Effects of SP1 site to hTERT promoter activity and its response to retinoid acid

Objective : To investigate the function of Spl consensus sites to human telomerase reverse transcriptase (hTERT) promoter in different cell lines and in TRA-treated Hela cell. Methods: Different length of hTERT promoter was cloned and inserted into pGL3/basic reporter plasmid. The last four Sp1 sites were deleted by PCR and pGL3B/TRTP413A reporter plasmid was constructed. All reporter plasmids were transiently transfected into 293, A549, Hela and HepG2 cell lines. 48 h after transfection, luciferase activity was analyzed, hTERT promoter activity of Hela cell which was treated with trans-retinoid acid (TRA) was tested too. Total RNA of these cells were extracted and reverse transcript, hTERT mRNA level was analyzed in all tested cells, c-Myc and Spl expression were examined in Hela ceil 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 ceil lines. 207bp promoter upstream of transcription start site maintained complete activity. Deletion of last 4 Sp1 sites greatly decreased activity of hTERT promoter, and almost eliminated its activity in HepG2. TRA increased the activity of different length hTERT promoters in Hela cell, but the activity of Spl site-deleted promoter decreased by 3 times. Unlike U937 cell, hTERT expression of Hela cell increased after TRA treatment, and c-Myc and Spl mRNA level were relatively stable. Conclusion: Spl site was required for transactivation of hTERT promoter and played an important role during TRA treatment.