Glutathione S-transferase-pi overexpression is closely associated with K-ras mutation during human colon carcinogenesis

Background & Aims: In colorectal adenoma and carcinoma, glutathione S-trans ferase-pi (GSTP1-1) is highly expressed. K-ras mutation is also known to oc cur frequently in colorectal adenoma and carcinoma, as well as in the putat ive precursor of adenoma, aberrant crypt foci (ACF). Further, forced expres sion of v-H-ras in rat liver epithelial cells has been shown to enhance rat Tr-class GST expression. The aim of the present study is, therefore, to in vestigate the causative relationship between GSTP1-1 overexpression and K-r as mutation in these lesions, Methods: Twenty-seven specimens of colorectal carcinoma, 24 of adenoma, and 28 of AGF were examined in this study. The e xpression of GSTP1-1 or p21(K-ras) was examined by immunohistochemistry. Th e GSTP1-1 messenger RNA levels were measured by Taq-Man reverse-transcripti on polymerase chain reaction (PCR). K-ras mutation was detected by two-step PCR restriction fragment length polymorphism. v-K-ras transfection to RPMI -4788 colon carcinoma cells was carried out by the lipofection method. Acti vities of GSTP1-1 promoters containing AP-1 and Sp1 responsive elements in the v-K-ras transfectants were measured by a secreted form of human placent al alkaline phosphatase (SEAP) assay. Nuclear protein from these transfecta nts bound to the GSTP1-1 promoter was analyzed by electrophoretic mobility shift assay (EMSA). Results: In human colorectal carcinoma, adenoma, and AC F, close association of increased expression of GSTP1-1 with K-ras mutation was observed. v-K-ras transfectants showed significantly higher SEAP activ ity than that of mock-transfectant activity. EMSA showed specific interacti on of AP-1 with promoter of GSTP1-1. Conclusions: It is highly plausible th at GSTP1-1 overexpression in ACF, colorectal adenoma, and carcinoma is indu ced by K-ras mutation via AP-1 activation.