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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Dong
Y B
YB
Department of Surgery, University of Louisville, James Graham Brown Cancer Center, Louisville, Kentucky, USA.
Yang
H L
HL
Elliott
M J
MJ
Liu
T J
TJ
Stilwell
A
A
Atienza
C
C
McMasters
K M
KM
D017502:Adenovirus E2 Proteins / Q000235:genetics*; D017209:Apoptosis / Q000235:genetics*; D002453:Cell Cycle / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D015536:Down-Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D008545:Melanoma / Q000473:pathology / Q000628:therapy*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
D017502:Adenovirus E2 Proteins; D019253:Proto-Oncogene Proteins c-bcl-2