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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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Effect of Chk1 depletion on mitosis and p53 activation in tetraploid cells.A. Efficient Chk1 depletion after transfection with a specific siRNA. Tetraploid RKO cells were transfected with a Chk1-specific siRNA (Chk1a) or scrambled (SCR) control siRNA and the abundance of Chk1 was determined by immunobloting. GAPDH was detected to control equal loading. B–D. Videomicroscopic analyses of tetraploid cell division. Tetraploid RKO cells stably transfected with a histone H2B-GFP fusion construct (green fluorescence marking chromosomes) were transfected with a Chk1-specific siRNA (Chk1a) and monitored 48 h later for abnormal mitosis. Representative sequences of pictures in B illustrate normal mitosis (0), delayed mitotic exit resulting in apparently normal division (I), apoptotic disintegration of daughter cells after cytokinesis (II), abnormal metaphase plates leading to formation of binucleated cells (III), multipolar mitoses (IV) and apoptosis during or shortly after the metaphase (V). The frequency of mitotic aberrations observed in cells that were transfected with a control siRNA (SCR) or the Chk1-depleting siRNA was calculated after having monitored 150 to 200 mitoses (C) and the length of mitosis was computed (D). E, F. Premature mitosis in Chk1-depleted tetraploid cells. Thirty-six hours after transfection with control siRNA (SCR) or Chk1-depleting siRNA, diploid or tetraploid cells were stained to measure DNA content (with DAPI) and histone H3 phosphorylation, followed by cytofluorometric analysis. The rectangle in E marks the population of cells showing the phosphorylation of mitotic histone H3. Numbers refer to percentage of diploid or tetraploid cells with less than 4N or 8N, respectively, that manifest premature mitosis. The quantification is represented in F. (X±SEM, n = 3).
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pone-0001337-g002: Effect of Chk1 depletion on mitosis and p53 activation in tetraploid cells.A. Efficient Chk1 depletion after transfection with a specific siRNA. Tetraploid RKO cells were transfected with a Chk1-specific siRNA (Chk1a) or scrambled (SCR) control siRNA and the abundance of Chk1 was determined by immunobloting. GAPDH was detected to control equal loading. B–D. Videomicroscopic analyses of tetraploid cell division. Tetraploid RKO cells stably transfected with a histone H2B-GFP fusion construct (green fluorescence marking chromosomes) were transfected with a Chk1-specific siRNA (Chk1a) and monitored 48 h later for abnormal mitosis. Representative sequences of pictures in B illustrate normal mitosis (0), delayed mitotic exit resulting in apparently normal division (I), apoptotic disintegration of daughter cells after cytokinesis (II), abnormal metaphase plates leading to formation of binucleated cells (III), multipolar mitoses (IV) and apoptosis during or shortly after the metaphase (V). The frequency of mitotic aberrations observed in cells that were transfected with a control siRNA (SCR) or the Chk1-depleting siRNA was calculated after having monitored 150 to 200 mitoses (C) and the length of mitosis was computed (D). E, F. Premature mitosis in Chk1-depleted tetraploid cells. Thirty-six hours after transfection with control siRNA (SCR) or Chk1-depleting siRNA, diploid or tetraploid cells were stained to measure DNA content (with DAPI) and histone H3 phosphorylation, followed by cytofluorometric analysis. The rectangle in E marks the population of cells showing the phosphorylation of mitotic histone H3. Numbers refer to percentage of diploid or tetraploid cells with less than 4N or 8N, respectively, that manifest premature mitosis. The quantification is represented in F. (X±SEM, n = 3).

Mentions: In view of the apparent difficulty of tetraploid cells to successfully complete mitosis, we wondered whether these cells might rely more heavily on the cell cycle checkpoint kinase Chk1 than diploid cells. Transient transfection with a Chk1-specific small interfering RNA (siRNA) (Fig. 2A) caused a dramatic increase in aberrant mitoses of tetraploid cells (one third of all mitoses), resulting in delayed mitotic exit, apoptotic disintegration of both daughter cells after cytokinesis, formation of binucleated cells, multipolar mitoses, or apoptosis during or shortly after the metaphase (Fig. 2B,C,D and videos S3, S4, S5, S6, S7, S8). This correlated with premature mitoses, detectable as events in which the phosphorylation of histone H3 by mitotic kinases occurs in cells that have not duplicated their genome, and hence have less than 8N DNA content (Fig. 2E,F). The percentage of premature mitoses was not affected by preincubation with the pancaspase inhibitor Z-VAD.fmk (not shown). The increase in frequency of aberrant or premature mitoses induced by Chk1 inhibition was more pronounced among tetraploid than among diploid cells (Fig. 2A–F).


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)

Effect of Chk1 depletion on mitosis and p53 activation in tetraploid cells.A. Efficient Chk1 depletion after transfection with a specific siRNA. Tetraploid RKO cells were transfected with a Chk1-specific siRNA (Chk1a) or scrambled (SCR) control siRNA and the abundance of Chk1 was determined by immunobloting. GAPDH was detected to control equal loading. B–D. Videomicroscopic analyses of tetraploid cell division. Tetraploid RKO cells stably transfected with a histone H2B-GFP fusion construct (green fluorescence marking chromosomes) were transfected with a Chk1-specific siRNA (Chk1a) and monitored 48 h later for abnormal mitosis. Representative sequences of pictures in B illustrate normal mitosis (0), delayed mitotic exit resulting in apparently normal division (I), apoptotic disintegration of daughter cells after cytokinesis (II), abnormal metaphase plates leading to formation of binucleated cells (III), multipolar mitoses (IV) and apoptosis during or shortly after the metaphase (V). The frequency of mitotic aberrations observed in cells that were transfected with a control siRNA (SCR) or the Chk1-depleting siRNA was calculated after having monitored 150 to 200 mitoses (C) and the length of mitosis was computed (D). E, F. Premature mitosis in Chk1-depleted tetraploid cells. Thirty-six hours after transfection with control siRNA (SCR) or Chk1-depleting siRNA, diploid or tetraploid cells were stained to measure DNA content (with DAPI) and histone H3 phosphorylation, followed by cytofluorometric analysis. The rectangle in E marks the population of cells showing the phosphorylation of mitotic histone H3. Numbers refer to percentage of diploid or tetraploid cells with less than 4N or 8N, respectively, that manifest premature mitosis. The quantification is represented in F. (X±SEM, n = 3).
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pone-0001337-g002: Effect of Chk1 depletion on mitosis and p53 activation in tetraploid cells.A. Efficient Chk1 depletion after transfection with a specific siRNA. Tetraploid RKO cells were transfected with a Chk1-specific siRNA (Chk1a) or scrambled (SCR) control siRNA and the abundance of Chk1 was determined by immunobloting. GAPDH was detected to control equal loading. B–D. Videomicroscopic analyses of tetraploid cell division. Tetraploid RKO cells stably transfected with a histone H2B-GFP fusion construct (green fluorescence marking chromosomes) were transfected with a Chk1-specific siRNA (Chk1a) and monitored 48 h later for abnormal mitosis. Representative sequences of pictures in B illustrate normal mitosis (0), delayed mitotic exit resulting in apparently normal division (I), apoptotic disintegration of daughter cells after cytokinesis (II), abnormal metaphase plates leading to formation of binucleated cells (III), multipolar mitoses (IV) and apoptosis during or shortly after the metaphase (V). The frequency of mitotic aberrations observed in cells that were transfected with a control siRNA (SCR) or the Chk1-depleting siRNA was calculated after having monitored 150 to 200 mitoses (C) and the length of mitosis was computed (D). E, F. Premature mitosis in Chk1-depleted tetraploid cells. Thirty-six hours after transfection with control siRNA (SCR) or Chk1-depleting siRNA, diploid or tetraploid cells were stained to measure DNA content (with DAPI) and histone H3 phosphorylation, followed by cytofluorometric analysis. The rectangle in E marks the population of cells showing the phosphorylation of mitotic histone H3. Numbers refer to percentage of diploid or tetraploid cells with less than 4N or 8N, respectively, that manifest premature mitosis. The quantification is represented in F. (X±SEM, n = 3).
Mentions: In view of the apparent difficulty of tetraploid cells to successfully complete mitosis, we wondered whether these cells might rely more heavily on the cell cycle checkpoint kinase Chk1 than diploid cells. Transient transfection with a Chk1-specific small interfering RNA (siRNA) (Fig. 2A) caused a dramatic increase in aberrant mitoses of tetraploid cells (one third of all mitoses), resulting in delayed mitotic exit, apoptotic disintegration of both daughter cells after cytokinesis, formation of binucleated cells, multipolar mitoses, or apoptosis during or shortly after the metaphase (Fig. 2B,C,D and videos S3, S4, S5, S6, S7, S8). This correlated with premature mitoses, detectable as events in which the phosphorylation of histone H3 by mitotic kinases occurs in cells that have not duplicated their genome, and hence have less than 8N DNA content (Fig. 2E,F). The percentage of premature mitoses was not affected by preincubation with the pancaspase inhibitor Z-VAD.fmk (not shown). The increase in frequency of aberrant or premature mitoses induced by Chk1 inhibition was more pronounced among tetraploid than among diploid cells (Fig. 2A–F).

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