| Summary: | 碩士 === 中山醫學大學 === 毒理學研究所 === 90 === 貳、英文摘要
Telomerase activity is not detectable in most somatic cell but is upregulated in 85~95% of human canner, which suggests important role of telomerase in neoplastic transformation. Telomerase revese transcriptase was the determinant rate-limiting step in telomerase regulation, and its active promoter activity was involved in telomerase reactivation. Therefore we have cloned the telomerase revese transcriptase promoter (h.TERT-p548). In addition , we co-transfected a series of constructs containing unidirectionally delected fragments： h.TERT-p-212~+50 (core promoter)、h.TERT-p -196~+50 (deleted SP1 site)、h.TERT-p-177~+50 (deleted SP1 and c-MYC site)、h.TERT-p-155~+50 (deleted SP1、c-MYC and AP-2 site), respectively, to examine whether B[a]P、1-NP、p14ARF and taxol regulated telomerase activation through those transcription factors. In the present study, we first employed TRAP and RT-PCR to elucidate whether the regulation of telomerase activity and h.TERT mRNA expression in human NSCLC cell by treatment with B[a]P、1-NP and p14ARF . The telomerase activation was not influence by treament with B[a]P、1-NP and p14ARF ；h.TERT mRNA level was increased 1~2 fold by low concentration of B[a]P and 1-NP , and that was increased 1~1.5 fold by treatment with p14ARF .h.TERT-p548 was activated by low concentration of B[a]P, and repressed by 1-NP、p14ARF and taxol when experiment with transient transfection and luciferase reporter assay . In addition , they did not modulate these transcription factor to regulate h.TERT transcription activation. Significantly, after we alone transfecting h.TERT-p-177 or h.TERT-p-155 plasmid DNA , each still expressed 50~60% transcription activation.It indicated that except for c-Myc, AP-2 and NF-E2 have the ability to regulate telomerase reactivation. Finally our flow cytometric studed show that B[a]P、1-NP and p14ARF slightly increase G0/G1 percentage , and taxol cause cell cycle arrest in G2/M phase leading apoptosis . Furthermore, B[a]P、1-NP、p14ARF and taxol can induce cytotoxicity in H1299 and casue cell death using MTT and colony formation assay .
In summary, this study provides data showing that B[a]P、1-NP、p14ARF and taxol all were able to regulate h.TERT transcriptionactivation , but independently of c-Myc . Maybe they dependent on other transcription factors to regulate telomerase reactivation.
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