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Characterization of Apt- cell lines exhibiting cross-resistance to glucocorticoid- and Fas-mediated apoptosis
D J Askew1, U Kuscuoglu, T Brunner
1Departments of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona, 85721 USA.
Abstract:
Apoptosis induction by staurosporine, ceramide, and Fas stimulation was investigated in the mouse thymoma cell line W7.2 and a panel of dexamethasone (dex)-resistant W7.2 mutant cell lines, Apt3.8, Apt4.8 and Apt5.8, and a Bcl-2 transfected W7.2 cell line (Wbcl2). While W7. 2 cells were found to be sensitive to these apoptosis inducers, the Apt- mutants and Wbcl2 cells were shown to be resistant to some or all of the treatments. Specifically, all three Apt- mutants and Wbcl2 cells were found to be resistant to ceramide and Fas-mediated apoptosis, whereas, Apt4.8 and Apt5.8 were sensitive to staurosporine-induced apoptosis under conditions in which Apt3.8 and Wbcl2 cells were resistant. Measurements of caspase activity and cytochrome c release in cytosolic extracts of dex and staurosporine-treated cells indicated that the recessive Apt- mutations effect steps upstream of mitochondrial dysfunction. Steady-state RNA levels of apoptosis-associated gene transcripts showed that the observed differential resistance of the Apt- cell lines could not be explained by altered expression of numerous Bcl-2 or Fas related genes. Transient transfection of human Fas gene coding sequences into the Apt- mutants and Wbcl2 cells did not induce apoptosis, even though these same cell lines were sensitive to ectopic expression of the FADD and caspase 8 genes. Taken together, these data provide genetic evidence for the existence of shared components in the dex- and Fas-mediated apoptotic pathways in W7.2 cells.
Insights
This study investigates apoptosis resistance in mouse thymoma cells. Genetic mutations and Bcl-2 transfection confer resistance to ceramide and Fas-mediated apoptosis, suggesting shared pathways.
Area of Science:
- Cell Biology
- Molecular Biology
- Immunology
Background:
- Apoptosis, or programmed cell death, is crucial for development and tissue homeostasis.
- Dysregulation of apoptosis is implicated in various diseases, including cancer and autoimmune disorders.
- Understanding the molecular mechanisms of apoptosis induction and resistance is vital for therapeutic development.
Purpose of the Study:
- To investigate apoptosis induction by staurosporine, ceramide, and Fas stimulation in mouse thymoma cells.
- To characterize apoptosis resistance in dexamethasone-resistant mutants and Bcl-2 transfected cells.
- To identify shared components in the dexamethasone- and Fas-mediated apoptotic pathways.
Main Methods:
- Utilized mouse thymoma cell line W7.2 and its dexamethasone-resistant mutants (Apt3.8, Apt4.8, Apt5.8) and Bcl-2 transfected cells (Wbcl2).
- Assessed apoptosis induction via staurosporine, ceramide, and Fas stimulation.
- Measured caspase activity and cytochrome c release.
- Analyzed steady-state RNA levels of apoptosis-associated genes.
- Performed transient transfection of human Fas, FADD, and caspase 8 genes.
Main Results:
- W7.2 cells were sensitive to apoptosis inducers, while mutants and Wbcl2 cells showed resistance.
- All Apt- mutants and Wbcl2 cells were resistant to ceramide and Fas-mediated apoptosis.
- Apt4.8 and Apt5.8 were sensitive to staurosporine, unlike Apt3.8 and Wbcl2.
- Recessive Apt- mutations affect steps upstream of mitochondrial dysfunction.
- Altered gene expression of Bcl-2 or Fas-related genes did not explain differential resistance.
- Transfection of Fas did not induce apoptosis, but FADD and caspase 8 did.
Conclusions:
- The recessive Apt- mutations impact early steps in apoptosis signaling, upstream of mitochondrial events.
- Genetic evidence suggests shared components exist in both dexamethasone- and Fas-mediated apoptotic pathways in W7.2 cells.
- These findings contribute to understanding the complex regulation of apoptosis and potential therapeutic targets.
