Inhibition of Chk1 Kills Tetraploid Tumor Cells through a p53-Dependent Pathway
Figure 8
Involvement of p53 and Bcl-2 family proteins in apoptosis induction by Chk1 inhibition.
A. Effect of p53 knockdown. Diploid or tetraploid HCT116 cells were transfected with control siRNA (SCR), a p53-specific siRNA, and/or Chk1-depleting siRNA and cultured in the presence of absence of cisplatin during the last 24 hours of the 72-hour experiment. Then, cells were stained with DiOC6(3)/PI. B,C. Effect of p53 knockout. Tetraploid HCT116 cells generated on a wild type (WT) or p53 knockout (p53−/−) background were treated with the indicated combination of Chk1-depleting siRNA and/or cisplatin (B) or were depleted from SAC proteins (C), followed by determination the frequency of dying (DiOC6(3)low PI−) or dead (DiOC6(3)low PI+). Results (X±SEM, n = 3) are representative of three independent determinations. Asterisks indicate a significant protection by p53 deletion. D. Contribution of p53 target genes to Chk1 depletion-induced apoptosis. Tetraploid HCT116 cells were transfected with the indicated siRNAs and the frequency of cell death was determined by DiOC6(3)/PI staining. E. Contribution of p53 and pro-apoptotic proteins of Bcl-2 family to the death of HCT116 cells induced by Chk1 depletion. Tetraploid HCT116 cells were subjected to the siRNA-mediated downregulation of Chk1 alone or together with the indicated gene products, followed by measurement of cell viability with a tetrazolium reduction assay. Negative values indicate sensitization to the cytotoxicity of Chk1 depletion while positive values indicate protective effects (X±SEM, n = 3). F. Effect of Bax knockout. Tetraploid HCT116 cells generated on a wild type (WT) or Bax knockout (Bax−/−) background were subjected to Chk1 inhibition, followed by determination of the frequency of dead and dying cells 48 h later. Results (X±SEM) are representative of three independent determinations.