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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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Apoptosis induction by Chk1 or SAC inhibition.A. Chk1 inhibition by a dominant-negative (DN) Chk1 mutant. Diploid or tetraploid HCT116 cells were transfected with vector only (Co) or vectors encoding wild type (WT) or DN Chk1 or Chk2, together with a DsRed-encoding construct. 48 h later, cells were stained with DiOC6(3) and the frequency of DiOC6(3)low cells was determined among the transfected (dsRed+) population. B–F. Chk1 inhibition by pharmacological agents. HCT116 cells were cultured for 48 h in the presence of the indicated concentrations of UCN-01 (B,D) or SD1825 (C,D) and the frequency of dead and dying cells was measured by co-staining with DiOC6(3) and PI (as in Fig. 3A). Alternatively, cells cultured on polylysin slides were stained for the simultaneous immunofluorescence detection of mitochondrial cytochrome c (Cyt c) release and caspase-3 activation (Casp-3a). Representative microfluorographs are shown in D and the frequency of cells showing diffuse Cyt c staining and caspase-3 activation are scored in E. Finally, phosphatidylserine exposure was measured in diploid and tetraploid RKO cells by annexin V-FITC staining (F). Asterisks denote significant (p<0.01) effects of Chk1 inhibition. G, H. Preferential mortality of tetraploid cells subjected to SAC inhibition. Diploid or tetraploid cells were depleted from the indicated SAC proteins. Forty-eight hours later, apoptotic events were scored by measuring the frequency of ΔΨm (with DiOC6(3)) and viability (with PI) (G) or sub-G1 cells (H). Columns represent the average of three independent experiments ±SEM.
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pone-0001337-g005: Apoptosis induction by Chk1 or SAC inhibition.A. Chk1 inhibition by a dominant-negative (DN) Chk1 mutant. Diploid or tetraploid HCT116 cells were transfected with vector only (Co) or vectors encoding wild type (WT) or DN Chk1 or Chk2, together with a DsRed-encoding construct. 48 h later, cells were stained with DiOC6(3) and the frequency of DiOC6(3)low cells was determined among the transfected (dsRed+) population. B–F. Chk1 inhibition by pharmacological agents. HCT116 cells were cultured for 48 h in the presence of the indicated concentrations of UCN-01 (B,D) or SD1825 (C,D) and the frequency of dead and dying cells was measured by co-staining with DiOC6(3) and PI (as in Fig. 3A). Alternatively, cells cultured on polylysin slides were stained for the simultaneous immunofluorescence detection of mitochondrial cytochrome c (Cyt c) release and caspase-3 activation (Casp-3a). Representative microfluorographs are shown in D and the frequency of cells showing diffuse Cyt c staining and caspase-3 activation are scored in E. Finally, phosphatidylserine exposure was measured in diploid and tetraploid RKO cells by annexin V-FITC staining (F). Asterisks denote significant (p<0.01) effects of Chk1 inhibition. G, H. Preferential mortality of tetraploid cells subjected to SAC inhibition. Diploid or tetraploid cells were depleted from the indicated SAC proteins. Forty-eight hours later, apoptotic events were scored by measuring the frequency of ΔΨm (with DiOC6(3)) and viability (with PI) (G) or sub-G1 cells (H). Columns represent the average of three independent experiments ±SEM.

Mentions: Since we worried about possible off-target effects of the Chk1-specific siRNAs, we repeated the experiments using alternative methods of Chk1 inhibition. Transfection with a dominant-negative (DN) Chk1 mutant (Fig. 5A) or inhibition of Chk1 with UCN-01 or SD1825 (Fig. 5B–F) resulted in an enhanced mortality of tetraploid HCT116 cells, with little or no effects on diploid cell. Efficient cell death of tetraploid cells was confirmed in RKO cells after pharmacological inhibition of Chk1 (Fig. S2 B). Moreover, alternative strategies to subvert SAC, by knockdown of Bub1, BubR1, Mad2 or Aurora B caused cell death much more efficiently in tetraploid than in diploid cancer cells (Fig. 5G, H).


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)

Apoptosis induction by Chk1 or SAC inhibition.A. Chk1 inhibition by a dominant-negative (DN) Chk1 mutant. Diploid or tetraploid HCT116 cells were transfected with vector only (Co) or vectors encoding wild type (WT) or DN Chk1 or Chk2, together with a DsRed-encoding construct. 48 h later, cells were stained with DiOC6(3) and the frequency of DiOC6(3)low cells was determined among the transfected (dsRed+) population. B–F. Chk1 inhibition by pharmacological agents. HCT116 cells were cultured for 48 h in the presence of the indicated concentrations of UCN-01 (B,D) or SD1825 (C,D) and the frequency of dead and dying cells was measured by co-staining with DiOC6(3) and PI (as in Fig. 3A). Alternatively, cells cultured on polylysin slides were stained for the simultaneous immunofluorescence detection of mitochondrial cytochrome c (Cyt c) release and caspase-3 activation (Casp-3a). Representative microfluorographs are shown in D and the frequency of cells showing diffuse Cyt c staining and caspase-3 activation are scored in E. Finally, phosphatidylserine exposure was measured in diploid and tetraploid RKO cells by annexin V-FITC staining (F). Asterisks denote significant (p<0.01) effects of Chk1 inhibition. G, H. Preferential mortality of tetraploid cells subjected to SAC inhibition. Diploid or tetraploid cells were depleted from the indicated SAC proteins. Forty-eight hours later, apoptotic events were scored by measuring the frequency of ΔΨm (with DiOC6(3)) and viability (with PI) (G) or sub-G1 cells (H). Columns represent the average of three independent experiments ±SEM.
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pone-0001337-g005: Apoptosis induction by Chk1 or SAC inhibition.A. Chk1 inhibition by a dominant-negative (DN) Chk1 mutant. Diploid or tetraploid HCT116 cells were transfected with vector only (Co) or vectors encoding wild type (WT) or DN Chk1 or Chk2, together with a DsRed-encoding construct. 48 h later, cells were stained with DiOC6(3) and the frequency of DiOC6(3)low cells was determined among the transfected (dsRed+) population. B–F. Chk1 inhibition by pharmacological agents. HCT116 cells were cultured for 48 h in the presence of the indicated concentrations of UCN-01 (B,D) or SD1825 (C,D) and the frequency of dead and dying cells was measured by co-staining with DiOC6(3) and PI (as in Fig. 3A). Alternatively, cells cultured on polylysin slides were stained for the simultaneous immunofluorescence detection of mitochondrial cytochrome c (Cyt c) release and caspase-3 activation (Casp-3a). Representative microfluorographs are shown in D and the frequency of cells showing diffuse Cyt c staining and caspase-3 activation are scored in E. Finally, phosphatidylserine exposure was measured in diploid and tetraploid RKO cells by annexin V-FITC staining (F). Asterisks denote significant (p<0.01) effects of Chk1 inhibition. G, H. Preferential mortality of tetraploid cells subjected to SAC inhibition. Diploid or tetraploid cells were depleted from the indicated SAC proteins. Forty-eight hours later, apoptotic events were scored by measuring the frequency of ΔΨm (with DiOC6(3)) and viability (with PI) (G) or sub-G1 cells (H). Columns represent the average of three independent experiments ±SEM.
Mentions: Since we worried about possible off-target effects of the Chk1-specific siRNAs, we repeated the experiments using alternative methods of Chk1 inhibition. Transfection with a dominant-negative (DN) Chk1 mutant (Fig. 5A) or inhibition of Chk1 with UCN-01 or SD1825 (Fig. 5B–F) resulted in an enhanced mortality of tetraploid HCT116 cells, with little or no effects on diploid cell. Efficient cell death of tetraploid cells was confirmed in RKO cells after pharmacological inhibition of Chk1 (Fig. S2 B). Moreover, alternative strategies to subvert SAC, by knockdown of Bub1, BubR1, Mad2 or Aurora B caused cell death much more efficiently in tetraploid than in diploid cancer cells (Fig. 5G, H).

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