title: Adenovirus-mediated E2F-1 gene transfer sensitizes melanoma cells to apoptosis induced by topoisomerase II inhibitors.
abstract: Melanoma has proven to be resistant to conventional chemotherapy; however,the mechanism of chemoresistance is still unclear. Recent reports show that the transcription factor, E2F-1, may play a role in mediating cytotoxicity of certain chemotherapeutic agents. We have shown in a previous study that adenovirus-mediated overexpression of E2F-1 can efficiently induce apoptosis in melanoma cells. In the present study, the effect of E2F-1 expression on drug sensitivity of melanoma cells was evaluated. Two human melanoma cell lines, SK-MEL-28 and SK-MEL-2, were treated with drugs (etoposide, Adriamycin, roscovitine, cisplatin, 5-fluorouracil, or cycloheximide), alone or in combination with adenoviral vectors expressing beta-galactosidase (Ad-LacZ) or E2F-1 (Ad-E2F-1) at a multiplicity of infection of 1 in vitro. E2F-1 expression was confirmed by Western blot analysis. Sublethal concentrations of each drug alone or infection with Ad-E2F-1 alone produced <5% apoptosis by 3 days posttreatment. Conversely, cotreatment with Ad-E2F-1 and low concentrations of etoposide or Adriamycin markedly sensitized melanoma cells to apoptotic cell death. A slight enhancement of the cytotoxicity of roscovitine was demonstrated in combination with E2F-1 overexpression, but not to cisplatin, 5-fluorouracil, or cycloheximide. Ad-LacZ infection showed no obvious effects on drug sensitivity. Overexpression of p21 can block apoptosis induced by the combination chemogene therapy of Ad-E2F-1 and topoisomerase II poisons and does not require its proliferating cell nuclear antigen-binding ability. The protein synthesis inhibitor cycloheximide also has a cytotoxicity-protective effect against topoisomerase II inhibitor/E2F-1-induced apoptosis and suggests that new protein synthesis is required for this process. Topoisomerase II inhibitors also cooperated with Ad-E2F-1 to enhance antitumor activity in an in vivo model using xenografts in nude mice. When combined with Adriamycin or etoposide, E2F-1 adenovirus therapy resulted in an 87% or 91% decrease in tumor size, respectively, compared with controls (P < 0.002). Our results show that adenovirus-mediated E2F-1 gene transfer can sensitize melanoma cells to some chemotherapeutic agents, particularly topoisomerase II poisons, in vitro and in vivo. These results suggest a new chemosensitization strategy for melanoma gene therapy.
authors.affiliation: Department of Surgery, University of Louisville, James Graham Brown Cancer Center, Louisville, Kentucky 40202, USA.
authors.forename: Yan Bin
authors.initials: YB
authors.lastname: Dong
authors.forename: Hai Liang
authors.initials: HL
authors.lastname: Yang
authors.forename: Mary Jane
authors.initials: MJ
authors.lastname: Elliott
authors.forename: Kelly M
authors.initials: KM
authors.lastname: McMasters
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D018797:Cell Cycle Proteins; D002945:Cisplatin / Q000494:pharmacology; D003131:Combined Modality Therapy; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins; D004317:Doxorubicin / Q000494:pharmacology; D004357:Drug Synergism; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005047:Etoposide / Q000494:pharmacology; D005472:Fluorouracil / Q000494:pharmacology; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008297:Male; D008545:Melanoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011687:Purines / Q000494:pharmacology; D000077546:Roscovitine; D059005:Topoisomerase II Inhibitors; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C493754:E2f1 protein, mouse; D004791:Enzyme Inhibitors; D018809:Proliferating Cell Nuclear Antigen; D011687:Purines; D059005:Topoisomerase II Inhibitors; D014157:Transcription Factors; D000077546:Roscovitine; D005047:Etoposide; D004317:Doxorubicin; D003513:Cycloheximide; D002945:Cisplatin; D005472:Fluorouracil