[Study on the relationship between TK gene regulated by enhanced suicide gene vector and telomerase activity in nasopharyngeal carcinoma cells].
OBJECTIVE
To explore the relationship between TK gene expression regulated by enhanced suicide gene vector and telomerase activity in nasopharyngeal carcinoma cells.


METHODS
The reformed reconstructed enhanced vector, pGL3-basic-EGFP-TK-hTRETp-CMV enhancer, and hTERT mono-promoter vector, pGL3-basic-EGFP-TK-hTRETp(as controls), were transfected into telomerase(+) nasopharyngeal carcinoma 5-8F cell lines, telomerase(+) human breast cancer MCF-7 cell lines and telomerase(-) normal vascular endothelium cell lines respectively. TK gene green fluorescent protein was observed by fluorescence microscope. The expression of TK gene mRNA was measured by the real-time fluorescent quantified PCR and the telomerase activity was determined by the method of TRAP argentation in malignant tumour cells pre- and post-transfected by enhanced vector. Meanwhile the relationship between TK and telomerase was analyzed.


RESULTS
(1) A strong TK gene fluorescent show and TK mRNA expression were displayed after the enhanced suicide gene vector was transfected into nasopharyngeal carcinoma 5-8F cell lines and human breast cancer MCF-7 cell line, which were more stronger than those of mono-promoter group, pGL3-basic-EGFP-TK-hTRETp, and ECV cells transfected by enhanced suicide gene vector. Meanwhile,real-time fluorescent quantified PCR showed that the A value of enhanced vector group was higher than that of controls. (2) Telomerase activity after transfection of enhanced vector and GCV was lower than those before by the method of TRAP argentation in nasopharyngeal carcinoma cell lines,but no change in normal control cells after transfection of enhanced vector and GCV. (3) After adding GCV, the obvious inhibitory effect of tumour cells growth induced by pGL3-basic-EGFP-TK-hTRETp-CMV enhancer were observed in nasopharyngeal carcinoma 5-8F cell lines and human breast cancer MCF-7 cell line, which was higher than those of mono-promoter, pGL3-basic-EGFP-TK-hTRETp, pGL3-basic-EGFP3 and blank controls, but without inhibitory effect in ECV cells transfected by enhanced vector.


CONCLUSIONS
TK gene expression is regulated by hTERT promoter and CMV enhancer, and then the telomerase activity is reduced and the cancer cells are specifically killed. But it is unclear how the telomerase are down-regulated by TK gene.
Shen
Congxiang
C
Department of Otolaryngology-Head and Neck Surgery, Pearl River Hospital, Nanfang Medical University, Guangzhou, 510282, China.
Wen
Zhong
Z
Qian
Yuhong
Y
Mu
Shaofeng
S
Guan
Xiaofang
X
D016923:Cell Death; D002460:Cell Line; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D041722:Genes, Transgenic, Suicide; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D009303:Nasopharyngeal Neoplasms / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection
D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase; D019098:Telomerase