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

Ilana Braunstein, Orit Cohen-Barak, Catherine Shachaf, Yael Ravel, Michal Yalon-Hacohen, Gordon Mills, Maty Tzukerman, Karl L. Skorecki

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

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 during tumorigenesis. Although expression of the RNA component of telomerase appears to be constitutive, the expression pattern of human telomerase reverse 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 gene expression in ovarian carcinoma, we have performed a detailed analysis of the 5′-flanking promoter region of the hTERT gene. We have reported previously the isolation and analysis of a 5.8-kb genomic fragment containing the human hTERT gene promoter (M. Tzukerman et al., Mol. Biol. Cell, 11: 4381-4391, 2000). Deletion analysis of this promoter was carried out using transient transfection of promoter-reporter constructs in four different telomerase-expressing, ovarian carcinoma-derived cell lines, the tumorigenic properties of which have been characterized, and was compared with telomerase-negative primary human fibroblasts and nontransformed ovarian epithelial cells. These assays have shown that the hTERT promoter is inactive in telomerase-negative cells and is active in telomerase-positive cell lines. A core promoter of 283 bp upstream of the transcription initiation site (TI) was found to be sufficient for maximum promoter activity, suggesting the presence of inhibitory elements within the larger promoter sequence. Gel shift analysis of the core promoter using nuclear extracts from the ovarian and control cell lines revealed specific transcription factor binding using extracts 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 differentiation systems. Site-directed mutagenesis was used to introduce mutations into this novel transcription factor binding element. These mutations significantly affect the transcriptional activity of hTERT promoter in a cell type-specific manner and suggest that the transcription factors that bind to the E-box and the novel element cooperatively function as major determinants of hTERT expression and telomerase activity in ovarian cancer. Further comparison of promoter activity, telomerase activity, and telomere length among 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.

Original languageEnglish (US)
Pages (from-to)5529-5536
Number of pages8
JournalCancer Research
Volume61
Issue number14
StatePublished - Jul 15 2001
Externally publishedYes

Fingerprint

Telomerase
Epithelial Cells
Ovarian Neoplasms
Cell Line
Transcription Factors
Telomere
human TERT protein
E-Box Elements
Carcinoma
Mutation
5' Flanking Region
Transcription Initiation Site
Electrophoretic Mobility Shift Assay
Site-Directed Mutagenesis
Genetic Promoter Regions
Genes
Transfection
Carcinogenesis
Fibroblasts
Chromosomes

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Braunstein, I., Cohen-Barak, O., Shachaf, C., Ravel, Y., Yalon-Hacohen, M., Mills, G., ... Skorecki, K. L. (2001). Human telomerase reverse transcriptase promoter regulation in normal and malignant human ovarian epithelial cells. Cancer Research, 61(14), 5529-5536.

Human telomerase reverse transcriptase promoter regulation in normal and malignant human ovarian epithelial cells. / Braunstein, Ilana; Cohen-Barak, Orit; Shachaf, Catherine; Ravel, Yael; Yalon-Hacohen, Michal; Mills, Gordon; Tzukerman, Maty; Skorecki, Karl L.

In: Cancer Research, Vol. 61, No. 14, 15.07.2001, p. 5529-5536.

Research output: Contribution to journalArticle

Braunstein, I, Cohen-Barak, O, Shachaf, C, Ravel, Y, Yalon-Hacohen, M, Mills, G, Tzukerman, M & Skorecki, KL 2001, 'Human telomerase reverse transcriptase promoter regulation in normal and malignant human ovarian epithelial cells', Cancer Research, vol. 61, no. 14, pp. 5529-5536.
Braunstein I, Cohen-Barak O, Shachaf C, Ravel Y, Yalon-Hacohen M, Mills G et al. Human telomerase reverse transcriptase promoter regulation in normal and malignant human ovarian epithelial cells. Cancer Research. 2001 Jul 15;61(14):5529-5536.
Braunstein, Ilana ; Cohen-Barak, Orit ; Shachaf, Catherine ; Ravel, Yael ; Yalon-Hacohen, Michal ; Mills, Gordon ; Tzukerman, Maty ; Skorecki, Karl L. / Human telomerase reverse transcriptase promoter regulation in normal and malignant human ovarian epithelial cells. In: Cancer Research. 2001 ; Vol. 61, No. 14. pp. 5529-5536.
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abstract = "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 during tumorigenesis. Although expression of the RNA component of telomerase appears to be constitutive, the expression pattern of human telomerase reverse 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 gene expression in ovarian carcinoma, we have performed a detailed analysis of the 5′-flanking promoter region of the hTERT gene. We have reported previously the isolation and analysis of a 5.8-kb genomic fragment containing the human hTERT gene promoter (M. Tzukerman et al., Mol. Biol. Cell, 11: 4381-4391, 2000). Deletion analysis of this promoter was carried out using transient transfection of promoter-reporter constructs in four different telomerase-expressing, ovarian carcinoma-derived cell lines, the tumorigenic properties of which have been characterized, and was compared with telomerase-negative primary human fibroblasts and nontransformed ovarian epithelial cells. These assays have shown that the hTERT promoter is inactive in telomerase-negative cells and is active in telomerase-positive cell lines. A core promoter of 283 bp upstream of the transcription initiation site (TI) was found to be sufficient for maximum promoter activity, suggesting the presence of inhibitory elements within the larger promoter sequence. Gel shift analysis of the core promoter using nuclear extracts from the ovarian and control cell lines revealed specific transcription factor binding using extracts 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 differentiation systems. Site-directed mutagenesis was used to introduce mutations into this novel transcription factor binding element. These mutations significantly affect the transcriptional activity of hTERT promoter in a cell type-specific manner and suggest that the transcription factors that bind to the E-box and the novel element cooperatively function as major determinants of hTERT expression and telomerase activity in ovarian cancer. Further comparison of promoter activity, telomerase activity, and telomere length among 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.",
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AU - Mills, Gordon

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AU - Skorecki, Karl L.

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