Figure 5

Regulation of MTAP expression in colon carcinoma cell lines and HT29M3 cells. (a) A reporter construct containing the luciferase gene under the control of the human MTAP promoter was transiently transfected into different colon carcinoma cell lines (HT29, HCT116, SW48, SW480, LoVo, Caco) and HT29M3 cells. The MTAP promoter showed strong activity in all colon carcinoma cell lines but only little activity in HT29M3. These findings are in accordance with the RNA expression levels. (b) Regulation of the MTAP promoter construct was analysed in HT29 cells. Cotransfection of TCF1, LEF1, TCF4 and β-catenin, revealed strong transactivation of the MTAP promoter by TCF1 and TCF1 plus β-catenin. (c) Gel shift analysis using the TCF1 binding site of the MTAP promoter showed a defined shift using HT29 nuclear extracts. Competition of this shift was achieved using the same unlabelled oligonucleotide (MTAPcold) and a TCF/LEF consensus site (TCF/LEFcold). Addition of an antibody against TCF1 led to suppression of the shift, while an antibody against β-catenin resulted in a supershift. (d) TCF1 and LEF1 expression was analysed in the colon carcinoma cell lines and CEC by RT-PCR. β-actin PCR was performed to ensure quality of RNA.