Feedback regulation of the hTERT promoter

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Telomere function has proven to be a crucial mechanism of preserving genomic stability. With each cell division, telomeres become progressively shorter, eventually resulting in senescence and apoptosis. Loss of telomere DNA in cancer cells is compensated by the activity of telomerase, a reverse transcriptase that synthesizes telomere repeats. Human telomerase reverse transcriptase (hTERT) is the catalytic protein subunit of the telomerase enzyme that, along with the telomerase RNA component (hTERC), makes up the ribonucleoprotein. hTERT mRNA levels have been shown to exhibit a strong correlation with telomerase activity in breast cancer, with expression of the TERT subunit being one of the most common features occurring in nearly all tumor types. In this context, it is important to understand the mechanisms by which TERT expression is regulated, and how it becomes misregulated in cancer cells.
 Recent studies suggest that the hTERT protein may be involved in its own regulation through the modulation of its upstream regulatory proteins p53 and p21. In these studies, knockdown of hTERT through RNA interference was shown to cause an increase in p53 and p21, both known regulators of hTERT. This would indicate that hTERT is involved in a feedback regulation loop. However, since both hTERT mRNA and protein levels were artificially altered because of the RNAi approach, it is difficult to determine the effect of hTERT knockdown on its own promoter activity.
 The purpose of this investigation was to silence hTERT using RNA interference and measure the effect on the activity of its own promoter. Since hTERT mRNA or protein cannot be used to measure hTERT promoter activity in knockdown experiments, we constructed an adenovirus containing the hTERT promoter driving a GFP reporter gene. This adenoviral construct allowed us to indirectly, yet accurately monitor hTERT promoter activity by level of GFP fluorescence. HEK293 cells with 0.1 uM siRNA, resulting in 80% knockdown of hTERT mRNA. We then infected these cells with the hTERT promoter-GFP adenovirus and measured fluorescence using flow cytometry. This method allowed for a better assessment of the hTERT feedback regulation loop. Understanding the mechanism by which hTERT controls transcription could reveal novel ways to manipulate hTERT in the treatment of cancer.