title: Adenovirus-mediated gene transfer of FKHRL1 triple mutant efficiently induces apoptosis in melanoma cells.
abstract: The PTEN/Akt signal pathway plays an important role in tumorigenesis. Mutations or deletions of PTEN have been observed in up to 60% of melanoma cell lines, resulting in PI3K/Akt activation. The Forkhead family of transcription factors induce apoptosis in their unphosphorylated forms and were recently reported to be a substrate of Akt kinase. In the present study, an adenovirus expressing a triple mutant (TM) of FKHRL1, which cannot be phosphorylated by Akt, was assessed for its ability to induce apoptosis in melanoma cells. Marked overexpression of FKHRL1/TM was evident in the SK-MEL-2 cell line 24 hours after infection with Ad-FKHRL1/TM by Western blot analysis. The expression of FKHRL1/ TM was moderately delayed in SK-MEL-28 cells. Overexpression of FKHRL1/TM can efficiently inhibit melanoma cell growth and result in rapid loss of cell viability. Cell cycle analysis showed overexpression of FKHRL1/TM in both melanoma cell lines resulted in development of a Sub-G1 population, indicating apoptosis by Ad-FKHRL1/TM infection. Apoptosis was confirmed by morphologic inspection, poly-ADP-ribosepolymerase (PARP) cleavage assay, and annexin V-PE analysis. After Ad-FKHRL1/TM infection, the expression of Bax and Bak did not differ markedly, whereas Mcl-1 and Bcl-x(L) levels decreased markedly. Involvement of caspase 3 and 6 in FKHRL1/TM-mediated apoptosis was demonstrated by cleavage of caspase 3/CPP32 and PARP as well as fragmentation of the caspase 6 substrate lamin B in SK-MEL-2 cells as early as 24 hours after Ad-FKHRL1/ TM infection, but those events were delayed 72 hours in SK-MEL-28. In addition, we found that p27(kip1) was cleaved in SK-MEL-2 cells at 24 hours after treatment with Ad-FKHRL1/TM. This cleavage was observed in SK-MEL-28 cells until 72 hours after infection with Ad-FKHRL1/TM. Our data suggest that adenovirus expressing a FKHRL1 triple mutant could be a useful vector for gene therapy of cancers resistant to chemotherapy and radiotherapy induced by hyperactivity of PI3K/Akt.
authors.affiliation: James Graham Brown Cancer Center, Department of Surgery, University of Louisville School of Medicine, Louisville, Kentucky 40292, USA.
authors.forename: Jorge G
authors.initials: JG
authors.lastname: Gomez-Gutierrez
authors.forename: Vinicius
authors.initials: V
authors.lastname: Souza
authors.forename: Hong Ying
authors.initials: HY
authors.lastname: Hao
authors.forename: Roberto
authors.initials: R
authors.lastname: Montes de Oca-Luna
authors.forename: Yan Bin
authors.initials: YB
authors.lastname: Dong
authors.forename: H Sam
authors.initials: HS
authors.lastname: Zhou
authors.forename: Kelly M
authors.initials: KM
authors.lastname: McMasters
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis / Q000235:genetics; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics; D045744:Cell Line, Tumor; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000235:genetics* / Q000378:metabolism; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D008545:Melanoma / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D009154:Mutation; D010766:Phosphorylation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012878:Skin Neoplasms / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D014161:Transduction, Genetic
chemical_list: C499517:FOXO3 protein, human; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D019253:Proto-Oncogene Proteins c-bcl-2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D020169:Caspases