Re-expression of endogenous p16ink4a 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 lines express specifically elevated cdk6 activity. One of the cell lines, SCC4, contains a cdk6 amplification and expresses functional p16ink4a, the other cell lines express undetectable levels of p16ink4a, despite a lack of coding-region mutations. Two of the cell lines, SCC15 and SCC40 have a hypermethylated p16ink4A promoter and a third cell line, SCC9, has a mutation in the p16ink4a promoter. Using the demethylation agent 5-aza-2'-deoxycytidine, we showed that the p16ink4a protein was re-expressed after a 5-day treatment with this chemical. One cell line, SCC15 expressed high levels of p16ink4a. 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 p16ink4a persisted, even after the drug was removed and the cells expressed senescence-associated beta-galactosidase activity. Ectopic expression of p16ink4a 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 p16ink4a in response to treatment with a demethylating agent.
Pathology Department and Harvard Center for Cancer Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
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Timmermann
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Hinds
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Münger
D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000235:genetics*; D016922:Cellular Senescence / Q000187:drug effects*; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000187:drug effects / Q000235:genetics*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors*; D000077209:Decitabine; D015972:Gene Expression Regulation, Neoplastic; D018014:Gene Transfer Techniques; D006801:Humans; D009062:Mouth Neoplasms / Q000188:drug therapy / Q000235:genetics*; D009154:Mutation; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000187:drug effects / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D019941:Cyclin-Dependent Kinase Inhibitor p16; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; D000077209:Decitabine; D015254:DNA Modification Methylases; D017346:Protein Serine-Threonine Kinases; C495900:CDK4 protein, human; C495911:CDK6 protein, human; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases; D001616:beta-Galactosidase; D001374:Azacitidine