Glutathione S-transferase-pi overexpression is closely associated with K-ras mutation during human colon carcinogenesis.
OBJECTIVE
In colorectal adenoma and carcinoma, glutathione S-transferase-pi (GSTP1-1) is highly expressed. K-ras mutation is also known to occur frequently in colorectal adenoma and carcinoma, as well as in the putative precursor of adenoma, aberrant crypt foci (ACF). Further, forced expression of v-H-ras in rat liver epithelial cells has been shown to enhance rat pi-class GST expression. The aim of the present study is, therefore, to investigate the causative relationship between GSTP1-1 overexpression and K-ras mutation in these lesions.


METHODS
Twenty-seven specimens of colorectal carcinoma, 24 of adenoma, and 28 of ACF were examined in this study. The expression of GSTP1-1 or p21(K-ras) was examined by immunohistochemistry. The GSTP1-1 messenger RNA levels were measured by TaqMan reverse-transcription 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. Activities 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 placental alkaline phosphatase (SEAP) assay. Nuclear protein from these transfectants bound to the GSTP1-1 promoter was analyzed by electrophoretic mobility shift assay (EMSA).


RESULTS
In human colorectal carcinoma, adenoma, and ACF, close association of increased expression of GSTP1-1 with K-ras mutation was observed. v-K-ras transfectants showed significantly higher SEAP activity than that of mock-transfectant activity. EMSA showed specific interaction of AP-1 with promoter of GSTP1-1.


CONCLUSIONS
It is highly plausible that GSTP1-1 overexpression in ACF, colorectal adenoma, and carcinoma is induced by K-ras mutation via AP-1 activation.
Miyanishi
K
K
Fourth Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
Takayama
T
T
Ohi
M
M
Hayashi
T
T
Nobuoka
A
A
Nakajima
T
T
Takimoto
R
R
Kogawa
K
K
Kato
J
J
Sakamaki
S
S
Niitsu
Y
Y
D000236:Adenoma / Q000201:enzymology / Q000473:pathology; D001483:Base Sequence; D002277:Carcinoma / Q000201:enzymology / Q000473:pathology; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000473:pathology; D015179:Colorectal Neoplasms / Q000201:enzymology / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D015971:Gene Expression Regulation, Enzymologic; D011905:Genes, ras; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D012150:Polymorphism, Restriction Fragment Length; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D012333:RNA, Messenger; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase