpressed. Our findings suggest that the opposing activities of CRMP2A and CRMP2B contribute to the cellular morphogenesis including neuronal axonogenesis through remodeling of microtubule organization. [source]

Overexpression of spermidine/spermine N1 -acetyltransferase in transgenic mice protects the animals from kainate-induced toxicity

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2000
Kyllikki Kaasinen
Abstract We recently generated a transgenic mouse line with activated polyamine catabolism through overexpression of spermidine/spermine N1 -acetyltransferase (SSAT). A detailed analysis of brain polyamine concentrations indicated that all brain regions of these animals showed distinct signs of activated polyamine catabolism, e.g. overaccumulation of putrescine (three- to 17-fold), appearance of N1 -acetylspermidine and decreases in spermidine concentrations. In situ hybridization analyses revealed a marked overexpression of SSAT-specific mRNA all over the brain tissue of the transgenic animals. The transgenic animals appeared to tolerate subcutaneous injections of high-dose kainate substantially better as their overall mortality was less than 50% of that of their syngenic littermates. We used the expression of glial fibrillary acidic protein (GFAP) as a marker of brain injury in response to kainate. In situ hybridization analysis with GFAP oligonucleotide up to 7 days after the administration of sublethal kainate doses showed reduced GFAP expression in transgenic animals in comparison with their non-transgenic littermates. This difference was especially striking in the cerebral cortex of the transgenic mice where the exposure to kainate hardly induced GFAP expression. The treatment with kainate likewise resulted in loss of the hippocampal (CA3) neurons in non-transgenic but not transgenic animals. These results support our earlier findings indicating that elevated concentrations of brain putrescine, irrespective whether derived from an overexpression of ornithine decarboxylase, or as shown here, from an overexpression of SSAT, play in all likelihood a neuroprotective role in brain injury. [source]

Expression of Osterix in mechanical stress-induced osteogenic differentiation of periodontal ligament cells in vitro

EUROPEAN JOURNAL OF ORAL SCIENCES, Issue 3 2008
Yanhong Zhao
Osterix (Osx) is an osteoblast-specific transcription factor required for the differentiation of pre-osteoblasts into functional osteoblasts. This study sought to examine the changes of Osx expression in periodontal ligament cells (PDLC) subjected to mechanical force, and to investigate whether Osx is involved in the mechanical stress-induced differentiation of PDLC. Human PDLC were exposed to centrifugal force for 1,12 h. Real-time polymerase chain reaction (PCR), western blot, and immunofluorescence assays were used to examine the mRNA and protein expression of Osx and its subcellular localization. Furthermore, PDLC were transfected with the expression vector pcDNA3.1 flag-Osx and subjected to mechanical force for 6 h. The changes in alkaline phosphatase (ALP) activity and in the expression of core-binding factor alpha1 (Cbfa1), ALP, osteopontin, bone sialoprotein, osteocalcin, and collagen I were measured. After the application of mechanical force, Osx was upregulated in a time-dependent manner at both mRNA and protein levels, and Osx protein was translocated from the cytosol into the cell nuclei. Overexpression of Osx did not affect the expression of Cbfa1, but it significantly enhanced the ALP activity and the mRNA expression of all the aforementioned osteogenic marker genes, all of which increased further under mechanical stress. These results suggest that Osx might play an important role in the mechanical stress-induced osteogenic differentiation of PDLC and therefore be involved in alveolar bone remodeling during orthodontic therapy. [source]

Efficient and selective tumor cell lysis and induction of apoptosis in melanoma cells by a conditional replication-competent CD95L adenovirus

EXPERIMENTAL DERMATOLOGY, Issue 8 2010
Lothar F. Fecker
Please cite this paper as: Efficient and selective tumor cell lysis and induction of apoptosis in melanoma cells by a conditional replication-competent CD95L adenovirus. Experimental Dermatology 2010; 19: e56,e66. Abstract:, The high mortality of melanoma demands the development of new strategies, and gene therapy may be considered provided improvements in efficacy and selectivity. Overexpression of the death ligand CD95L/FasL has been shown in previous studies as highly effective for apoptosis induction in melanoma cells. For efficient and selective targeting of melanoma, a conditional replication-competent adenoviral vector was constructed (Ad5-FFE-02), which drives CD95L expression by a tetracycline-inducible promoter. For restricting its replication to melanoma cells, the adenoviral E1A gene is controlled by a tyrosinase-derived promoter. Furthermore, adenoviral E1B was deleted and a mutated E1A was used to preferentially support replication in tumor cells. Proving its high selectivity and efficiency, strong expression of E1A and doxycycline-dependent induction of CD95L were characteristic for tyrosinase-positive melanoma cells after Ad5-FFE-02 transduction, whereas absent in non-melanoma cell lines. Importantly, Ad5-FFE-02-mediated cell lysis was restricted to melanoma cells, and induction of apoptosis was found only in tyrosinase and CD95 expressing cells. Finally, the combination of adenoviral replication and CD95L-mediated apoptosis resulted in an enhanced repression of melanoma cell growth. This new adenoviral vector may provide a basis for an efficient targeting of melanoma. [source]

Osteopontin and the skin: multiple emerging roles in cutaneous biology and pathology

EXPERIMENTAL DERMATOLOGY, Issue 9 2009
Franziska Buback
Abstract:, Osteopontin (OPN) is a glycoprotein expressed by various tissues and cells. The existence of variant forms of OPN as a secreted (sOPN) and intracellular (iOPN) protein and its modification through post-translational modification and proteolytic cleavage explain its broad range of functions. There is increasing knowledge which receptors OPN isoforms can bind to and which signaling pathways are activated to mediate different OPN functions. sOPN interacts with integrins and CD44, mediates cell adhesion, migration and tumor invasion, and has T helper 1 (Th1) cytokine functions and anti-apoptotic effects. iOPN has been described to regulate macrophage migration and interferon-, secretion in plasmacytoid dendritic cells. Both sOPN and iOPN, through complex functions for different dendritic cell subsets, participate in the regulation of Th cell lineages, among them Th17 cells. For skin disease, OPN from immune cells and tumor cells is of pathophysiological relevance. OPN is secreted in autoimmune diseases such as lupus erythematosus, and influences inflammation of immediate and delayed type allergies and granuloma formation. We describe that OPN is overexpressed in psoriasis and propose a model to study OPN function in psoriatic inflammation. Through cytokine functions, OPN supports immune responses against Mycobacteria and viruses such as herpes simplex virus. OPN is also implicated in skin tumor progression. Overexpression of OPN influences invasion and metastasis of melanoma and squamous cell carcinoma cells, and OPN expression in melanoma is a possible prognostic marker. As OPN protein preparations and anti-OPN antibodies may be available in the near future, in-depth knowledge of OPN functions may open new therapeutic approaches for skin diseases. [source]

Receptors for calcitonin gene-related peptide and adrenomedullin: implications for skin cell biology

EXPERIMENTAL DERMATOLOGY, Issue 9 2004
J. A. Fischer
The specificity of a G-protein-coupled calcitonin receptor (CTR) and a CT receptor-like receptor (CLR) for calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and amylin is defined by the heterodimeric non-covalent association with three receptor-activity-modifying proteins (RAMPs). Chemical cross-linking of proteins at the cell surface and immunoprecipitation have identified [125I]CGRP/CLR/RAMP1, [125I]AM/CLR/RAMP2 and -3 as well as [125I]CGRP/CTR/RAMP1, [125I]amylin/CTR/RAMP1 and -RAMP3 complexes. CLR/RAMP1 defines a CGRP receptor. CLR/RAMP2 and -3 correspond to AM1 and AM2 receptor isotypes, respectively. The AM1 receptor cross-reacts with CGRP at high and the AM2 receptor at low concentrations. With the N-terminal deletion of amino acids 14,20 of the mouse, CLR-selective inactivation of AM over CGRP receptor function was obtained. As a result, functional interaction with AM was no longer possible. Overexpression of the CLR in transgenic mice together with the endogenous RAMP2 results in thinning of the hairs during postnatal development (L. M. Ittner et al. conference poster). In conclusion, the extreme N-terminus of the CLR and the extracellular N-terminal domains of RAMP1 and -2 contain amino acid residues that provide AM- or CGRP-binding selectivity of the CLR/RAMP complexes. Hair development is attenuated, resulting in the thinning of the hairs and eventually alopecia during postnatal development. [source]

Golgi reassembly stacking protein 55 interacts with membrane-type (MT) 1-matrix metalloprotease (MMP) and furin and plays a role in the activation of the MT1-MMP zymogen

FEBS JOURNAL, Issue 15 2010
Christian Roghi
Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a proteinase involved in the remodelling of extracellular matrix and the cleavage of a number of substrates. MT1-MMP is synthesized as a zymogen that requires intracellular post-translational cleavage to gain biological activity. Furin, a member of the pro-protein convertase family, has been implicated in the proteolytic removal of the MT1-MMP prodomain sequence. In the present study, we demonstrate a role for the peripheral Golgi matrix protein GRASP55 in the furin-dependent activation of MT1-MMP. MT1-MMP and furin were found to co-localize with Golgi reassembly stacking protein 55 (GRASP55). Further analysis revealed that GRASP55 associated with the cytoplasmic domain of both proteases and that the LLY573 motif in the MT1-MMP intracellular domain was crucial for the interaction with GRASP55. Overexpression of GRASP55 was found to enhance the formation of a complex between MT1-MMP and furin. Finally, we report that disruption of the interaction between GRASP55 and furin led to a reduction in pro-MT1-MMP activation. Taken together, these data suggest that GRASP55 may function as an adaptor protein coupling MT1-MMP with furin, thus leading to the activation of the zymogen. Structured digital abstract ,,MINT-7897990: Furin (uniprotkb:P09958) and GRASP55 (uniprotkb:Q9H8Y8) colocalize (MI:0403) by fluorescence microscopy (MI:0416) ,,MINT-7897801: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with MT2-MMP (uniprotkb:P51511) by two hybrid (MI:0018) ,,MINT-7897821: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with MT3-MMP (uniprotkb:P51512) by two hybrid (MI:0018) ,,MINT-7897577: GRASP55 (uniprotkb:Q9R064) and MT1-MMP (uniprotkb:P50281) colocalize (MI:0403) by fluorescence microscopy (MI:0416) ,,MINT-7897366: MT1-MMP (uniprotkb:P50281) physically interacts (MI:0915) with GRASP55 (uniprotkb:Q9H8Y8) by anti bait coimmunoprecipitation (MI:0006) ,,MINT-7897617, MINT-7897659, MINT-7897681, MINT-7897702, MINT-7897725, MINT-7898032, MINT-7898011, MINT-7897907, MINT-7897884: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with MT1-MMP (uniprotkb:P50281) by two hybrid (MI:0018) ,,MINT-7898002: MT1-MMP (uniprotkb:P50281) physically interacts (MI:0914) with Furin (uniprotkb:P09958) by anti bait coimmunoprecipitation (MI:0006) ,,MINT-7897500: MT1-MMP (uniprotkb:P50281) and Giantin (uniprotkb:Q14789) colocalize (MI:0403) by fluorescence microscopy (MI:0416) ,,MINT-7897750, MINT-7897394: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with MT1-MMP (uniprotkb:P50281) by anti tag coimmunoprecipitation (MI:0007) ,,MINT-7897562: MT1-MMP (uniprotkb:P50281) and GRASP55 (uniprotkb:Q9H8Y8) colocalize (MI:0403) by fluorescence microscopy (MI:0416) ,,MINT-7897512: TGN46 (uniprotkb:O43493) and MT1-MMP (uniprotkb:P50281) colocalize (MI:0403) by fluorescence microscopy (MI:0416) ,,MINT-7897921, MINT-7897975: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with Furin (uniprotkb:P09958) by two hybrid (MI:0018) ,,MINT-7898052, MINT-7897410: MT1-MMP (uniprotkb:P50281) physically interacts (MI:0915) with GRASP55 (uniprotkb:Q9R064) by anti bait coimmunoprecipitation (MI:0006) ,,MINT-7897951: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with PC7 (uniprotkb:Q16549) by two hybrid (MI:0018) ,,MINT-7897866: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with MT5-MMP (uniprotkb:Q9Y5R2) by two hybrid (MI:0018) ,,MINT-7897633: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with TGFA (uniprotkb:P01135) by two hybrid (MI:0018) ,,MINT-7897551: GRASP55 (uniprotkb:Q9H8Y8) and Giantin (uniprotkb:Q14789) colocalize (MI:0403) by fluorescence microscopy (MI:0416) ,,MINT-7897938: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with PC5/6B (uniprotkb:Q04592) by two hybrid (MI:0018) [source]

Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation

FEBS JOURNAL, Issue 16 2009
Lilian C. Russo
Thimet oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide-metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium-dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca2+, with an estimated Kd value of 0.52 ,m. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187-stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187-stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells. Structured digital abstract ,,MINT-7148420: EP24.15 (uniprotkb:P52888) and Calmodulin (uniprotkb:P62161) bind (MI:0407) by surface plasmon resonance (MI:0107) ,,MINT-7148437: EP24.15 (uniprotkb:P52888) and Calmodulin (uniprotkb:P62158) colocalize (MI:0403) by surface plasmon resonance (MI:0107) ,,MINT-7148406: Calmodulin (uniprotkb:P62161) binds (MI:0407) to EP24.15 (uniprotkb:P52888) by pull down (MI:0096) [source]

Vanadium-induced apoptosis of HaCaT cells is mediated by c-fos and involves nuclear accumulation of clusterin

FEBS JOURNAL, Issue 14 2009
Soultana Markopoulou
Vanadium exerts a variety of biological effects, including antiproliferative responses through activation of the respective signaling pathways and the generation of reactive oxygen species. As epidermal cells are exposed to environmental insults, human keratinocytes (HaCaT) were used to investigate the mechanism of the antiproliferative effects of vanadyl(IV) sulfate (VOSO4). Treatment of HaCaT cells with VOSO4 inhibited proliferation and induced apoptosis in a dose-dependent manner. Inhibition of proliferation was associated with downregulation of cyclins D1 and E, E2F1, and the cyclin-dependent kinase inhibitors p21Cip1/Waf1 and p27Kip1. Induction of apoptosis correlated with upregulation of the c-fos oncoprotein, changes in the expression of clusterin (CLU), an altered ratio of antiapoptotic to proapoptotic Bcl-2 protein family members, and poly(ADP-ribose) polymerase-1 cleavage. Forced overexpression of c-fos induced apoptosis in HaCaT cells that correlated with secretory CLU downregulation and upregulation of nuclear CLU (nCLU), a pro-death protein. Overexpression of Bcl-2 protected HaCaT cells from vanadium-induced apoptosis, whereas secretory CLU overexpression offered no cytoprotection. In contrast, nCLU sensitized HaCaT cells to apoptosis. Our data suggest that vanadium-mediated apoptosis was promoted by c-fos, leading to alterations in CLU isoform processing and induction of the pro-death nCLU protein. [source]