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Lysosomal membrane permeabilization induces cell death in a mitochondrion-dependent fashion.

Boya P, Andreau K, Poncet D, Zamzami N, Perfettini JL, Metivier D, Ojcius DM, Jäättelä M, Kroemer G - J. Exp. Med. (2003)

Bottom Line: A number of diseases are due to lysosomal destabilization, which results in damaging cell loss.The single knockout of Bax or Bak (but not Bid) or the transfection-enforced expression of mitochondrion-targeted (but not endoplasmic reticulum-targeted) Bcl-2 conferred protection against CPX (but not NFX*)-induced MMP and death.Altogether, our data indicate that mitochondria are indispensable for cell death initiated by lysosomal destabilization.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique, UMR 8125, Institut Gustave Roussy, Pavillon de Recherche 1, 39 rue Camille-Desmoulins, F-94805 Villejuif, France.

ABSTRACT
A number of diseases are due to lysosomal destabilization, which results in damaging cell loss. To investigate the mechanisms of lysosomal cell death, we characterized the cytotoxic action of two widely used quinolone antibiotics: ciprofloxacin (CPX) or norfloxacin (NFX). CPX or NFX plus UV light (NFX*) induce lysosomal membrane permeabilization (LMP), as detected by the release of cathepsins from lysosomes. Inhibition of the lysosomal accumulation of CPX or NFX suppresses their capacity to induce LMP and to kill cells. CPX- or NFX-triggered LMP results in caspase-independent cell death, with hallmarks of apoptosis such as chromatin condensation and phosphatidylserine exposure on the plasma membrane. LMP triggers mitochondrial membrane permeabilization (MMP), as detected by the release of cytochrome c. Both CPX and NFX* cause Bax and Bak to adopt their apoptotic conformation and to insert into mitochondrial membranes. Bax-/- Bak-/- double knockout cells fail to undergo MMP and cell death in response to CPX- or NFX-induced LMP. The single knockout of Bax or Bak (but not Bid) or the transfection-enforced expression of mitochondrion-targeted (but not endoplasmic reticulum-targeted) Bcl-2 conferred protection against CPX (but not NFX*)-induced MMP and death. Altogether, our data indicate that mitochondria are indispensable for cell death initiated by lysosomal destabilization.

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Requirement of Bax and/or Bak for NFX*- or CPX-induced cell death. (A) SV40-transformed WT MEF or Bax−/− Bak−/− DKO cells were exposed to NFX* and CPX for the indicated period, followed by determination of the frequency of DiOC6(3)low and annexin V–positive cells. (B) MEF lacking Bax, Bak, Bax and Bak (DKO), or Bid were exposed to either NFX* or CPX and the indicated parameters were measured. (C) NFX*- and CPX-induced cathepsin B release in control and Bax−/− Bak−/− DKO cells, as determined by immunostaining. The percentage of cells exhibiting diffuse cathepsin B staining as well as those with diffuse cytochrome c staining has been quantified. (D) Failure of chemical cathepsin inhibition to prevent NFX*- or CPX-induced cell death. HeLa cells were pretreated with the indicated cathepsin inhibitors, followed by determination of the frequency of DiOC6(3)low and PIhigh cells. (E) Cathepsins are dispensable for NFX*- or CPX-induced cell death. MEF with the indicated genotypes were treated with NFX* or CPX, and apoptotic parameters were measured as in D. Results are means ± SD of three independent determinations.
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fig6: Requirement of Bax and/or Bak for NFX*- or CPX-induced cell death. (A) SV40-transformed WT MEF or Bax−/− Bak−/− DKO cells were exposed to NFX* and CPX for the indicated period, followed by determination of the frequency of DiOC6(3)low and annexin V–positive cells. (B) MEF lacking Bax, Bak, Bax and Bak (DKO), or Bid were exposed to either NFX* or CPX and the indicated parameters were measured. (C) NFX*- and CPX-induced cathepsin B release in control and Bax−/− Bak−/− DKO cells, as determined by immunostaining. The percentage of cells exhibiting diffuse cathepsin B staining as well as those with diffuse cytochrome c staining has been quantified. (D) Failure of chemical cathepsin inhibition to prevent NFX*- or CPX-induced cell death. HeLa cells were pretreated with the indicated cathepsin inhibitors, followed by determination of the frequency of DiOC6(3)low and PIhigh cells. (E) Cathepsins are dispensable for NFX*- or CPX-induced cell death. MEF with the indicated genotypes were treated with NFX* or CPX, and apoptotic parameters were measured as in D. Results are means ± SD of three independent determinations.

Mentions: Both NFX* and CPX induced a conformational change of Bax and Bak (detectable with monoclonal antibodies specific for the NH2 terminus of these molecules), which are exposed only in the “apoptotic conformation” (35, 36). Both NFX* and CPX treatment resulted in a punctate cytoplasmic staining of apoptotic Bax and Bak (Fig. 5 A), which were found to colocalize with the mitochondrial marker Hsp60 (not depicted). The activation of Bax and Bak occurs after LMP (as assessed by measuring the LysoTracker red incorporation and cathepsin B release), but before the mitochondrial release of cytochrome c and before caspase-3–activation (Fig. 5, B and C). These alterations were not due to transcriptional activation of p53 because NFX* and CPX (in contrast to the positive control etoposide) failed to induce a p53-inducible GFP reporter gene (Fig. 5 D). Moreover, NFX*- and CPX-induced MMP and cell death were not inhibited by pifithrin-α, a chemical inhibitor of p53-mediated transactivation (Fig. 5 E) or cycloheximide, a general inhibitor of protein synthesis (not depicted). To directly address the contribution of Bax and Bak to cell death induced by lysomotropic agents, we took advantage of SV40–immortalized MEF lacking Bax, Bak, Bid, or both Bax and Bak. Bax−/−Bak−/− DKO were found to be profoundly resistant to the induction of MMP and PS exposure by NFX* or CPX (Fig. 6 A). Only the DKO of Bax and Bak led to cytoprotection against NFX* (Fig. 6 B). In contrast, the absence of either Bax or Bak (but not Bid) was sufficient to prevent cell death induction by CPX (Fig. 6 B). WT and Bax−/−Bak−/− cells exposed to CPX or NFX* underwent a similar degree of lysosomal disruption, as measured with cathepsin B immunostaining (Fig. 6 C). However, Bax−/− Bak−/− cells manifested much less diffuse cytochrome c staining induced by CPX or NFX*, as compared with controls (Fig. 6 C). The knockout of the genes coding for cathepsin B, D, L, or S and several cathepsin inhibitors failed to prevent CPX- or NFX-induced MMP and cell death (Fig. 6, D and E). Altogether, these data demonstrate that Bax and/or Bak, but not cathepsins, control cell death induced by lysosomotropic agents.


Lysosomal membrane permeabilization induces cell death in a mitochondrion-dependent fashion.

Boya P, Andreau K, Poncet D, Zamzami N, Perfettini JL, Metivier D, Ojcius DM, Jäättelä M, Kroemer G - J. Exp. Med. (2003)

Requirement of Bax and/or Bak for NFX*- or CPX-induced cell death. (A) SV40-transformed WT MEF or Bax−/− Bak−/− DKO cells were exposed to NFX* and CPX for the indicated period, followed by determination of the frequency of DiOC6(3)low and annexin V–positive cells. (B) MEF lacking Bax, Bak, Bax and Bak (DKO), or Bid were exposed to either NFX* or CPX and the indicated parameters were measured. (C) NFX*- and CPX-induced cathepsin B release in control and Bax−/− Bak−/− DKO cells, as determined by immunostaining. The percentage of cells exhibiting diffuse cathepsin B staining as well as those with diffuse cytochrome c staining has been quantified. (D) Failure of chemical cathepsin inhibition to prevent NFX*- or CPX-induced cell death. HeLa cells were pretreated with the indicated cathepsin inhibitors, followed by determination of the frequency of DiOC6(3)low and PIhigh cells. (E) Cathepsins are dispensable for NFX*- or CPX-induced cell death. MEF with the indicated genotypes were treated with NFX* or CPX, and apoptotic parameters were measured as in D. Results are means ± SD of three independent determinations.
© Copyright Policy
Related In: Results  -  Collection

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fig6: Requirement of Bax and/or Bak for NFX*- or CPX-induced cell death. (A) SV40-transformed WT MEF or Bax−/− Bak−/− DKO cells were exposed to NFX* and CPX for the indicated period, followed by determination of the frequency of DiOC6(3)low and annexin V–positive cells. (B) MEF lacking Bax, Bak, Bax and Bak (DKO), or Bid were exposed to either NFX* or CPX and the indicated parameters were measured. (C) NFX*- and CPX-induced cathepsin B release in control and Bax−/− Bak−/− DKO cells, as determined by immunostaining. The percentage of cells exhibiting diffuse cathepsin B staining as well as those with diffuse cytochrome c staining has been quantified. (D) Failure of chemical cathepsin inhibition to prevent NFX*- or CPX-induced cell death. HeLa cells were pretreated with the indicated cathepsin inhibitors, followed by determination of the frequency of DiOC6(3)low and PIhigh cells. (E) Cathepsins are dispensable for NFX*- or CPX-induced cell death. MEF with the indicated genotypes were treated with NFX* or CPX, and apoptotic parameters were measured as in D. Results are means ± SD of three independent determinations.
Mentions: Both NFX* and CPX induced a conformational change of Bax and Bak (detectable with monoclonal antibodies specific for the NH2 terminus of these molecules), which are exposed only in the “apoptotic conformation” (35, 36). Both NFX* and CPX treatment resulted in a punctate cytoplasmic staining of apoptotic Bax and Bak (Fig. 5 A), which were found to colocalize with the mitochondrial marker Hsp60 (not depicted). The activation of Bax and Bak occurs after LMP (as assessed by measuring the LysoTracker red incorporation and cathepsin B release), but before the mitochondrial release of cytochrome c and before caspase-3–activation (Fig. 5, B and C). These alterations were not due to transcriptional activation of p53 because NFX* and CPX (in contrast to the positive control etoposide) failed to induce a p53-inducible GFP reporter gene (Fig. 5 D). Moreover, NFX*- and CPX-induced MMP and cell death were not inhibited by pifithrin-α, a chemical inhibitor of p53-mediated transactivation (Fig. 5 E) or cycloheximide, a general inhibitor of protein synthesis (not depicted). To directly address the contribution of Bax and Bak to cell death induced by lysomotropic agents, we took advantage of SV40–immortalized MEF lacking Bax, Bak, Bid, or both Bax and Bak. Bax−/−Bak−/− DKO were found to be profoundly resistant to the induction of MMP and PS exposure by NFX* or CPX (Fig. 6 A). Only the DKO of Bax and Bak led to cytoprotection against NFX* (Fig. 6 B). In contrast, the absence of either Bax or Bak (but not Bid) was sufficient to prevent cell death induction by CPX (Fig. 6 B). WT and Bax−/−Bak−/− cells exposed to CPX or NFX* underwent a similar degree of lysosomal disruption, as measured with cathepsin B immunostaining (Fig. 6 C). However, Bax−/− Bak−/− cells manifested much less diffuse cytochrome c staining induced by CPX or NFX*, as compared with controls (Fig. 6 C). The knockout of the genes coding for cathepsin B, D, L, or S and several cathepsin inhibitors failed to prevent CPX- or NFX-induced MMP and cell death (Fig. 6, D and E). Altogether, these data demonstrate that Bax and/or Bak, but not cathepsins, control cell death induced by lysosomotropic agents.

Bottom Line: A number of diseases are due to lysosomal destabilization, which results in damaging cell loss.The single knockout of Bax or Bak (but not Bid) or the transfection-enforced expression of mitochondrion-targeted (but not endoplasmic reticulum-targeted) Bcl-2 conferred protection against CPX (but not NFX*)-induced MMP and death.Altogether, our data indicate that mitochondria are indispensable for cell death initiated by lysosomal destabilization.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique, UMR 8125, Institut Gustave Roussy, Pavillon de Recherche 1, 39 rue Camille-Desmoulins, F-94805 Villejuif, France.

ABSTRACT
A number of diseases are due to lysosomal destabilization, which results in damaging cell loss. To investigate the mechanisms of lysosomal cell death, we characterized the cytotoxic action of two widely used quinolone antibiotics: ciprofloxacin (CPX) or norfloxacin (NFX). CPX or NFX plus UV light (NFX*) induce lysosomal membrane permeabilization (LMP), as detected by the release of cathepsins from lysosomes. Inhibition of the lysosomal accumulation of CPX or NFX suppresses their capacity to induce LMP and to kill cells. CPX- or NFX-triggered LMP results in caspase-independent cell death, with hallmarks of apoptosis such as chromatin condensation and phosphatidylserine exposure on the plasma membrane. LMP triggers mitochondrial membrane permeabilization (MMP), as detected by the release of cytochrome c. Both CPX and NFX* cause Bax and Bak to adopt their apoptotic conformation and to insert into mitochondrial membranes. Bax-/- Bak-/- double knockout cells fail to undergo MMP and cell death in response to CPX- or NFX-induced LMP. The single knockout of Bax or Bak (but not Bid) or the transfection-enforced expression of mitochondrion-targeted (but not endoplasmic reticulum-targeted) Bcl-2 conferred protection against CPX (but not NFX*)-induced MMP and death. Altogether, our data indicate that mitochondria are indispensable for cell death initiated by lysosomal destabilization.

Show MeSH
Related in: MedlinePlus