Methylation and mutational analysis of p27(kipl) in prostate carcinoma

BACKGROUND. We have previously identified 12p12-13 as a region of frequent genetic loss in prostate carcinoma. A candidate tumor suppressor gene at th is locus is the cyclin dependent kinase inhibitor p27(kip1), which has been implicated as a marker of aggressive prostate carcinoma. Herein, we examin e metastatic prostate tumors, xenografts, and cell lines for gene inactivat ion via mutational inactivation or promoter hypermethylation. METHODS. Mutation analysis was performed on metastatic prostate tumors of 1 8 patients, eight prostate carcinoma cell lines, and 18 xenografts by PCR a mplification of the entire open reading frame of p27(kip1). PCR products we re sequenced directly using internal primers. Methylation analysis was perf ormed on four cell lines and nine xenografts using direct sequencing of clo ned PCR products of bisulfite treated DNA. Presence of a CpG was consistent with methylation of that cytosine in the original sample. RESULTS. With the exception of the previously reported homozygous deletion, no additional mutations were identified. Methylated CpG residues were iden tified in three xenografts (LuCAP23, LuCAP35, and PC82) and the methylated residues clustered at six sites; the cytosines 69, 149, 191, 286, 349, and 487 base pairs 5' of the ATG start codon. However, no sample demonstrated p romotor methylation in all sequenced clones and the number of methylated ba se pairs ranged from seven to three, not the level usually associated with gene silencing. CONCLUSIONS. Mutational inactivation of p27(kip1) is a rare event in metast atic prostate carcinoma. While CpG methylation does occur, it is an infrequ ent event and does not appear to be the mechanism of p27(kip1) down regulat ion in prostate carcinoma.