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      Telomerase Inhibition by Everolimus Suppresses Smooth Muscle Cell Proliferation and Neointima Formation Through Epigenetic Gene Silencing

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          Summary

          Proliferation of smooth muscle cells (SMCs) during neointima formation is prevented by drug-eluting stents. The replicative capacity of mammalian cells is enhanced by telomerase expression; however, the contribution of telomerase to the proliferative response underlying neointima formation and its potential role as a pharmacological target are unknown. The present study investigated the mechanisms underlying the mitogenic function of telomerase, and tested the hypothesis that everolimus, which is commonly used on drug-eluting stents, suppresses SMC proliferation by targeting telomerase. Inhibition of neointima formation by everolimus was lost in mice overexpressing telomerase reverse transcriptase (TERT), indicating that repression of telomerase confers the anti-proliferative efficacy of everolimus. Everolimus reduced TERT expression in SMC through an Ets-1-dependent inhibition of promoter activation. The inhibition of TERT-dependent SMC proliferation by everolimus occurred in the absence of telomere shortening but rather as a result of a G1→S-phase arrest. Although everolimus failed to inhibit phosphorylation of the retinoblastoma protein as the gatekeeper of S-phase entry, it potently repressed downstream target genes. Chromatin immunoprecipitation assays demonstrated that TERT induced E2F binding to S-phase gene promoters and supported histone acetylation. These effects were sensitive to inhibition by everolimus. These results characterize telomerase as a previously unrecognized target for the antiproliferative activity of everolimus, and further identify a novel mitogenic pathway in SMC that depends on the epigenetic activation of S-phase gene promoters by TERT.

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          • The proliferative capacity of smooth muscle cells (SMC) during neointima formation is prevented by everolimus-coated drug-eluting stents.

          • Everolimus failed to inhibit neointima formation by in mice overexpressing telomerase reverse transcriptase (TERT).

          • Everolimus reduced TERT-dependent SMC proliferation through inhibition of Ets-1–dependent promoter activation.

          • The inhibition of TERT-dependent SMC proliferation by everolimus occurred as a result of a G1→S-phase arrest, rather than telomerase shortening.

          • Chromatin immunoprecipitation assays demonstrated that TERT induced E2F binding to S-phase gene promoters and supported histone acetylation.

          • These studies identify a novel mitogenic pathway in SMC that depends on the epigenetic activation of S-phase gene promoters by TERT.

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          Most cited references24

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          Extension of life-span by introduction of telomerase into normal human cells.

          Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced straining for beta-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.
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            Essential role of mouse telomerase in highly proliferative organs.

            We have investigated the role of the enzyme telomerase in highly proliferative organs in successive generations of mice lacking telomerase RNA. Late-generation animals exhibited defective spermatogenesis, with increased programmed cell death (apoptosis) and decreased proliferation in the testis. The proliferative capacity of haematopoietic cells in the bone marrow and spleen was also compromised. These progressively adverse effects coincided with substantial erosion of telomeres (the termini of eukaryotic chromosomes) and fusion and loss of chromosomes. These findings indicate an essential role for telomerase, and hence telomeres, in the maintenance of genomic integrity and in the long-term viability of high-renewal organ systems.
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              Molecular mechanisms underlying RB protein function.

              Inactivation of the RB protein is one of the most fundamental events in cancer. Coming to a molecular understanding of its function in normal cells and how it impedes cancer development has been challenging. Historically, the ability of RB to regulate the cell cycle placed it in a central role in proliferative control, and research focused on RB regulation of the E2F family of transcription factors. Remarkably, several recent studies have found additional tumour-suppressor functions of RB, including alternative roles in the cell cycle, maintenance of genome stability and apoptosis. These advances and new structural studies are combining to define the multifunctionality of RB.
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                Author and article information

                Contributors
                Journal
                JACC Basic Transl Sci
                JACC Basic Transl Sci
                JACC: Basic to Translational Science
                Elsevier
                2452-302X
                15 February 2016
                Jan-Feb 2016
                15 February 2016
                : 1
                : 1-2
                : 49-60
                Affiliations
                [1]Division of Cardiovascular Medicine, Gill Heart Institute and Saha Cardiovascular Research Center, University of Kentucky, Lexington, Kentucky
                Author notes
                [] Reprint requests and correspondence: Dr. Dennis Bruemmer, Division of Cardiovascular Medicine, Gill Heart Institute and Saha Cardiovascular Research Center, University of Kentucky, B257 BBSRB, 741 South Limestone Street, Lexington, Kentucky 40536-0200. dennis.bruemmer@ 123456uky.edu
                Article
                S2452-302X(16)00006-1
                10.1016/j.jacbts.2016.01.002
                4843168
                27127803
                5c4b4207-849a-4859-8356-062e1022f7c4
                © 2016 The Authors

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 9 October 2015
                : 3 December 2015
                Categories
                PRE-CLINICAL RESEARCH

                everolimus,neointima,smooth muscle cell,telomerase,chip, chromatin immunoprecipitation,mcm7, minichromosome maintenance protein 7,mtor, mammalian target of rapamycin,pcna, proliferating cell nuclear antigen,rb, retinoblastoma protein,smc, vascular smooth muscle cells,tbp, tata binding protein,tert, telomerase reverse transcriptase,terttg, telomerase reverse transcriptase–overexpressing transgenic,wt, wild type

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