Methylation and mutational analysis of p27(kip1) 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 this locus is the cyclin dependent kinase inhibitor p27(kip1), which has been implicated as a marker of aggressive prostate carcinoma. Herein, we examine metastatic prostate tumors, xenografts, and cell lines for gene inactivation via mutational inactivation or promoter hypermethylation.


METHODS
Mutation analysis was performed on metastatic prostate tumors of 18 patients, eight prostate carcinoma cell lines, and 18 xenografts by PCR amplification of the entire open reading frame of p27(kip1). PCR products were sequenced directly using internal primers. Methylation analysis was performed on four cell lines and nine xenografts using direct sequencing of cloned 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 identified 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 promotor methylation in all sequenced clones and the number of methylated base 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 metastatic prostate carcinoma. While CpG methylation does occur, it is an infrequent event and does not appear to be the mechanism of p27(kip1) down regulation in prostate carcinoma.
Kibel
A S
AS
Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63105, USA. kibela@msnotes.wustl.edu
Christopher
M
M
Faith
D A
DA
Bova
G S
GS
Goodfellow
P J
PJ
Isaacs
W B
WB
D018797:Cell Cycle Proteins / Q000235:genetics*; D050760:Cyclin-Dependent Kinase Inhibitor p27; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D018816:Genes, cdc; D006801:Humans; D008297:Male; D009154:Mutation; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D004273:DNA, Neoplasm; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27