OBJECTIVE: The cell cycle regulatory protein p16 (CDKN2/cyclin dependent kinase 4 inhibitor/multiple tumor suppressor-1) causes cell cycle arrest at the G1 checkpoint by inhibiting activity of cyclin D-CDK4 complexes. The purpose of this study is to assess the effect of introduction of the p16 gene into two ovarian cancer cell lines via a recombinant adenoviral vector (Ad5CMV-p16). METHODS: Cells lines used were SKOV3, which has a p16 deletion, and OVCA420, which has normal p16. Transduction efficiency was established by infecting cells with an adenovirus containing the Escherichia coli beta-galactosidase gene (Ad5CMV-beta-gal) at multiplicity of infection from 0 to 1000 and staining for X-gal. Cells were infected with Ad5CMV-p16 and cell growth was assessed by counting cells every other day for up to 7 days. Western blotting was done to assess for p16 expression after infection. Fluorescence-activated cell sorting after staining with propidium iodide was done to assess the effect of p16 on the cell cycle. RESULTS: The SKOV3 cell line was transduced with the adenovirus at a slightly lower MOI than the OVCA420 cell line. Growth of the Ad5CMV-p16-infected cells was suppressed 75-80% by cell count in both cell lines and caused morphologic changes of the cells consistent with apoptosis. The p16 protein expression was seen to increase within 24 h after introduction of the p16 gene. G1 arrest of cells occurred beginning 24 h after introduction of the p16 gene. CONCLUSIONS: These results suggest that Ad5CMV-p16 may be further studied as a potential therapeutic agent for ovarian cancer as introduction of the p16 gene into ovarian cancer cell lines causes a G1 arrest and attenuation of growth, regardless of the endogenous p16 status of the cells. Copyright 1999 Academic Press.
OBJECTIVE: The cell cycle regulatory protein p16 (CDKN2/cyclin dependent kinase 4 inhibitor/multiple tumor suppressor-1) causes cell cycle arrest at the G1 checkpoint by inhibiting activity of cyclin D-CDK4 complexes. The purpose of this study is to assess the effect of introduction of the p16 gene into two ovarian cancer cell lines via a recombinant adenoviral vector (Ad5CMV-p16). METHODS: Cells lines used were SKOV3, which has a p16 deletion, and OVCA420, which has normal p16. Transduction efficiency was established by infecting cells with an adenovirus containing the Escherichia coli beta-galactosidase gene (Ad5CMV-beta-gal) at multiplicity of infection from 0 to 1000 and staining for X-gal. Cells were infected with Ad5CMV-p16 and cell growth was assessed by counting cells every other day for up to 7 days. Western blotting was done to assess for p16 expression after infection. Fluorescence-activated cell sorting after staining with propidium iodide was done to assess the effect of p16 on the cell cycle. RESULTS: The SKOV3 cell line was transduced with the adenovirus at a slightly lower MOI than the OVCA420 cell line. Growth of the Ad5CMV-p16-infected cells was suppressed 75-80% by cell count in both cell lines and caused morphologic changes of the cells consistent with apoptosis. The p16 protein expression was seen to increase within 24 h after introduction of the p16 gene. G1 arrest of cells occurred beginning 24 h after introduction of the p16 gene. CONCLUSIONS: These results suggest that Ad5CMV-p16 may be further studied as a potential therapeutic agent for ovarian cancer as introduction of the p16 gene into ovarian cancer cell lines causes a G1 arrest and attenuation of growth, regardless of the endogenous p16 status of the cells. Copyright 1999 Academic Press.
Authors: Thanh U Barbie; David A Barbie; David T MacLaughlin; Shyamala Maheswaran; Patricia K Donahoe Journal: Proc Natl Acad Sci U S A Date: 2003-12-11 Impact factor: 11.205