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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 depletion of Chk1 in tetraploid HCT116 cells.A, B. Loss of the mitochondrial transmembrane potential (ΔΨm) and viability induced by Chk1 depletion. Diploid (D) or tetraploid (T) cells were transfected with either of two siRNAs specific for Chk1 (Chk1a, Chk1b) or Chk2 (Chk2a, Chk2b) and stained 48 h later to measure ΔΨm (with DiOC6(3)) and viability (with PI). Representative FACS pictograms are shown in A and data are quantified in B. C, D. Apoptotic DNA loss induced by Chk1 depletion. Cells were stained with DAPI to measure DNA content, 24, 48 or 72 h after transfection with Chk1-depleting siRNA. Data shown in C have been obtained 48 h after transfection. The numbers in C refer to the percentage of cells with a sub-G1 DNA content. Inserts in D demonstrate siRNA-mediated Chk1 and Chk2 depletion, as detected by immunoblots. * p<0.01 and ** p<0.001 as compared to untreated or control siRNA-transfected (SCR) controls. Data represent the mean of three independent experiments in triplicate. (X± SEM).
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pone-0001337-g004: Apoptosis induction by depletion of Chk1 in tetraploid HCT116 cells.A, B. Loss of the mitochondrial transmembrane potential (ΔΨm) and viability induced by Chk1 depletion. Diploid (D) or tetraploid (T) cells were transfected with either of two siRNAs specific for Chk1 (Chk1a, Chk1b) or Chk2 (Chk2a, Chk2b) and stained 48 h later to measure ΔΨm (with DiOC6(3)) and viability (with PI). Representative FACS pictograms are shown in A and data are quantified in B. C, D. Apoptotic DNA loss induced by Chk1 depletion. Cells were stained with DAPI to measure DNA content, 24, 48 or 72 h after transfection with Chk1-depleting siRNA. Data shown in C have been obtained 48 h after transfection. The numbers in C refer to the percentage of cells with a sub-G1 DNA content. Inserts in D demonstrate siRNA-mediated Chk1 and Chk2 depletion, as detected by immunoblots. * p<0.01 and ** p<0.001 as compared to untreated or control siRNA-transfected (SCR) controls. Data represent the mean of three independent experiments in triplicate. (X± SEM).

Mentions: In view of the mitotic or post-mitotic apoptosis induced by Chk1 depletion (Fig. 2A–C), we quantified the frequency of cell death induced by Chk1 inhibition by cytofluorometric methods. Simultaneous detection of dying cells (which exhibit a dissipated mitochondrial transmembrane potential, i.e. ΔΨm, and hence a reduced DiOC6(3) incorporation) and dead cells (which possess permeabilized plasma membranes and hence incorporate the vital dye propidium iodide, i.e. PI) revealed that depletion of Chk1 with either of two distinct siRNAs killed tetraploid HCT116 cells more efficiently than diploid cells. This effect was less pronounced when instead of Chk1, Chk2 was depleted (Fig. 4A,B). The downregulation of Chk1 was more efficient in inducing an apoptosis-associated DNA loss (sub-G1 DNA content, as determined by staining with DAPI) among tetraploid than among diploid cells (Fig. 4C,D). Similar results were obtained for another colon carcinoma cell line, namely RKO (Fig. S2 A) in thus far that Chk1 depletion was more efficient in killing tetraploid than diploid 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)

Apoptosis induction by depletion of Chk1 in tetraploid HCT116 cells.A, B. Loss of the mitochondrial transmembrane potential (ΔΨm) and viability induced by Chk1 depletion. Diploid (D) or tetraploid (T) cells were transfected with either of two siRNAs specific for Chk1 (Chk1a, Chk1b) or Chk2 (Chk2a, Chk2b) and stained 48 h later to measure ΔΨm (with DiOC6(3)) and viability (with PI). Representative FACS pictograms are shown in A and data are quantified in B. C, D. Apoptotic DNA loss induced by Chk1 depletion. Cells were stained with DAPI to measure DNA content, 24, 48 or 72 h after transfection with Chk1-depleting siRNA. Data shown in C have been obtained 48 h after transfection. The numbers in C refer to the percentage of cells with a sub-G1 DNA content. Inserts in D demonstrate siRNA-mediated Chk1 and Chk2 depletion, as detected by immunoblots. * p<0.01 and ** p<0.001 as compared to untreated or control siRNA-transfected (SCR) controls. Data represent the mean of three independent experiments in triplicate. (X± SEM).
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pone-0001337-g004: Apoptosis induction by depletion of Chk1 in tetraploid HCT116 cells.A, B. Loss of the mitochondrial transmembrane potential (ΔΨm) and viability induced by Chk1 depletion. Diploid (D) or tetraploid (T) cells were transfected with either of two siRNAs specific for Chk1 (Chk1a, Chk1b) or Chk2 (Chk2a, Chk2b) and stained 48 h later to measure ΔΨm (with DiOC6(3)) and viability (with PI). Representative FACS pictograms are shown in A and data are quantified in B. C, D. Apoptotic DNA loss induced by Chk1 depletion. Cells were stained with DAPI to measure DNA content, 24, 48 or 72 h after transfection with Chk1-depleting siRNA. Data shown in C have been obtained 48 h after transfection. The numbers in C refer to the percentage of cells with a sub-G1 DNA content. Inserts in D demonstrate siRNA-mediated Chk1 and Chk2 depletion, as detected by immunoblots. * p<0.01 and ** p<0.001 as compared to untreated or control siRNA-transfected (SCR) controls. Data represent the mean of three independent experiments in triplicate. (X± SEM).
Mentions: In view of the mitotic or post-mitotic apoptosis induced by Chk1 depletion (Fig. 2A–C), we quantified the frequency of cell death induced by Chk1 inhibition by cytofluorometric methods. Simultaneous detection of dying cells (which exhibit a dissipated mitochondrial transmembrane potential, i.e. ΔΨm, and hence a reduced DiOC6(3) incorporation) and dead cells (which possess permeabilized plasma membranes and hence incorporate the vital dye propidium iodide, i.e. PI) revealed that depletion of Chk1 with either of two distinct siRNAs killed tetraploid HCT116 cells more efficiently than diploid cells. This effect was less pronounced when instead of Chk1, Chk2 was depleted (Fig. 4A,B). The downregulation of Chk1 was more efficient in inducing an apoptosis-associated DNA loss (sub-G1 DNA content, as determined by staining with DAPI) among tetraploid than among diploid cells (Fig. 4C,D). Similar results were obtained for another colon carcinoma cell line, namely RKO (Fig. S2 A) in thus far that Chk1 depletion was more efficient in killing tetraploid than diploid 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