| Suicide gene therapy has been recognized as an effective strategy for cancer treatment with potential application in clinic. Nowadays the major problems encountered tumor gene therapy is the specific expression of the therapeutic genes in cancer cells,including suicide gene therapy. Utilizing the expression regulation sequences of a gene encoding a tumor-specific protein to drive the suicide gene may be promising to restrict the expression of the therapeutic gene in tumor cells,thus to improve the safety and effect of gene therapy.Recently,telomerase has been recognized as a new wide-range tumor marker. Telomerase is silent in normal cells but active in more than 90% of cancers and correlates well with the degree of malignancy. The core component of telomerase-human telomerase reverse transcriptase (hTERT) is a key determinant of telomerase activity. Further research has demonstrated that one important reason for telomerase reactivation is that hTERT is constitutively activated at the transcriptional level. It is reported that in cancer cells,there has been forming a set of genomic expression model different from normal cells,leading to activation and expression of some specific transcription factors in cancer cells,thus resulting in the significant discrepancy in hTERT promoter activity between tumor and normal cells. Recent studies also found that the 5'-flanking sequences containing the region of 181bp upstream the transcription start site functions as a core promoter,which is sufficient to drive the expression of downstream exoticgene and retain its tumor specificity. In the present study,the hTERT regulation sequence containing the core promoter region is cloned and its tumor specificity is studied,and the cw-element of E box and the c-Myc transcription factor are also preliminary studied;and then the hTERT promoter was used to induce the expression of cytosine deaminase (CD) suicide gene,and the feasibility was tested in cancer gene therapy.Our results showed that:the hTERT promoter (-378-+78) we cloned correlated with the telomerase activity,both of which showed high tumor specificity. They were upregulated in seven tumor cells tested,but repressed in normal cells,which was consistent with the results reported by other independent studies. Furthermore,the transcription activity of the hTERT promoter containing the deletion of the E box at -165 was greatly decreased,with 70%,60% and 60% decrease in HeLa,A549 and LoVo cells respectively,indicating that this E box (-165) is indispensible for the promoter activity. Interestingly,the activity of the promoter containing the deletion of the E box at +44 site did not show any changes in A549 and LoVo cells,but was 3-fold decreased in Hela cells,indicating that the role of this E box (+44) depends on cell type. These results were first reported in similar studies. EMS A showed that the activity of c-Myc binding to E box (-165) was stronger than that of binding to E box (+44),suggesting that the role of the E box is correlated to the binding activity of c-Myc. By Western blot,higher expression of c-Myc is observed in Bcap-37 Hela cells with higher hTERT promoter activity and lower expression of c-Myc in A549 with lower hTERT promoter,while weak expression of c-Myc is found in normal cells WI38,indicating the high correlation of c-Myc expression and hTERT transcription activity. These results suggested that the effect of c-Myc through its target element E box may involved in the regulation of hTERT promoter.Based on the above results,an expression plasmid,phTERT-CD,was constructed,in which the E. coli. cytosine deaminase (CD) gene was controlled by the hTERT promoter. A colorectal cancer cell line (LoVo) and anormal amnion cell line (WISH) were transfected by this plasmid. It was shown that the expression of the CD gene increased the sensitivity of LoVo cells to the prodrug,5-fluorocytosine (5FC),over 800 folds,and a strong "bystander effect" was also observed;while the sensitivity of WISH cells to 5FC was increased only 6 folds with no se...
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