Expression of a mutant hTERT in human bladder carcinoma cell line T24 and its clinical significance

Abstract

Objective: To construct a mutant pEGFP- hTERT expression vector, to observe its steady expression in transfected human bladder carcinoma cell line T24 and its role in molecular regulatory mechanisms of telomerase, and to provide a new target gene for bladder cancer. Methods: PCR amplification was performed by using primers based on the known gene sequence of hTERT. PCR production was cloned into plasmid pGEMT-T easy and the sequence of mutant hTERT gene was analyzed. A recombinant mutant hTERT vector (pEGFP-hTERT) was constructed at the EcoR I and Sal I sites of the pEGFP-C1 vector. After transfecting the fusion gene into bladder carcinoma cell line T24 by calcium phosphate-DNA coprecipitation, the steady expression of GFP-hTERT fusion protein was tested by fluorescent light microscopy. The proliferation changes of bladder carcinoma cell line T24 were detected by light microscopy and senescence correlated β-galactosidase staining. Results: Identification of pEGFP-hTERT by enzyme digestion showed that mutant hTERT fragment had been cloned into EcoR I and Sal I sites of the pEGFP-C1 vector. The steady expression of GFP-hTERT fusion protein was localized in the nucleus of transfected cells. Expression of senescence-associated β-galactosidase in transfected cells gradually increased with extended cultured time and cell growth was suppressed. Conclusion: The mutant-type hTERT gene suppresses the proliferation of bladder carcinoma cell line T24 by competitive effect on telomerase activity. This suggests that hTERT gene might be a suitable gene target for bladder cancer therapy.