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 ...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.展开更多
基金Supported by Shanghai Science and Technology Development Foundation (01JC14025) and the 4th phase financial support of Shanghai EducationCommittee.
文摘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.
