TELOMERASE ACTIVITY IS MODULATED BY ESTROGENS IN HUMAN EPITHELIAL CELLS WITH HIGHLY RENEWING POTENTIAL

Eukaryotic chromosomes terminate in specialized structures, telomeres, that are necessary for their stability and function. Telomere replication is catalyzed by telomerase, a ribonucleoprotein that utilizes its own RNA component as the template for de novo synthesis of telomeric DNA. Introduction of an active telomerase gene into normal mortal cells causes a lenghtening of telomeres and a corresponding marked increase in the life span of the cells, making them potentially immortal. Because most of human somatic cells lack detectable levels of telomerase, it has been suggested that the resulting telomere erosion produces chromosome instability, causing senescence and subsequent death of the cell. The correlation between telomerase activity, telomere maintenance, and cellular replicative capacity strongly indicates that activation of telomerase is essential for cell immortality. However, little is known about the molecular mechanisms underlying regulation of the enzyme. We are currently investigating whether hormones and nuclear hormone receptors play a role in modulating telomerase activity in human epithelial tissues or cell lines known to be target of hormone action (e.g.ovary, endometrium, prostate). Telomerase activity in diploid ovary epithelial cells (GRO, LLO, LEA) grown in the absence or presence of estrogen was measured by a highly sensitive PCR-based assay, the Telomeric Repeats Amplification Protocol (TRAP). In the absence of the hormone all samples were telomerase-negative. However, addition of 17?-estradiol (10-7M) induced telomerase activity within 3 hours of treatment. This effect was paralleled by a similar induction of mRNA levels of hTERT, the protein catalytic component of telomerase, as measured by RNase protection assays. This prompt estrogen modulation of telomerase is in favour of a regulatory mechanism acting at the transcriptional level. The recently cloned 5'-flanking region of hTERT (Cong et al., Hum Mol Gen, in press) was analysed in search of putative hormone-response elements. A non canonical estrogen response element (ERE) was identified at position (-947/-935) relative to the translation start site (+1), and its sequence shown to bind baculovirus-expressed human estrogen receptor?? (ER?) by electrophoretic mobility shift assays. Genomic footprint analysis using DNA prepared from the ER-positive breast cancer (MCF7) cell line was performed at 0, 6 and 12 hours of estrogen treatment. At least two distinct regions of the hTERT promoter sequences, encompassing the putative ERE, appeared specifically altered upon estrogen stimulation, suggesting an estrogen-dependent recruitment of transcription factors onto the hTERT 5' flanking region. Our findings substantiate a hormone-dependent transcriptional regulation of the human telomerase catalytic subunit gene. This is the first demonstration of hormonal regulation of a non-traditional target, as that of the human telomerase gene, a suitable model for extending our knowledge on the juvenilizing effects of steroid hormones with interesting implications in the process of cell senescence and oncogenesis.