Molecular Mechanisms Of Transcriptional Regulation And Signal Transduction Of Telomerase Expression

Objective Human telomerase reverse transcriptase (hTERT) has been generally acknowledged as a rate-limiting determinant of the telomerase only expressed in immortal or tumor cells, and this may be one of the reason that tumor cells differ from somatic cells. Many observations indicate that transcriptional regulation of the hTERT gene plays a key role in the activation of telomerase, and transcriptional factors of c-Myc and Spl are obviously involved in telomerase expression. In Jurkat T cells, telomerase could be induced by PHA. The purpose of this dissertation is: (1) to investigate the effect of c-Myc and Spl on expression of telomerase and hTERT; (2) to study the signal transduction pathway of telomerase induced by PHA in Jurkat T. Methods Antisense Spl oligodeoxynucleotide (ODN) and antisense c-Myc (ODN) or Spl and Sp3 expression vector(pCMV-Spl, pCMV-Sp3) were delivered to Jurkat T cells by lipofectamin. Telomerase PCR ELISA was used to detect telomerase activity; RT-PCR analysis was used to assess the mRNA expression of Spl ,c-Myc and hTERT; Western blot was used to analyze the levels of Spl and Sp3 protein and the cells proliferation was measured by MTT. The expression vector of dominant negative p21ras (pZIPras17N)and raf-l(pCGNraf(l-130)) or antisense H-ras ODN and antisense c-raf ODN were transferred to Jurkat T cells by lipofectamin, or using selective inhibitors of kinases blocked different signal cascades such as PKC and MEK, and then cells were stimulated with PHA. Telomerase PCR ELISA was used to detect telomerase activity. Results (1) Treatment of Jurkat T with Spl antisense inhibitorC 1 μ mol ·L-1)resulted in dramatically reduced Spl mRNA and protein levels. The inhibition rate were 44.8%(P<0.05) and 57% (P<0.01) respectively. Treatment of Jurkat T with c-Myc antisense inhibitor (1 u mol -L-1) also resulted in significantly reduced c-Myc mRNA with an inhibition rate of 36.2%(P<0.01). Following the transcriptional factor Spl and c-Myc functionally altering, the telomerase activity were significantly suppressed with a 43.1% (P<0.01) and a 27.2% (P<0.01) inhibition rate respectively. From 0.25-2.0 n mol -L-l, a dose-dependent inhibition of telomerase activity by antisense Spl ODN was discovered, but the similar dose-dependent inhibition did not occurred by antisense c-Myc ODN. (2) Treatment of Jurkat T with 1 μmol·L-1 Spl antisense ODN or c-Myc ODN, hTERT mRNA expression were significantly decreased by 43.6% (P<0.01) and a 23.4% (P<0.05) respectively. (3) Treatment of Jurkat T cells with Spl or Sp3 expressing vectors for 36h result in significantly increase of Spl and Sp3 protein levels by 59.6%( P<0.01) and 36.8% (P<0.05) respectively. Enhancement of Spl expression obviously increased telomerase activity and hTERT mRNA levels with a rate of 38.5%(P<0.05) and 25.4% (P<0.05) respectively, whereas Sp3 had no significant effect on both telomerase activity and hTERT mRNA levels. (4) Dose-effect relationship of cells survival rate indicated a dose-dependent survival decreasing after treated with 0.25, 0.5, 1.0, 2.0(μmol·L-l) antisense Sp1 ODN for 48h, the similar dose-dependent inhibition did not occurred by antisense c-Myc ODN, but antisense c-Myc ODN of different concentrations inhibited cells proliferation more significantly than antisense Spl ODN. Time-effect relationship of cells survival rate indicated that antisense c-Myc ODN had an earlier and more powerful effect on cells proliferation than antisense Spl ODN. (5) Telomerase activity was greatly stimulated after exposure to PHA, and treatment of Jurkat T cells with dominant negative p21ras and raf-1 for 36h result in significantly decrease of telomerase by 38.7% (PO.05) and 34.3% (P<0.05) respectively. (6) Transferring to Jurkat T with 1 v mol -L-l H-ras or c-raf antisense inhibitor resulted in dramaticallyreduced H-ras mRNA and c-raf mRNA by 52.1%(P<0.05) and 42.8% (P<0.01) respectively. From 0.5-2.0 μmol·L-1, a dose-dependent inhibition of telomerase activity by antisense H-ras ODN and antisense c-raf ODN. (7) The increase of telomerase activit...