In vitro expansion of erythroid progenitors from polycythemia vera patients leads to decrease in JAK2 V617F allele

Abstract

Objectives: A G>T transversion in a tyrosine kinase JAK2 (V617F) was reported in over 80% of patients with polycythemia vera (PV). Current evidence suggests that JAK2(V617F) somatic mutation is involved in the pathogenesis of PV, as it confers erythropoietin-independent proliferation to erythroid progenitor cells. However, several unanswered questions regarding the essential role of JAK2(V617F) arose as 1) it is not a dominant mutation, 2) it is not PV-specific as it is found in several myeloproliferative disorders, and 3) some ( approximately 20%) PV patients lack the JAK2(V617F) mutation. We investigated the relative frequency of JAK2(V617F) in in vitro-expanded PV progenitors.

Methods: In vitro expansion of erythroid progenitors from mononuclear cells was optimized. Frequency of JAK2(V617F) allele was measured by using allele-specific real-time polymerase chain reaction. Clonality was performed using established procedure.

Results: In vitro expansion of PV erythroid progenitors and differentiated dendritic cells resulted in a decrease of the frequency of JAK2(V617F) allele compared with granulocytes or CD235(+) erythroid progenitors. Clonality analysis demonstrated that although granulocytes of these PV patients were clonal, expanded erythroid cells were polyclonal. However, in vitro-expanded PV erythroid progenitors still had approximately a twofold increased proliferative capacity in comparison with erythroid progenitors from healthy individuals. Erythropoietin favors the cells without JAK2(V617F) allele. Dendritic cells in one out of three patients remained clonal.

Conclusion: JAK2(V617F) mutation does not provide a proliferative/survival advantage to the PV clone during in vitro expansion. These data suggest that the JAK2(V617F) mutation plays an important role in the biology of PV, yet it may not be the PV-initiating event.

Figure 1

(A) Fluorescent activator flow cytometer analysis of in vitro expanded erythroid progenitors: Mononuclear cells were isolated from PV patients and expanded as described in the Methods. About 5 × 10⁵ cells were stained with PE-conjugated anti-CD235A antibodies (glycophorin) and FITC-conjugated anti-human-CD71 antibodies (transferrin receptor). Regions R2 to R6 are defined by characteristic staining pattern of erythroid cells. These are primitive progenitor cells plus mature BFU-Es and CFU-Es in R2 (CD71med CD235Alow), pro-erythroblasts and early basophilic erythroblasts in R3 (CD71high CD235Alow), early and late basophilic erythroblasts in R4 (CD71high CD235Ahigh), polychromatophilic and orthochromatophilic erythroblasts in R5 (CD71med CD235Ahigh), and late orthochromatophilic erythroblasts and reticulocytes in R6 (CD71low CD235Ahigh). (B) Percent JAK2^V617F in granulocytes and in vitro expanded erythroid progenitors: Granulocytes (GNC) were isolated from peripheral blood of PV patients. JAK2^V617F was measured in genomic DNA of granulocytes as well as in vitro expanded erythroid progenitors. M and F represent male and female patients, respectively.

Figure 2

A. Flow cytometric separation of glycophorin positive cells from MNC of PV patients: Mononuclear cells from peripheral blood of PV patients were either unstained (A/I) or immuno-stained with anti-human CD235a antibodies (A/II). Flow cytometric sorting was performed as described in the methods. Cells in the R2 region (CD235a⁺) were collected. B: JAK2^V617F in glycophorin positive and PV erythroid expanded cells: Genomic DNA from CD235a⁺ was used to measure the JAK2^V617F (Gly⁺) to compare with the JAK2^V617F in corresponding in vitro expanded erythroid progenitors (S3) from three PV patients.

Figure 3

Clonal analysis of granulocytes and PV erythroid progenitors after in vitro expansion: (A) MNC and granulocytes from peripheral blood of five female PV patients was isolated. Expansion of the erythroid progenitors from mononuclear cells was performed as described earlier. Assays for clonality were performed in granulocytes (GNC) and the in vitro expanded erythroid progenitors (S3) using the markers as described. Skewed polyclonal: Ambiguous or unclear distinction of clonality due to mixed proportion of clonal and polyclonal cells(B) Auto radiographic representation of the clonality analysis. Total RNA was prepared using Trizol reagent from the granulocytes (GNC) isolated from peripheral blood of female PV patients and from the in vitro expanded erythroid cells (S3). The T, C, and TC alleles in the control panel are representative of the standard exonic polymorphism BTK marker described previously

Figure 4

Apoptosis and proliferation of in vitro expanded erythroid progenitors: (A) Expansion of the erythroid progenitors from control and PV mononuclear cells was performed as described in methods. Whole cell lysates resolved on SDS12%PAGE were probed for Bclxl, caspase3, and β-actin proteins of control (C) and PV cell lysates. Position of the molecular weight markers is shown on the left. (B) Proliferation of erythroid progenitors was assessed from the total number of cells at the end of S3 step during the in vitro expansion of control and PV erythroid progenitors. Fold increase in the number of cells was determined from the initial total number of MNCs used for expansion. Values from five normal and six PV patients were used to analyze the rate of proliferation. P value was determined using students t-test. *P<0.05. The JAK2^V617F allelic frequency of the PV cells at the end of expansion is shown on top.

Figure 5

The frequency of JAK2^V617F and clonal analysis of dendritic cells: (A) JAK2^V617F frequency in granulocytes and dendritic cells was measured as described in methods. (B) Clonal analysis of dendritic cells from three female patients was performed.

Figure 6

Effect of Epo on JAK2^V617F frequency: MNC from five PV patients (PV#10–14) were used for the expansion of erythroid progenitors as described earlier. At the end of S1 step, the cells were divided into two groups. Half the cells were grown in S2 medium without any Epo (S2-Epo: lane1) while the other half was grown in S2 containing 3units/ml of Epo (S2+Epo: lane2). At the end of S2 step of erythroid expansion, both the groups were grown separately, in complete S3 medium (cells from S2-Epo are labeled as S3 (lane3) and cells from S2+Epo are labeled as S3+Epo: lane4, respectively). JAK2^V617F frequency was measured at the end of expansion and expressed as percent change in their JAK2^V617F frequency using the granulocyte JAK2^V617F at a value of 100. Significance was determined using the students’ t-test. * P<0.05.