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William Vainchenker

Publications and source records attributed to William Vainchenker.

71 records · Page 4Linked to original sources
PubMed2003

Spontaneous STAT5 activation induces growth factor independence in idiopathic myelofibrosis: possible relationship with FKBP51 overexpression.

Spontaneous growth of megakaryocyte progenitors is one of the biologic hallmarks of idiopathic myelofibrosis (IMF). The molecular mechanisms underlying this hypersensitivity to cytokines are poorly understood. Using a differential display approach, we previously observed FK506 binding protein 51 (FKBP51) overexpression in pathologic megakaryocytes from IMF. Using an FKBP51-overexpressing cell line, we found sustained STAT5 activation associated with JAK2 phosphorylation. We subsequently tested whether this transcription factor was activated in patient samples. We detected a STAT5 nuclear translocation and activation in spontaneously grown megakaryocytes and in circulating CD34(+) cells from the majority of patients studied. The biologic role of this JAK/STAT pathway activation was demonstrated by inhibiting both the anti-apoptotic phenotype mediated by FKBP51 overexpression in UT7 cells and the spontaneous megakaryocytic growth by addition in culture of the JAK2 inhibitor AG490 or overexpression of a STAT5b dominant negative or SOCS-1. These results demonstrate that a constitutive STAT5 activation in IMF is indispensable for spontaneous growth of megakaryocytes. They also suggest that FKBP51 overexpression could be involved in STAT5 activation in IMF cells and in subsequent abnormal growth.

Blotting, Western↗
PubMed2003

Expression of osteoprotegerin mRNA and protein in murine megakaryocytes.

OBJECTIVE: Osteoprotegerin (OPG) is a soluble member of the tumor necrosis factor receptor superfamily critically involved in the regulation of bone resorption. Within the bone microenvironment, OPG is abundantly produced by osteoblast/stromal cells, and its expression is regulated by transforming growth factor-beta(1) (TGF-beta(1)). However, OPG expression and regulation in primary hematopoietic cells have not been fully investigated. MATERIALS AND METHODS: Opg mRNA was studied in murine hematopoietic cells by semiquantitative reverse transcriptase-polymerase chain reaction. The OPG protein was identified by immunofluorescence labeling and secretion was assessed by enzyme-linked immunosorbent assay. RESULTS: Opg transcripts were detected in platelets, megakaryocytes (MK), monocytes, and B lymphocytes, but not in erythroblasts, neutrophils, and T lymphocytes. Mature MK and proplatelets exhibited strong immunostaining for OPG outside the storage alpha-granules, and secretion was detected in the conditioned medium. To analyze whether opg transcription in MK was influenced by TGF-beta(1), the opg/GpIIb mRNA ratio was compared in cultured MK derived from TGF-beta(1) null mutants and wild-type littermates without or after the addition of bioactive TGF-beta(1). No difference was seen, indicating that opg expression in MK was not modulated by TGF-beta(1). However, mRNA levels were increased when thrombopoietin was present in the culture medium, suggesting that MK maturation was correlated with enhanced opg expression. CONCLUSIONS: With these results we document for the first time that murine MK and platelets express OPG. This suggests a novel role for MK in bone homeostasis, in addition to its role in vascular homeostasis.

Animals↗
PubMed2002

Stimulation of osteoprotegerin production is responsible for osteosclerosis in mice overexpressing TPO.

Myelofibrosis and osteosclerosis are prominent features arising in mice overexpressing thrombopoietin (TPO). The pivotal role of transforming growth factor beta 1 (TGF-beta 1) in the pathogenesis of myelofibrosis has been documented, but the mechanisms mediating osteosclerosis remain unclear. Here, we used mice deficient in osteoprotegerin (OPG), a secreted inhibitor of bone resorption, to determine whether osteosclerosis occurs through a deregulation of osteoclastogenesis. Marrow cells from opg-deficient mice (opg(-/-)) or wild-type (WT) littermates were infected with a retrovirus encoding TPO and engrafted into an opg(-/-) or WT background for long-term reconstitution. The 4 combinations of graft/host (WT/WT, opg(-/-)/opg(-/-), opg(-/-)/WT, and WT/opg(-/-)) were studied. Elevation of TPO and TGF-beta 1 levels in plasma was similar in the 4 experimental groups and all the mice developed a similar myeloproliferative syndrome associated with severe myelofibrosis. Osteosclerosis developed in WT hosts engrafted with WT or opg(-/-) hematopoietic cells and was associated with increased OPG levels in plasma and decreased osteoclastogenesis. In contrast, opg(-/-) hosts exhibited an osteoporotic phenotype and a growth of bone trabeculae was rarely seen. These findings suggest that osteosclerosis in mice with TPO overexpression occurs predominantly via an up-regulation of OPG in host stromal cells leading to disruption of osteoclastogenesis.

Animals↗
PubMed2002

Telomere dysfunction and telomerase reactivation in human leukemia cell lines after telomerase inhibition by the expression of a dominant-negative hTERT mutant.

As activation of telomerase represents a key step in the malignant transformation process, experimental models to develop anti-telomerase drugs provide a rational basis for anticancer strategies. We analysed the short and long-term efficacy of a stably expressed dominant-negative mutant (DN) of the telomerase catalytic unit (hTERT) in UT-7 and U937 human leukemia cell lines by using an IRES-e-GFP retrovirus. As expected, telomerase inactivation resulted in drastic telomere shortening, cytogenetic instability and cell growth inhibition in all e-GFP positive DN clones after 15-35 days of culture. However, despite this initial response, 50% of e-GFP positive DN clones with short telomeres escaped from crisis after 35 days of culture and recovered a proliferation rate similar to the control cells. This rescue was associated with a telomerase reactivation inducing telomere lengthening. We identified two pathways, one involving the loss of the DN transgene expression and the other the transcriptional up-regulation of endogenous hTERT with persistence of the DN transgene expression. Although this second mechanism appears to be a very rare event (one clone), these findings suggest that genomic instability induced by short telomeres after telomerase inhibition might enhance the probability of activation or selection of telomere maintenance mechanisms dependent on hTERT transcription.

Cell Division↗
PubMed2002

Overexpression of FKBP51 in idiopathic myelofibrosis regulates the growth factor independence of megakaryocyte progenitors.

Idiopathic myelofibrosis (IMF) is a chronic myeloproliferative disorder characterized by megakaryocyte hyperplasia and bone marrow fibrosis. Biologically, an autonomous megakaryocyte growth and differentiation is noticed, which contributes to the megakaryocyte accumulation. To better understand the molecular mechanisms involved in this spontaneous growth, we searched for genes differentially expressed between normal megakaryocytes requiring cytokines to grow and IMF spontaneously proliferating megakaryocytes. Using a differential display technique, we found that the immunophilin FKBP51 was 2 to 8 times overexpressed in megakaryocytes derived from patients' CD34(+) cells in comparison to normal megakaryocytes. Overexpression was moderate and confirmed in 8 of 10 patients, both at the mRNA and protein levels. Overexpression of FKBP51 in a UT-7/Mpl cell line and in normal CD34(+) cells induced a resistance to apoptosis mediated by cytokine deprivation with no effect on proliferation. FKBP51 interacts with both calcineurin and heat shock protein (HSP)70/HSP90. However, a mutant FKBP51 deleted in the HSP70/HSP90 binding site kept the antiapoptotic effect, suggesting that the calcineurin pathway was responsible for the FKBP51 effect. Overexpression of FKBP51 in UT-7/Mpl cells induced a marked inhibition of calcineurin activity. Pharmacologic inhibition of calcineurin by cyclosporin A mimicked the effect of FKBP51. The data support the conclusion that FKBP51 inhibits apoptosis through a calcineurin-dependent pathway. In conclusion, FKBP51 is overexpressed in IMF megakaryocytes and this overexpression could be, in part, responsible for the megakaryocytic accumulation observed in this disorder by regulating their apoptotic program.

Base Sequence↗
PubMed2002

Megakaryocyte polyploidization is associated with a functional gene amplification.

It is believed that polyploidy induces an orchestrated increase in gene expression. To know whether all alleles remain functional during megakaryocyte polyploidization, we used a well-established fluorescence in situ hybridization technique which allows one to simultaneously detect pre-mRNAs and assess ploidy level in a single cell. All alleles of GPIIb, GPIIIa, VWF, beta-actin, hsp70, c-mpl, Fli-1, and FOG-1 genes are transcriptionally active in megakaryocytes from 4N to 32N. All X chromosomes in male cells are transcriptionally active but only half of them are transcriptionally active in female megakaryocytes, as revealed by the transcriptional activity of the GATA-1 gene. Nuclear untranslated XIST RNA accumulates on the inactivated X chromosomes, indicating that they are subjected to a normal inactivation process. Altogether, our results demonstrate that megakaryocyte polyploidization results in a functional gene amplification whose likely function is an increase in protein synthesis parallel with cell enlargement.

Alleles↗
PubMed2002

Platelet formation is the consequence of caspase activation within megakaryocytes.

Platelets are formed from mature megakaryocytes (MKs) and arise from the development of long and thin cytoplasmic extensions called proplatelets. After platelet release, the senescent MKs (nucleus surrounded by some cytoplasm) undergo cell death by apoptosis. To explore the precise role of apoptosis in proplatelet formation, we grew human MKs from CD34(+) cells and assessed the possible role of caspases. Proteolytic maturation of procaspase-3 and procaspase-9 was detected by immunoblots in maturing MKs as well as in proplatelet-bearing MKs and senescent MKs. Cleavage of caspase substrates such as gelsolin or poly adenosine diphosphate (ADP)-ribose polymerase (PARP) was also detected. Interestingly, activated forms of caspase-3 were detected in maturing MKs, before proplatelet formation, with a punctuate cytoplasmic distribution, whereas a diffuse staining pattern was seen in senescent and apoptotic MKs. This localized activation of caspase-3 was associated with a mitochondrial membrane permeabilization as assessed by the release of cytochrome c, suggesting an activation of the intrinsic pathway. Moreover, these MKs with localized activated caspase-3 had no detectable DNA fragmentation. In contrast, when apoptosis was induced by staurosporine, diffuse caspase activation was seen; these MKs had signs of DNA fragmentation, and no proplatelet formation occurred. The pan-caspase inhibitor z-VAD.fmk as well as more specific inhibitors of caspase-3 and caspase-9 blocked proplatelet formation, whereas an inhibitor of calpeptin had no effect. Overexpression of Bcl-2 also inhibited proplatelet formation in maturing MKs. Thus, localized caspase activation is causal to proplatelet formation. We conclude that proplatelet formation is regulated by a caspase activation limited to only some cellular compartments.

Antigens, CD34↗
PubMed2002

Specific involvement of caspases in the differentiation of monocytes into macrophages.

Caspases are cysteine proteases involved in apoptosis and cytokine maturation. In erythroblasts, keratinocytes, and lens epithelial cells undergoing differentiation, enucleation has been regarded as a caspase-mediated incomplete apoptotic process. Here, we show that several caspases are activated in human peripheral blood monocytes whose differentiation into macrophages is induced by macrophage colony-stimulating factor (M-CSF). This activation is not associated with cell death and cannot be detected in monocytes undergoing dendritic cell differentiation in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The mechanisms and consequences of caspase activation were further studied in U937 human monocytic cells undergoing phorbol ester-induced differentiation into macrophages. Differentiation-associated caspase activation involves the release of cytochrome c from the mitochondria and leads to the cleavage of the protein acinus while the poly(ADP-ribose)polymerase remains uncleaved. Inhibition of caspases by either exposure to the broad-spectrum inhibitor benzyloxycarbonyl-Val-Ala-(DL)-Asp-fluoromethylketone (z-VAD-fmk) or expression of the p35 baculovirus inhibitory protein or overexpression of Bcl-2 inhibits the differentiation process. In addition, z-VAD-fmk amplifies the differentiation-associated production of radical oxygen species in both phorbol ester-differentiated U937 cells and M-CSF-treated monocytes, shifting the differentiation process to nonapoptotic cell death. Altogether, these results indicate that caspase activation specifically contributes to the differentiation of monocytes into macrophages, in the absence of cell death.

Adult↗
PubMed2002

Prominent role of TGF-beta 1 in thrombopoietin-induced myelofibrosis in mice.

Several studies suggest an implication of transforming growth factor-beta1 (TGF-beta1) in the promotion of myelofibrosis associated with hematopoietic malignancies, but the involvement of this cytokine is not fully investigated. To test directly the impact of TGF-beta1 in the pathogenesis of myelofibrosis, bone marrow stem cells from homozygous TGF-beta1 null (TGF-beta1(-/-)) and wild-type (WT) littermates were infected with a retrovirus encoding the murine thrombopoietin (TPO) protein and engrafted into lethally irradiated wild-type hosts for long-term reconstitution. Over the 4 months of follow-up, TPO levels in plasma were markedly elevated in both groups of mice, and animals typically developed a myeloproliferative syndrome characterized by thrombocytosis, leukocytosis, splenomegaly, increased numbers of progenitors in blood, and extramedullary hematopoiesis. Severe fibrosis was observed in spleen and marrow from all the mice engrafted with WT cells. In contrast, none of the mice repopulated with TGF-beta1(-/-) cells (chimerism > 70%) showed deposition of reticulin fibers at any time during the follow-up. In accordance with the development of fibrosis, latent TGF-beta1 levels in plasma and extracellular fluid of the spleen from mice engrafted with WT cells were increased 6-fold and 4-fold, respectively, over levels found in normal hosts, whereas no increase over baseline levels could be demonstrated in animals undergoing transplantation with TGF-beta1(-/-) cells. These data provide evidence that TGF-beta1 produced by hematopoietic cells is pivotal for the pathogenesis of myelofibrosis that develops in mice with TPO overexpression.

Animals↗
PubMed2002

Distinct effects of thrombopoietin depending on a threshold level of activated Mpl in BaF-3 cells.

Thrombopoietin (TPO) plays a critical role in megakaryopoiesis through binding to its receptor Mpl. This involves activation of various intracellular signaling pathways, including phosphoinositide 3-kinase (PI3K) and the mitogen-activated protein kinase (MAPK) pathways. Their precise role in TPO-mediated proliferation, survival and differentiation is not fully understood. In the present study, we show that TPO induces different biological responses in Mpl-transduced BaF-3 cells, depending on the cell surface density of Mpl and the resulting activation level of signaling pathways. TPO mediates cell proliferation in cells expressing high levels of Mpl but only mediates survival without proliferation in cells expressing low levels of the receptor. By using the kinase inhibitors PD98059 and LY294002, we further showed that the activation level of the PI3K and MAPK p42/44 pathways is a determining factor for the proliferative effect. In cells expressing low levels of Mpl, the survival effect was strongly dependent on the activation level of the PI3K/AKT, but not the MAPK p42/44 pathway. Moreover, this effect was correlated with the phosphorylation level of BAD but not with the expression level of Bcl-X(L). However, PI3K pathway inhibition did not increase apoptosis when BaF-3 cells proliferated in response to TPO, indicating a compensating mechanism from other Mpl signaling pathways in this case.

Animals↗
PubMed2002

Antiviral agent Cidofovir restores p53 function and enhances the radiosensitivity in HPV-associated cancers.

High-risk human papillomaviruses (HPVs) have been associated to the development of cervical and some other human cancers. Most of them express E6 and E7 oncoproteins, able to bind to p53 and retinoblastoma (pRb) tumor suppressor proteins respectively and neutralize their function. Restoration of these pathways by blocking E6 and E7 expression would provide a selective therapeutic effect. Here, we show that a clinically approved antiviral agent Cidofovir reduced E6 and E7 expression in cervical carcinoma Me180 and head and neck squamous cell carcinoma HEP2 cells at the transcriptional level. Cidofovir induced the accumulation of active p53 and pRb associated to induction of cyclin dependent kinase inhibitor p21(WAF1/CIP1) in Me180 and HEP2 cells. p53 induction was also shown in Hela HPV-positive cervical carcinoma cell line. In addition, S phase cell cycle accumulation with concomitant decrease of cyclin A expression were associated to the antiproliferative activity of Cidofovir in HPV-treated cells. Combining Cidofovir to irradiation both in vivo and in nude mice xenografts resulted in a marked radiosensitization in HPV-positive cells, which was not observed in virus negative cells. This study provides the basis for a new anticancer strategy to enhance the antitumor effect of ionizing radiation in HPV-related cancers, without increase deleterious effects.

Animals↗
PubMed2002

Requirement for mitogen-activated protein kinase activation in the response of embryonic stem cell-derived hematopoietic cells to thrombopoietin in vitro.

Enforced expression of c-mpl in embryonic stem (ES) cells inactivated for this gene results in protein expression in all the ES cell progeny, producing cells that do not belong to the megakaryocytic lineage and are responsive to PEG-rhuMGDF, a truncated form of human thrombopoietin (TPO) conjugated to polyethylene glycol. These include a primitive cell called BL-CFC, thought to represent the equivalent of the hemangioblast, and all myeloid progenitor cells. In this model, PEG-rhuMGDF was able to potentiate the stimulating effects of other growth factors, including vascular endothelial growth factor, on BL-CFC and a combination of cytokines on the growth of granulocyte macrophage-colony-forming units. The importance of the C-terminal domain of Mpl and of mitogen-activated protein kinase (MAPK) activation in TPO-dependent megakaryocytic differentiation has been well studied in vitro. Here, the role of this domain and the involvement of MAPK in upstream and nonmegakaryocytic cells are examined by using 2 truncated mutants of Mpl (Delta34, deletion of residues 71 to 121 in the C-terminal domain; and Delta3, deletion of residues 71-94) and specific inhibitors of the MAPK pathway. The 2 deleted regions support different functions, mediated by different signals. Residues 71 to 121 were required for PEG-rhuMGDF-dependent growth of BL-CFC, for megakaryocytic and other myeloid progenitors, and for megakaryocyte polyploidization. These responses were mediated by the ERK1-ERK2 MAPK pathway. In contrast, the only function of the sequence comprising residues 71 to 94 was to mediate the synergistic effects of PEG-rhuMGDF with other hematopoietic growth factors. This function is not mediated by MAPK activation.

Animals↗
PubMed2002

MplK, a natural variant of the thrombopoietin receptor with a truncated cytoplasmic domain, binds thrombopoietin but does not interfere with thrombopoietin-mediated cell growth.

OBJECTIVE: Interaction of thrombopoietin (TPO) with its receptor c-Mpl is responsible for the formation of megakaryocytes and platelets. In humans, there are two major c-mpl molecules, MplP and MplK, which are generated by alternative splicing. In contrast to MplP, MplK has none of the intracellular sequences required for typical signal transduction but instead has a unique 27 amino acid sequence that is coded by intron 10. We tested to determine if MplK exerts a negative effect on TPO Mpl signal transduction by interfering with the normal homodimerization of MplP. MATERIALS AND METHODS: A cassette coding for MplK cDNA was introduced into parental and MplP-expressing BaF3 cells and TPO-mediated cell growth studied. RESULTS: Cells expressing MplK alone did not respond to TPO compared to cells that expressed MplP. When MplK was coexpressed with MplP on the cell surface of BaF3, no modification in cell growth was observed when compared to those expressing MplP alone. To determine if the normal homodimerization process was negatively influenced, two genetically engineered variants of c-Mpl, one lacking the box1 sequence and the other containing only the first nine amino acids of the intracellular domain, were introduced into MplP-expressing cells. In contrast to MplK, these mutants had a dominant negative effect on TPO-mediated cell growth. CONCLUSIONS: MplK does not influence TPO-mediated growth of Mpl-expressing cells. Our data suggest that the absence of a dominant negative effect of MplK most probably is due to the inability of MplK to dimerize with the MplP receptor.

Animals↗
PubMed2002

Absence of mutations in the HoxA10, HoxA11 and HoxD11 nucleotide coding sequences in thrombocytopenia with absent radius syndrome.

Recent studies have suggested the HoxA10, HoxA11 and HoxD11 homeobox genes as candidate loci for the thrombocytopenia with absent radius (TAR) syndrome. For example, targeted disruptions of these Hox genes result in abnormal development of the mouse radius, while overexpression of HoxA10 stimulates mouse megakaryocyte (MK) development in vitro. To examine the expression of Hox genes in human MK cells, we utilized reverse transcription polymerase chain reaction with degenerate oligonucleotides to study megakaryocytic cell lines (MEG-01, DAMI), and primary human MK purified from adult and cord blood. Using this approach, 13 out of 40 clones isolated from cell lines, 10 out of 21 from cord MK, and 11 out of 21 from adult MK were identified as HoxA10, while HoxA11 and HoxD11 sequences were not detected. The normal genomic sequences for the human HoxA10, -A11, and -D11 genes were then determined and sequenced in 10 unrelated individuals with TAR syndrome. In all patients the derived amino acid sequence for the three Hox genes was identical to normal controls. Southern blotting did not reveal genomic rearrangements or deletions at these loci, and in two patients intact HoxA10 transcripts were detected by amplification in myeloid cells. Although these studies cannot completely exclude the possibility that the TAR syndrome results from non-coding mutations that affect the level of Hox gene expression in megakaryocytes, mutations in the coding sequence of the Hox genes known to affect radial development are not a common cause of TAR syndrome.

Adolescent↗
PubMed2002

Ex vivo expansion marginally amplifies repopulating cells from baboon peripheral blood mobilized CD34+ cells.

The ability of ex vivo expansion to increase the long-term repopulating capacity of a graft is still unknown. One problem is the most reliable way to quantify transplantable cells. We addressed this point in a baboon model based on autologous transplantation of serial limiting doses of non-manipulated or ex vivo-expanded mobilized CD34+ cells and determined the threshold doses of non-manipulated and expanded cells which supported long-term multilineage engraftment. In the expansion group, CD34+ cells were cultured for 6 d with a combination of early acting cytokines (Flt3-ligand, stem cell factor, thrombopoietin and interleukin 3). Grafted cells were characterized by their surface antigens and biological properties [semisolid assays, long-term culture-initiating cells (LTC-IC) and non-obese diabetic severe combined immunodeficient reconstituting cells (SRC)]. Animals were followed for at least 12 months post transplantation. The expansion protocol yielded 12.3-fold, 16.9-fold, 3.7-fold, 3.5-fold and 2.2-fold increases in CD34+ cells, granulocyte-macrophage colony-forming units (CFU-GM), megakaryocyte CFU (CFU-MK), LTC-IC and SRC respectively. It induced a modest increase in the long term reconstitutive ability of the graft; the threshold value for long-term engraftment was 0.5 x 10(6)/kg CD34+ cells in the control group and 0.3 x 10(6)/kg CD34+ cells in the expansion group, although one animal in this latter group remained hypoplastic. Frequencies of SRC had a high predictive value of long-term engraftment (r > 0.80). The main advantage of the protocol was the acceleration of granulocyte recovery, achieved at the different doses tested. In conclusion, these experiments suggest that this ex vivo expansion protocol marginally amplifies long-term reconstituting cells.

Animals↗
PubMed2002

Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells.

In this study, we have characterized the early steps of hematopoiesis during embryonic stem cell differentiation. The immunophenotype of hematopoietic progenitor cells derived from murine embryonic stem cells was determined using a panel of monoclonal antibodies specific for hematopoietic differentiation antigens. Surprisingly, the CD41 antigen (alphaIIb integrin, platelet GPIIb), essentially considered to be restricted to megakaryocytes, was found on a large proportion of cells within embryoid bodies although very few megakaryocytes were detected. In clonogenic assays, more than 80% of all progenitors (megakaryocytic, granulo-macrophagic, erythroid and pluripotent) derived from embryoid bodies expressed the CD41 antigen. CD41 was the most reliable marker of early steps of hematopoiesis. However, CD41 remained a differentiation marker because some CD41(-) cells from embryoid bodies converted to CD41(+) hematopoietic progenitors, whereas the inverse switch was not observed. Immunoprecipitation and western blot analysis confirmed that CD41 was present in cells from embryoid bodies associated with CD61 (beta3 integrin, platelet GPIIIa) in a complex. Analysis of CD41 expression during ontogeny revealed that most yolk sac and aorta-gonad-mesonephros hematopoietic progenitor cells were also CD41(+), whereas only a minority of bone marrow and fetal liver hematopoietic progenitors expressed this antigen. Differences in CD34 expression were also observed: hematopoietic progenitor cells from embryoid bodies, yolk sac and aorta-gonad-mesonephros displayed variable levels of CD34, whereas more than 90% of fetal liver and bone marrow progenitor cells were CD34(+). Thus, these results demonstrate that expression of CD41 is associated with early stages of hematopoiesis and is highly regulated during hematopoietic development. Further studies concerning the adhesive properties of hematopoietic cells are required to assess the biological significance of these developmental changes.

Animals↗