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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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Combined effects of Chk1 inhibition and cisplatin on tetraploid tumor cells in vitro and in vivo.A. Cytotoxic effects of Chk1 depletion in combination with cisplatin. Diploid (D) and tetraploid (T) HCT116 cells were knocked down for Chk1 (or transfected with control siRNA SCR) for 24 h and then cultured in absence or presence of cisplatin (20 µM) for further 48 h. Finally, the frequency of dying (DiOC6(3)low PI−) or dead (DiOC6(3)low PI+) cells was monitored by DiOC6(3)/PI staining. B. Cell killing by pharmacological inhibition of Chk1 plus cisplatin. Cells were cultured with cisplatin (20 µM), UCN-01 (500 nM) and/or SD1825 (500 nM) for 48 h, and dead and dying cells were determined as in A. C–E. Combined effects of Chk1 inhibition and cisplatin on tetraploid tumors established in vivo. Diploid or tetraploid HCT116 tumors were established in vivo and their growth was monitored continuously from day 18 (when tumors measured 125 to 250 mm3), when animals were injected with PBS alone (controls in C), cisplatin (D), UCN-01 (E), or with a combination of both (F). Asterisks indicate significant differences between diploid and tetraploid cells (p<0.05, unpaired Student t test). The results shown in Fig. 6C–E are representative for three different experiments.
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pone-0001337-g006: Combined effects of Chk1 inhibition and cisplatin on tetraploid tumor cells in vitro and in vivo.A. Cytotoxic effects of Chk1 depletion in combination with cisplatin. Diploid (D) and tetraploid (T) HCT116 cells were knocked down for Chk1 (or transfected with control siRNA SCR) for 24 h and then cultured in absence or presence of cisplatin (20 µM) for further 48 h. Finally, the frequency of dying (DiOC6(3)low PI−) or dead (DiOC6(3)low PI+) cells was monitored by DiOC6(3)/PI staining. B. Cell killing by pharmacological inhibition of Chk1 plus cisplatin. Cells were cultured with cisplatin (20 µM), UCN-01 (500 nM) and/or SD1825 (500 nM) for 48 h, and dead and dying cells were determined as in A. C–E. Combined effects of Chk1 inhibition and cisplatin on tetraploid tumors established in vivo. Diploid or tetraploid HCT116 tumors were established in vivo and their growth was monitored continuously from day 18 (when tumors measured 125 to 250 mm3), when animals were injected with PBS alone (controls in C), cisplatin (D), UCN-01 (E), or with a combination of both (F). Asterisks indicate significant differences between diploid and tetraploid cells (p<0.05, unpaired Student t test). The results shown in Fig. 6C–E are representative for three different experiments.

Mentions: As compared to their diploid counterparts, tetraploid cancer cells are relatively resistant against DNA damaging agents including cisplatin, both in vitro (Ref. [8], Fig. 6A,B for HCT116 and Fig. S2 C for RKO) and in vivo (Fig. 6C,D). Although there was no difference in the growth tumors originating from diploid versus tetraploid HCT116 cells inoculated into immunodeficient mice (Fig. 6C), tetraploid tumors responded far less to chemotherapy with cisplatin than diploid cancers (Fig. 6D). Chk1 depletion (Fig. 6A, Fig. S2 C) or inhibition (Fig. 6B) had an additive cytotoxic effect on tetraploid cells in vitro, in short-term assays. Treatment of tetraploid HCT116 tumors that had been established in xenotransplanted immunodeficient mice with a subtoxic dose of UCN-01 had no growth-inhibitory effect (Fig. 6E). However, the combination of cisplatin plus UCN-01 had a synergistic anti-cancer effect, which was particularly pronounced for tetraploid tumors (Fig. 6F). Based on these observations, we decided to explore the effect of cisplatin, Chk1 inhibition and the combination of both on the transcriptome of diploid and tetraploid colon cancer (HCT116) cells. The number of cisplatin-modulated genes showing a statistically relevant altered expression (p value<10−5) was higher among diploid than among tetraploid cells (Fig. 7A,B), correlating with the higher susceptibility of diploid cells to cisplatin-induced killing (Fig. 6D). In contrast, the number of genes modulated by Chk1 inhibition was significantly higher among tetraploid (152 genes) than among diploid cells (20 genes), with only six genes that were modulated in both diploid and tetraploid cells (Fig. 7A,B), again correlating with the enhanced killing of tetraploid cells by Chk1 inhibitors. The combination of cisplatin and Chk1 inhibition modified a large pool of transcripts that were common to tetraploid and diploid cells, with a higher number of tetraploid- than diploid- specific transcripts (Fig. 7A,B), in correlation with the particularly dramatic effects of the combined therapeutic regimen on tetraploid cells in vivo (Fig. 6F).


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)

Combined effects of Chk1 inhibition and cisplatin on tetraploid tumor cells in vitro and in vivo.A. Cytotoxic effects of Chk1 depletion in combination with cisplatin. Diploid (D) and tetraploid (T) HCT116 cells were knocked down for Chk1 (or transfected with control siRNA SCR) for 24 h and then cultured in absence or presence of cisplatin (20 µM) for further 48 h. Finally, the frequency of dying (DiOC6(3)low PI−) or dead (DiOC6(3)low PI+) cells was monitored by DiOC6(3)/PI staining. B. Cell killing by pharmacological inhibition of Chk1 plus cisplatin. Cells were cultured with cisplatin (20 µM), UCN-01 (500 nM) and/or SD1825 (500 nM) for 48 h, and dead and dying cells were determined as in A. C–E. Combined effects of Chk1 inhibition and cisplatin on tetraploid tumors established in vivo. Diploid or tetraploid HCT116 tumors were established in vivo and their growth was monitored continuously from day 18 (when tumors measured 125 to 250 mm3), when animals were injected with PBS alone (controls in C), cisplatin (D), UCN-01 (E), or with a combination of both (F). Asterisks indicate significant differences between diploid and tetraploid cells (p<0.05, unpaired Student t test). The results shown in Fig. 6C–E are representative for three different experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2131784&req=5

pone-0001337-g006: Combined effects of Chk1 inhibition and cisplatin on tetraploid tumor cells in vitro and in vivo.A. Cytotoxic effects of Chk1 depletion in combination with cisplatin. Diploid (D) and tetraploid (T) HCT116 cells were knocked down for Chk1 (or transfected with control siRNA SCR) for 24 h and then cultured in absence or presence of cisplatin (20 µM) for further 48 h. Finally, the frequency of dying (DiOC6(3)low PI−) or dead (DiOC6(3)low PI+) cells was monitored by DiOC6(3)/PI staining. B. Cell killing by pharmacological inhibition of Chk1 plus cisplatin. Cells were cultured with cisplatin (20 µM), UCN-01 (500 nM) and/or SD1825 (500 nM) for 48 h, and dead and dying cells were determined as in A. C–E. Combined effects of Chk1 inhibition and cisplatin on tetraploid tumors established in vivo. Diploid or tetraploid HCT116 tumors were established in vivo and their growth was monitored continuously from day 18 (when tumors measured 125 to 250 mm3), when animals were injected with PBS alone (controls in C), cisplatin (D), UCN-01 (E), or with a combination of both (F). Asterisks indicate significant differences between diploid and tetraploid cells (p<0.05, unpaired Student t test). The results shown in Fig. 6C–E are representative for three different experiments.
Mentions: As compared to their diploid counterparts, tetraploid cancer cells are relatively resistant against DNA damaging agents including cisplatin, both in vitro (Ref. [8], Fig. 6A,B for HCT116 and Fig. S2 C for RKO) and in vivo (Fig. 6C,D). Although there was no difference in the growth tumors originating from diploid versus tetraploid HCT116 cells inoculated into immunodeficient mice (Fig. 6C), tetraploid tumors responded far less to chemotherapy with cisplatin than diploid cancers (Fig. 6D). Chk1 depletion (Fig. 6A, Fig. S2 C) or inhibition (Fig. 6B) had an additive cytotoxic effect on tetraploid cells in vitro, in short-term assays. Treatment of tetraploid HCT116 tumors that had been established in xenotransplanted immunodeficient mice with a subtoxic dose of UCN-01 had no growth-inhibitory effect (Fig. 6E). However, the combination of cisplatin plus UCN-01 had a synergistic anti-cancer effect, which was particularly pronounced for tetraploid tumors (Fig. 6F). Based on these observations, we decided to explore the effect of cisplatin, Chk1 inhibition and the combination of both on the transcriptome of diploid and tetraploid colon cancer (HCT116) cells. The number of cisplatin-modulated genes showing a statistically relevant altered expression (p value<10−5) was higher among diploid than among tetraploid cells (Fig. 7A,B), correlating with the higher susceptibility of diploid cells to cisplatin-induced killing (Fig. 6D). In contrast, the number of genes modulated by Chk1 inhibition was significantly higher among tetraploid (152 genes) than among diploid cells (20 genes), with only six genes that were modulated in both diploid and tetraploid cells (Fig. 7A,B), again correlating with the enhanced killing of tetraploid cells by Chk1 inhibitors. The combination of cisplatin and Chk1 inhibition modified a large pool of transcripts that were common to tetraploid and diploid cells, with a higher number of tetraploid- than diploid- specific transcripts (Fig. 7A,B), in correlation with the particularly dramatic effects of the combined therapeutic regimen on tetraploid cells in vivo (Fig. 6F).

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