Hypomethylation of WNT5A, CRIP1 and S100P in prostate cancer.

Oligoarray analysis of a matched pair of prostate cancer and normal cell lines derived from the same radical prostatectomy specimen identified 113 candidate hypomethylated genes that were overexpressed in the cancer cells and contained CpG islands. Hypomethylation of wingless-related MMTV integration site 5A (WNT5A), S100 calcium-binding protein P (S100P) and cysteine-rich protein 1(CRIP1) was confirmed in the cancer cells by bisulfite sequencing. Treatment of the corresponding normal prostate epithelial cells 1542-NPTX with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-aza-CdR) induced higher levels of mRNA expression and partial loss of methylation on these genes. Primary prostate cancers were tested using methylation-specific polymerase chain reaction. WNT5A was hypomethylated in 11/17 (65%) tumors, S100P in 8/16 (50%) and CRIP1 in 13/20 (65%). Bisulfite sequencing of a section of the 5' untranslated region (UTR) of WNT5A revealed that three CpG sites (15, 24 and 35) were consistently methylated (93%) in the normal cell line and normal tissues, but not in the prostate cancer cell line and eight primary prostate cancers. Multiple putative binding sites for the transcription factors SP1 and AP-2 were found adjacent to CpG sites 15 and 24. A putative c-Myb binding site was located within the CpG site 35. Anti-c-Myb antibody co-precipitation with WNT5A was methylation-sensitive in 1542-NPTX cells. It is likely that an epigenetic mechanism regulates WNT5A expression in prostate cancer.

Genome-wide screening for genetic changes in a matched pair of benign and prostate cancer cell lines using array CGH.

Copy number alterations in a matched pair of benign epithelial and prostate cancer cell lines derived from the same patient were assessed using array-based comparative genomic hybridisation (aCGH). The cancer cell line showed a gain of chromosome 7, deletion of chromosome 8, gains (including high level) and losses on chromosome 11, loss of 18p and gain of 20q. Deletions on chromosome 8 were confirmed with microsatellite markers. The aCGH results were compared to gene expression data obtained using DNA microarrays and suggested the involvement of caspases and ICEBERG on 11q and E2F1 on chromosome 20q.

Isolation and sequencing of the cDNA of a novel cytochrome P450 from rat oesophagus.

RT-PCR was used to find whether cytochromes P450 of the 2A, 2B and 2E sub-families are expressed in the rat oesophagus. This showed that this tissue expresses a previously unknown member of the CYP2B sub-family, now designated CYP2B21. Using a combination of 5'- and 3'-RACE (rapid amplification of cDNA ends) and library screening, the cDNA was amplified and sequenced. The cDNA sequence (GenBank accession no. AF159245) covers the whole of the coding region and the whole of the 3'-untranslated region (UTR), but only 17 nt of the 5'-UTR. The DNA sequence has strong similarity to those of CYP2B1 and CYP2B2, with the derived amino acid sequence being 84 and 83% identical, respectively. The ease with which this cDNA was found in the cDNA library suggests that CYP2B21 is a major P450 of the oesophagus. The catalytic activity of this new CYP2B is not yet known, but as previous authors have reported that other members of this sub-family (CYP2B1 or 2B2) metabolize the selective oesophageal carcinogen N:-nitrosomethylbutylamine with the chemical selectivity necessary for carcinogenesis, i.e. they preferentially hydroxylate the alpha-carbon of the butyl chain, this new CYP2B may be the nitrosamine-activating enzyme of the oesophagus.