CDKN2 gene silencing in lung cancer by DNA hypermethylation and kinetics of p16INK4 protein induction by 5-aza 2'deoxycytidine.
Absent expression of the cyclin dependent kinase-inhibitor, p16INK4, is observed in a wide range of primary human cancers. Although homozygous deletions and point mutations have been reported in a subset of these tumors, the molecular basis for absent p16INK4 in other samples is unknown. We have examined 33 tumor cell lines and have shown that hypermethylation of a G:C-rich region within exon 1 of the CDKN2 gene was present in 100% of samples with wildtype RB expression and no detectable CDKN2 mutations. Treatment for at least 4 hours with the demethylating agent 5-aza 2'deoxycytidine, but not 5-azacytidine or 6-azacytidine, induces the prolonged expression of p16INK4 protein in each of these samples following a discrete 24-48 hour lag period. Consistent with the hypothesis that hypermethylation of the CDKN2 gene is a tumor-specific mechanism for gene inactivation, we observed hypomethylation at the exon 1 site exclusively in tumor lines that expressed p16INK4 or that had sustained inactivating point mutations within the CDKN2 open reading frame. These findings demonstrate a link between DNA methylation and the p16INK4:RB tumor suppressor pathway.
Otterson
G A
GA
NCI-Navy Oncology Branch, National Cancer Institute, Bethesda, MD 20889, USA.
Khleif
S N
SN
Chen
W
W
Coxon
A B
AB
Kaye
F J
FJ
D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics*; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004247:DNA / Q000378:metabolism; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors*; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D047428:Protein Kinase Inhibitors; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D000077209:Decitabine; D004247:DNA; D015254:DNA Modification Methylases; D001374:Azacitidine