Mutations in P21RAS and B-catenin in Azoxymethane-Induced Rat Colonic Tumors Differentially Regulate the ERKS, COX-2 and Cyclin D1

PTEN gene expression is suggested to be due to hypermethylation of precoding region, but direct evidence has not been reported.Using colon cancer cell line, we purposed to certify this possibility that expression of PTEN gene is regulated by methylation of DNA.MaterialslMethods: We have analyzed 6 colon cancer cell lines, T-84, HT-29, SW480, SW620, CaC02, and Col0320DM provided from ATCC.Expression of PTEN gene transcript and protein expression were examined by RT-PCR-Southern analysis and Western blot with polyclonal goat anti-PTEN antibody (Santa Cruz Biotechnology), respectively.Analysis of DNA sequence was performed by direct sequencing technique.Results: RT-PCR-Southern analysis showed that PTEN gene transcript was expressed in CaC02, HT-29, SW480, and SW620 cells, but significantly reduced in T-84 and Col0320DM cells.Protein expressions of PTEN examined by Western blot were also reduced in these two cell lines.However, mRNA and protein expression of PTEN were augmented by demethylating agent 5-Azadeoxycitidine, added to the culture medium in T-84 and Col0320DM cells.Direct sequencing of 800 bps upstream to start codon in exon I of PTEN gene, which was treated with bisulfite-converting reagents revealed that over 90% of CpG sequence of PTEN gene in T-84 cells were methylated.In contrast, CpGs in this region of CaC02 cells were not methylated, and those of HT-29 cells were partially (54%) methylated.Conclusions: These results indicate that expression of PTEN gene is regulated by hypermethylation of precoding region in some colon cancer cell lines.However, PTEN gene product is weakly expressed even in T-84 cells.This result suggests that suppression of PTEN gene seems to be not essential in carcinogenesis, but it may be important to tumor cell survival in colon cancer.