Characterizing the effects of N/NRTIs on human telomerase activity in vitro and telomere maintenance in a transformed human cell model

Telomeres are nucleoprotein structures found at the ends of most linear chromosomes. Telomeric DNA shortens with each cell division, effectively restricting the proliferative capacity of most human cells. Telomerase, a specialized reverse transcriptase (RT), is responsible for de novo synthesis of telomeric DNA, and is the only physiological mechanism through which some human cells extend their telomere length. Disruption in telomerase activity results in accelerated telomere attrition, which manifests as a loss in tissue regenerative capacity. In individuals infected with the human immunodeficiency virus (HIV), current clinical treatment guidelines prescribe the use of a long-term, combination drug therapy known as highly active anti-retroviral therapy (HAART). Nucleoside and non-nucleoside reverse transcriptase inhibitors (N/NRTIs) inhibit HIV RT and are integral components of HAART. There are both reported structural and mechanistic similarities between telomerase RT and HIV RT. Based on these observations, we hypothesized that N/NRTIs will inhibit telomerase in the same ways that they inhibit HIV RT, and that long-term exposure to these agents will limit telomere maintenance in telomerase-dependent cells. We tested our hypothesis using two approaches. First, N/NRTIs were tested against telomerase activity in vitro using a primer extension assay. All NRTIs tested in this assay inhibited human telomerase, and their relative potencies were compared to their respective dideoxynucleotide analog counterparts. The NNRTIs, which are non-competitive inhibitors of HIV RT, did not inhibit telomerase. In our second approach, we tested the effects of long-term, continuous treatment with N/NRTIs on telomere length maintenance in a transformed human cell model with constitutive telomerase activity. The rates of telomere length attrition in the presence of high doses of several NRTIs were consistent with maximal telomerase inhibition. In contrast, I observed minimal effects on telomere maintenance in cells treated with NNRTIs. My primer extension assay data corroborate conclusions from previous studies on telomerase biochemistry and support mechanistic conservation between telomerase RT and HIV RT. Collectively, my biochemical and cell culture studies demonstrated that telomerase inhibition by NRTIs could potentially lead to treatment complications in current antiretroviral therapies and encourage large-scale clinical and epidemiological studies on the effects of telomerase inhibition by these drugs.Medicine, Faculty ofMedical Genetics, Department ofGraduat