Human telomerase reverse transcriptase promoter regulation in normal and malignant human ovarian epithelial cells

The telomerase RNA-protein complex responsible for maintenance of telomeric DNA at chromosome ends, is usually inactive in most primary somatic human cells, but is specifically activated with in vitro immortalization and duri ng tumorigenesis, Although expression of the RNA component of telomerase ap pears to be constitutive, the expression pattern of human telomerase revers e transcriptase (hTERT), the catalytic subunit of telomerase, is correlated with measured enzyme activity. In particular, a > 80% concordance has been reported between telomerase activity and hTERT mRNA expression in ovarian tumors. Accordingly, to learn more about the mechanism regulating hTERT gen e expression in ovarian carcinoma, we have performed a detailed analysis of the 5 ' -flanking promoter region of the hTERT gene. We have reported prev iously the isolation and analysis of a 5.8-kb genomic fragment containing t he human hTERT gene promoter (M, Tzukerman el al., Mel. Biol, Cell, 11: 438 1-4391, 2000). Deletion analysis of this promoter was carried out using tra nsient transfection of promoter-reporter constructs in four different telom erase-expressing, ovarian carcinoma-derived cell lines, the tumorigenic pro perties of which have been characterized, and was compared with telomerase- negative primary human fibroblasts and nontransformed ovarian epithelial ce lls. These assays have shown that the hTERT promoter is inactive in telomer ase-negative cells and is active in telomerase-positive cell lines. A core promoter of 283 bp upstream of the transcription initiation site (TI) was F ound to be sufficient for maximum promoter activity, suggesting the presenc e of inhibitory elements within the larger promoter sequence. Gel shift ana lysis of the core promoter using nuclear extracts from the ovarian and cont rol cell Lines revealed specific transcription factor binding using extract s from telomerase-positive cells. Among the binding elements, we identified two E-boxes (CACGTG) as well as a novel element (MT-box), which we identified recently in a number of differ entiation systems. Site-directed mutagenesis was used to introduce mutation s into this novel transcription factor binding element. These mutations sig nificantly affect the transcriptional activity of hTERT promoter in a cell type-specific manner and suggest that the transcription factors that bind t o the E-box and the novel element cooperatively function as major determina nts of hTERT expression and telomerase activity in ovarian cancer. Further comparison of promoter activity, telomerase activity, and telomere length a mong the different ovarian cancer cells indicated that a threshold level of telomerase activity is apparently sufficient to protect telomere integrity and permit the immortal state of the different ovarian cancer cell lines.