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Regulation of Fas-mediated apoptosis by N-ras in melanoma
Jean L Urquhart1, Sandra J Meech, David G Marr
1University of Colorado Health Sciences Center, Department of Dermatology, Denver, Colorado 80262, USA. jean.urquhart@uchsc.edu
Abstract:
Oncogenic ras has been shown to downregulate Fas receptor expression and increase Fas ligand expression and thus contribute to resistance to Fas-mediated cell death in several cell types. The effects of ras on Fas-mediated apoptosis have not been studied in melanoma. We studied the effects of activated N-ras by measuring Fas, Fas ligand, and FLIP expression as well as susceptibility to Fas-ligand-induced cell death in transfectants of WM35, a radial growth phase human melanoma cell line. Based on quantitative polymerase chain reaction and fluorescence-activated cell sorter analysis, we found that the ras transfectants expressed less Fas mRNA and surface Fas receptor. Cr51 release cytotoxicity assays demonstrated less susceptibility to Fas-mediated apoptosis in ras transfectants, correlating with the Fas mRNA and protein expression results. Ras inhibition with the specific inhibitor FTI-277 showed that downregulation of Fas in the ras transfectants could be reversed. This correlates with cytotoxicity experiments showing that ras inhibition increases susceptibility to Fas-mediated apoptosis. The control transfectants expressed FLIP but ras did not affect FLIP expression. The control and ras transfectants did not express Fas ligand as demonstrated by reverse transcriptase polymerase chain reaction and fluorescence-activated cell sorter analysis. Cytotoxicity assays further confirmed that these melanoma ras transfectants do not express functional Fas ligand. These results suggest that ras contributes to tumor progression by decreasing susceptibility to Fas-mediated cell death at least in part through downregulation of Fas receptor at the transcriptional level.
Insights
Oncogenic ras signaling in melanoma cells reduces Fas receptor expression, leading to resistance against Fas-mediated cell death. Inhibiting ras restores Fas expression and increases melanoma cell susceptibility to apoptosis.
Area of Science:
- Oncology
- Molecular Biology
- Cell Death Research
Background:
- Oncogenic ras signaling is implicated in resistance to Fas-mediated cell death in various cell types.
- The specific impact of ras on Fas-mediated apoptosis in melanoma remains largely uncharacterized.
Purpose of the Study:
- To investigate the effects of activated N-ras on Fas receptor, Fas ligand, and FLIP expression in human melanoma cells.
- To determine the susceptibility of melanoma cells with activated ras to Fas-ligand-induced apoptosis.
Main Methods:
- Utilized quantitative polymerase chain reaction (qPCR) and fluorescence-activated cell sorter (FACS) analysis to measure gene and protein expression.
- Employed Cr51 release cytotoxicity assays to assess apoptosis susceptibility.
- Investigated the effects of the ras inhibitor FTI-277.
Main Results:
- Ras transfectants exhibited decreased Fas mRNA and surface Fas receptor expression.
- Melanoma cells with activated ras showed reduced susceptibility to Fas-mediated apoptosis.
- Ras inhibition reversed Fas downregulation and increased apoptosis susceptibility.
- Ras activation did not affect FLIP expression or Fas ligand expression in these melanoma cells.
Conclusions:
- Activated ras contributes to melanoma progression by downregulating Fas receptor expression at the transcriptional level.
- This downregulation of Fas receptor decreases melanoma cell susceptibility to Fas-mediated cell death.
- Targeting ras may represent a therapeutic strategy to enhance apoptosis in melanoma.