[Effects of hFRNK on E-cadherin/beta-catenin in colon cancer cells in vitro].
OBJECTIVE
To explore the effects of human FRNK gene on E-cadherin/beta-catenin complex in colon cancer cell line Colo320WT cells stimulated with extrinsic gastrinl7.


METHODS
AdEasy system was used to construct pAdhFRNK expressing human FRNK gene by recombination in E. coli. BJ5283. pCR3.1/GR plasmid expressing gastrin receptor CCK-2 was transfected into colon cancer cell line Colo320 cells by Lipofectamine 2000 and expressing stably CCK-2R clones were screened by G418 (500 pg/ml). The expression levels of gastrin receptor in Colo320 cells and the transfected Colo320WT cells were assayed by RT-PCR. Colo320WT cells were treated by 10(-8) mol/L gastrinl7 for 12 h; and after Colo320WT cells were infected by pAdhFRNK (MOI: 100) for 2 d the cells were treated by gastrin17 for 12 h again. The expression levels of E-cadherin and beta-catenin in TX-100 soluble fraction and TX-100 insoluble fraction of Colo320WT cells were assayed by co-immunoprecipation and Western blot. E-cadherin and beta-catenin's distribution in Colo320WT cells were detected by immunocytochemistry.


RESULTS
When 10(-8) mol/L gastrin17 stimulated Colo320WT cells for 12 h, the expression levels of E-cadherin and beta-catenin in TX-100-soluble fraction decreased apparently, while the expression levels of E-cadherin and beta-catenin in TX-100-insoluble fraction increased markedly. When pAdhFRNK infected Colo320WT cells for 2 d and 10(-8) mol/L gastrin17 treated the cells for 12 h, the expression levels of E-cadherin and beta-catenin in TX-100-soluble fraction increased apparently again, and the expression levels of E-cadherin and beta-catenin in TX-100-insolutble fraction decreased markedly. Immunocytochemistry showed that the distribution of E-cadherin and beta-catenin was translocated from plasma membrane into cytoplasm and nucleus in the cells stimulated with gastrinl7, and after the cells were infected with pAdhFRNK and stimulated by gastrinl7 again. beta-catenin was mainly observed in cytoplasm and little nuclear immunoreactivity.


CONCLUSIONS
An adenovirus vector pAdhFRNK can inhibit abnormal distribution of E-cadherin and beta-catenin in the gastrin17-stimulated cells. The mechanism is probably that hFRNK can disphosphorylate phosphorylated FAK and block FAK pathway.
Cao
Jun
J
Department of Gastroenterology, People's Hospital of Wuhan University, Wuhan 430060, China.
Yu
Jie-ping
JP
Liu
Chao-hong
CH
Chen
Xin-wen
XW
Liu
Song
S
Luo
He-sheng
HS
Yu
Hong-gang
HG
D000256:Adenoviridae / Q000235:genetics; D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D003593:Cytoplasm / Q000378:metabolism; D005755:Gastrins / Q000494:pharmacology*; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008055:Lipids / Q000737:chemistry; D011485:Protein Binding; D021381:Protein Transport / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D043887:Receptor, Cholecystokinin B / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods; D051176:beta Catenin / Q000378:metabolism*
D015820:Cadherins; D005755:Gastrins; D008055:Lipids; C086724:Lipofectamine; D043887:Receptor, Cholecystokinin B; D051176:beta Catenin; C013550:gastrin 17; C113183:FAK-related nonkinase; D011505:Protein-Tyrosine Kinases