Judy M. Y. Wong

Telomere maintenance and disease

The proliferative capacity of human cells is regulated by telomerase, an enzyme uniquely specialised for telomeric DNA synthesis. The critical role of telomerase activation in tumour progression and tumour maintenance has been well established in studies of cancer and of oncogenic transformation in cell culture. New evidence suggests that telomerase activation has an important role in normal somatic cells, and that failure to activate sufficient telomerase also promotes disease. We review the evidence for premature telomere attrition in proliferative deficiencies of the human haemopoietic system, and discuss the potential use of telomerase activation in telomere-restorative gene therapy.

Subnuclear shuttling of human telomerase induced by transformation and DNA damage

The telomerase ribonucleoprotein complex caps chromosome ends by adding telomeric repeats. Here we show that catalytically active human telomerase has a regulated intranuclear localization that is dependent on the cell-cycle stage, transformation and DNA damage. In primary cell lines, low expression of a fusion protein of green fluorescent protein and telomerase reverse transcriptase (GFP–hTERT) increases telomerase activity and stabilizes the maintenance of telomere length. Confocal microscopy shows that the release of telomerase to the nucleoplasm from sequestration at nucleolar sites is enhanced at the expected time of telomere replication. By contrast, in tumour and transformed cells, there is an almost complete dissociation of telomerase from nucleoli at all stages of the cell cycle. Transfection of the simian virus 40 genome into a primary cell line is sufficient to mobilize telomerase from nucleoli to the nucleoplasm. Conversely, ionizing radiation induces the reassociation of telomerase with nucleoli in both primary and transformed cells. These findings show that transformation and DNA damage have opposite effects on the cellular regulation of active telomerase, affecting the enzymes access to both telomeric and nontelomeric substrates.

Polymorphisms in the human AH receptor.

The AH receptor (AHR) mediates toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as well as induction of three cytochrome P450 enzymes and certain Phase II enzymes. In laboratory animals, genetic variations in the AHR lead to substantial differences in sensitivity to biochemical and toxic effects of TCDD and related compounds. Relatively few polymorphisms have been discovered in the human AHR gene; these occur predominantly in exon 10, a region that encodes a major portion of the transactivation domain of the receptor that is responsible for regulating expression of other genes. In human populations there is a wide range of variation in responses regulated by the AHR for example, induction of CYP1A1. Some variation in human responsiveness likely is due to genetically based variations in AHR structure. Thus far, however, only one pair of polymorphisms, those at codons 517 and 570, has been shown to have a clear cut and strong effect on the phenotype of an AHR-mediated response. The search continues for polymorphisms that alter AHR function because this receptor is a central factor in determining responses to important environmental contaminants and also plays a physiologic role in early development in mammals.

Visualization of RNA-Quadruplexes in Live Cells

Visualization of DNA and RNA quadruplex formation in human cells was demonstrated recently with different quadruplex-specific antibodies. Despite the significant interest in these immunodetection approaches, dynamic detection of quadruplex in live cells remains elusive. Here, we report on NaphthoTASQ (N-TASQ), a next-generation quadruplex ligand that acts as a multiphoton turn-on fluorescent probe. Single-step incubation of human and mouse cells with N-TASQ enables the direct detection of RNA-quadruplexes in untreated cells (no fixation, permeabilization or mounting steps), thus offering a unique, unbiased visualization of quadruplexes in live cells.

Analysis of epinephrine from fifteen different dental anesthetic formulations by capillary electrophoresis.

A robust method for the quantification of epinephrine from 15 different commercial dental anesthetic formulations is developed using CE. This work presents an extension to a method reported earlier. The solvability of several anesthetic compounds was improved through appropriate dilutions and the addition of sodium dodecyl sulfate to the separation background electrolyte. By controlling the mobility of the analyte at different pH values, a dilute solution of epinephrine is focused into a sharp zone with the injection of about 150 nl of anesthetic solution into the capillary. This on-column concentration technique extended the concentration detection limit of epinephrine to about 5.0 x 10(-7) M using a commercially available UV detector. A correlation plot between the measured and listed epinephrine concentration for the 15 dental anesthetic solutions demonstrated excellent accuracy of this method.

Telomerase RNA deficiency in peripheral blood mononuclear cells in X-linked dyskeratosis congenita

Compromised renewal and eventual failure of the hematopoietic system in dyskeratosis congenita (DC) have been proposed to arise from a deficiency in telomerase function. Previously, cultured cell lines from patients with X-linked DC were shown to accumulate less telomerase RNA than cell lines from unaffected family members. Here, we report that telomerase RNA deficiency is also present in the circulating lymphocytes of DC patients. We have compared the accumulation levels of telomerase RNA and a panel of other small RNAs in peripheral blood mononuclear cells from an X-linked DC patient and an unaffected maternal carrier and similarly analyzed cultured lymphoblasts from an X-linked DC patient and maternal carrier in a second family. The DC-patient lymphoid cells show a specific defect in telomerase RNA accumulation with or without cell culture. Our findings support the clinical significance of telomerase deficiency and encourage the use of telomerase activation as a disease therapy.

Human telomerase reverse transcriptase motifs required for elongation of a telomeric substrate.

The reverse transcriptase telomerase copies an internal RNA template to synthesize telomeric simple-sequence repeats. In the cellular context, telomerase must elongate its few intended substrates (authentic chromosome ends) without spurious activity on other potential substrates (chromosome ends created by damage, repair, or recombination). Many mechanisms have been proposed to account for the biological substrate specificity of telomerase, with most models focusing on protein-protein interactions between telomerase and telomeric chromatin. Telomerase activity assays testing the elongation of model oligonucleotide substrates have revealed that in addition to hybridization with the RNA template, optimal DNA substrates also engage telomerase protein-based interaction sites. The physiological significance of these non-template interaction sites has not been established. We used in vivo reconstitution to assemble telomerase enzymes with variant telomerase reverse transcriptase proteins. Several telomerase enzyme variants retained a wild-type level of catalytic function in vitro when assayed using an artificial sequence substrate but exhibited reduced activity on a more physiological telomeric-sequence substrate. Telomerases that demonstrated this defect in telomeric substrate usage in vitro also failed to support telomere length maintenance in vivo. Our findings suggest that non-template interactions of the telomerase ribonucleoprotein with telomeric DNA play a critical role in supporting telomerase function on its appropriate cellular substrates.

Dyskeratosis congenita: The first NIH clinical research workshop

Dyskeratosis congenita (DC) is a heterogeneous inherited bone marrow failure syndrome, characterized by abnormally short telomeres and mutations in telomere biology genes. The spectrum of telomere biology disorders is growing and the clinical management of these patients is complex. A DC‐specific workshop was held at the NIH on September 19, 2008; participants included physicians, patients with DC, their family members, and representatives from other support groups. Data from the UKs DC Registry and the NCIs DC cohort were described. Updates on the function of the known DC genes were presented. Clinical aspects discussed included androgen therapy, stem cell transplant, cancer risk, and cancer screening. Families with DC met for the first time and formed a family support group (http://www.dcoutreach.com/). Ongoing, open collaboration between the clinical, scientific, and family communities is required for continued improvement in our understanding of DC and the clinical consequences of telomeric defects. Pediatr Blood Cancer 2009;53:520–523.

Telomerase Inhibition Potentiates the Effects of Genotoxic Agents in Breast and Colorectal Cancer Cells in a Cell Cycle–Specific Manner

Previous studies have shown that telomerase facilitates DNA-damage repair and cell survival following stress. It is not clear how telomerase promotes DNA repair, or whether short-term telomerase inhibition, combined with genotoxic stress, can be exploited for cancer therapy. Here, we show that transient inhibition of telomerase activity by the specific inhibitor, GRN163L, increases the cytotoxicity of some, but not all, DNA-damaging agents. Such synergistic inhibition of growth requires the use of DNA-damaging agents that are toxic in the S/G(2) phase of the cell cycle. Notably, inhibition of Ataxia Telangiectasia Mutated (ATM) kinase, together with telomerase inhibition, synergistically increases the cytotoxicity induced by the G(2)-specific topoisomerase II inhibitor etoposide. By varying the timing of telomerase inhibition, relative to the timing of DNA damage, it is apparent that the prosurvival functions of telomerase occur at early stages of DNA damage recognition and repair. Our results suggest that the protective role of telomerase in cell cycle-restricted DNA damage repair could be exploited for combined anticancer chemotherapy.

In Vitro and Ex Vivo Inhibition of Human Telomerase by Anti-HIV Nucleoside Reverse Transcriptase Inhibitors (NRTIs) but Not by Non-NRTIs

Telomerase is a specialized reverse transcriptase responsible for the de novo synthesis of telomeric DNA repeats. In addition to its established reverse transcriptase and terminal transferase activities, recent reports have revealed unexpected cellular activities of telomerase, including RNA-dependent RNA polymerization. This telomerase characteristic, distinct from other reverse transcriptases, indicates that clinically relevant reverse transcriptase inhibitors might have unexpected telomerase inhibition profiles. This is particularly important for the newer generation of RT inhibitors designed for anti-HIV therapy, which have reported higher safety margins than older agents. Using an in vitro primer extension assay, we tested the effects of clinically relevant HIV reverse transcriptase inhibitors on cellular telomerase activity. We observed that all commonly used nucleoside reverse transcriptase inhibitors (NRTIs), including zidovudine, stavudine, tenofovir, didanosine and abacavir, inhibit telomerase effectively in vitro. Truncated telomere synthesis was consistent with the expected mode of inhibition by all tested NRTIs. Through dose-response experiments, we established relative inhibitory potencies of NRTIs on in vitro telomerase activity as compared to the inhibitory potencies of the corresponding dideoxynucleotide triphosphates. In contrast to NRTIs, the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine and efavirenz did not inhibit the primer extension activity of telomerase, even at millimolar concentrations. Long-term, continuous treatment of human HT29 cells with select NRTIs resulted in an accelerated loss of telomere repeats. All tested NRTIs exhibited the same rank order of inhibitory potencies on telomerase and HIV RT, which, according to published data, were orders-of-magnitude more sensitive than other DNA polymerases, including the susceptible mitochondria-specific DNA polymerase gamma. We concluded that telomerase activity could be inhibited by common NRTIs, including currently recommended RTI agents tenofovir and abacavir, which warrants large-scale clinical and epidemiological investigation of the off-target effects of long-term highly active antiretroviral therapy (HAART) with these agents.