EFFECTS OF SP1 SITE TO hTERT PROMOTER ACTIVITY AND ITS RESPONSE TO RETINOID ACID

Objective To investigate the junction of Sp1 consensus sites to human telomerase reverse tran-scriptase (hTERT) promoter in different cell lines and in TRA-treated Hela cell. Methods Different length ofhTERT promoter was cloned and inserted into pGL3/basic reporter plasmid. The last four Sp1 sites were deleted byPCR and pGL3B/TRTP413Δ reporter plasmid was constructed. All reporter plasmids were transiently transfected in-to 293, A549, Hela and HepG2 cell lines. 48 h after transfection, luciferase activity was analyzed. hTERT promoteractivity of Hela cell which was treated with trans-retinoid acid (TRA) was tested too. Total RNA of these cells wereextracted and reverse transcript. hTERT mRNA level was analyzed in all tested cells. c-Myc and Sp1 expression wereexamined in Hela cell before and after TRA treatment. U937 was used as a positive control in TRA treatment.Results hTERT was expressed at different level in all tested cell lines. 207bp promoter upstream of transcriptionstart site maintained complete activity. Deletion of last 4 Sp1l sites greatly decreased activity of hTERT promoter, andalmost eliminated its activity in HepG2. TRA increased the activity of different length hTERT promoters in Hela cell,but the activity of Sp1 site-deleted promoter decreased by 3 times. Unlike U937 cell, hTERT expression of Hela cellincreased after TRA treatment, and c-Myc and Sp1 mRNA level were relatively stable. Conclusion Sp1 site wasrequired for transactivation of hTERT promoter and played an important role during TRA treatment.

Increased Activity of the Human Telomerase Catalytic Subunit Promoter by the VEGF Enhancer in Human Cancer Cells

We attempted to improve the activity of hTERT promoter by fusing the vascular endothelial growth factor （VEGF） enhancer. To determine the potential as cancer specific promoters, we measured the reporter gene transfection assay driven by the hTERT promoter and the VEGF enhancer in human cancer cells. We found that the hTERT promoter containing VEGF enhancer conferred strong expression of the reporter gene only in different cancer cell lines but not in normal human cells. Retrovirus vector expressing HSV-TK controlled by the hTERT promoter and the VEGF enhancer was constructed. A549 cells infected with LN-enh-hT-TK was significantly suppressed and induced to apoptosis more than those infected with LN-hT-TK. The apoptosis ratio ofA549 cell infected with two kinds of retrovirus cell with GCV in lower concentration is 20.94% and 50.7%. It suggested that there is significant differentiation between the assay groups. Our results demonstrated the possible application of hTERT promoter and the VEGF enhancer in targeted cancer gene therapy.

hTERT-targeted E.coli purine nucleoside phosphorylase gene/ 6-methylpurine deoxyribose therapy for pancreatic cancer

Background Pancreatic cancer is one of the most common tumors and has a 5-year survival for all stages of less than 5%. Most patients with pancreatic cancer are diagnosed at an advanced stage and therefore are not candidates for surgical resection. In recent years, investigation into alternative treatment strategies for this aggressive disease has led to advances in the field of gene therapy for pancreatic cancer. E. coil purine nucleoside phosphorylase/6-methylpurine deoxyribose （ePNP/MePdR） is a suicide gene/prodrug system where PNP enzyme cleaves nontoxic MePdR into cytotoxic membrane-permeable compounds 6-methylpurine （MeP） with high bystander activity, hTERT is expressed in cell lines and tissues for telomerase activity. In this study we examined the efficacy of ePNP under the control of hTERT promoter sequences and assessed the selective killing effects of the ePNP/prodrug MePdR system on pancreatic tumors. Methods Recombinant pET-PNP was established. The protein of E. coil PNPase was expressed and an antibody to E. coil PNPase was prepared. Transcriptional activities of hTERT promoter sequences were analyzed using a luciferase reporter gene. A recombinant phTERT-ePNP vector was constructed. The ePNP/MePdR system affects SW1990 human pancreatic cancer cell lines in vitro. Results The hTERT promoter had high transcriptional activity and conferred specificity on cancer cell lines. The antibody to E. coil PNPase was demonstrated to be specific for the ePNP protein. The MePdR treatment induced a high in vitro cytotoxicity on the sole hTERT-ePNP-producing cell lines and affected SW1990 cells in a dose-dependent manner. Conclusions The hTERT promoter control of the ePNP/MePdR system can provide a beneficial anti-tumor treatment in pancreatic cancer cell lines including a good bystander killing effect.