EFFECTS OF ADENOVIRUS-MEDIATED P16(INK4A) EXPRESSION ON CELL-CYCLE ARREST ARE DETERMINED BY ENDOGENOUS P16 AND RB STATUS IN HUMAN CANCER-CELLS

We constructed an adenoviral vector containing human p16 cDNA in order to evaluate the cytotoxic effects of exogenous p16 expression on canc er cell proliferation and to explore the potential use of p16 in cance r gene therapy, Following infection of human breast (MCF-7, MDA-MB-231 , and BT549), osteosarcoma (U-2 OS and Saos-2), cervical (C33a), and l ung cancer (H358) cell lines with the recombinant adenovirus Adp16, hi gh levels of p16 expression were observed in all cell lines. Cancer ce ll lines which were mutant or null for p16 but wild-type for the retin oblastoma gene product (pRb) (MCF-7, MDA-MB-231, BT549 and U-2 OS) wer e 7-22-fold more sensitive to the cytotoxic effects of Adp16 than to a control virus, In contrast, cancer cell lines which were wild-type fo r p16 but mutant or null for pRb (Saos-2, C33a and H358) were <threefo ld more sensitive to Adp16 when compared to a control virus, Analysis of 5-bromodeoxyuridine incorporation into DNA following infection with Adp16 showed a loss of S phase in those cell lines which were null or mutant for p16 but expressed a functional pRb. This cell cycle arrest was associated with binding of the p16 protein to cyclin-dependent ki nase 4 and dephosphorylation of pRb. In contrast, human cancer cell li nes expressing a wild-type p16 and a mutant pRb or no pRb showed no su bstantial loss of S phase following Adp16 infection. Based on these st udies, we conclude that p16-mediated cytotoxicity is tightly associate d with the presence of functional pRb in human cancer cells, and that tumor cells which are mutant or null for p16 are candidates for Adp16 mediated cancer gene therapy.