Expression of a recombinant vector of a mutant human telomerase reverse transcriptase gene in human bladder cancer cell line T24, and its clinical significance.
OBJECTIVE
To construct a mutant enhanced green fluorescence protein (pEGFP) human telomerase reverse transcriptase (hTERT) gene expression vector (pEGFP-hTERT), to observe its expression in transfected human bladder cancer cell line T24 and its role in the molecular regulatory mechanisms of telomerase, and to provide a new target gene for bladder cancer therapy.


METHODS
Polymerase chain reaction (PCR) amplification was performed using primers based on the gene sequence of hTERT. The PCR product was cloned into plasmid pGEMT-T Easy and the sequence of mutant hTERT gene analysed. 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 T24 cells by the method of calcium phosphate-DNA co-precipitation, we detected steady expression of the GFP-hTERT fusion protein by fluorescent-light microscopy. Changes in the proliferation of T24 cells were detected by light microscopy, and beta-galactosidase staining correlated with senescence.


RESULTS
Identification of pEGFP-hTERT by enzyme digestion showed that the mutant hTERT fragment had been cloned into EcoR I and Sal I sites of the pEGFP-C1 vector. Steady expression of GFP-hTERT fusion protein was located in the nucleus of transfected cells. Positive expression senescence-associated beta-galactosidase staining in transfected cells increased gradually with extended cultured time, and their growth was suppressed.


CONCLUSIONS
The recombinant mutant vector (pEGFP-hTERT) was successfully constructed and expressed steadily in T24 cells. The mutant-type hTERT gene suppresses the proliferation of T24 cells by a competitive effect on telomerase activity. This suggests that the hTERT gene might be a suitable gene target for bladder cancer therapy.
Fu
Wei-Jun
WJ
Department of Urology, Chinese People's Liberation Army General Hospital, Military Postgraduate Medical College, Beijing, China. fuweijun@hotmail.com
Hong
Bao-Fa
BF
Huang
Jun-Jian
JJ
Xu
Bing
B
Gao
Jiang-Ping
JP
Wang
Xiao-Xiong
XX
Huang
Cui-Feng
CF
D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004268:DNA-Binding Proteins / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins; D019098:Telomerase / Q000235:genetics*; D014162:Transfection; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000378:metabolism / Q000628:therapy*
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D019098:Telomerase