b Graph depicting the increasing levels of the sub-G1 fraction determined from their PI profile, according to the time of TdR treatment, for each cell line MIFF1, MIFF3, and MIFF4. apoptotic response was properly restored during reprogramming with mRNA, and that apoptosis is an important mechanism shared by hiPS and hES cells to maintain their genomic integrity when a replication stress occurs. test was used to evaluate the statistical differences between control and experimental groups. alpha-fetoprotein, undifferentiated cells, embryoid body, fetal bovine serum, human embryonic stem, mRNA-induced foreskin fibroblast, paired box 6, stage-specific embryonic antigen The apoptotic response following DNA replication stress was investigated in three iPS cell lines (MIFF1, MIFF3, and MIFF4). Activation of the S-phase checkpoint was induced by adding excess TdR to the culture environment. The propidium iodide (PI) profile of MIFF3 and MIFF4 showed a significant increase in the sub-G1 population after 16?hours of TdR, and all three cell lines showed a significant increase after 24?hours of TdR treatment (Table?1, Fig.?2a, b). Concomitant with this increase in the sub-G1 population, the number of cells in the G1, S, and G2 phases were reduced in all three iPS cell lines (Table?1, Fig.?2a). In MIFF3 cells, an increase in active caspase 3 expression and an IU1-47 increment in annexinV+/PI? cells in MIFF1 cells were both indicative of apoptotic cells (Fig.?2c, d). Similarly, Shef5N hES cells showed an increase in active caspase 3 expression after TdR treatment. These data suggest that iPS cells, like hES cells but unlike somatic tumor cells, undergo apoptosis after replication stress but do not sustain a cell cycle arrest. Table 1 Cell cycle distribution of iPS cells treated with thymidine 0.05, ** 0.001, *** 0.0001 induced pluripotent stem, mRNA-induced foreskin fibroblast Open in a separate window Fig. 2 hiPS cells undergo apoptosis and no cell cycle arrest in response to replication inhibitor. a hiPS cell lines MIFF1, MIFF3, and MIFF4 show an increase in the sub-G1 fraction after TdR treatment as reflected by stacked PI profiles obtained by flow cytometry at different time points. hiPS cells show an early accumulation in the S phase but fail to reach G2 phase. b Graph depicting the increasing levels of the sub-G1 fraction determined from their PI profile, according to the time of TdR treatment, for each cell line MIFF1, MIFF3, and MIFF4. c Western blots showing an increased activation of caspase 3 protein level following TdR treatment. Beta-actin is presented as the control. Shef5N, a normal hES cell line, also show this increase in caspase 3 activation, while the somatic cell line HCT116 does not, in response to TdR. d Increased proportions annexinV+/PIC MIFF1 cells, a marker of apoptosis, after TdR treatment. * 0.05, ** 0.001, *** 0.0001. mRNA-induced foreskin fibroblast, propidium iodide, thymidine Next, we analyzed the activation status of the proteins CHK1, histone 2AX (H2AX), and replication protein A (RPA), known to be signaling through the ATR pathway and S-phase checkpoint . All three iPS cell lines displayed reduced levels of pSer345-CHK1 following TdR, compared with the levels observed in the HCT116 control cell line (Fig.?3aCc). The low levels of pSer345-CHK1 were comparable with those observed in Shef5N (Fig.?3a, b). Despite the absence of CHK1 activation, the total CHK1 protein was expressed at constant levels after TdR treatment. Open in a separate window Fig. 3 Activation of DNA damage response pathways in iPS cell lines in response to TdR. a MIFF1, b MIFF3, and c MIFF4 iPS cell lines show a reduced CHK1 activation in western blots, comparable with IU1-47 what is observed Rabbit Polyclonal to Cytochrome P450 2A13 in the Shef5N normal hES cell line (b) and in contrast to the strong activation observed in HCT116 cells (b). In all iPS cell lines, there is a reduced H2AX phosphorylation compared with that observed in HCT116 treated with the CHK1 inhibitor G?6976 (b), indicating that DNA IU1-47 damage is not enhanced in these cell lines in response to replication inhibitor TdR, despite the absence of a clear CHK1 activation. In addition, RPA is not hyperphosphorylated in any iPS cell lines, suggesting that ssDNA formation is suppressed. In contrast, HCT116 cells treated with the CHK1 inhibitor G?6976 (b) show a marked hyperphosphorylation of RPA. d MIFF1 and f MIFF4 activate ATM by phosphorylation of Ser1981 after TdR treatment. This is accompanied by the phosphorylation.