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.