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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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Phosphorylation of p53 on serine 15 in response to SAC inhibition.A, B. Abolition of SAC by Chk1 depletion. Tetraploid cells were either transfected with a scrambled control siRNA (SCR) or with a Chk1-specific siRNA and then subjected 48 h later to immunofluorescence to determine the centromeric location of BubR1 or Bub1 (as in Fig. 1B) as a sign of SAC activation. Note that depletion of Chk1 fully abolished SAC. C. p53 activation by Chk1 inhibition in diploid versus tetraploid cells. Cells treated with 15 µM cisplatin, a Chk1-depleting siRNA (or its control SCR) or the Chk1 inhibitor UCN-01 were stained 48 h later to detect p53 phosphorylation on serine 15. Cisplatin treatment was used as an internal positive control. D. Efficacy of siRNAs directed against SAC proteins, as determined by immunoblot, 48 hours after transfection. E. p53 phosphorylation on serine 15 after depletion of SAC proteins. (X±SEM, n = 3).
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pone-0001337-g003: Phosphorylation of p53 on serine 15 in response to SAC inhibition.A, B. Abolition of SAC by Chk1 depletion. Tetraploid cells were either transfected with a scrambled control siRNA (SCR) or with a Chk1-specific siRNA and then subjected 48 h later to immunofluorescence to determine the centromeric location of BubR1 or Bub1 (as in Fig. 1B) as a sign of SAC activation. Note that depletion of Chk1 fully abolished SAC. C. p53 activation by Chk1 inhibition in diploid versus tetraploid cells. Cells treated with 15 µM cisplatin, a Chk1-depleting siRNA (or its control SCR) or the Chk1 inhibitor UCN-01 were stained 48 h later to detect p53 phosphorylation on serine 15. Cisplatin treatment was used as an internal positive control. D. Efficacy of siRNAs directed against SAC proteins, as determined by immunoblot, 48 hours after transfection. E. p53 phosphorylation on serine 15 after depletion of SAC proteins. (X±SEM, n = 3).

Mentions: The knockdown of Chk1 resulted in a complete abolition of SAC resulting in the failure to recruit BubR1 and Bub1 to kinetochores during the prometaphase and aberrant metaphases for diploid and tetraploid cells (Fig. 3A,B for tetraploid cells). Indeed, after Chk1 knockdown less than 30% of prometaphases and aberrant metaphases exhibited the localization of Bub or BubR1 to kinetochores, indicating abolition of SAC in more than 70% of the cells. The percentage of SAC inhibition, as detectable among aberrant metaphases, was similar in diploid and tetraploid cells (although the number of aberrant metaphases is much higher in tetraploid cells, see Fig. 1). Small interfering RNA (siRNA)-mediated depletion of Chk1 or its pharmacological inhibition by UCN-01 (7-hydroxystaurosporine) also resulted in the activating phosphorylation of p53, more frequently in tetraploid than in diploid cells (Fig. 3C). Similarly, the knockdown of the essential SAC proteins Bub1, BubR1, Mad2 or Aurora B induced the phosphorylation of p53 on serine 15, more so in tetraploid than in diploid cells (Fig. 3D,E). Taken together, these data indicate that Chk1 inhibition abolishes SAC, and that SAC inhibition exacerbates mitotic defects coupled to p53 activation in tetraploid tumor cells.


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)

Phosphorylation of p53 on serine 15 in response to SAC inhibition.A, B. Abolition of SAC by Chk1 depletion. Tetraploid cells were either transfected with a scrambled control siRNA (SCR) or with a Chk1-specific siRNA and then subjected 48 h later to immunofluorescence to determine the centromeric location of BubR1 or Bub1 (as in Fig. 1B) as a sign of SAC activation. Note that depletion of Chk1 fully abolished SAC. C. p53 activation by Chk1 inhibition in diploid versus tetraploid cells. Cells treated with 15 µM cisplatin, a Chk1-depleting siRNA (or its control SCR) or the Chk1 inhibitor UCN-01 were stained 48 h later to detect p53 phosphorylation on serine 15. Cisplatin treatment was used as an internal positive control. D. Efficacy of siRNAs directed against SAC proteins, as determined by immunoblot, 48 hours after transfection. E. p53 phosphorylation on serine 15 after depletion of SAC proteins. (X±SEM, n = 3).
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pone-0001337-g003: Phosphorylation of p53 on serine 15 in response to SAC inhibition.A, B. Abolition of SAC by Chk1 depletion. Tetraploid cells were either transfected with a scrambled control siRNA (SCR) or with a Chk1-specific siRNA and then subjected 48 h later to immunofluorescence to determine the centromeric location of BubR1 or Bub1 (as in Fig. 1B) as a sign of SAC activation. Note that depletion of Chk1 fully abolished SAC. C. p53 activation by Chk1 inhibition in diploid versus tetraploid cells. Cells treated with 15 µM cisplatin, a Chk1-depleting siRNA (or its control SCR) or the Chk1 inhibitor UCN-01 were stained 48 h later to detect p53 phosphorylation on serine 15. Cisplatin treatment was used as an internal positive control. D. Efficacy of siRNAs directed against SAC proteins, as determined by immunoblot, 48 hours after transfection. E. p53 phosphorylation on serine 15 after depletion of SAC proteins. (X±SEM, n = 3).
Mentions: The knockdown of Chk1 resulted in a complete abolition of SAC resulting in the failure to recruit BubR1 and Bub1 to kinetochores during the prometaphase and aberrant metaphases for diploid and tetraploid cells (Fig. 3A,B for tetraploid cells). Indeed, after Chk1 knockdown less than 30% of prometaphases and aberrant metaphases exhibited the localization of Bub or BubR1 to kinetochores, indicating abolition of SAC in more than 70% of the cells. The percentage of SAC inhibition, as detectable among aberrant metaphases, was similar in diploid and tetraploid cells (although the number of aberrant metaphases is much higher in tetraploid cells, see Fig. 1). Small interfering RNA (siRNA)-mediated depletion of Chk1 or its pharmacological inhibition by UCN-01 (7-hydroxystaurosporine) also resulted in the activating phosphorylation of p53, more frequently in tetraploid than in diploid cells (Fig. 3C). Similarly, the knockdown of the essential SAC proteins Bub1, BubR1, Mad2 or Aurora B induced the phosphorylation of p53 on serine 15, more so in tetraploid than in diploid cells (Fig. 3D,E). Taken together, these data indicate that Chk1 inhibition abolishes SAC, and that SAC inhibition exacerbates mitotic defects coupled to p53 activation in tetraploid tumor cells.

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