JAK2R564Q-expressing cells exhibit increased cell growth with a pronounced antiapoptotic effect and a mild proliferative effect. (A) 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide assay to measure proliferation in the 4 mutant JAK2 cell lines with increasing TPO concentration under starved conditions. Each data point is expressed as a percentage of proliferation stimulated by a maximal dose of murine interleukin 3 and represents 6 repeats. All mutant JAK2-expressing cells show significantly increased proliferation compared with WTJAK2-expressing cells in the absence of, and at all concentrations of, TPO. **P < .01; ***P < .001. (B) Viable cell counts every 24 hours under starved conditions, for a total of 72 hours. Data shown are from 3 independent repeats. JAK2V617F and double-mutant cells are able to proliferate in the absence of cytokine and show a significant increase in cell number, compared with 0 hours, at both 48 and 72 hours (*P < .05). The number of viable WTJAK2-expressing cells, compared with starting number, was significantly decreased at all points (*P < .05; **P < .01). No significant difference was seen in the number of viable JAK2R564Q cells, compared with the starting number, for the 72-hour period. (C) Apoptosis measured by Annexin V staining of cells under starved conditions for 72 hours. By 48 hours, the number of apoptotic cells in the mutant JAK2 cell lines was significantly less than the number of WTJAK2-expressing apoptotic cells. (D) Western blot analysis over 36 hours of starvation demonstrates increased levels of uncleaved PARP in all 3 of the JAK2 mutants compared with WTJAK2. (E) BrdU assay to measure proliferation during 48 hours of starvation. Proliferation in both WTJAK2 and JAK2R564Q cells decreased during the 48-hour period, although proliferation in JAK2R564Q cells was significantly increased compared with WTJAK2 controls after 48 hours (*P < .05). Proliferation continued in the cell lines with the JAK2V617F mutation, however, and the percentage of BrdU-positive cells in these cell lines was significantly more than in the WTJAK2 controls after both 24 and 48 hours (***P < .001). (F) Western blot analysis during starved conditions showed an increase in p27/Kip1 protein levels during the starvation period in the WTJAK2 and JAK2R564Q cell lines. p27/Kip1 levels were much reduced in the JAK2V617F-expressing mutants, however. p21CIP/WAF1 levels remained fairly constant in each cell line throughout the starvation period.
