טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBraunstein Ilana
SubjectTranscriptional Regulation of Human Telomerase Reverse
Transcriptase Gene in Normal and Malignant Human
Ovraian Cells
DepartmentDepartment of Medicine
Supervisors Professor Karl Skorecki
Dr. Maty Zukerman


Abstract

The telomerase complex is usually inactive in primary somatic human cells but is specifically activated with in vitro immortalization and during tumorigenesis. While expression of the RNA component of telomerase appears to be constitutive, the expression pattern of hTERT, the catalytic subunit of telomerase, is correlated with measured enzyme activity. In order to learn more about hTERT gene activation in cancer, we examined the transcriptional regulation of the gene in ovarian-derived normal and cancer cell lines. A direct correlation was found between hTERT mRNA expression and telomerase activity in the different ovarian cell lines. In order to understand the regulatory mechanisms that affect the transcription of the hTERT gene, we have isolated a 5868 bp genomic fragment containing the 5’-flanking sequence of the human hTERT gene. Transient transfection of promoter-reporter constructs in ovarian-derived normal and cancer cell lines have shown that the hTERT promoter is inactive in telomerase negative cells, and is active in telomerase positive cell lines and a core promoter of 361 bp upstream of the translation initiation site (ATG) was sufficient for high promoter activity. Gel shift analysis of the core promoter revealed specific transcription factor binding using extracts from telomerase positive cells. Among the binding elements, we identified SP1 sites, two E- boxes (CACGTG) as well as a novel element (MT-box). Mutations introduced into these transcription factor binding elements significantly affect the transcriptional activity of hTERT promoter in a cell type specific manner and suggest that the transcription factors that bind to these elements, cooperatively function as major determinants of hTERT expression and telomerase activity in ovarian cancer.