Regulation of Fas-Mediated Apoptosis by N-ras in Melanoma

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.

Role of reactive oxygen species in proapoptotic ability of oncogenic H-Ras to increase human bladder cancer cell susceptibility to histone deacetylase inhibitor for caspase induction

“…In contrast, FasL treatment induced caspase-8 activation, downregulation of FLIP, and cell death in the human urinary bladder cancer T24 cell line, which hosts the endogenous, oncogenic H-ras mutant gene (Taparowsky et al 1982). Our results showed that expression of oncogenic H-Ras did not result in any changes in FasL, Fas, or FLIP protein level, which is consistent with another report (Urquhart et al 2002); expression of oncogenic H-Ras is unlikely to play any roles in regulation of FasL, Fas, and FLIP expression in J82 cells. In addition, FR901228 treatment failed to induce any detectable changes in the protein levels of TNFand TRAIL in either J82 or J82-Ras cells (data not shown).…”

Oncogenic RAS directs silencing of tumor suppressor genes through ordered recruitment of transcriptional repressors

“…For example, as in murine cells, activated RAS silences FAS in human cells (Urquhart et al 2002;Gazin et al 2007). Moreover, FAS silencing also occurs in some transformed cells, human tumors, and mouse models of cancer and has been shown to be relevant to both tumor progression (for example, see Hopkins-Donaldson et al 2003) and chemotherapeutic resistance (Maecker et al 2002).…”

Chemoprevention of melanoma

“…In particular, ras has been shown to protect cells from Fas-mediated apoptosis [42]. Ras decreased melanoma susceptibility to Fas-mediated cell death through downregulation of Fas receptor gene expression [40]. These data suggest that ras inhibitors may increase the susceptibility of melanoma cells to Fas-mediated cell death.…”

“…The functional contribution of MAP kinase activation to melanoma development has been described by Govindarajan et al [39••]. Another important aspect of ras signaling is that ras mutations promote resistance to apoptosis, thus contributing to tumor progression [40,41]. In particular, ras has been shown to protect cells from Fas-mediated apoptosis [42].…”