Relevant Degree Programs
Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2019)
Telomeres, the protective caps at the end of human chromosomes, are shortened during cellular proliferation in normal aging. Telomere biology disorders (TBDs) refer to a spectrum of tissue degenerative disorders caused by accelerated shortening of telomeres secondary to genetic defects in telomere biology genes. Defects in eleven genes involved in telomere length maintenance have been found to cause TBDs. Accelerated telomere shortening leads to premature aging at the cellular level and regenerative defects at the tissue level. TBD-related genetic defects, in concert with intrinsic tissue turn-over rate and various environmental insults that precipitate telomere shortening, determine the aging process of specific tissues and the clinical presentations of TBDs. The main objective of this dissertation is to investigate the genetic factors that contribute to phenotypic variations of TBDs. The thesis is divided into two sections based on the presentations of TBDs in fast- and slow-turnover tissues. In Chapter 2, using patient-derived cell models and DNA samples, I comprehensively assessed the molecular and cellular phenotypes of X-linked dyskeratosis congenita (X-DC) in female DKC1 mutation carriers. I demonstrated that successful X chromosome inactivation (XCI) in their blood cells led to normal dyskerin expression and function and thus normal telomere length maintenance. These populations should be free of hematopoietic disease manifestations. In contrast, protection from XCI in tissue compartments other than the hematopoietic system may not be complete, and DC manifestations could be observed in a patient-specific manner depending on the sum total of the environmental and inherited telomere lengths as confounding factors.Extending from the observation with phenotypic variations in female DKC1 mutation carriers, I further investigated how incomplete genetic perturbations of telomerase activity may impact clinical presentations of a common disease. In Chapter 3, using a combination of cell and clinical disease models, I showed that functional defects in telomerase catalytic activity, caused by selected genetic polymorphisms in TERT, led to suboptimal telomere length maintenance. Rapid progression of chronic obstructive pulmonary diseases is associated with patients’ carrying status of the minor allele of rs61748181. Collectively, my study revealed two genetic modifiers for potential causes of phenotypic variations in TBDs.
Master's Student Supervision (2010 - 2018)
Telomerase is the ribonucleoprotein reverse transcriptase that catalyzes the synthesis of TTAGGG nucleotide repeats at the ends of linear chromosomes, contributing to proper telomeric structure and cap formation. Most human somatic cells have low or undetectable telomerase expression. In contrast, telomerase overexpression is found in over 85% of human cancers allowing cancer cells to replicate indefinitely. Telomerase inhibition by GRN163L (Imetelstat) has previously been observed to potentiate genotoxic stress in a cell-cycle (S/G2) specific manner, through an unknown mechanism. We hypothesized that GRN163L treatment alters cell-cycle kinetics and that this effect depends upon active signaling through ataxia telangiectasia mutated (ATM). Here we tested the effects of combining GRN163L and the topoisomerase II inhibitor etoposide, together with pharmacological ATM inhibition on MCF-7 breast cancer cells, to assess dependence of telomerase’s cyto-protective function on this DNA-damage repair transducer. Additive increased cytotoxicity and cell-cycle profile alterations depended upon the order of treatment addition. Investigating possible causes of these cell-cycle distribution changes we observed that telomerase inhibition alone induces γH2AX DNA-damage foci in a subset of telomerase-positive cells but not telomerase-negative primary human fibroblasts. Additional FACS and immunocytochemistry experiments indicate that GRN163L-treated cells were reversibly stalled but not arrested at G2/M. Our results suggest that treatment with GRN163L sensitizes telomerase-positive cells to cell-cycle specific DNA-damaging agents through the engagement of an ATM-dependent DNA-damage signal, which may represent a separate mechanism by which telomerase inhibition could affect DNA repair homeostasis in telomerase-positive cancer cells. In addition to its telomere-maintenance function telomerase has recently been reported to participate in non-canonical activities such as protection from DNA-damaging agents, apoptosis, cellular proliferation rate, and resistance to oxidative stress. In a separate study, we hypothesized that overexpression of telomerase in transformed human cells would increase their survival following exposure to DNA-damaging agents. Our results indicate that telomerase expression protects cells from a variety of DNA-damaging drugs by improving the kinetics of DNA-repair. Telomerase expression also allows surviving cells to tolerate increased levels of chromosomal instability following drug exposure. This work has implications on improving the design of future telomerase inhibition strategies to also target non-canonical effects of this enzyme.
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.
Telomerase is the specialized reverse transcriptase responsible for the de novo synthesis of telomeric repeats at chromosome ends. Telomerase plays important roles in tumor development and is responsible for the indefinite growth phenotype in cancer. Telomerase over-expression is found in more than 85% of human tumors surveyed. In contrast, normal somatic cells have low or undetectable telomerase expression, making the enzyme an appealing target for the development of anticancer therapy. However, there is a significant time lag between the start of telomerase inhibition therapy and growth inhibition effects, restricting the use of telomerase inhibitors in clinical applications. In addition to telomere maintenance, telomerase participates in cellular recovery processes following genotoxic insults. Genetic suppression of the human telomerase catalytic subunit, telomerase reverse transcriptase (hTERT), diminishes cellular DNA repair capability following double-stranded DNA damage induction, suggesting that the enzyme is involved in the regulation of DNA repair response. I hypothesize that transient telomerase inhibition at the time of genotoxic stimulus will increase cytotoxicity in tumor cells. My studies showed that short-term telomerase inhibition potentiates the cytotoxic effects of DNA damage inducing agents in MCF-7 breast cancer and HT29 colorectal cancer cells, in a cell-cycle dependent and DNA damage mechanism-specific manner. Additionally, I found that the Ataxia Telangiectasia Mutated kinase may interact with telomerase dependent DNA damage repair pathways to further augment cancer cell death. This study provides new mechanistic insight into the roles of telomerase function in cancer cell survival and impetus to design new telomerase-based clinical therapies against breast and colorectal cancers.