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Expression of the apoptosis-mediator Fas is enhanced by dysfunctional mitochondria
Abstract:
We applied an antibody against an apoptosis mediator, Fas/APO-1/CD95, to HeLa-derived cells that completely lack mitochondrial DNA (mtDNA) or have mutant mtDNAs. The anti-Fas antibody killed the cells completely lacking mtDNA (EB8), at concentrations as low as 1 ng/ml, but not control cells harboring wild-type mtDNA (Ft2-11). TUNEL (terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end-labeling) and analysis of fragmented DNA indicated that the cell death of EB8 was due to apoptosis. The antibody was cytotoxic to other two cell lines harboring mutant mtDNA with a point mutation or a large-scale deletion. RT-PCR (reverse transcriptase-polymerase chain reaction) showed that the mRNA content of the Fas gene was 2 to 19-fold higher in the cells with deficient mtDNA than in the control cells. In addition, the expressed Fas protein was detected by immunohistochemical staining in the cells without mtDNA but not in the control cells. Incubating the cells containing wild-type mtDNA with the respiratory inhibitors rotenone and antimycin A enhanced the content of mRNA of the Fas gene 2 to 4-fold and sensitized cells to the antibody. Thus, defects in mitochondria caused apoptotic cell death by anti-Fas antibody and enhanced Fas gene expression.
Insights
Mitochondrial DNA defects trigger apoptosis in cells treated with an anti-Fas antibody. This study reveals enhanced Fas gene expression and increased sensitivity to Fas-mediated cell death in cells lacking functional mitochondrial DNA.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Mitochondrial DNA (mtDNA) plays a crucial role in cellular respiration and survival.
- Apoptosis, or programmed cell death, is a tightly regulated process essential for development and tissue homeostasis.
- Fas (also known as APO-1/CD95) is a cell surface receptor that mediates apoptosis upon ligand binding.
Purpose of the Study:
- To investigate the role of mitochondrial DNA integrity in Fas-mediated apoptosis.
- To determine if defects in mtDNA affect Fas gene expression and cellular sensitivity to Fas-induced cell death.
Main Methods:
- Utilized HeLa-derived cell lines with complete absence or mutations in mtDNA.
- Applied anti-Fas antibody to induce apoptosis.
- Performed TUNEL assays and DNA fragmentation analysis to confirm apoptosis.
- Quantified Fas gene mRNA levels using RT-PCR.
- Detected Fas protein expression via immunohistochemical staining.
- Treated cells with respiratory inhibitors (rotenone, antimycin A) to assess their effect on Fas expression.
Main Results:
- Cells lacking mtDNA (EB8) underwent apoptosis upon anti-Fas antibody treatment at low concentrations.
- Control cells with wild-type mtDNA (Ft2-11) were resistant to anti-Fas antibody-induced cell death.
- Cell lines with mutant mtDNA (point mutation or large deletion) were sensitive to the antibody.
- Fas gene mRNA levels were significantly elevated (2-19 fold) in mtDNA-deficient cells.
- Fas protein was detected in mtDNA-deficient cells but not in controls.
- Respiratory inhibitors increased Fas mRNA levels and sensitized wild-type cells to the antibody.
Conclusions:
- Defects in mitochondrial DNA render cells susceptible to Fas-mediated apoptotic cell death.
- mtDNA deficiency leads to enhanced Fas gene expression.
- Mitochondrial dysfunction is a key factor in regulating Fas-mediated apoptosis.