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

Abstract

We have previously reported that a set of oral squamous cell carcinoma lines express specifically elevated cdk6 activity. One of the cell lines, SCC4, contains a cdk6 amplification and expresses functional p16(ink4a), the other 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 hypermethylated p16(ink4a) promoter and a third cell line, SCC9, has a mutation in the p16(ink4a) promoter. Using the demethylation agent 5-aza-2'-deoxycytidine, we showed that the p16(ink4a) protein was re-expressed after a 5-day treatment with this chemical. One cell line, SCC15 expressed high levels of p16(ink4a). In this line, cdk6 activity was decreased after 5-aza-2'deoxycytidine treatment, and the hypophosphorylated, growth suppressive form of the retinoblastoma tumor suppressor protein pRB was detected. Expression of p16(ink4a) persisted, even after the drug was removed and the cells expressed senescence-associated β-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 functional pRB pathway in an oral squamous cell carcinoma line by inducing re-expression of endogenous p16(ink4a) in response to treatment with a demethylating agent.