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 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.

Identification of a novel transcription factor binding element involved in the regulation by differentiation of the human telomerase (hTERT) promoter.

Three different cell differentiation experimental model systems (human embryonic stem cells, mouse F9 cells, and human HL-60 promyelocytic cells) were used to determine the relationship between the reduction in telomerase activity after differentiation and the regulation of the promoter for the hTERT gene. Promoter constructs of three different lengths were subcloned into the PGL3-basic luciferase reporter vector. In all three experimental systems, all three promoter constructs drove high levels of reporter activity in the nondifferentiated state, with a marked and time-dependent reduction after the induction of differentiation. In all cases, the smallest core promoter construct (283 nt upstream of the ATG) gave the highest activity. Electrophoretic mobility shift assays revealed transcription factor binding to two E-box domains within the core promoter. There was also a marked time-dependent reduction in this binding with differentiation. In addition, a distinct and novel element was identified within the core promoter, which also underwent time-dependent reduction in transcription factor binding with differentiation. Site-directed mutagenesis of this novel element revealed a correlation between transcription factor binding and promoter activity. Taken together, the results indicate that regulation of overall telomerase activity with differentiation is mediated at least in part at the level of the TERT promoter and provides new information regarding details of the regulatory interactions that are involved in this process.

Zinc metabolism in rheumatoid arthritis: plasma and urinary zinc and relationship to disease activity.

OBJECTIVE: To study zinc absorption in patients with active rheumatoid arthritis (RA). METHODS: We studied zinc tolerance tests and 24 hour urinary zinc excretion before and after ingestion of 50 mg elemental zinc in 8 healthy volunteers (Group 1) and 13 patients with low RA activity (Group 2) and 16 patients with high RA activity (Group 3). RESULTS: In Group 1, plasma zinc rose from 111 +/- 7 micrograms/dl to a peak of 200 +/- 24 micrograms/dl (mean +/- SEM) in 2 h. In Groups 2 and 3, plasma zinc before zinc ingestion was significantly lower than that of the control group (p < 0.00001 for both groups) and showed no significant increase in plasma after ingestion. Twenty-four hour urinary zinc excretions before and after zinc ingestion were significantly lower (p < 0.01, p < 0.0001 for Group 2; p < 0.05, p < 0.01 for Group 3, respectively) than those in the control group. CONCLUSION: These results are compatible with zinc malabsorption and consequent zinc deficiency in patients with RA. Whether zinc deficiency contributes to perpetuation of disease activity by compromising cellular immune function needs further investigation.