Re-expression of endogenous p16(ink4a) in oral squamous cell carcinoma lines by 5-aza-2 '-deoxycytidine treatment induces a senescence-like state

We have previously reported that a set of oral squamous cell carcinoma line s express specifically elevated cdk6 activity. One of the cell lines, SCC4, contains a cdk6 amplification and expresses functional p16(ink4a), the oth er cell lines express undetectable levels of p16(ink4a), despite a lack of coding-region mutations. Two of the cell lines, SCC15 and SCC40 have a hype rmethylated p16(ink4A) promoter and a third cell line, SCC9, has a mutation in the p16(ink4a) promoter. Using the demethylation agent 5-aza-2'-deoxycy tidine, we showed that the p16(ink4a) protein was re-expressed after a 5-da y treatment with this chemical, One cell line, SCC15 espressed high levels of p16(ink4a). In this line, cdk6 activity was decreased after 5-aza-2'deox ycytidine treatment, and the hypophosphorylated, growth suppressive form of the retinoblastoma tumor suppressor protein PRE was detected. Expression o f p16(ink4a) persisted, even after the drug was removed and the cells espre ssed senescence-associated beta-galactosidase activity. Ectopic expression of p16(ink4a) with a recombinant retrovirus in this cell line also induced a similar senescence-like phenotype, Hence, it was possible to restore a fu nctional pRB pathway in an oral squamous cell carcinoma line by inducing re -expression of endogenous p16(ink4a) in response to treatment with a demeth ylating agent.