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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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Generation of stable Bid deficient HeLa cells.A) Cells were stably transfected with plasmids encoding one of three different bid-specific shRNAs or a scrambled control shRNA as described in Materials and Methods. For analysis of the bid knockdown, cells were subjected to Western blotting with a polyclonal Bid antibody. α-Tubulin served as a loading control. B) Control cells expressing the scrambled (Ctrl 1, Ctrl 2), or the bid specific shRNA (Bid kd 4) and clone (Bid kd 1) were incubated with Cycloheximide (CHX) (1 µg/ml) and an agonistic Fas antibody or vehicle (clone CH11) at the indicated concentrations or vehicle for 4 h. Caspase-3 like activity was measured by cleavage of the fluorigenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05: difference from control cells (Ctrl). C) Cell lysates were subjected to Western blotting with antibodies for the indicated proteins, as described in Materials and Methods. D) HeLa Bid kd cells were stably transfected with a vector coding for YFP-bid-CFP (GFP-Bid); cell lysates were subjected to Western blotting with GFP and Bid antibodies. E) HeLa Bid kd cells expressing GFP-Bid were treated with CHX (1 µg/ml) and an activating Fas antibody (100 ng/ml). Controls were treated with vehicle for 4 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 control cells (Ctrl).
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pone-0002844-g001: Generation of stable Bid deficient HeLa cells.A) Cells were stably transfected with plasmids encoding one of three different bid-specific shRNAs or a scrambled control shRNA as described in Materials and Methods. For analysis of the bid knockdown, cells were subjected to Western blotting with a polyclonal Bid antibody. α-Tubulin served as a loading control. B) Control cells expressing the scrambled (Ctrl 1, Ctrl 2), or the bid specific shRNA (Bid kd 4) and clone (Bid kd 1) were incubated with Cycloheximide (CHX) (1 µg/ml) and an agonistic Fas antibody or vehicle (clone CH11) at the indicated concentrations or vehicle for 4 h. Caspase-3 like activity was measured by cleavage of the fluorigenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05: difference from control cells (Ctrl). C) Cell lysates were subjected to Western blotting with antibodies for the indicated proteins, as described in Materials and Methods. D) HeLa Bid kd cells were stably transfected with a vector coding for YFP-bid-CFP (GFP-Bid); cell lysates were subjected to Western blotting with GFP and Bid antibodies. E) HeLa Bid kd cells expressing GFP-Bid were treated with CHX (1 µg/ml) and an activating Fas antibody (100 ng/ml). Controls were treated with vehicle for 4 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 control cells (Ctrl).

Mentions: In order to study the role of Bid in death receptor- and drug-induced apoptosis, gene silencing using three different shRNA constructs targeting different regions of bid was performed as described in Materials and Methods. Western blot analysis of established stable cell lines demonstrated different levels of reduction in Bid protein expression compared to cells transfected with a scrambled control sequence (Fig. 1 A). These reduced Bid levels correlated with reduced executioner caspase activity in response to an activating Fas antibody (clone CH11), as determined by analyzing the cleavage of the fluorogenic caspase substrate Ac-DEVD-AMC (Fig. 1 B). The Bid knockdown clone 1 (hereafter designated as HeLa Bid kd) was further analyzed as this clone demonstrated maximal knockdown of Bid expression, whereas the expression of other BH3-only proteins, Bcl-2 family members, or other key proteins involved in caspase activation were not altered (Fig. 1 C). Furthermore, re-introduction of ectopically expressed bid (Fig. 1 D) was able to re-sensitize this clone to Fas-induced caspase activation and apoptosis (Fig. 1 E), indicating that the reduction in caspase activity after death receptor activation was due to the lack of Bid expression.


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)

Generation of stable Bid deficient HeLa cells.A) Cells were stably transfected with plasmids encoding one of three different bid-specific shRNAs or a scrambled control shRNA as described in Materials and Methods. For analysis of the bid knockdown, cells were subjected to Western blotting with a polyclonal Bid antibody. α-Tubulin served as a loading control. B) Control cells expressing the scrambled (Ctrl 1, Ctrl 2), or the bid specific shRNA (Bid kd 4) and clone (Bid kd 1) were incubated with Cycloheximide (CHX) (1 µg/ml) and an agonistic Fas antibody or vehicle (clone CH11) at the indicated concentrations or vehicle for 4 h. Caspase-3 like activity was measured by cleavage of the fluorigenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05: difference from control cells (Ctrl). C) Cell lysates were subjected to Western blotting with antibodies for the indicated proteins, as described in Materials and Methods. D) HeLa Bid kd cells were stably transfected with a vector coding for YFP-bid-CFP (GFP-Bid); cell lysates were subjected to Western blotting with GFP and Bid antibodies. E) HeLa Bid kd cells expressing GFP-Bid were treated with CHX (1 µg/ml) and an activating Fas antibody (100 ng/ml). Controls were treated with vehicle for 4 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 control cells (Ctrl).
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pone-0002844-g001: Generation of stable Bid deficient HeLa cells.A) Cells were stably transfected with plasmids encoding one of three different bid-specific shRNAs or a scrambled control shRNA as described in Materials and Methods. For analysis of the bid knockdown, cells were subjected to Western blotting with a polyclonal Bid antibody. α-Tubulin served as a loading control. B) Control cells expressing the scrambled (Ctrl 1, Ctrl 2), or the bid specific shRNA (Bid kd 4) and clone (Bid kd 1) were incubated with Cycloheximide (CHX) (1 µg/ml) and an agonistic Fas antibody or vehicle (clone CH11) at the indicated concentrations or vehicle for 4 h. Caspase-3 like activity was measured by cleavage of the fluorigenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05: difference from control cells (Ctrl). C) Cell lysates were subjected to Western blotting with antibodies for the indicated proteins, as described in Materials and Methods. D) HeLa Bid kd cells were stably transfected with a vector coding for YFP-bid-CFP (GFP-Bid); cell lysates were subjected to Western blotting with GFP and Bid antibodies. E) HeLa Bid kd cells expressing GFP-Bid were treated with CHX (1 µg/ml) and an activating Fas antibody (100 ng/ml). Controls were treated with vehicle for 4 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 control cells (Ctrl).
Mentions: In order to study the role of Bid in death receptor- and drug-induced apoptosis, gene silencing using three different shRNA constructs targeting different regions of bid was performed as described in Materials and Methods. Western blot analysis of established stable cell lines demonstrated different levels of reduction in Bid protein expression compared to cells transfected with a scrambled control sequence (Fig. 1 A). These reduced Bid levels correlated with reduced executioner caspase activity in response to an activating Fas antibody (clone CH11), as determined by analyzing the cleavage of the fluorogenic caspase substrate Ac-DEVD-AMC (Fig. 1 B). The Bid knockdown clone 1 (hereafter designated as HeLa Bid kd) was further analyzed as this clone demonstrated maximal knockdown of Bid expression, whereas the expression of other BH3-only proteins, Bcl-2 family members, or other key proteins involved in caspase activation were not altered (Fig. 1 C). Furthermore, re-introduction of ectopically expressed bid (Fig. 1 D) was able to re-sensitize this clone to Fas-induced caspase activation and apoptosis (Fig. 1 E), indicating that the reduction in caspase activity after death receptor activation was due to the lack of Bid expression.

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