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Bid participates in genotoxic drug-induced apoptosis of HeLa cells and is essential for death receptor ligands' apoptotic and synergistic effects.

Köhler B, Anguissola S, Concannon CG, Rehm M, Kögel D, Prehn JH - PLoS ONE (2008)

Bottom Line: While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin.Similar effects were observed using the Bid inhibitor BI6C9.They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland.

ABSTRACT

Background: The BH3-only protein Bid is an important component of death receptor-mediated caspase activation. Bid is cleaved by caspase-8 or -10 into t-Bid, which translocates to mitochondria and triggers the release of caspase-activating factors. Bid has also been reported to be cleaved by other proteases.

Methodology/principal findings: To test the hypothesis that Bid is a central mediator of stress-induced apoptosis, we investigated the effects of a small molecule Bid inhibitor on stress-induced apoptosis, and generated HeLa cells deficient for Bid. Stable knockdown of bid lead to a pronounced resistance to Fas/CD95- and TRAIL-induced caspase activation and apoptosis, and significantly increased clonogenic survival. While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin. Similar effects were observed using the Bid inhibitor BI6C9. Interestingly, Bid-deficient cells were dramatically protected from apoptosis when subtoxic concentrations of ER stressors, Etoposide or Oxaliplatin were combined with subtoxic TRAIL concentrations.

Conclusions/significance: Our data demonstrate that Bid is central for death receptor-induced cell death and participates in anti-cancer drug-induced apoptosis in human cervical cancer HeLa cells. They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling.

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Additive effects of the BH3-only proteins PUMA and Bid during genotoxic stress-induced apoptosis.A) HeLa Control and HeLa Bid kd cells were treated with Oxaliplatin (30 µg/ml); puma mRNA levels were evaluated by qPCR at the indicated time points. Data are means+/−SD from n = 3 separate experiments. n.s. = not significant versus control (Ctrl). B) After transient transfection with scrambled siRNA or puma siRNA, HeLa control and HeLa Bid kd cells were treated with the indicated concentrations of Oxaliplatin for 24 h. Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. ∗ p<0.05: difference from vehicle treated control cultures transfected with control siRNA (Ctrl SiRNA). # p<0.05 difference between respective HeLa control and Bid kd cells.
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pone-0002844-g011: Additive effects of the BH3-only proteins PUMA and Bid during genotoxic stress-induced apoptosis.A) HeLa Control and HeLa Bid kd cells were treated with Oxaliplatin (30 µg/ml); puma mRNA levels were evaluated by qPCR at the indicated time points. Data are means+/−SD from n = 3 separate experiments. n.s. = not significant versus control (Ctrl). B) After transient transfection with scrambled siRNA or puma siRNA, HeLa control and HeLa Bid kd cells were treated with the indicated concentrations of Oxaliplatin for 24 h. Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. ∗ p<0.05: difference from vehicle treated control cultures transfected with control siRNA (Ctrl SiRNA). # p<0.05 difference between respective HeLa control and Bid kd cells.

Mentions: Since the BH3-only gene puma has been shown to be a pivotal regulator of apoptosis induced by DNA-damaging anticancer drugs in many types of cancer cells and in particular to mediate Oxaliplatin-induced apoptosis [42], we finally determined the relative contributions of Bid and PUMA in apoptosis induced by oxaliplatin. We compared the effects of Oxaliplatin on puma gene expression in HeLa control cells and HeLa Bid kd cells. puma mRNA levels were potently induced by Oxaliplatin as evaluated by qPCR (Fig. 11 A). We could not observe significant differences in puma mRNA upregulation between HeLa control and HeLa Bid kd cells (Fig. 11 A), indicating that the stress signaling pathways mediating puma expression were similarly activated in control and Bid kd cells, and that the induction of puma occurred independently of Bid. Transient RNA interference against puma was able to reduce puma mRNA expression by 47.58+/−8.7% and 64.61+/−3.2% after 24 h and by 59.71+/−1.1% and 69.67+/−0.1% after 48 h in the HeLa control and HeLa Bid kd cells, respectively (n = 3 experiments; normalized to β-actin mRNA expression and compared to cultures transfected with a scrambled siRNA sequence). Treatment with puma siRNA significantly reduced levels of apoptosis in HeLa control cells exposed to Oxaliplatin (Fig. 11 B). The knockdown of puma led to a further decrease of apoptosis in the HeLa Bid kd cells, indicating that both BH3-only proteins cooperate in mediating apoptosis triggered by Oxaliplatin.


Bid participates in genotoxic drug-induced apoptosis of HeLa cells and is essential for death receptor ligands' apoptotic and synergistic effects.

Köhler B, Anguissola S, Concannon CG, Rehm M, Kögel D, Prehn JH - PLoS ONE (2008)

Additive effects of the BH3-only proteins PUMA and Bid during genotoxic stress-induced apoptosis.A) HeLa Control and HeLa Bid kd cells were treated with Oxaliplatin (30 µg/ml); puma mRNA levels were evaluated by qPCR at the indicated time points. Data are means+/−SD from n = 3 separate experiments. n.s. = not significant versus control (Ctrl). B) After transient transfection with scrambled siRNA or puma siRNA, HeLa control and HeLa Bid kd cells were treated with the indicated concentrations of Oxaliplatin for 24 h. Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. ∗ p<0.05: difference from vehicle treated control cultures transfected with control siRNA (Ctrl SiRNA). # p<0.05 difference between respective HeLa control and Bid kd cells.
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Related In: Results  -  Collection

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pone-0002844-g011: Additive effects of the BH3-only proteins PUMA and Bid during genotoxic stress-induced apoptosis.A) HeLa Control and HeLa Bid kd cells were treated with Oxaliplatin (30 µg/ml); puma mRNA levels were evaluated by qPCR at the indicated time points. Data are means+/−SD from n = 3 separate experiments. n.s. = not significant versus control (Ctrl). B) After transient transfection with scrambled siRNA or puma siRNA, HeLa control and HeLa Bid kd cells were treated with the indicated concentrations of Oxaliplatin for 24 h. Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. ∗ p<0.05: difference from vehicle treated control cultures transfected with control siRNA (Ctrl SiRNA). # p<0.05 difference between respective HeLa control and Bid kd cells.
Mentions: Since the BH3-only gene puma has been shown to be a pivotal regulator of apoptosis induced by DNA-damaging anticancer drugs in many types of cancer cells and in particular to mediate Oxaliplatin-induced apoptosis [42], we finally determined the relative contributions of Bid and PUMA in apoptosis induced by oxaliplatin. We compared the effects of Oxaliplatin on puma gene expression in HeLa control cells and HeLa Bid kd cells. puma mRNA levels were potently induced by Oxaliplatin as evaluated by qPCR (Fig. 11 A). We could not observe significant differences in puma mRNA upregulation between HeLa control and HeLa Bid kd cells (Fig. 11 A), indicating that the stress signaling pathways mediating puma expression were similarly activated in control and Bid kd cells, and that the induction of puma occurred independently of Bid. Transient RNA interference against puma was able to reduce puma mRNA expression by 47.58+/−8.7% and 64.61+/−3.2% after 24 h and by 59.71+/−1.1% and 69.67+/−0.1% after 48 h in the HeLa control and HeLa Bid kd cells, respectively (n = 3 experiments; normalized to β-actin mRNA expression and compared to cultures transfected with a scrambled siRNA sequence). Treatment with puma siRNA significantly reduced levels of apoptosis in HeLa control cells exposed to Oxaliplatin (Fig. 11 B). The knockdown of puma led to a further decrease of apoptosis in the HeLa Bid kd cells, indicating that both BH3-only proteins cooperate in mediating apoptosis triggered by Oxaliplatin.

Bottom Line: While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin.Similar effects were observed using the Bid inhibitor BI6C9.They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland.

ABSTRACT

Background: The BH3-only protein Bid is an important component of death receptor-mediated caspase activation. Bid is cleaved by caspase-8 or -10 into t-Bid, which translocates to mitochondria and triggers the release of caspase-activating factors. Bid has also been reported to be cleaved by other proteases.

Methodology/principal findings: To test the hypothesis that Bid is a central mediator of stress-induced apoptosis, we investigated the effects of a small molecule Bid inhibitor on stress-induced apoptosis, and generated HeLa cells deficient for Bid. Stable knockdown of bid lead to a pronounced resistance to Fas/CD95- and TRAIL-induced caspase activation and apoptosis, and significantly increased clonogenic survival. While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin. Similar effects were observed using the Bid inhibitor BI6C9. Interestingly, Bid-deficient cells were dramatically protected from apoptosis when subtoxic concentrations of ER stressors, Etoposide or Oxaliplatin were combined with subtoxic TRAIL concentrations.

Conclusions/significance: Our data demonstrate that Bid is central for death receptor-induced cell death and participates in anti-cancer drug-induced apoptosis in human cervical cancer HeLa cells. They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling.

Show MeSH
Related in: MedlinePlus