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Inhibition of Chk1 kills tetraploid tumor cells through a p53-dependent pathway.

Vitale I, Galluzzi L, Vivet S, Nanty L, Dessen P, Senovilla L, Olaussen KA, Lazar V, Prudhomme M, Golsteyn RM, Castedo M, Kroemer G - PLoS ONE (2007)

Bottom Line: Depletion of checkpoint kinase-1 (Chk1) by siRNAs, transfection with dominant-negative Chk1 mutants or pharmacological Chk1 inhibition killed tetraploid colon cancer cells yet had minor effects on their diploid counterparts.Chk1 inhibition activated p53-regulated transcripts including Puma/BBC3 in tetraploid but not in diploid tumor cells.Altogether, our results demonstrate that, in tetraploid tumor cells, the inhibition of Chk1 sequentially triggers aberrant mitosis, p53 activation and Puma/BBC3-dependent mitochondrial apoptosis.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U848, Cancer and Immunity, Villejuif, France.

ABSTRACT
Tetraploidy constitutes an adaptation to stress and an intermediate step between euploidy and aneuploidy in oncogenesis. Tetraploid cells are particularly resistant against genotoxic stress including radiotherapy and chemotherapy. Here, we designed a strategy to preferentially kill tetraploid tumor cells. Depletion of checkpoint kinase-1 (Chk1) by siRNAs, transfection with dominant-negative Chk1 mutants or pharmacological Chk1 inhibition killed tetraploid colon cancer cells yet had minor effects on their diploid counterparts. Chk1 inhibition abolished the spindle assembly checkpoint and caused premature and abnormal mitoses that led to p53 activation and cell death at a higher frequency in tetraploid than in diploid cells. Similarly, abolition of the spindle checkpoint by knockdown of Bub1, BubR1 or Mad2 induced p53-dependent apoptosis of tetraploid cells. Chk1 inhibition reversed the cisplatin resistance of tetraploid cells in vitro and in vivo, in xenografted human cancers. Chk1 inhibition activated p53-regulated transcripts including Puma/BBC3 in tetraploid but not in diploid tumor cells. Altogether, our results demonstrate that, in tetraploid tumor cells, the inhibition of Chk1 sequentially triggers aberrant mitosis, p53 activation and Puma/BBC3-dependent mitochondrial apoptosis.

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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.
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pone-0001337-g008: 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.

Mentions: Close inspection of the microarray data led us to the discovery that Chk1 inhibition caused the modulation of a particularly elevated percentage (12.3%) of p53 target genes in tetraploid cells (18 p53 target genes among a total of 146 Chk1-modulated genes) but not in diploid cells (none among 14 genes). This percentage is higher than that observed among the set of genes modulated by cisplatin (7.6%) or cisplatin plus Chk1 inhibitor (7.0%) in both diploid and tetraploid cells (Fig. 7B, Tables S1). These results corroborate the observation that Chk1 inhibition causes the activating phosphorylation of p53 (Fig. 3C). To clarify whether the transcription of p53 target genes is an epiphenomenon or if it is directly responsible for cell killing by Chk1 inhibition, we depleted p53 with a specific siRNA. p53 knockdown strongly reduced the cytotoxic effect of Chk1 depletion (Fig. 8A). Similarly, tetraploid derivatives of p53−/− HCT116 cells, generated as previously described [8], died significantly less than the p53 proficient tetraploid cells in response to the knockdown of Chk1 (Fig. 8B) or that of Bub1, BubR1, Mad2 or AuroraB (Fig. 8C). In each case, the inhibition of p53 strongly attenuated the cytotoxic effects of cisplatin and/or SAC inhibition.


Inhibition of Chk1 kills tetraploid tumor cells through a p53-dependent pathway.

Vitale I, Galluzzi L, Vivet S, Nanty L, Dessen P, Senovilla L, Olaussen KA, Lazar V, Prudhomme M, Golsteyn RM, Castedo M, Kroemer G - PLoS ONE (2007)

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.
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pone-0001337-g008: 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.
Mentions: Close inspection of the microarray data led us to the discovery that Chk1 inhibition caused the modulation of a particularly elevated percentage (12.3%) of p53 target genes in tetraploid cells (18 p53 target genes among a total of 146 Chk1-modulated genes) but not in diploid cells (none among 14 genes). This percentage is higher than that observed among the set of genes modulated by cisplatin (7.6%) or cisplatin plus Chk1 inhibitor (7.0%) in both diploid and tetraploid cells (Fig. 7B, Tables S1). These results corroborate the observation that Chk1 inhibition causes the activating phosphorylation of p53 (Fig. 3C). To clarify whether the transcription of p53 target genes is an epiphenomenon or if it is directly responsible for cell killing by Chk1 inhibition, we depleted p53 with a specific siRNA. p53 knockdown strongly reduced the cytotoxic effect of Chk1 depletion (Fig. 8A). Similarly, tetraploid derivatives of p53−/− HCT116 cells, generated as previously described [8], died significantly less than the p53 proficient tetraploid cells in response to the knockdown of Chk1 (Fig. 8B) or that of Bub1, BubR1, Mad2 or AuroraB (Fig. 8C). In each case, the inhibition of p53 strongly attenuated the cytotoxic effects of cisplatin and/or SAC inhibition.

Bottom Line: Depletion of checkpoint kinase-1 (Chk1) by siRNAs, transfection with dominant-negative Chk1 mutants or pharmacological Chk1 inhibition killed tetraploid colon cancer cells yet had minor effects on their diploid counterparts.Chk1 inhibition activated p53-regulated transcripts including Puma/BBC3 in tetraploid but not in diploid tumor cells.Altogether, our results demonstrate that, in tetraploid tumor cells, the inhibition of Chk1 sequentially triggers aberrant mitosis, p53 activation and Puma/BBC3-dependent mitochondrial apoptosis.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U848, Cancer and Immunity, Villejuif, France.

ABSTRACT
Tetraploidy constitutes an adaptation to stress and an intermediate step between euploidy and aneuploidy in oncogenesis. Tetraploid cells are particularly resistant against genotoxic stress including radiotherapy and chemotherapy. Here, we designed a strategy to preferentially kill tetraploid tumor cells. Depletion of checkpoint kinase-1 (Chk1) by siRNAs, transfection with dominant-negative Chk1 mutants or pharmacological Chk1 inhibition killed tetraploid colon cancer cells yet had minor effects on their diploid counterparts. Chk1 inhibition abolished the spindle assembly checkpoint and caused premature and abnormal mitoses that led to p53 activation and cell death at a higher frequency in tetraploid than in diploid cells. Similarly, abolition of the spindle checkpoint by knockdown of Bub1, BubR1 or Mad2 induced p53-dependent apoptosis of tetraploid cells. Chk1 inhibition reversed the cisplatin resistance of tetraploid cells in vitro and in vivo, in xenografted human cancers. Chk1 inhibition activated p53-regulated transcripts including Puma/BBC3 in tetraploid but not in diploid tumor cells. Altogether, our results demonstrate that, in tetraploid tumor cells, the inhibition of Chk1 sequentially triggers aberrant mitosis, p53 activation and Puma/BBC3-dependent mitochondrial apoptosis.

Show MeSH
Related in: MedlinePlus