Ligands that stabilize the formation of telomeric DNA G-quadruplexes have potential as cancer treatments, because the G-quadruplex structure cannot be extended by telomerase, an enzyme over-expressed in many cancer cells. Understanding the kinetic, thermodynamic and mechanical properties of small-molecule binding to these structures is therefore important, but classical ensemble assays are unable to measure these simultaneously. Here, we have used a laser tweezers method to investigate such interactions. With a force jump approach, we observe that pyridostatin promotes the folding of telomeric G-quadruplexes. The increased mechanical stability of pyridostatin-bound G-quadruplex permits the determination of a dissociation constant Kd of 490 ± 80 nM. The free-energy change of binding obtained from a Hess-like process provides an identical Kd for pyridostatin and a Kd of 42 ± 3 μM for a weaker ligand RR110. We anticipate that this single-molecule platform can provide detailed insights into the mechanical, kinetic and thermodynamic properties of liganded bio-macromolecules, which have biological relevance.

BackgroundAs the multipotent progenitor population of the airway epithelium, human airway basal cells (BC) replenish the specialized differentiated cell populations of the mucociliated airway epithelium during physiological turnover and repair. Cultured primary BC divide a limited number of times before entering a state of replicative senescence, preventing the establishment of long-term replicating cultures of airway BC that maintain their original phenotype.MethodsTo generate an immortalized human airway BC cell line, primary human airway BC obtained by brushing the airway epithelium of healthy nonsmokers were infected with a retrovirus expressing human telomerase (hTERT). The resulting immortalized cell line was then characterized under non-differentiating and differentiating air-liquid interface (ALI) culture conditions using ELISA, TaqMan quantitative PCR, Western analysis, and immunofluorescent and immunohistochemical staining analysis for cell type specific markers. In addition, the ability of the cell line to respond to environmental stimuli under differentiating ALI culture was assessed.ResultsWe successfully generated an immortalized human airway BC cell line termed BCi-NS1 via expression of hTERT. A single cell derived clone from the parental BCi-NS1 cells, BCi-NS1.1, retains characteristics of the original primary cells for over 40 passages and demonstrates a multipotent differentiation capacity into secretory (MUC5AC, MUC5B), goblet (TFF3), Clara (CC10) and ciliated (DNAI1, FOXJ1) cells on ALI culture. The cells can respond to external stimuli such as IL-13, resulting in alteration of the normal differentiation process.ConclusionDevelopment of immortalized human airway BC that retain multipotent differentiation capacity over long-term culture should be useful in understanding the biology of BC, the response of BC to environmental stress, and as a target for assessment of pharmacologic agents.

BackgroundAging involves multiple biologically complex processes characterized by a decline in cellular homeostasis over time leading to a loss and impairment of physiological integrity and function. Specific cellular hallmarks of aging include abnormal gene expression patterns, shortened telomeres and associated biological dysfunction. Like all organs, the lung demonstrates both physiological and structural changes with age that result in a progressive decrease in lung function in healthy individuals. Cigarette smoking accelerates lung function decline over time, suggesting smoking accelerates aging of the lung. Based on this data, we hypothesized that cigarette smoking accelerates the aging of the small airway epithelium, the cells that take the initial brunt of inhaled toxins from the cigarette smoke and one of the primary sites of pathology associated with cigarette smoking.MethodsUsing the sensitive molecular parameters of aging-related gene expression and telomere length, the aging process of the small airway epithelium was assessed in age matched healthy nonsmokers and healthy smokers with no physical manifestation of lung disease or abnormalities in lung function.ResultsAnalysis of a 73 gene aging signature demonstrated that smoking significantly dysregulates 18 aging-related genes in the small airway epithelium. In an independent cohort of male subjects, smoking significantly reduced telomere length in the small airway epithelium of smokers by 14% compared to nonsmokers.ConclusionThese data provide biologic evidence that smoking accelerates aging of the small airway epithelium.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-014-0094-1) contains supplementary material, which is available to authorized users.

Telomerase is the specialized reverse transcriptase responsible for DNA synthesis at chromosomal termini. 1 The two core components of human telomerase that are essential for catalytic activity are human telomerase RNA (hTR) 2 and human telomerase reverse transcriptase (hTERT). 3 hTR contains the template for reverse transcription and acts as a scaffold for assembly of the telomerase holoenzyme. Two regions of the hTR primary sequence that are conserved among vertebrates, CR2 and CR3, are predicted to fold into a type of structure called a pseudoknot, in which the single-stranded terminal loop of an RNA stem-loop is base-paired with a single-stranded region elsewhere in the RNA molecule. 4 The presence of a pseudoknot element in close proximity to the template, within a template/ pseudoknot domain, has been proposed as a universal feature of telomerase RNAs from all species. 5 Conservation of this motif is suggestive of an important role in telomerase function, and this has been supported by mutational analysis. 6 -9 A minimal but nonfunctional model of the hTR pseudoknot, ~50 nucleotides in length and formed essentially from sequences CR2 and CR3 alone, does fold into a pseudoknot. 8 , 10 -13 However, there is a lack of direct physical evidence to support the existence of a pseudoknot in full-length wild-type (wt) telomerase RNA from humans or any other species. Herein we provide biophysical evidence to support the existence of a pseudoknot in full-length hTR, but only as part of a catalytically competent telomerase complex.For our studies, we first synthesized a dual-labeled full-length hTR construct, DL hTR, that was site-specifically modified with a donor and an acceptor fluorophore suitable for Förster resonance energy transfer (FRET) analysis specifically to report on pseudoknot formation (Figure 1). In particular, formation of helix P3 and therefore the hTR pseudoknot was predicted, on the basis of an NMR structure of the minimal wt hTR pseudoknot and a molecular model of the entire template/pseudoknot domain, to result in formation of a high-FRET species (FRET efficiency close to 0.68) (Figure 1a; for details of the interfluorophore distance calculation, see the Supporting Information). 13 , 14 A scenario in which the hTR pseudoknot was not present was predicted to result in formation of a low-or zero-FRET species ( Figure 1b). We assembled DL hTR from three pieces of RNA; two were generated by runoff in vitro transcription, and the third was produced by chemical synthesis. DL hTR Supporting Information Available: General experimental protocols, experimental details for the synthesis of DL hTR, experimental details and data for the TRAP assay, details of the interfluorophore distance calculation, and additional FRET data. This material is available free of charge via the Internet at http://pubs.acs.org. Because of the low quantities of sample we were able to produce, which precluded classical ensemble biophysical approaches, and the potential for conformational heterogeneity, it was advantageous to study...

It has been proposed that human telomerase RNA (hTR) interacts with dyskerin, prior to assembly of the telomerase holoenzyme. The direct interaction of dyskerin and hTR has not been demonstrated and is an experimentally challenging research problem because of difficulties in expressing and purifying dyskerin in quantities that are useful for biophysical analysis. By orthogonally labeling dyskerin and hTR, we have been able to employ single-molecule two-color coincidence detection (TCCD) to observe directly the formation of a dyskerin·hTR complex. By systematic deletion of hTR subdomains, we have gained insights into the RNA sites required for interaction with dyskerin. We then investigated mutated forms of hTR and dyskerin that are associated with dyskeratosis congenita (DC), on the basis of clinical genetics studies, for their effects on the dyskerin·hTR interaction. Dyskerin mutations associated with X-linked DC resulted in significant impairment of the dyskerin·hTR interaction, whereas mutations in hTR associated with autosomal dominant (AD) DC did not affect the interaction. We propose that disruption of the dyskerin·hTR interaction may contribute to X-linked DC.

Cord blood (CB) leukocytes have inherent telomere length (TL) variation, and CB hematopoietic stem cells (HSC) can maintain high telomerase levels preventing telomere attrition in vitro. We evaluated TL changes in 13 adult double unit CB transplantation (CBT) recipients. In the 26 units, we observed a marked variation in CB TL at thaw (median 9.99 kb, range 6.85-13.5). All 13 patients engrafted. Of 11 engrafting with one unit, there was no correlation between unit dominance and TL (mean dominant unit TL 8.84 kb ± 1.76, mean non-engrafting unit TL 10.3 kb ± 1.81, P = 0.77). Serial measurements of TL up to 1-year post-CBT demonstrated an overall mean 3.04 kb ± 0.16 TL decrease with only one patient exhibiting telomere maintenance. In summary, initial TL does not predict CB unit dominance. Moreover, our analysis suggests neonatal hematopoiesis makes a transition to an HSC characterized by changes in average TL and potentially low telomerase asymmetric cell division in adult CBT recipients. Further investigation of alterations in telomere length and its clinical implications post-transplant of this observation are indicated.

4661 Background: Umbilical cord blood (CB) is an alternative source of allogeneic hematopoietic stem cells for transplantation and has the advantage of a reduced stringency of the required human leukocyte antigen-match. It is limited, however, by low total nucleated cell (TNC) dose and a low progenitor number in single CB units, thus restricting the use of CB transplantation (CBT) in larger children and adults. One strategy to augment engraftment is to combine 2 units from 2 different donors in a double-unit graft. We have previously shown that in most cases, one unit emerges as the sole source of hematopoiesis long-term, but as yet the mechanism of unit dominance remains unknown (Blood, 2010, 116(19):3999–4006). CB units are known to have an inherent biological variation in telomere length, the repeat sequence capping the ends of chromosomes. Telomere length variation and progressive shortening of telomere could later hematopoietic potential. Methods: We evaluated if telomere length has a role in unit dominance and how telomere length progresses over time post-transplant. We purified mononuclear cells from small aliquots of each unit of double-unit grafts and post-transplant peripheral blood (days +28, 100, 180 and 1 year) in 12 adult double-unit CBT recipients transplanted for hematologic malignancies at MSKCC. Average telomere length was measured using the TeloTTAGGG Telomere Length Assay (Roche) which utilizes Southern analysis of terminal restriction fragments (TRF) that are obtained by the digestion of isolated genomic DNA. Results: All 12 patients engrafted with one unit showing predominance. While there was a range of telomere length (5.57–12.09 kb) in the 24 units evaluated on the day of transplant, when comparing the telomere length of engrafting to non-engrafting units, there was no association between telomere length at day 0 and subsequent unit dominance. In 5 patients the engrafting unit had longer telomeres and in 7 patients the non-engrafting unit had longer telomeres. In serial assays, 7 of 12 patients demonstrated telomere length stabilization post-transplant in the dominant unit with a mean percentage loss of telomere length of 7.15% ± 2.33%. A second group (n = 5) demonstrated a decrease in telomere length with a mean percentage loss of 29.48% ± 5.12%. While this difference is significant (p = 0.0015) the clinical significance of this finding is uncertain. We are currently following these patients in order to correlate telomere length (stabilization versus decrease) with clinical outcomes. Conclusions: This data suggests that unit dominance is not influenced by telomere length. It is likely, based on emerging data from our laboratory as well as others, that unit dominance is immune mediated. However, the influence of telomere length on the quality of engraftment is of interest and analysis of this is ongoing with larger numbers of patients required to also consider the infused cell dose. It is notable that 7/12 patients had telomere length stabilization which correlates with the high levels of telomerase activity previously reported in the in vitro expansion of CB (Blood, 2004, 103(12):4440-8). Disclosures: No relevant conflicts of interest to declare.