Analysis of cyclin-dependent kinase inhibitor expression and methylation patterns in human prostate cancers.
BACKGROUND
Downregulation of genes which negatively control cell cycle progression represents a possible mechanism for prostate tumorigenesis. We examined the expression levels of the p16, p15, p14, and retinoblastoma-susceptibility (RB) genes in primary prostate cancers and human prostate cancer cell lines, and correlated this with the DNA methylation levels of two loci in p16.


METHODS
The mRNA levels of p16, p15, and p14 were examined by reverse transcriptase-PCR (RT-PCR). DNA methylation of the p16 5' CpG island was determined by bisulfite genomic sequencing, while methylation of exon 2 shared by the p16 and p14 genes was measured by a quantitative bisulfite-based technique, methylation-sensitive single-nucleotide primer extension (Ms-SNuPE). RB protein levels were assessed by immunohistochemical staining of histologic sections of normal and tumor prostate tissues, using a monoclonal antibody (mAB).


RESULTS
Overexpression of p16 mRNA was found in 6/9 (67) of prostate tumors compared to the adjacent normal prostate, whereas elevated p14 and p15 levels were only observed in 2/9 (22) and 1/6 (17) of prostate cases, respectively. There was no statistically significant association of grade (P = 0.18) and stage (P = 1.00) of prostate cancer to the elevated p16 levels in the tumors. The p16 5' CpG island was completely unmethylated in these tissues. In contrast, exon 2 of p16/p14 was methylated in both the tumor and normal adjacent prostates, and was increased in 8/11 (73) of tumors relative to normal tissues. There was no association between p16 overexpression and increased p16/p14 exon 2 methylation in these tumors (P = 1.00). Diminished RB levels in prostate tumors that had upregulated p16 mRNA were found, although absent RB was also detected in tumors without elevated p16 levels. The expression levels of the two genes, RB and p16, were not correlated statistically (P = 0.16).


CONCLUSIONS
Our studies show that although the levels of the cell cycle regulators p16, p15, p14, and Rb are altered in prostate cancers, there is no apparent correlation to grade, stage, or any pattern of regulation between the related genes. Exon 2 of p16/p14 is methylated in a majority of prostate tumors compared to the unmethylated upstream 5' region, and may be a potential tumor marker for human prostate cancer.
Nguyen
T T
TT
Department of Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles, CA 90089-9181, USA.
Nguyen
C T
CT
Gonzales
F A
FA
Nichols
P W
PW
Yu
M C
MC
Jones
P A
PA
D018899:CpG Islands / Q000235:genetics; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics* / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D019175:DNA Methylation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005091:Exons; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D019941:Cyclin-Dependent Kinase Inhibitor p16; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D016160:Retinoblastoma Protein; D018844:Cyclin-Dependent Kinases