Cloning Of Human TERT Promoter For Treating Tumors

Objective: In cancer gene therapy, restricted expression of the therapeutic gene, especially toxic gene, in tumor cells is important. As proved, telomerase activity is undetectable in most human somatic cells, while it is generally presented in highly proliferative cells and in most human cancers. Its exclusive expression in most tumor cells makes it a strong candidate for targeting cancer gene therapy and it is a good approach to use telomerase promoter to target the expression of toxic gene(s) in the telomerase positive tumors and kill them. For this purpose, an hTERT promoter was cloned from HepG2 genome and then a set of expression vectors encoding LacZ or thymidine kinase (tk) gene under the control of the hTERT promoter, and hCMV promoter for comparison, were constructed to check out whether this system could work well. Methods: According to Horikawa, a core hTERT promoter sequence is located at -208~+40 bp upstream the telomerase, a pair of primers were designed and the core hTERT promoter containing the -208~+40 sequence flanking Kpn I restriction site in 5'-terminal and EcoR Ⅰ site in 3'-terminal were cloned by PCR method from HepG2 genome. The correction of the promoter sequence was comfirmed via sequencing. A set of expression vectors, including pDC511 hTERT/LacZ, pDC518 hCMV/LacZ, pDC511 hTERT/tk, and pDC518 hCMV/tk, were constructed through standard molecular cloning methods. After transfecting pDC511 hTERT/LacZ and pDC518 hCMV/LacZ into human tumor cells A549 and normal cell MRC5, β-Gal staining was performed to check out whether hTERT promoter could properly work. After transfection of pDC511 hTERT/tk and pDC518 hCMV/tk into A549 and MRC5 cells with Lipofectamine 2000, the mRNA transcripted by tk gene was assessed using RT-PCR. Furthermore, the cytotoxic effects of GCV/tk on A549 and MRC5 cells transfected with pDC511 hTERT/tk...