Adenovirus-mediated E2F-1 gene transfer efficiently induces apoptosis in melanoma cells.

Abstract

E2F-1 is a transcription factor that stimulates cellular proliferation and cell cycle progression from G(1) to S-phase. Somewhat paradoxically, E2F-1 also has the properties of a tumor suppressor. Overexpression of E2F-1 has been shown to induce apoptosis in some cancer cells. In the current study, the effect of adenovirus-mediated E2F-1 gene transfer on human melanoma cell growth was investigated. Two human melanoma cell lines, SK-MEL-28 (wild-type p53) and SK-MEL-2 (mutant p53), were treated by mock infection, infection with a control vector expressing the beta-galactosidase gene (Ad5CMV-LacZ), or infection with a vector expressing E2F-1 (Ad5CMV-E2F-1) at a multiplicity of infection of 100. Cell proliferation and viability were determined by WST-1 assay and trypan blue exclusion, respectively. Apoptosis was assessed by cell flow cytometry and confirmed by cell morphology, in situ terminal deoxynucleotidyl nick end labeling assay, and poly(ADP-ribose) polymerase cleavage assay. Marked overexpression of E2F-1 was evident in both cell lines 24 hours after infection with Ad5CMVE2F-1 by Western blot analysis. E2F-1 overexpression resulted in growth inhibition and rapid loss of cell viability. Overexpression of E2F-1 also resulted in premature S-phase entry and G(2) arrest at 24 hours followed by apoptotic cell death at 48 hours. After Ad5CMVE2F-1 infection, expression of Bax and Bak was unchanged, whereas Mcl-1 levels decreased markedly. In SK-MEL-28 cells, Bcl-XL levels also declined after E2F-1 expression. Bcl-2 was undetectable in SK-MEL-28 cells but was increased in SK-MEL-2 cells in response to E2F-1 overexpression. Adenovirus-mediated E2F-1 gene transfer efficiently induces widespread apoptosis in human melanoma cells. E2F-1 overexpression induced apoptosis in cell lines containing wild-type and mutant p53, suggesting that this effect does not require wild-type p53 function. Anti-apoptotic proteins of the Bcl-2 family, notably Mcl-1 and Bcl-XL, may be involved in mediating the response to E2F-1. These data suggest that adenovirus-mediated E2F-1 gene therapy may be effective in the treatment of melanoma.