JAK2(V617F): Prevalence in a large Chinese hospital population.

Recently, the JAK2(V617F) mutation was found in patients with myeloproliferative disorders (MPDs), including most with polycythemia vera (PV). The mutant JAK2 has increased kinase activity, and it was shown to be pathogenic in mouse models. Herein, we analyzed blood samples randomly collected from a clinical laboratory. Surprisingly, as many as 37 samples from a total of 3935 were found positive for the JAK2 mutation. However, only one of these samples had blood test results indicative for probable PV, but several had nonhematologic diseases. On average, samples with the mutation had normal red cell counts but significantly higher white blood cell and platelet counts, although most were within the normal range. The data suggest that the JAK2(V617F) mutation is apparently much more common than MPDs. Its occurrence may be a prelude to full blood cell abnormalities and other diseases, but it cannot by itself diagnose MPDs.

Role of tyrosine kinases and phosphatases in polycythemia vera.

Protein tyrosine kinases (PTKs) and phosphatases (PTPs) play a crucial role in normal cell development, and dysfunction of these enzymes has been implicated in human cancers. Polycythemia vera (PV) is a clonal hematologic disease characterized by hypersensitivity of hematopoietic progenitor cells to growth factors and cytokines. Recently, a unique and clonal mutation in the JAK homology 2 (JH2) domain of JAK2 that results in a valine to phenylalanine substitution at position 617 (V617F) was found in the majority of PV patients. This mutation leads to constitutive JAK2 activation and abnormal signaling and induces erythrocytosis in an animal model. The mutation is also found in a significant percentage of patients with idiopathic myelofibrosis (50%) and essential thrombocythemia (30%). Thus, it seems probable that this mutation associates with other molecular genetic events to cause different myeloproliferative disorders (MPDs). One of these secondary events is the transition to homozygosity of the mutated gene in 30% of the PV patients. Other events may include defects in PTPs, but these remain to be characterized. Recent studies represent a great step forward in the molecular pathogenesis in PV and the development of targeted new drugs to treat the disease.

Identification of an acquired JAK2 mutation in polycythemia vera.

Polycythemia vera (PV) is a human clonal hematological disorder. The molecular etiology of the disease has not been identified. PV hematopoietic progenitor cells exhibit hypersensitivity to growth factors and cytokines, suggesting possible abnormalities in protein-tyrosine kinases and phosphatases. By sequencing the entire coding regions of cDNAs of candidate enzymes, we identified a G:C--> T:A point mutation of the JAK2 tyrosine kinase in 20 of 24 PV blood samples but none in 12 normal samples. The mutation has varying degrees of heterozygosity and is apparently acquired. It changes conserved Val(617) to Phe in the pseudokinase domain of JAK2 that is known to have an inhibitory role. The mutant JAK2 has enhanced kinase activity, and when overexpressed together with the erythropoietin receptor in cells, it caused hyperactivation of erythropoietin-induced cell signaling. This gain-of-function mutation of JAK may explain the hypersensitivity of PV progenitor cells to growth factors and cytokines. Our study thus defines a molecular defect of PV.

Increased erythropoiesis in polycythemia vera is associated with increased erythroid progenitor proliferation and increased phosphorylation of Akt/PKB.

OBJECTIVE: The aim of this study was to explore the mechanism by which increased erythropoiesis occurs in polycythemia vera (PV). METHODS: CD34(+) and erythroid colony-forming cells (ECFC) were purified from normal or PV peripheral blood and then incubated in the presence of erythropoietin (EPO) to generate erythroid progenitor cells. Measurement of proliferation by Ki-67 staining, TUNEL assays to measure apoptosis, and Western blots for detection of Akt/PKB and glycogen synthase kinase 3 (GSK3) phosphorylation were performed in both normal and PV erythroid progenitors. RESULTS: Polycythemia vera erythroid progenitor cells generated 60% more cells compared to normal cells in liquid medium cell cultures. TUNEL assays revealed no difference between PV and normal erythroid progenitors, but Ki-67 staining for cell proliferation showed many more positive cells in the PV samples. A marked increase of phosphorylation of Akt/PKB occurred in the day-8 erythroid progenitors of 4/5 PV patients, compared to normal cells, after incubation with either stem cell factor (SCF) or EPO. PV cells also had much greater glycogen synthase kinase 3 (GSK3) alpha,beta phosphorylation compared to normal cells after incubation with SCF or EPO. These results are parallel to the cellular hypersensitivity of PV cells to SCF and EPO previously reported. CONCLUSIONS: Increased erythropoiesis in PV is associated with increased cellular proliferation and increased phosphorylation of Akt/PKB and GSK3. This study provides additional insight into the pathogenesis of PV and the regulation of normal erythropoiesis, even though a specific molecular defect of the disease is still not apparent.

In human immature BFU-E tumor necrosis factor-alpha not only downregulates CDK6 but also directly produces apoptosis which is prevented by stem cell factor.

OBJECTIVE: The aim of this study was to reveal the mechanisms by which tumor necrosis factor-alpha (TNF-alpha) inhibits immature human day-4 burst-forming units-erythroid (BFU-E) and the effect of stem cell factor (SCF) on this process. METHODS: Sequential density-gradient centrifugation, depletion of lymphocytes, removal of adherent cells, and negative selection with CD2, CD11b, CD16, and CD45 monoclonal antibodies were used to purify day-1 BFU-E, which were then incubated for 3 days to generate day-4 cells. The day-4 cells were incubated with TNF-alpha, and/or SCF, and the extent of apoptosis was gauged by morphologic observations, TUNEL assays, and Western blots. RESULTS: The cell number and the number and size of erythroid colonies were significantly reduced when day-4 cells were incubated with TNF-alpha. Apoptosis was observed in single-cell plasma clot assays. TUNEL assays showed 20% +/- 6% apoptotic cells with TNF-alpha while controls had 2.8% +/- 2.2%. Caspases 3 and 8 were strongly activated while the amount of CDK6 was reduced by TNF-alpha. When SCF, a potent stimulator of cell growth, was added with TNF-alpha, cell growth inhibition was reduced and the apoptotic cells decreased to 0.9% +/- 1.2%. The activations of caspase 3 and caspase 8 were almost completely blocked by SCF while CDK6 and the FLICE-inhibitory protein (FLIP) were increased. CONCLUSIONS: Our results indicate that in immature human BFU-E, TNF-alpha downregulates CDK6 but also directly produces apoptosis which is prevented by SCF.

The LTR enhancer of ERV-9 human endogenous retrovirus is active in oocytes and progenitor cells in transgenic zebrafish and humans.

The solitary LTRs of ERV-9 human endogenous retrovirus are middle repetitive DNAs associated with 3,000-4,000 human gene loci including the beta-globin gene locus where the ERV-9 LTR is juxtaposed to the locus control region (beta-LCR) far upstream of the globin genes. The ERV-9 LTRs are conserved during primate evolution, but their function in the primate genomes is unknown. Here, we show that in transgenic zebrafish harboring the beta-globin ERV-9 LTR coupled to the GFP gene, the LTR enhancer was active and initiated synthesis of GFP mRNA in oocytes but not in spermatozoa, and GFP expression in the embryos was maternally inherited. The LTR enhancer was active also in stem/progenitor cell regions of adult tissues of transgenic zebrafish. In human tissues, ERV-9 LTR enhancer was active also in oocytes and stem/progenitor cells but not in spermatozoa and a number of differentiated, adult somatic cells. Transcriptional analyses of the human beta-globin gene locus showed that the beta-globin ERV-9 LTR enhancer initiated RNA synthesis from the LTR in the direction of the downstream beta locus control region and globin genes in ovary and erythroid progenitor cells. The findings suggest that, during oogenesis, ERV-9 LTR enhancers in the human genome could activate the cis-linked gene loci to synthesize maternal mRNAs required for early embryogenesis. Alternatively, the ERV-9 LTR enhancers, in initiating RNA syntheses into the downstream genomic DNAs, could transcriptionally potentiate and preset chromatin structure of the cis-linked gene loci in oocytes and adult stem/progenitor cells.

PTP-MEG2 is activated in polycythemia vera erythroid progenitor cells and is required for growth and expansion of erythroid cells.

Polycythemia vera (PV) is a human clonal hematologic disorder. Previously we demonstrated that erythroid colony-forming cells (ECFCs) from PV patients contained a hyperactive membrane-associated tyrosine phosphatase. We now show that this phosphatase corresponded to protein tyrosine phosphatase (PTP)-MEG2, an intracellular enzyme with a putative lipid-binding domain. The increased activity of PTP-MEG2 in PV cells is due to its elevated distribution in the membrane fraction. With the development of ECFCs to mature red cells, the protein level of PTP-MEG2 decreased gradually, but membrane-associated PTP-MEG2 was sustained for a longer period of time in PV cells, which correlated with an enhanced colony-forming capability of the cells. Importantly, expression of dominant-negative mutant forms of PTP-MEG2 suppressed in vitro growth and expansion of both normal and PV ECFCs. The data indicate that PTP-MEG2 has an important role in the development of erythroid cells.

Reduction of cell cycle progression in human erythroid progenitor cells treated with tumour necrosis factor alpha occurs with reduced CDK6 and is partially reversed by CDK6 transduction.

Tumor necrosis factor alpha (TNFalpha) potently inhibits the in vitro growth of highly purified human d-6 erythroid colony forming cells (ECFC). Unlike the inhibitory effect of TNFalpha on other cells, including more immature ECFC, this antiproliferative effect of TNFalpha is not related to apoptosis because the d-6 cell descendants were morphologically normal, without apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling assay and without caspase activation by Western blots after TNFalpha treatment. TNFalpha did not appear to affect the cell cycle distribution, but the cell cycle duration was significantly longer in TNFalpha-treated cells. DNA synthesis was also significantly reduced by TNFalpha. Studies of various proteins that regulate the cell cycle showed that cyclin-dependent kinase 6 (CDK6) protein and mRNA levels were concomitantly decreased in the presence of TNFalpha, suggesting that inhibition of cell growth was related to reduced CDK6. To evaluate this, the CDK6 gene was transferred into ECFC using green fluorescence protein-retrovirus-mediated gene transfer. The results showed that the level of cell growth produced by TNFalpha was increased by 30% when the cells were transfected with CDK6. Therefore, the modification of cell cycle progression in the presence of TNFalpha through a reduction of CDK6 is an important mechanism in the TNFalpha inhibition of human ECFC expansion.

Specific interaction of protein tyrosine phosphatase-MEG2 with phosphatidylserine.

Protein tyrosine phosphatase (PTP)-MEG2 is an intracellular tyrosine phosphatase that contains a Sec14 homology domain. We have purified the full-length and truncated forms of the enzyme from recombinant adenovirus-infected human 293 cells. By using lipid-membrane overlay and liposome binding assays, we demonstrated that PTP-MEG2 specifically binds phosphatidylserine among over 20 lipid compounds tested. The binding is mediated by its N-terminal Sec14 domain. In intact cells, the Sec14 domain is responsible for localization of PTP-MEG2 to the perinuclear region, and uploading of PS into the cell membrane causes translocation of PTP-MEG2 to the plasma membrane. Phosphatidylserine is a relatively abundant cell membrane phospholipid non-symmetrically distributed in the outer layer and inner layer of cell membranes. It has recently been defined as an important ligand for clearance of apoptotic cells. By specifically binding phosphatidylserine, PTP-MEG2 may play an important role in regulating signaling processes associated with phagocytosis of apoptotic cells.

Stem cell factor increases the expression of FLIP that inhibits IFNgamma -induced apoptosis in human erythroid progenitor cells.

Interferon gamma (IFNgamma) acts on human erythroid colony-forming cells (ECFCs) to up-regulate Fas, without a demonstrable change of Fas ligand (FasL) or Fas-associated DD-containing protein (FADD) expression and activates caspase-8 plus caspase-3, which produce apoptosis. Our previous data showed that stem cell factor (SCF) reduced the inhibitory effect of IFNgamma on human ECFCs when both factors were present in the cultures. However, the mechanism by which SCF prevents IFNgamma-induced apoptosis in ECFCs is unclear. In this study we used highly purified human ECFCs to investigate the mechanism of the effect of SCF on IFNgamma-induced apoptosis. Because the binding of FasL to Fas is the first step of the apoptosis cascade and IFNgamma strongly up-regulates Fas expression, we added FasL (50 ng/mL) to the cultures with IFNgamma to accentuate the IFNgamma-induced activation of caspase-8 and caspase-3 plus subsequent apoptosis. SCF (100 ng/mL) clearly inhibited the activation of caspase-8 and caspase-3 induced by IFNgamma and/or FasL, and it also reduced apoptosis as measured by the terminal dUTP nick-end labeling (TUNEL) assay. SCF did not decrease the surface expression of Fas on the ECFCs. FADD-like interleukin 1 beta (IL-1beta)-converting enzyme (FLICE)-inhibitory protein (FLIP) has been reported to interact with FADD and/or caspase-8 at the death-inducing signaling complex (DISC) level following Fas stimulation and acts as a dominant-negative caspase-8. SCF increased FLIP mRNA and protein expression, concomitant with reduced apoptosis, whereas IFNgamma and/or FasL did not change FLIP expression. Reduction of FLIP expression with antisense oligonucleotides decreased the capacity of SCF to inhibit IFNgamma-induced apoptosis, demonstrating a definite role for FLIP in the SCF-induced protection of ECFCs from IFNgamma-initiated apoptosis.

Purification and characterization of protein tyrosine phosphatase PTP-MEG2.

PTP-MEG2 is an intracellular protein tyrosine phosphatase with a putative lipid-binding domain at the N-terminus. The present study reports expression, purification, and characterization of the full-length form of the enzyme plus a truncated form containing the catalytic domain alone. Full-length PTP-MEG2 was expressed with an adenovirus system and purified from cytosolic extracts of human 293 cells infected with the recombinant adenovirus. The purification scheme included chromatographic separation of cytosolic extracts on fast flow Q-Sepharose, heparin-agarose, l-histidyldiazobenzylphosphonic acid agarose, and hydroxylapatite. The enrichment of PTP-MEG2 from the cytosol was about 120-fold. The truncated form of PTP-MEG2 was expressed in E. coli cells as a non-fusion protein and purified by using a chromatographic procedure similar to that used for the full-length enzyme. The purified full-length and truncated enzymes showed single polypeptide bands on SDS-polyacrylamide gel electrophoresis under reducing conditions and behaved as monomers on gel exclusion chromatography. With para-nitrophenylphosphate and phosphotyrosine as substrates, both forms of the enzyme exhibited classical Michaelis-Menten kinetics. Their responses to pH, ionic strength, metal ions, and protein phosphatase inhibitors are similar to those observed with other characterized tyrosine phosphatases. Compared with full-length PTP-MEG2, the truncated DeltaPTP-MEG2 displayed significantly higher V(max) and lower K(m) values, suggesting that the N-terminal putative lipid-binding domain may have an inhibitory role. The full-length and truncated forms of PTP-MEG2 were also expressed as GST fusion proteins in E. coli cells and purified to near homogeneity through affinity columns. However, the specific phosphatase activities of the GST fusion proteins were 10-25-fold below those obtained with the correspondent non-fusion proteins.

The solitary long terminal repeats of ERV-9 endogenous retrovirus are conserved during primate evolution and possess enhancer activities in embryonic and hematopoietic cells.

The solitary long terminal repeats (LTRs) of ERV-9 endogenous retrovirus contain the U3, R, and U5 regions but no internal viral genes. They are middle repetitive DNAs present at 2,000 to 4,000 copies in primate genomes. Sequence analyses of the 5" boundary area of the erythroid beta-globin locus control region (beta-LCR) and the intron of the embryonic axin gene show that a solitary ERV-9 LTR has been stably integrated in the respective loci for at least 15 million years in the higher primates from orangutan to human. Functional studies utilizing the green fluorescent protein (GFP) gene as the reporter in transfection experiments show that the U3 region of the LTRs possesses strong enhancer activity in embryonic cells of widely different tissue origins and in adult cells of blood lineages. In both the genomic LTRs of embryonic placental cells and erythroid K562 cells and transfected LTRs of recombinant GFP plasmids in K562 cells, the U3 enhancer activates synthesis of RNAs that are initiated from a specific site 25 bases downstream of the AATAAA (TATA) motif in the U3 promoter. A second AATAAA motif in the R region does not serve as the TATA box or as the polyadenylation signal. The LTR-initiated RNAs extend through the R and U5 regions into the downstream genomic DNA. The results suggest that the ERV-9 LTR-initiated transcription process may modulate transcription of the associated gene loci in embryonic and hematopoietic cells.