In silico analysis and DHPLC screening strategy identifies novel apoptotic gene targets of aberrant promoter hypermethylation in prostate cancer.
BACKGROUND
Aberrant DNA methylation has been implicated as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many cellular processes including apoptosis. Limited data is available on the methylation profile of apoptotic genes in prostate cancer (CaP). The aim of this study was to profile methylation of apoptotic-related genes in CaP using denaturing high performance liquid chromatography (DHPLC).


METHODS
Based on an in silico selection process, 13 genes were screened for methylation in CaP cell lines using DHPLC. Quantitative methylation specific PCR was employed to determine methylation levels in prostate tissue specimens (n = 135), representing tumor, histologically benign prostate, high-grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. Gene expression was measured by QRT-PCR in cell lines and tissue specimens.


RESULTS
The promoters of BIK, BNIP3, cFLIP, TMS1, DCR1, DCR2, and CDKN2A appeared fully or partially methylated in a number of malignant cell lines. This is the first report of aberrant methylation of BIK, BNIP3, and cFLIP in CaP. Quantitative methylation analysis in prostate tissues identified 5 genes (BNIP3, CDKN2A, DCR1, DCR2 and TMS1) which were frequently methylated in tumors but were unmethylated in 100% of benign tissues. Furthermore, 69% of tumors were methylated in at least one of the five-gene panel. In the case of all genes, except BNIP3, promoter hypermethylation was associated with concurrent downregulation of gene expression.


CONCLUSIONS
Future examination of this "CaP apoptotic methylation signature" in a larger cohort of patients is justified to further evaluate its value as a diagnostic and prognostic marker.
Murphy
Therese M
TM
Prostate Molecular Oncology, Institute of Molecular Medicine, Trinity College, Dublin, Ireland. murphyth@tcd.ie
Sullivan
Linda
L
Lane
Caroline
C
O'Connor
Lisa
L
Barrett
Ciara
C
Hollywood
Donal
D
Lynch
Thomas
T
Lawler
Mark
M
Perry
Antoinette S
AS
D000258:Adenovirus Infections, Human / Q000235:genetics; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D017209:Apoptosis / Q000235:genetics*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D015331:Cohort Studies; D019175:DNA Methylation; D015972:Gene Expression Regulation, Neoplastic; D018390:Gene Targeting; D056426:Genetic Loci; D006801:Humans; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D009691:Nucleic Acid Denaturation; D011401:Promoter Regions, Genetic; D011470:Prostatic Hyperplasia / Q000235:genetics; D019048:Prostatic Intraepithelial Neoplasia / Q000235:genetics*; D011471:Prostatic Neoplasms / Q000235:genetics*