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:C3 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. Polycythemia vera (PV)1 is a clonal myeloproliferative disorder characterized by increased production of red cells, granulocytes, and platelets (1-3). It mainly affects people between 40 and 60 years of age with an annual incidence of about 14 per million in the population. Thus far there is no effective cure for the disease. Phlebotomy is the mainstay of treatment for the disease, and hydroxyurea, interferon-␣, and anagrelide drug therapies and 32 P radiation therapy have commonly been used (3). The mortality rate is high if the disease is untreated or is associated with leukemia. Despite extensive studies in recent years, the molecular etiology of PV remains unknown.A major feature of PV is that hematopoietic progenitors in patients display hypersensitive responses to many growth factors and cytokines (1-3). Despite these abnormal responses, the numbers of receptors for the growth factors and cytokines on the surface of these cells are normal, suggesting a primary defect in a shared signaling pathway in these cells. Tyrosine phosphorylation is a fundamental regulatory mechanism for cell growth and development, and this process is controlled by coordinate actions of protein-tyrosine kinases (PTKs) and phosphatases (PTPs) (4). Both families of enzymes have highly diverse structures and functions. Their activities are tightly regulated under normal conditions, and deregulation of the enzymes produces human diseases. According to the human genomic data base, there are about 90 PTKs and 38 phosphotyrosine-specific PTPs (5-7). Both mutation of PTKs and PTPs have been implicated in human cancers. Recent studies using a large scale sequencing-based approach revealed that PTK and PTP genes are mutated more often in human cancers than previously anticipated. A minimum of 30% of colorectal cancers contains at least one mutation in PTKs, and 26% of them has a mutation in PTPs (8, 9). It is highly expected that mutations of PTKs and PTPs may also be a major manifestation of other types o...

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Previous studies have shown that approximately 40% of patients with myelodysplastic syndrome (MDS) and anaemia respond to treatment with human recombinant granulocyte-CSF (G-CSF) plus erythropoietin (epo). The present study was designed to investigate pre-treatment variables for their ability to predict erythroid responses to this treatment. 98 patients with MDS (30 RA, 31 RARS, 32 RAEB, five RAEB-t) were treated with a combination of G-CSF (0.3-3.0 microg/kg/d, s.c.) and epo (60-300 U/kg/d, s.c.) for at least 10 weeks. Minimum criteria for erythroid response was a 100% reduction of red blood cell (RBC) transfusion need or an increase in haemoglobin level of > or = 1.5 g/dl. 35 patients (36%) showed responses to treatment. Medium duration of response was 11-24 months. In multivariate analysis, serum erythropoietin levels and initial RBC-transfusion need retained high statistical significance (P < 0.01). Using pre-treatment serum epo levels as a ternary variable (< 100, 100-500 or > 500 U/l) and RBC transfusion need as a binary variable (< 2 or > or = 2 units per month), the analysis provided a predictive score for erythroid response. This score divided patients into three groups: one group with a high probability of erythroid responses (74%), one intermediate group (23%) and one group with poor responses to treatment (7%). This predictive scoring system could be used in decisions regarding use of these cytokines for treating the anaemia of MDS, both for defining patients who should not be given the treatment and for selecting patients for inclusion in prospective trials.

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. IntroductionProtein tyrosine kinases are central regulators of signaling pathways and are attractive therapeutic targets. [1][2][3] Recently, several groups identified a recurrent somatic activating mutation in the JAK2 tyrosine kinase in polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis. 4-8 Infrequent occurrence of this mutation has been also reported in chronic myelomonocytic leukemia, atypical myeloproliferative disorders (MPDs), myelodysplastic syndrome, systemic mastocytosis, chronic neutrophilic leukemia, and acute myeloid leukemia. [9][10][11][12][13] This mutation results in a valine to phenylalanine substitution within the pseudokinase domain of JAK2. The mutant JAK2 possesses enhanced tyrosine kinase activity and causes a PV-like phenotype in mouse bone marrow transplantation models. 6,14,15 However, it remains unclear whether the JAK2 V617F mutation is solely responsible for these diseases and why it is associated with such a wide spectrum of phenotypes. Previous investigations were carried out to identify JAK2 V617F in samples from specific diseases. In this study, we analyzed blood samples randomly collected from a clinical laboratory. Materials and methods Blood samplesDeidentified, consecutive peripheral blood samples from donors (all Chinese) of any age, sex, and disease were randomly collected from the clinical laboratory of China Japan Union Hospital, Changchun, China, over a period of about 6 months. Institutional Review Board approvals were obtained from both Jilin University and the University of Oklahoma Health Science Center. PCR amplificationGenomic DNA was extracted from the whole blood. A genomic DNA fragment containing the exon 14 of the JAK2 gene which bears the V617F mutation site was amplified by polymerase chain reaction (PCR) with primers P1 and P1r ( Figure 1A). The PCR was run with Taq DNA polymerase or Phusion polymerase (for confirmation of the positive samples) for 35 cycles with 94°C for 20 seconds, 60°C for 20 seconds, and 72°C for 30 seconds. For allele-specific PCR, 0.2 L of the initial PCR product was used for further PC...

Morphological and biochemical studies of human colony-forming units-erythroid (CFU-E) have been hindered by their extreme rarity. Since burst-forming units-erythroid (BFU-E) develop into CFU-E, we used normal human blood BFU-E to generate large numbers of highly purified CFU-E in vitro. Using density centrifugation, sheep erythrocyte rosetting, surface immunglobulin positive cell depletion, adherence to plastic, and negative pn with monoclonal antibodies, human blood BFU-E were purified from 0.017 to 0368%, a 22-fold purification with a 43% yield. The panned cells were cultured in methylcellulose with recombinant erythropoietin (rEp) and conditioned medium for 9 d. These cells were then collected and CFU-E were further purified using adherence and density centrifugation. This yielded almost 107 erythroid colony forming cells with a purity of 70±18%.Analysis of these cells by light and electron microscopy showed 94% erythroid cells. The prominent cell was a primitive blast with hih nuclear/cytoplasmic ratio, dispersed nuclear chromatin and a distinct large nucleolus. The relation between the number of erythroid colonies and the number of day 9 cells plated in plasma clots was a straight line through the origin with a maximum number of erythroid colonies at 1 U/ml of rEp and no erythroid colonies without rEp. Specific binding with ', SI-rEp showed that 60% of the binding was inhibited by excess pure erythropoietin (Ep), but not by albumin, fetal calf serum, and a variety of growth factors or glycoproteins. By days 12-13 of cell culture, when the progenitor cells matured to late erythroblasts, specific binding markedly declined. In this study, human CFU-E have been isolated in sufficient purity to characterize the morphology of these rare cells and in sufficient numbers to measure specific binding of Ep.

To facilitate the direct study of the molecular events that control the development of human burst-forming units-erythroid (BFU-E), we have developed a method to purify BFU-E from peripheral blood. Using density centrifugation, rosetting with a mixture of neuraminidase-treated and IgG-coated sheep erythrocytes, positive panning with anti-My10 monoclonal antibody, overnight adherence to plastic dishes, negative panning with monoclonal antibodies, and density centrifugation, human blood BFU-E were purified from 0.04% to 56.6%, a 1,400-fold purification with a 13% yield. More than 90% of purified BFU-E were recombinant interleukin-3 (rIL-3) dependent, which survived for 48 h with rIL-3 in the absence of recombinant erythropoietin (rEP), and 80% gave rise to erythroid bursts of more than 500 hemoglobinized cells. rEP dependency was not evident until after 72 h of incubation in vitro. The purified cells (day 1) were incubated with rIL-3 and rEP in liquid culture for 24 (day 2), 48 (day 3), and 72 (day 4) h and then were transferred into semisolid cultures and incubated until day 15. The size of the erythroid colonies observed in semisolid cultures decreased continuously in association with the incubation time of day 1 purified cells in liquid cultures. The first appearance of colony-forming units-erythroid (CFU-E) that gave rise to colonies of 8 to 49 cells was observed after 72 h of incubation of day 1 cells in the liquid culture. 125I-rEP was incubated for 5 h at 37 degrees C with purified cells (day 1) or with the cells that had been incubated in liquid culture for an additional 24-72 h, and the presence of erythropoietin (EP) receptors was investigated using autoradiography. Specific binding of 125I-rEP was detected in 19 +/- 7% of the initial day 1 BFU-E. The percentage of 125I-rEP-binding to erythroid progenitor cells and the amount of binding continuously increased as day 1 BFU-E matured. 125I-rEP specific binding was observed with all of the erythroid progenitor cells that had been incubated in liquid culture for 72 h. These data demonstrate that primitive BFU-E have a much lower number of EP receptors than CFU-E and develop an increased concentration of EP receptors in association with their maturation and loss of proliferative capacity.

Erythropoietin (EP), insulin-like growth factor I (IGF-I) and stem cell factor (SCF) each reduce apoptosis of human erythroid progenitor cells. To determine if these growth factors have additional roles in stimulating erythropoiesis, the proliferation, maturation, and survival of highly purified human erythroid colony-forming cells (ECFCs) were studied during the application of different combinations of these growth factors in a serum-free liquid culture. EP maintained cell viability and supported heme synthesis during erythroid maturation, with little increase in viable cell number or stimulation of DNA synthesis. The addition of SCF with EP resulted in a substantial increase in DNA synthesis, which was greater than that seen with the addition of EP and was associated with a large expansion in the number of ECFCs. Thus EP, by itself, produces little increase in cell proliferation, and expansion of the number of erythroid cells depends upon the presence of SCF with EP. The addition of IGF-I with EP led to enhanced heme synthesis and moderate cellular proliferation, but also greatly enhanced nuclear condensation and enucleation in the late erythroblasts. Thus EP, by itself, is not sufficient for complete end-terminal nuclear condensation/enucleation and the presence of IGF-I is necessary for this complete process. While EP greatly reduced apoptosis during 16 h of incubation at 370C, the addition of SCF and IGF-I with EP had little additional effect, but these additions enhanced DNA synthesis > 3.4-fold. Thus SCF may have an additional role in directly stimulating proliferation through a process that is distinct from apoptosis. Our observations indicate that EP prevents apoptosis and maintains erythroid cell viability and development. IGF-I enhances erythroid maturation and proliferation, but the proliferation of erythroid progenitors is mainly controlled by the addition of SCF with EP, independent of an effect on apoptosis. (J. Clin. Invest. 1994. 94:34-43.)

Our data support the hypothesis that the sympathetic nervous system stimulates erythropoiesis in humans because anemia is a frequent occurrence in patients with severe autonomic failure and is associated with a blunted erythropoietin response.