The effects of Peg-IFNα 2a and ABT-737 alone and in combination on hematopoietic colony formation by JAK2V617F-positive PV CD34⁺ cells. (A) CFU-GM and BFU-E derived colony formation by CD34⁺ cells isolated from 6 different CB, 6 different bone marrow samples, and peripheral blood from 14 different patients with PV in the presence of 200 U/mL Peg-IFNα 2a and 250 nM of ABT-737 alone or in combination; hematopoietic colony numbers were enumerated after 14 days of incubation. Data were analyzed by the Student t test (*P < .05, **P < .01; ***P < .001; n = 14). (B) Effect of 200 and 500 U/mL Peg-IFNα 2a and 250 nM ABT-737 alone or combination treatment on the JAK2 V617F-positive colony formation by PV HPC. Individual hematopoietic colonies were randomly plucked from the cultures; JAK2V617F was detected using a nested allele-specific PCR. At least 50% of colonies were examined if the number of colonies exceeded 50 in an individual dish. If the number of colonies in an individual dish were less than 50, all colonies were plucked and examined. In these experiments, more than 78% of the total number of colonies were plucked and analyzed. Data were analyzed by the Student t test (**P < .01; n = 14). (C) Treatment with 200 U/mL Peg-IFNα 2a combined with 250 nM of ABT-737 significantly decreased the number of JAK2V617F homozygous hematopoietic colonies compared with treatment with Peg-IFNα 2a alone (*P < .05; n = 9). In panels B and C, different symbols stand for the different samples.

PV CD34⁺ cells are more sensitive to treatment with ABT-737 than CB and normal bone marrow CD34⁺ cells. (A) Western blotting demonstrates greater expression of Bcl-xL in PV CD34⁺ cells than CB CD34⁺ cells (CB1, CB2, PV1, and PV2). (B) The flow-cytometric analysis results represent the percentage of apoptotic-positive cells observed after the treatment of CB and PV CD34⁺ cells with ABT-737 at various doses for 3 days. Cells were stained with annexin V. The data were analyzed by the Student t test (*P < .05, **P < .01; n = 6). (C) Effects of 200 and 500 U/mL Peg-IFNα 2a and 250 nM ABT-737 alone and in combination on CB and PV CD34⁺ cell apoptosis. The numbers of apoptotic cells were also determined flow cytometrically after 3 days of incubation. The data were analyzed by the Student t test (*P < .05, **P < .01; n = 6). (D) Western blotting showed that the cleaved forms of caspase-3 were increased in PV CD34⁺ cells after treatment with 500 U/mL Peg-IFNα 2a and 250 nM ABT-737 in combination compared with that observed with Peg-IFNα 2a and ABT-737 alone. The α-tubulin serves as the loading control in the Western blot analysis. (E) Effect of Peg-IFNα 2a and ABT-737 alone or in combination on mitochondrial membrane potential in progenitor cells of PV patients. Cells were stained with the JC-1 mitochondrial membrane potential dye after 2 days of treatment with 200 U/mL Peg-IFNα 2a alone, 250 nM of ABT-737 alone, or a combination of 2 drugs. The cells were observed immediately with a fluoroscope (Nikon Eclipse-E600; 10 × .45 objective lens) using a dual-bandpass filter designed to simultaneously detect fluorescein and Texas Red dyes. In live nonapoptotic cells, JC-1 accumulates as aggregates in the mitochondrial membrane which stain red. In apoptotic cells, JC-1 exists in the monomeric form because of the low mitochondrial membrane potential, staining the cytosol green.