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Bolzán AD. Mutagen-induced telomere instability in human cells. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2021; 868-869:503387. [PMID: 34454696 DOI: 10.1016/j.mrgentox.2021.503387] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/29/2021] [Revised: 07/13/2021] [Accepted: 07/18/2021] [Indexed: 11/27/2022]
Abstract
Telomere instability is one of the main sources of genome instability and may result from chromosome end loss (due to chromosome breakage at one or both ends) or, more frequently, telomere dysfunction. Dysfunctional telomeres arise when they lose their end-capping function or become critically short, which causes chromosomal termini to behave like a DNA double-strand break. Telomere instability may occur at the chromosomal or at the molecular level, giving rise, respectively, to telomere-related chromosomal aberrations or the loss or modification of any of the components of the telomere (telomere DNA, telomere-associated proteins, or telomere RNA). Since telomeres play a fundamental role in maintaining genome stability, the study of telomere instability in cells exposed to mutagens is of great importance to understand the telomere-driven genomic instability present in those cells. In the present review, we will focus on the current knowledge about telomere instability induced by physical, chemical, and biological mutagens in human cells.
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Affiliation(s)
- Alejandro D Bolzán
- Laboratorio de Citogenética y Mutagénesis, Instituto Multidisciplinario de Biología Celular (IMBICE, CONICET-CICPBA-UNLP), calle 526 y Camino General Belgrano, B1906APO La Plata, Buenos Aires, Argentina; Universidad Nacional de La Plata, Facultad de Ciencias Naturales y Museo, calle 60 y 122, La Plata, Buenos Aires, Argentina.
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Kitsou C, Lazaros L, Papoudou-Bai A, Sakaloglou P, Mastora E, Lykovardakis T, Giaka K, Vartholomatos G, Bouba I, Markoula S, Batistatou A, Georgiou I. Reverse Transcriptase Affects Gametogenesis and Preimplantation Development in Mouse. In Vivo 2021; 34:2269-2276. [PMID: 32871749 DOI: 10.21873/invivo.12037] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2020] [Revised: 05/04/2020] [Accepted: 05/05/2020] [Indexed: 11/10/2022]
Abstract
BACKGROUND/AIM The expression of reverse transcriptase (RT) in ovaries, testes, gametes and embryos highlights its critical role in cell growth and differentiation. We sought to investigate the effects of the potent RT inhibitor lamivudine in gametogenesis and mouse embryo preimplantation development. MATERIALS AND METHODS Male and female FVB/N mice were treated with the reverse transcriptase inhibitor Lamivudine for seven consecutive weeks. Following treatment, mouse sperm parameters, testicular and ovarian morphology as well as post-IVF embryo development were evaluated. RESULTS Lamivudine impaired the sperm parameters and the testicular structure in male mice, the number of primordial germ cells and primary oocytes in ovaries of female mice, and the embryos' morphology and development up to the blastocyst stage during in vitro culture. CONCLUSION The administration of lamivudine affected the processes of spermatogenesis and oogenesis as well as the in vitro preimplantation development of mouse embryos.
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Affiliation(s)
- Chrysoula Kitsou
- Laboratory of Medical Genetics of Human Reproduction, Medical School, Ioannina University, Ioannina, Greece
| | - Leandros Lazaros
- Laboratory of Medical Genetics of Human Reproduction, Medical School, Ioannina University, Ioannina, Greece.,Genesis Genoma Lab, Genetic Diagnosis-Clinical Genetics-Research, Athens, Greece
| | | | - Prodromos Sakaloglou
- Laboratory of Medical Genetics of Human Reproduction, Medical School, Ioannina University, Ioannina, Greece
| | - Eirini Mastora
- Laboratory of Medical Genetics of Human Reproduction, Medical School, Ioannina University, Ioannina, Greece
| | - Theodoros Lykovardakis
- Laboratory of Medical Genetics of Human Reproduction, Medical School, Ioannina University, Ioannina, Greece
| | - Katerina Giaka
- Laboratory of Medical Genetics of Human Reproduction, Medical School, Ioannina University, Ioannina, Greece
| | - Georgios Vartholomatos
- Laboratory of Hematology, Molecular Biology Unit, Ioannina University Hospital, Ioannina, Greece
| | - Ioanna Bouba
- Laboratory of Medical Genetics of Human Reproduction, Medical School, Ioannina University, Ioannina, Greece
| | - Sofia Markoula
- Department of Neurology, Ioannina University Hospital, Ioannina, Greece
| | - Anna Batistatou
- Department of Pathology, Ioannina University Hospital, Ioannina, Greece
| | - Ioannis Georgiou
- Laboratory of Medical Genetics of Human Reproduction, Medical School, Ioannina University, Ioannina, Greece
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3
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Association between zidovudine-containing antiretroviral therapy exposure in utero and leukocyte telomere length at birth. AIDS 2019; 33:2091-2096. [PMID: 31335808 DOI: 10.1097/qad.0000000000002317] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
OBJECTIVES Zidovudine (ZDV) is a nucleoside reverse transcriptase inhibitor that could cause telomere shortening through inhibition of telomerase. We examined the association between in utero exposure to ZDV and telomere length at birth in HIV-exposed-uninfected (HEU) newborns. METHODS We selected 94 ZDV-exposed HEU children and 85 antiretroviral therapy (ART)-unexposed HEU children from the Surveillance Monitoring for ART Toxicities Study and the Women and Infants Transmission Study. We assessed relative telomere length in stored peripheral blood mononuclear cells taken in the first 7 days of life using quantitative polymerase chain reaction. We used linear regression to compare relative telomere length between ZDV-exposed and ART-unexposed children. We additionally evaluated relative telomere length according to maternal and infant characteristics. RESULTS Relative telomere length was longer in ZDV-exposed children compared with ART-unexposed individuals (adjusted mean ratio difference 0.21, 95% confidence interval 0.15-0.28, P < 0.001). We found an inverse correlation between maternal HIV RNA levels and infant relative telomere length (-0.06 per log10 copies, 95% confidence interval -0.08 to -0.03, P < 0.001). Relative telomere length was not associated with maternal CD4 cell count, maternal age, gestational age, sex, sample storage time, or maternal substance use (P > 0.05). CONCLUSION Relative telomere length was longer in ZDV-exposed infants. This difference may reflect beneficial health effects of ART during pregnancy, as we observed an inverse association with maternal HIV RNA levels.
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Gomez DLM, Armando RG, Cerrudo CS, Ghiringhelli PD, Gomez DE. Telomerase as a Cancer Target. Development of New Molecules. Curr Top Med Chem 2017; 16:2432-40. [PMID: 26873194 PMCID: PMC4997958 DOI: 10.2174/1568026616666160212122425] [Citation(s) in RCA: 59] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2015] [Revised: 09/15/2015] [Accepted: 10/25/2015] [Indexed: 12/26/2022]
Abstract
Telomeres are the terminal part of the chromosome containing a long repetitive and non-codifying sequence that has as function protecting the chromosomes. In normal cells, telomeres lost part of such repetitive sequence in each mitosis, until telomeres reach a critical point, triggering at that time senescence and cell death. However, in most of tumor cells in each cell division a part of the telomere is lost, however the appearance of an enzyme called telomerase synthetize the segment that just has been lost, therefore conferring to tumor cells the immortality hallmark. Telomerase is significantly overexpressed in 80–95% of all malignant tumors, being present at low levels in few normal cells, mostly stem cells. Due to these characteristics, telomerase has become an attractive target for new and more effective anticancer agents. The capability of inhibiting telomerase in tumor cells should lead to telomere shortening, senescence and apoptosis. In this work, we analyze the different strategies for telomerase inhibition, either in development, preclinical or clinical stages taking into account their strong points and their caveats. We covered strategies such as nucleosides analogs, oligonucleotides, small molecule inhibitors, G-quadruplex stabilizers, immunotherapy, gene therapy, molecules that affect the telomere/telomerase associated proteins, agents from microbial sources, among others, providing a balanced evaluation of the status of the inhibitors of this powerful target together with an analysis of the challenges ahead.
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Affiliation(s)
| | | | | | | | - D E Gomez
- Laboratory of Molecular Oncology, Department of Science and Technology. Quilmes National University, Bernal, Buenos Aires, Argentina. R. Saenz Peña 352, (1876) Buenos Aires, Argentina.
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5
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Varshney A, Bala J, Santosh B, Bhaskar A, Kumar S, Yadava PK. Identification of an RNA aptamer binding hTERT-derived peptide and inhibiting telomerase activity in MCF7 cells. Mol Cell Biochem 2016; 427:157-167. [PMID: 28004350 DOI: 10.1007/s11010-016-2907-7] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2016] [Accepted: 12/03/2016] [Indexed: 01/30/2023]
Abstract
Human telomerase reverse transcriptase is an essential rate-limiting component of telomerase complex. hTERT protein in association with other proteins and the human telomerase RNA (hTR) shows telomerase activity, essential for maintaining genomic integrity in proliferating cells. hTERT binds hTR through a decapeptide located in the RID2 (RNA interactive domain 2) domain of N-terminal region. Since hTERT is essential for telomerase activity, inhibitors of hTERT are of great interest as potential anti-cancer agent. We have selected RNA aptamers against a synthetic peptide from the RID2 domain of hTERT by employing in vitro selection protocol (SELEX). The selected RNAs could bind the free peptide, as CD spectra suggested conformational change in aptamer upon RID2 binding. Extracts of cultured breast cancer cells (MCF7) expressing this aptamer showed lower telomerase activity as estimated by TRAP assay. hTERT-binding RNA aptamers hold promise as probable anti-cancer therapeutic agent.
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Affiliation(s)
- Akhil Varshney
- Applied Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India
| | - Jyoti Bala
- Applied Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India
| | - Baby Santosh
- Applied Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India
| | - Ashima Bhaskar
- Applied Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India
| | - Suresh Kumar
- Applied Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India.,Molecular Genetics Laboratory, Institute of Cytogenetic and Preventive Oncology, Indian Council of Medical Research, Noida, Uttar Pradesh, 201301, India
| | - Pramod K Yadava
- Applied Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India.
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Armando RG, Gomez DM, Gomez DE. AZT exerts its antitumoral effect by telomeric and non-telomeric effects in a mammary adenocarcinoma model. Oncol Rep 2016; 36:2731-2736. [DOI: 10.3892/or.2016.5094] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2016] [Accepted: 07/29/2016] [Indexed: 11/06/2022] Open
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7
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Bryan C, Rice C, Hoffman H, Harkisheimer M, Sweeney M, Skordalakes E. Structural Basis of Telomerase Inhibition by the Highly Specific BIBR1532. Structure 2015; 23:1934-1942. [PMID: 26365799 DOI: 10.1016/j.str.2015.08.006] [Citation(s) in RCA: 100] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2015] [Revised: 08/12/2015] [Accepted: 08/13/2015] [Indexed: 01/03/2023]
Abstract
BIBR1532 is a highly specific telomerase inhibitor, although the molecular basis for inhibition is unknown. Here we present the crystal structure of BIBR1532 bound to Tribolium castaneum catalytic subunit of telomerase (tcTERT). BIBR1532 binds to a conserved hydrophobic pocket (FVYL motif) on the outer surface of the thumb domain. The FVYL motif is near TRBD residues that bind the activation domain (CR4/5) of hTER. RNA binding assays show that the human TERT (hTERT) thumb domain binds the P6.1 stem loop of CR4/5 in vitro. hTERT mutations of the FVYL pocket alter wild-type CR4/5 binding and cause telomere attrition in cells. Furthermore, the hTERT FVYL mutations V1025F, N1028H, and V1090M are implicated in dyskeratosis congenita and aplastic anemia, further supporting the biological and clinical relevance of this novel motif. We propose that CR4/5 contacts with the telomerase thumb domain contribute to telomerase ribonucleoprotein assembly and promote enzymatic activity.
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Affiliation(s)
- Christopher Bryan
- The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA; Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104, USA
| | - Cory Rice
- The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA; Department of Biochemistry, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Hunter Hoffman
- The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA
| | | | - Melanie Sweeney
- The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA; Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104, USA
| | - Emmanuel Skordalakes
- The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA; Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104, USA; Department of Biochemistry, University of Pennsylvania, Philadelphia, PA 19104, USA.
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Matteucci C, Minutolo A, Marino-Merlo F, Grelli S, Frezza C, Mastino A, Macchi B. Characterization of the enhanced apoptotic response to azidothymidine by pharmacological inhibition of NF-kB. Life Sci 2015; 127:90-7. [PMID: 25744407 DOI: 10.1016/j.lfs.2015.01.038] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2014] [Revised: 11/10/2014] [Accepted: 01/28/2015] [Indexed: 12/26/2022]
Abstract
AIMS The present study addresses the issue of enhanced apoptotic response to AZT following co-treatment with an NF-kB inhibitor. MAIN METHODS To investigate this issue, different cell lines were assayed for susceptibility to AZT-mediated apoptosis without or with the addition of the NF-kB inhibitor Bay-11-7085. For further investigation, U937 cells were selected as good-responder cells to the combination treatment with 32 or 128 μM AZT, and 1 μM Bay-11-7085. Inhibition of NF-kB activation by Bay-11-7085 in cells treated with AZT was assayed through Western blot analysis of p65 expression and by EMSA. Involvement of the mitochondrial pathway of apoptosis in mechanisms underlying the improved effect of AZT following Bay-11-7085 co-treatment, was evaluated by assaying the cytochrome c release and the mitochondrial membrane potential (MMP) status using the JC-1 dye. Moreover, the transcriptional activity of both anti- and pro-apoptotic genes in U937 cells after combination treatment was quantitatively evaluated through real-time PCR. KEY FINDINGS We found that the combined treatment induced high levels of cytochrome c release and of MMP collapse in association with evident changes in the expression of both anti- and pro-apoptotic genes of the Bcl-2 family. Overexpression of Bcl-2 significantly suppressed the sensitization of U937 cells to an enhanced apoptotic response to AZT following co-treatment with the NF-kB inhibitor. SIGNIFICANCE The new findings suggest that a combination regimen based on AZT plus an NF-kB inhibitor could represent a new chemotherapeutic tool for retrovirus-related pathologies.
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Affiliation(s)
- Claudia Matteucci
- Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", Rome, Italy
| | - Antonella Minutolo
- Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", Rome, Italy
| | - Francesca Marino-Merlo
- Department of Biological and Environmental Sciences, University of Messina, Messina, Italy
| | - Sandro Grelli
- Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", Rome, Italy
| | - Caterina Frezza
- Department of Systems Medicine, University of Rome "Tor Vergata", Rome, Italy
| | - Antonio Mastino
- Department of Biological and Environmental Sciences, University of Messina, Messina, Italy; The Institute of Translational Pharmacology, CNR, Rome, Italy.
| | - Beatrice Macchi
- Department of Systems Medicine, University of Rome "Tor Vergata", Rome, Italy
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Olivero OA, Ongele MO, Braun HM, Marrogi A, Divi K, Mitchell JB, Poirier MC. Selective protection of zidovudine-induced DNA-damage by the antioxidants WR-1065 and tempol. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2014; 55:566-572. [PMID: 24833597 PMCID: PMC7673230 DOI: 10.1002/em.21872] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/23/2014] [Revised: 04/03/2014] [Accepted: 04/23/2014] [Indexed: 06/03/2023]
Abstract
The cytokinesis-block micronucleus cytome (CBMN) assay, introduced by Fenech, was used to demonstrate different types of DNA damage in MOLT-3 human lymphoblastoid cells exposed to 10 μM zidovudine (AZT). In addition, we explored the cytoprotective potential of two antioxidants, WR-1065 and Tempol, to decrease AZT-induced genotoxicity. Binucleated cells, arrested by Cytochalasin B (Cyt B), were evaluated for micronuclei (MN), caused by DNA damage or chromosomal loss, and chromatin nucleoplasmic bridges (NPBs), caused by telomere attrition. Additionally, nuclear buds (NBUDs), caused by amplified DNA, and apoptotic and necrotic (A/N) cells were scored. We hypothesized that AZT exposure would increase the frequency of genotoxic end points, and that the antioxidants Tempol and WR-1065 would protect against AZT-induced genotoxicity. MOLT-3 cells were exposed to 0 or 10 µM AZT for a total of 76 hr. After the first 24 hr, 0 or 5 µM WR-1065 and/or 0 or 200 µM Tempol were added for the remainder of the experiment. For the last 28 hr (of 76 hr), Cyt B was added to arrest replication after one cell division, leaving a predominance of binucleated cells. The nuclear division index (NDI) was similar for all treatment groups, indicating that the exposures did not alter cell viability. MOLT-3 cells exposed to AZT alone had significant (P < 0.05) increases in MN and NBs, compared to unexposed cells. Both Tempol and WR-1065 protected against AZT-induced MN formation (P < 0.003 for both), and WR-1065, but not Tempol, reduced the levels of A/N (P = 0.041). In cells exposed to AZT/Tempol there were significantly reduced levels of NBUDs, compared to cells exposed to AZT alone (P = 0.015). Cells exposed to AZT/WR-1065 showed reduced levels of NPBs, compared to cells exposed to AZT alone (P = 0.037). Thus WR-1065 and Tempol protected MOLT-3 cells against specific types of AZT-induced DNA damage.
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Affiliation(s)
- Ofelia A. Olivero
- Carcinogen–DNA Interactions Section, Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
| | - Michael O. Ongele
- Carcinogen–DNA Interactions Section, Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
| | - Hannan M. Braun
- Carcinogen–DNA Interactions Section, Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
| | - Ariadna Marrogi
- Carcinogen–DNA Interactions Section, Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
| | - Kathyiani Divi
- Carcinogen–DNA Interactions Section, Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
| | - James B. Mitchell
- Tumor Biology Section, Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
| | - Miriam C. Poirier
- Carcinogen–DNA Interactions Section, Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
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Aging and HIV/AIDS: pathogenetic role of therapeutic side effects. J Transl Med 2014; 94:120-8. [PMID: 24336070 PMCID: PMC4144856 DOI: 10.1038/labinvest.2013.142] [Citation(s) in RCA: 61] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2013] [Revised: 10/17/2013] [Accepted: 10/22/2013] [Indexed: 12/22/2022] Open
Abstract
The intersection of aging and HIV/AIDS is a looming 'epidemic within an epidemic.' This paper reviews how HIV/AIDS and its therapy cause premature aging or contribute mechanistically to HIV-associated non-AIDS illnesses (HANA). Survival with HIV/AIDS has markedly improved by therapy combinations containing nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors, and protease inhibitors (PIs) called HAART (highly active antiretroviral therapy). Because NRTIs and PIs together prevent or attenuate HIV-1 replication, and prolong life, the population of aging patients with HIV/AIDS increases accordingly. However, illnesses frequently associated with aging in the absence of HIV/AIDS appear to occur prematurely in HIV/AIDS patients. Theories that help to explain biological aging include oxidative stress (where mitochondrial oxidative injury exceeds antioxidant defense), chromosome telomere shortening with associated cellular senescence, and accumulation of lamin A precursors (a nuclear envelop protein). Each of these has the potential to be enhanced or caused by HIV/AIDS, antiretroviral therapy, or both. Antiretroviral therapy has been shown to enhance events seen in biological aging. Specifically, antiretroviral NRTIs cause mitochondrial dysfunction, oxidative stress, and mitochondrial DNA defects that resemble features of both HANA and aging. More recent clinical evidence points to telomere shortening caused by NRTI triphosphate-induced inhibition of telomerase, suggesting telomerase reverse transcriptase (TERT) inhibition as being a pathogenetic contributor to premature aging in HIV/AIDS. PIs may also have a role in premature aging in HIV/AIDS as they cause prelamin A accumulation. Overall, toxic side effects of HAART may both resemble and promote events of aging and are worthy of mechanistic studies.
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Bollmann FM. Telomerase inhibition may contribute to accelerated mitochondrial aging induced by anti-retroviral HIV treatment. Med Hypotheses 2013; 81:285-7. [PMID: 23679995 DOI: 10.1016/j.mehy.2013.04.028] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2012] [Revised: 04/08/2013] [Accepted: 04/16/2013] [Indexed: 01/27/2023]
Abstract
HIV-infected individuals undergoing long-term anti-retroviral treatment tend to show premature senescence. Accelerated mitochondrial aging induced by nucleoside reverse transcriptase inhibitors (NRTIs) has been implicated as a part of this phenomenon. Traditionally, this has been attributed to inhibition of mtDNA polymerase γ by these drugs, but alternative explanations have been proposed. It is known that NRTIs can not only inhibit viral reverse transcriptase, but also human telomerase. A number of extratelomeric roles of telomerase, including protection of mitochondrial DNA and function, have emerged recently. In this paper, I propose that inhibition of mitochondrial telomerase activity by NRTI drugs contributes to the mitochondrial toxicity and premature aging seen in treated HIV patients, and discuss objections and experimental testing of the hypothesis.
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Affiliation(s)
- F M Bollmann
- University Medical Center Tübingen, Wilhelmstr 27, 72016 Tübingen, Germany.
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12
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Hukezalie KR, Thumati NR, Côté HCF, Wong JMY. In vitro and ex vivo inhibition of human telomerase by anti-HIV nucleoside reverse transcriptase inhibitors (NRTIs) but not by non-NRTIs. PLoS One 2012; 7:e47505. [PMID: 23166583 PMCID: PMC3499584 DOI: 10.1371/journal.pone.0047505] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2012] [Accepted: 09/14/2012] [Indexed: 02/03/2023] Open
Abstract
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.
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Affiliation(s)
- Kyle R. Hukezalie
- Genetics Graduate Program, The University of British Columbia, Vancouver, British Columbia, Canada
- Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada
| | - Naresh R. Thumati
- Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada
| | - Hélène C. F. Côté
- Genetics Graduate Program, The University of British Columbia, Vancouver, British Columbia, Canada
- Department of Pathology and Laboratory Medicine (HCFC), The University of British Columbia, Vancouver, British Columbia, Canada
| | - Judy M. Y. Wong
- Genetics Graduate Program, The University of British Columbia, Vancouver, British Columbia, Canada
- Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada
- * E-mail:
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Leukocyte telomere length in HIV-infected pregnant women treated with antiretroviral drugs during pregnancy and their uninfected infants. J Acquir Immune Defic Syndr 2012; 60:495-502. [PMID: 22580562 DOI: 10.1097/qai.0b013e31825aa89c] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
OBJECTIVES HIV disease can lead to accelerated telomere attrition, although certain drugs used as part of antiretroviral therapy (ART) can inhibit telomerase reverse transcriptase activity. This could in turn lead to shorter telomeres. We hypothesized that HIV and ART exposure would be associated with shorter leukocyte telomere length (TL) in exposed mother/infant pairs compared with controls. METHODS In these retrospective and prospective observational cohort studies, TL was evaluated in peripheral blood leukocytes obtained from HIV-infected pregnant women treated with ART and their uninfected infants, and compared with HIV untreated (retrospective cohort) or HIV mothers and their infants (prospective cohort). RESULTS In HIV-infected ART-exposed mothers, leukocyte TL was not significantly shorter than that in HIV untreated mothers or HIV controls, nor was their infants' TL significantly different. Cord blood of ART-exposed infants exhibited TL shorter than that from infants born to HIV-negative mothers. Placenta also showed evidence of shorter TL after adjustment for relevant covariates. Factors associated with shorter maternal and infant TL included smoking and the use of drugs of addiction in pregnancy. CONCLUSIONS These results suggest that maternal HIV infection or exposure to ART has minimal effect on infant leukocyte TL, a reassuring finding. In contrast, tissues that express higher telomerase activity such as umbilical cord blood and placenta appear comparatively more affected by ART. Smoking and the use of drugs of addiction have a negative impact on maternal and infant leukocyte TL, possibly through oxidative telomere damage.
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Gomez DE, Armando RG, Alonso DF. AZT as a telomerase inhibitor. Front Oncol 2012; 2:113. [PMID: 22973556 PMCID: PMC3434370 DOI: 10.3389/fonc.2012.00113] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2012] [Accepted: 08/17/2012] [Indexed: 01/23/2023] Open
Abstract
Telomerase is a highly specialized reverse transcriptase (RT) and the maintenance of telomeric length is determined by this specific enzyme. The human holoenzyme telomerase is a ribonucleoprotein composed by a catalytic subunit, hTERT, an RNA component, hTR, and a group of associated proteins. Telomerase is normally expressed in embryonic cells and is repressed during adulthood. The enzyme is reexpressed in around 85% of solid tumors. This observation makes it a potential target for developing drugs that could be developed for therapeutic purposes. The identification of the hTERT as a functional catalytic RT prompted studies of inhibiting telomerase with the HIV RT inhibitor azidothymidine (AZT). Previously, we have demonstrated that AZT binds preferentially to telomeres, inhibits telomerase and enhances tumor cell senescence, and apoptosis after AZT treatment in breast mammary adenocarcinoma cells. Since then, several studies have considered AZT for telomerase inhibition and have led to potential clinical strategies for anticancer therapy. This review covers present thinking of the inhibition of telomerase by AZT and future treatment protocols using the drug.
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Affiliation(s)
- Daniel E Gomez
- Laboratory of Molecular Oncology, Department of Science and Technology, Quilmes National University, Bernal Buenos Aires, Argentina
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The combined use of known antiviral reverse transcriptase inhibitors AZT and DDI induce anticancer effects at low concentrations. Neoplasia 2012; 14:44-53. [PMID: 22355273 DOI: 10.1593/neo.11426] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2011] [Revised: 03/20/2011] [Accepted: 12/13/2011] [Indexed: 12/31/2022] Open
Abstract
A hallmark of tumor cell survival is the maintenance of elongated telomeres. It is known that antiviral reverse transcriptase inhibitors (RTIs) such as azidothymidine (AZT) and didanosine (ddI) lead to telomere shortening at high, potentially toxic concentrations. We hypothesized that those drugs might have synergistic effects enabling successful therapy with low, nontoxic concentrations. Biologic effects of AZT and ddI were analyzed at concentrations that correspond to minimal plasma levels achieved during human immunodeficiency virus therapy. Long-term coapplication of low-dose AZT and ddI induced a significant shortening of telomeres in the tumor cell lines HCT-116, SkMel-28, MelJuso, and Jurkat. Treatment of cells with both RTI, but not with single RTI, led to a significant accumulation of γH2AX, to p53 phosphorylation, and to cell apoptosis in all cell lines. Oral low-dose dual RTI application but not low-dose single RTI application was associated with a significantly reduced tumor growth of HCT-116 cells in mice. This antiproliferative activity of the combined use of AZT and ddI at low, clinically applicable concentrations warrants clinical testing in human solid cancer.
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Glukhov AI, Svinareva LV, Severin SE, Shvets VI. Telomerase inhibitors as novel antitumor drugs. APPL BIOCHEM MICRO+ 2011. [DOI: 10.1134/s0003683811070039] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
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Guittat L, Alberti P, Gomez D, De Cian A, Pennarun G, Lemarteleur T, Belmokhtar C, Paterski R, Morjani H, Trentesaux C, Mandine E, Boussin F, Mailliet P, Lacroix L, Riou JF, Mergny JL. Targeting human telomerase for cancer therapeutics. Cytotechnology 2011; 45:75-90. [PMID: 19003245 DOI: 10.1007/s10616-004-5127-z] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2004] [Accepted: 09/21/2004] [Indexed: 01/28/2023] Open
Abstract
The enzyme telomerase is involved in the replication of telomeres, specialized structures that cap and protect the ends of chromosomes. Its activity is required for maintenance of telomeres and for unlimited lifespan, a hallmark of cancer cells. Telomerase is overexpressed in the vast majority of human cancer cells and therefore represents an attractive target for therapy. Several approaches have been developed to inhibit this enzyme through the targeting of its RNA or catalytic components as well as its DNA substrate, the single-stranded 3'-telomeric overhang. Telomerase inhibitors are chemically diverse and include modified oligonucleotides as well as small diffusable molecules, both natural and synthetic. This review presents an update of recent investigations pertaining to these agents and discusses their biological properties in the context of the initial paradigm that the exposure of cancer cells to these agents should lead to progressive telomere shortening followed by a delayed growth arrest response.
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Affiliation(s)
- Lionel Guittat
- Laboratoire de Biophysique, Muséum National d'Histoire Naturelle USM503, INSERM U 565, CNRS UMR 5153, 43, rue Cuvier, 75231, Paris cedex 05, France
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18
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Marconett CN, Sundar SN, Tseng M, Tin AS, Tran KQ, Mahuron KM, Bjeldanes LF, Firestone GL. Indole-3-carbinol downregulation of telomerase gene expression requires the inhibition of estrogen receptor-alpha and Sp1 transcription factor interactions within the hTERT promoter and mediates the G1 cell cycle arrest of human breast cancer cells. Carcinogenesis 2011; 32:1315-23. [PMID: 21693539 DOI: 10.1093/carcin/bgr116] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Indole-3-carbinol (I3C), a naturally occurring hydrolysis product of glucobrassicin from cruciferous vegetables such as broccoli, cabbage and Brussels sprouts, is an anticancer phytochemical that triggers complementary sets of antiproliferative pathways to induce a cell cycle arrest of estrogen-responsive MCF7 breast cancer cells. I3C strongly downregulated transcript expression of the catalytic subunit of the human telomerase (hTERT) gene, which correlated with the dose-dependent indole-mediated G(1) cell cycle arrest without altering the transcript levels of the RNA template (hTR) for telomerase elongation. Exogenous expression of hTERT driven by a constitutive promoter prevented the I3C-induced cell cycle arrest and rescued the I3C inhibition of telomerase enzymatic activity and activation of cellular senescence. Time course studies showed that I3C downregulated expression of estrogen receptor-alpha (ERα) and cyclin-dependent kinase-6 transcripts levels (which is regulated through the Sp1 transcription factor) prior to the downregulation of hTERT suggesting a mechanistic link. Chromatin immunoprecipitation assays demonstrated that I3C disrupted endogenous interactions of both ERα and Sp1 with an estrogen response element-Sp1 composite element within the hTERT promoter. I3C inhibited 17β-estradiol stimulated hTERT expression and stimulated the production of threonine-phosphorylated Sp1, which inhibits Sp1-DNA interactions. Exogenous expression of both ERα and Sp1, but not either alone, in MCF7 cells blocked the I3C-mediated downregulation of hTERT expression. These results demonstrate that I3C disrupts the combined ERα- and Sp1-driven transcription of hTERT gene expression, which plays a significant role in the I3C-induced cell cycle arrest of human breast cancer cells.
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Affiliation(s)
- Crystal N Marconett
- Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California, Berkeley, CA 94720-3200, USA
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Abstract
Inappropriate activation of a single enzyme, telomerase, is associated with the uncontrollable proliferation of cells observed in as many as 90% of all of human cancers. Since the mid-1990s, when telomerase activity was detected in human tumors, scientists have eyed the enzyme as an ideal target for developing broadly effective anticancer drugs. One of the missing links in the effort to identify such therapies has been the high-resolution structure of the enzyme, a powerful tool used for the identification and development of clinical drugs. A recent structure of the catalytic subunit of teleomerase from the Skordalakes laboratory, a major advancement in the field of telomeres, has opened the door to the development of new, broadly effective cancer drugs, as well as anti-aging therapies. Here we present a brief description of telomerase biology, current efforts to identify telomerase function modulators and the potential importance of the telomerase structure in future drug development.
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Affiliation(s)
- Emmanuel Skordalakes
- Gene Expression & Regulation Program, The Wistar Institute, 3601 Spruce St, Philadelphia, PA 19104, USA
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Pang LY, Argyle DJ. Using naturally occurring tumours in dogs and cats to study telomerase and cancer stem cell biology. Biochim Biophys Acta Mol Basis Dis 2009; 1792:380-91. [PMID: 19254761 DOI: 10.1016/j.bbadis.2009.02.010] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2008] [Revised: 02/15/2009] [Accepted: 02/17/2009] [Indexed: 01/06/2023]
Abstract
The recently described cancer stem cell theory opens up many new challenges and opportunities to identify targets for therapeutic intervention. However, the majority of cancer related therapeutic studies rely upon rodent models of human cancer that rarely translate into clinical success in human patients. Naturally occurring cancers in dogs, cats and humans share biological features, including molecular targets, telomerase biology and tumour genetics. Studying cancer stem cell biology and telomere/telomerase dynamics in the cancer bearing pet population may offer the opportunity to develop a greater understanding of cancer biology in the natural setting and evaluate the development of novel therapies targeted at these systems.
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Affiliation(s)
- Lisa Y Pang
- University of Edinburgh, Midlothian EH25 9RG, Scotland, UK
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Relevance of experimental models for investigation of genotoxicity induced by antiretroviral therapy during human pregnancy. Mutat Res 2008; 658:184-90. [PMID: 18295533 DOI: 10.1016/j.mrrev.2007.12.001] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2007] [Revised: 12/12/2007] [Accepted: 12/13/2007] [Indexed: 11/21/2022]
Abstract
The current incidence of human immunodeficiency virus (HIV-1)/AIDS affects around 7000 pregnant women in the United States. When given during pregnancy, the nucleoside analog 3'-azido-3'-deoxythymidine (AZT) significantly reduces maternal-fetal transmission. It has been previously shown that AZT is incorporated into DNA, where it causes mutations in the HPRT and TK genes. It also changes cell cycle gene expression, and induces S-phase arrest, micronuclei, chromosomal aberrations, sister chromatid exchanges, telomeric attrition, and other genotoxic effects in cultured cells. A predicted consequence of these events is genomic instability that together, with clastogenicity may contribute to the carcinogenic potency of AZT. Various aspects of genotoxicity are explored in this contribution seeking to understand the multiple effects of this antiretroviral agent in animal models and humans. This mini-review describes some of the experimental models used to elucidate the genotoxicity induced by antiretroviral therapy during human pregnancy. The use of diverse methods to detect biomarkers of exposure, such as an AZT-specific radioimmunoassay, micronuclei bearing intact chromosomes, and telomeric DNA attrition highlight the role of in vitro models to elucidate exposure and risk. The relevance of the in vitro models is followed by the introduction of the role of the nucleoside analogs in transplacental carcinogenesis along with the description of a transplacental perfusion model and a transplacental carcinogenesis rodent model. In a more direct clinical application the use of AZT-DNA incorporation as a biomarker of exposure, in experiments conducted in vivo in Erythrocebus patas monkeys and in humans, addresses the possibility of elucidation of potential cancer risk in those infants exposed in utero. Two relevant aspects of this contribution are the potential application of some of the models described in this mini-review, as diagnostic tools in antiretroviral-exposed populations, and the use of these models to understand the nature of the genotoxicities and minimize the undesirable side effects of the antiretroviral therapy.
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Liu X, Takahashi H, Harada Y, Ogawara T, Ogimura Y, Mizushina Y, Saneyoshi M, Yamaguchi T. 3'-Azido-2',3'-dideoxynucleoside 5'-triphosphates inhibit telomerase activity in vitro, and the corresponding nucleosides cause telomere shortening in human HL60 cells. Nucleic Acids Res 2007; 35:7140-9. [PMID: 17942424 PMCID: PMC2175342 DOI: 10.1093/nar/gkm859] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Telomerase adds telomeric DNA repeats to the ends of linear chromosomal DNA. 3′-Azido-3′-deoxythymidine 5′-triphosphate (AZTTP) is a known telomerase inhibitor. To obtain more selective and potent inhibitors that can be employed as tools for studying telomerase, we investigated the telomerase-inhibitory effects of purine nucleosides bearing a 3′-down azido group: 3′-azido-2′,3′-dideoxyguanosine (AZddG) 5′-triphosphate (AZddGTP), 3′-azido-2′,3′-dideoxy-6-thioguanosine (AZddSG) 5′-triphosphate (AZddSGTP), 3′-azido-2′,3′-dideoxyadenosine (AZddA) 5′-triphosphate (AZddATP) and 3′-azido-2′,3′-dideoxy-2-aminoadenosine (AZddAA) 5′-triphosphate (AZddAATP). Of these, AZddGTP showed the most potent inhibitory activity against HeLa cell telomerase. AZddGTP was significantly incorporated into the 3′-terminus of DNA by partially purified telomerase. However, AZddGTP did not exhibit significant inhibitory activity against DNA polymerases α and δ, suggesting that AZddGTP is a selective inhibitor of telomerase. We also investigated whether long-term treatment with these nucleosides could alter telomere length and growth rates of human HL60 cells in culture. Southern hybridization analysis of genomic DNA prepared from cells cultured in the presence of AZddG and AZddAA revealed reproducible telomere shortening.
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Affiliation(s)
- Xiaohong Liu
- Biotechnology Research Center, Teikyo University of Science and Technology, Uenohara, Yamanashi, Japan
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Tárkányi I, Aradi J. Pharmacological intervention strategies for affecting telomerase activity: future prospects to treat cancer and degenerative disease. Biochimie 2007; 90:156-72. [PMID: 17945408 DOI: 10.1016/j.biochi.2007.09.002] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2007] [Accepted: 09/04/2007] [Indexed: 12/20/2022]
Abstract
Telomerase enzyme is a ribonucleoprotein maintaining the length of the telomeres by adding G-rich repeats to the end of the eukaryotic chromosomes. Normal human somatic cells, cultured in vitro, have a strictly limited proliferative potential undergoing senescence after about 50-70 population doublings. In contrast, most of the tumor cells have unlimited replicative potential. Although the mechanisms of immortalization are not understood completely at a genetic level, the key role of the telomere/telomerase system in the process is clear. The DNA replication machinery is not able to replicate fully the DNA at the very end of the chromosomes; therefore, about 50-200 nucleotides are lost during each of the replication cycles resulting in a gradual decrease of telomere length. Critically short telomere induces senescence, subsequent crisis and cell death. In tumor cells, however, the telomerase enzyme prevents the formation of critically short telomeres, adding GGTTAG repeats to the 3' end of the chromosomes immortalizing the cells. Immortality is one of the hallmarks of cancer. Besides the catalytic activity dependent telomere maintenance, catalytic activity-independent effects of telomerase may also be involved in the regulation of cell cycle. The telomere/telomerase system offers two possibilities to intervene the proliferative activity of the cell: (1) inhibition the telomere maintenance by inhibiting the telomerase activity; (2) activating the residual telomerase enzyme or inducing telomerase expression. Whilst the former approach could abolish the limitless replicative potential of malignant cells, the activation of telomerase might be utilized for treating degenerative diseases. Here, we review the current status of telomerase therapeutics, summarizing the activities of those pharmacological agents which either inhibit or activate the enzyme. We also discuss the future opportunities and challenges of research on pharmacological intervention of telomerase activity.
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Affiliation(s)
- I Tárkányi
- 3rd Department of Internal Medicine, University of Debrecen, 22 Moricz Zsigmond Krt., Debrecen 4004, Hungary
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25
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Sun YQ, Guo TK, Xi YM, Chen C, Wang J, Wang ZR. Effects of AZT and RNA-protein complex (FA-2-b-β) extracted from Liang Jin mushroom on apoptosis of gastric cancer cells. World J Gastroenterol 2007; 13:4185-91. [PMID: 17696246 PMCID: PMC4250616 DOI: 10.3748/wjg.v13.i31.4185] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the synergistic effects of 3'-azido-3'-deoxythymidine (AZT) and FA-2-b-β extracted from Ling Jin mushroom on apoptosis of gastric cancer cells MKN45 in vitro.
METHODS: MTT analysis was made to examine the inhibition rate of MKN45 cells treated with AZT (2.5, 5, 10 and 20 mg/L) and FA-2-b-β (5, 10, 20 and 40 mg/L) singly and combinatively for 24, 48 and 72 h. Apoptotic effects were evaluated by morphological methods, DNA agarose gel electrophoresis and flow cytometry, respectively. Telomerase activity was estimated by TRAP-ELISA. The mRNA expression of caspase-3 and Bcl-2 were detected by RT-PCR.
RESULTS: AZT and FA-2-b-β could significantly inhibit MKN45 cell proliferation and induce its apoptosis. MKN45 cells were inhibited in dose- and time- dependent manner. The inhibition effect of AZT combined with FA-2-b-β was obviously better than that used singly (0.469 ±0.022 vs 1.075 ± 0.055, P < 0.05, 0.325 ± 0.029 vs 0.469± 0.022 P < 0.01). AZT used singly and combination of FA-2-b-β could decrease the activity of tumor cell telomerase, and AZT has synergistic function with FA-2-b-β. A certain concentration of AZT could up-regulate the expression of caspase-3 mRNA (r = 0.9969, P < 0.01), which was positively related to apoptosis rate, and could down-regulate the expression of Bcl-2 mRNA, which was negatively related to apoptosis rate (r = 0.926, P < 0.01). Furthermore, the effect of AZT combined with FA-2-b-β was significantly higher than that used singly.
CONCLUSION: Combination of AZT and FA-2-b-β has an obviously synergetic effect in the gastric cancer cells MKN45, which has provided a new approach to the treatment of gastric cancer clinically.
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Affiliation(s)
- Yan-Qing Sun
- School of Life Science, Lanzhou University, Lanzhou 730000, Gansu Province, China
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26
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Walker DM, Malarkey DE, Seilkop SK, Ruecker FA, Funk KA, Wolfe MJ, Treanor CP, Foley JF, Hahn FF, Hardisty JF, Walker VE. Transplacental carcinogenicity of 3'-azido-3'-deoxythymidine in B6C3F1 mice and F344 rats. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2007; 48:283-98. [PMID: 17358026 DOI: 10.1002/em.20297] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/14/2023]
Abstract
The prophylactic use of zidovudine (3'-azido-3'-deoxythymidine, AZT) during pregnancy greatly reduces transmission of HIV-1 from infected mothers to their infants; however, the affinity of host cell DNA polymerases for AZT also allows for its incorporation into host cell DNA, predisposing to cancer development. To expand upon previous transplacental carcinogenesis assays performed in CD-1 mice, the transplacental carcinogenicity of AZT was evaluated in a second mouse strain and a second rodent species. Date-mated female mice and rats were gavaged daily with 0, 80, 240, or 480 mg AZT/kg bw during the last 7 days of gestation. At 2 years postpartum, male and female B6C3F1 mouse and F344 rat offspring (n = 44-46 of each sex and species/treatment group) were necropsied for gross and microscopic tissue examinations. Under the conditions of these two-year studies, there was clear evidence of carcinogenic activity based upon significant dose-related trends and increases in the incidences of hemangiosarcoma in male mice and mononuclear cell leukemia in female rats. There was some evidence of carcinogenic activity in the livers of male mice based upon a positive trend and an increased incidence of hepatic carcinoma in the high-dose AZT group. The incidence of gliomas in female rats exceeded the historical background rates for gliomas in F344 rats. P53 overexpression was detected in some AZT-treated mouse neoplasms. These and other cancer-related findings confirm and extend those of previous transplacental carcinogenicity studies of AZT in mice, support the need for long-term follow-up of nucleoside reverse transcriptase inhibitor (NRTI)-exposed children, and indicate the necessity for effective protective strategies against NRTI-induced side effects.
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Affiliation(s)
- Dale M Walker
- Experimental Pathology Laboratories, Inc., Herndon, Virginia, USA
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27
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Moxibustion on telomerase activity in aging rat. JOURNAL OF ACUPUNCTURE AND TUINA SCIENCE 2007. [DOI: 10.1007/s11726-007-0074-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
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Olivero OA. Mechanisms of genotoxicity of nucleoside reverse transcriptase inhibitors. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2007; 48:215-23. [PMID: 16395695 DOI: 10.1002/em.20195] [Citation(s) in RCA: 74] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/06/2023]
Abstract
Nucleoside analogs were first approved by the U.S. Food and Drug Administration for use against HIV-AIDS in 1987. Since then, these agents, now commonly referred to as nucleoside reverse transcriptase inhibitors (NRTIs), have become essential components of the Highly Active Antiretroviral Therapy (HAART) drug combinations used for treatment of Human Immunodeficiency Virus-1 (HIV-1) infections. Their antiretroviral activity is likely two-fold: incorporation of the drug into viral DNA and inhibition of the viral reverse transcriptase. However, incorporation of the drug into host nuclear and mitochondrial DNA may be largely responsible for dose-limiting toxicities. Azidothymidine (AZT, 3'-azido-3'-deoxythymidine, zidovudine), the first NRTI approved for the therapy of HIV-1, is incorporated into DNA, causes mutations in the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) and thymidine kinase (TK) genes, and induces micronuclei, chromosomal aberrations, sister chromatid exchange, shortened telomeres, and other genotoxic effects in cultured cells. Genomic instability would be predicted as a consequence of these events. Metabolic pathways that result in the phosphorylation of AZT play a crucial role in AZT-DNA incorporation, and may be altered after prolonged treatment. For example, thymidine kinase 1, the enzyme responsible for AZT mono-phosphorylation, is down-regulated during long-term exposure and appears to be associated with AZT-induced replication inhibition and the accumulation of cells in S-phase. Detailed information on the mechanisms underlying NRTI-associated antiretroviral efficacy, toxicity, and metabolic resistance were not available when AZT was first approved for use as an antiretroviral agent. Current insights, based on 15 years of research, may lead to intervention strategies to attenuate toxicity without altering drug efficacy.
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Affiliation(s)
- Ofelia A Olivero
- Carcinogen-DNA Interactions Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, NIH, Bethesda, Maryland, USA.
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Liu T, Hu B, Chung MJ, Ullenbruch M, Jin H, Phan SH. Telomerase regulation of myofibroblast differentiation. Am J Respir Cell Mol Biol 2006; 34:625-33. [PMID: 16424384 PMCID: PMC2644224 DOI: 10.1165/rcmb.2005-0252oc] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Telomerase activity, which has wide expression in cancerous cells, is induced in lung proliferating fibroblasts. It is preferentially expressed in fibroblasts versus myofibroblasts. It is unknown whether regulation of telomerase expression is related to the process of fibroblast differentiation into myofibroblasts. The objective of this study was to clarify such a potential link between telomerase expression and myofibroblast differentiation. Telomerase inhibitor, 3'-azido-2',3'-dideoxythymidine, or antisense oligonucleotide to the telomerase RNA component was used to inhibit the induced fibroblast telomerase activity. The results showed that inhibition of induced telomerase increased alpha-smooth muscle actin expression, an indicator of myofibroblast differentiation. In contrast, induction of telomerase by basic fibroblast growth factor inhibited alpha-smooth muscle actin expression. These findings suggest that the loss of telomerase activity is closely associated with myofibroblast differentiation and possibly functions as a trigger for myofibroblast differentiation. Conversely, expression of telomerase suppresses myofibroblast differentiation.
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Affiliation(s)
- Tianju Liu
- Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
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Ji HJ, Rha SY, Jeung HC, Yang SH, An SW, Chung HC. Cyclic induction of senescence with intermittent AZT treatment accelerates both apoptosis and telomere loss. Breast Cancer Res Treat 2005; 93:227-36. [PMID: 16132531 DOI: 10.1007/s10549-005-5156-0] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
BACKGROUND 3'-azido-2',3'-dideoxythymidine (AZT) is phosphorylated intracellularly to 3'-azido-3'-deoxythymidine-5'-triphosphate (AZT-TP), which is incorporated into telomeric DNA, thereby blocking chain elongation. AZT is also known to inhibit reverse transcriptase, as well as other cellular enzymes including DNA polymerase gamma, thymidine kinase, and telomerase. METHODS We induced cancer cell senescence by treating MCF-7 cells with AZT in dosages of IC10 and IC20 for an extended period (about 120 population doublings (PD)). We then investigated the sequential changes in cellular growth, expression of telomerase subunits and transcription factors (c-Myc, Mad1), telomerase activity and telomere length. RESULTS Senescence, apoptosis, growth delay, inhibition of telomerase activity and shortening of telomere length were all observed in a dose- and time-dependent manner. After the onset of senescence, the apoptosis rate increased slowly during early PDs. In contrast to senescence, the apoptotic rate showed little change after AZT removal, while it increased suddenly and significantly in a dose-dependent manner upon the second introduction of AZT. Continuous shortening of the telomeric length was observed with AZT, and, upon re-exposure to AZT, shortening of the telomere occurred more rapidly than with first exposure. Of the telomerase subunits, telomerase reverse transcriptase (hTERT) and c-Myc were the first to show a reduction in activity after AZT treatment, followed by changes in hTER , Mad1 and hTEP-1. CONCLUSION Cyclic treatment with AZT initially suppressed hTERT and c-Myc, followed by suppression of hTER, Mad1 and hTEP-1. Furthermore, the treatment accelerated both telomere loss and apoptosis, even when administered at a senescence-inducing dosage level.
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Affiliation(s)
- Hyun Jung Ji
- Cancer Metastasis Research Center, Young Cancer Center, Yonsei University College of Medicine, Seoul, Seodaemun-Ku, Korea
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Yeo M, Rha SY, Jeung HC, Hu SX, Yang SH, Kim YS, An SW, Chung HC. Attenuation of telomerase activity by hammerhead ribozyme targeting human telomerase RNA induces growth retardation and apoptosis in human breast tumor cells. Int J Cancer 2005; 114:484-9. [PMID: 15551309 DOI: 10.1002/ijc.20720] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Ribozyme possesses specific endoribonuclease activity and catalyzes the hydrolysis of specific phosphodiester bonds, which results in the cleavage of target RNA sequences. Here, we evaluated the ability of hammerhead ribozymes targeting human telomerase RNA (hTR) to inhibit the catalytic activity of telomerase and the proliferation of cancer cells. Hammerhead ribozymes were designed against 7 NUX sequences located in open loops of the hTR secondary structure. We verified the ribozyme specificity by in vitro cleavage assay by using a synthetic RNA substrate. Subsequently, we introduced ribozyme expression vector into human breast tumor MCF-7 cells and assessed the biologic effects of ribozyme. Hammerhead ribozyme R1 targeting the template region of hTR efficiently cleaved hTR in vitro, and stable transfectants of this ribozyme induced the degradation of target hTR RNA and attenuated telomerase activity in MCF-7 cells. Moreover, the ribozyme R1 transfectant displayed a significant telomere shortening and a lower proliferation rate than parental cells. Clones with reduced proliferation capacity showed enlarged senescence-like shapes or highly differentiated dendritic morphologies of apoptosis. In conclusion, the inhibition of telomerase activity by hammerhead ribozyme targeting the template region of the hTR presents a promising strategy for inhibiting the growth of human breast cancer cells.
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Affiliation(s)
- Marie Yeo
- Cancer Metastasis Research Center, Yonsei Cancer Center, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea
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33
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Poirier MC, Divi RL, Al-Harthi L, Olivero OA, Nguyen V, Walker B, Landay AL, Walker VE, Charurat M, Blattner WA. Long-term mitochondrial toxicity in HIV-uninfected infants born to HIV-infected mothers. J Acquir Immune Defic Syndr 2003; 33:175-83. [PMID: 12794551 DOI: 10.1097/00126334-200306010-00010] [Citation(s) in RCA: 122] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Although children born to HIV-infected (HIV+) women receiving antiretroviral therapy during pregnancy show virtually no adverse clinical effects at birth, the antiretroviral nucleoside analog drugs are known to damage nuclear and mitochondrial DNA. In this study, biomarkers of mitochondrial toxicity and genotoxicity have been examined in a well-characterized sample set consisting of infants born to HIV-uninfected (HIV-) mothers (n = 30), and HIV- infants (n = 20) born to HIV-infected (HIV+) mothers who received either no antiretroviral therapy (n = 10) or zidovudine (3'-azido-3'-deoxythymidine [AZT]) during pregnancy (n = 10). DNA from cord blood leukocytes and peripheral blood leukocytes taken at 1 and 2 years of age was examined for loss of mitochondrial DNA (mtDNA) and telomere integrity. Telomere length, a measure of nuclear DNA damage, was the same in all infants at birth and at age 1 year. The quantity of mtDNA was assessed relative to nuclear DNA using a polymerase chain reaction-based chemiluminescence detection (PCR-CID) method that determined mitochondrial D Loop gene copies relative to nuclear 18S RNA gene copies by comparison with a standard curve. MtDNA quantity was expressed as a ratio of gene copy numbers. In infants of uninfected mothers (AZT-/HIV-) at the three time points, the ratios were 442 to 515, whereas in infants of untreated AZT-/HIV+ mothers the ratios were 261 to 297, and in infants of AZT-treated (AZT+/HIV+) mothers the ratios were 146 to 203. At all three time points, differences between the AZT-/HIV- group and the two HIV+ groups were statistically significant (p <.05), and differences between the AZT-/HIV+ and AZT+/HIV+ groups were also statistically significant (p <.05), demonstrating that AZT exposure causes a persistent depletion of mtDNA. The study shows that children of HIV+ mothers are at risk for mitochondrial damage that is further increased in infants of mothers receiving AZT during pregnancy.
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MESH Headings
- Adult
- Child, Preschool
- DNA, Mitochondrial/analysis
- DNA, Mitochondrial/drug effects
- Female
- Fetal Blood/immunology
- Genetic Markers
- HIV Infections/blood
- HIV Infections/drug therapy
- HIV Infections/genetics
- Humans
- Infant
- Infant, Newborn
- Leukocytes, Mononuclear/chemistry
- Leukocytes, Mononuclear/drug effects
- Pilot Projects
- Pregnancy
- Pregnancy Complications, Infectious/drug therapy
- Prenatal Exposure Delayed Effects
- Prospective Studies
- RNA, Ribosomal, 18S/analysis
- RNA, Ribosomal, 18S/drug effects
- Reverse Transcriptase Inhibitors/adverse effects
- Telomere/drug effects
- Telomere/ultrastructure
- Zidovudine/adverse effects
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Affiliation(s)
- Miriam C Poirier
- Carcinogen-DNA Interactions Section, National Cancer Institute, National Institutes of Health, Building 37, Room 4032, 37 Convent Drive, Bethesda, MD 20892-4255, USA.
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34
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Teulade-Fichou MP, Carrasco C, Guittat L, Bailly C, Alberti P, Mergny JL, David A, Lehn JM, Wilson WD. Selective recognition of G-qQuadruplex telomeric DNA by a bis(quinacridine) macrocycle. J Am Chem Soc 2003; 125:4732-40. [PMID: 12696891 DOI: 10.1021/ja021299j] [Citation(s) in RCA: 144] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
The interaction of G-quadruplex DNA with the macrocyclic compound BOQ1, which possesses two dibenzophenanthroline (quinacridine) subunits, has been investigated by a variety of methods. The oligonucleotide 5'-A(GGGT(2)A)(3)G(3), which mimics the human telomeric repeat sequence and forms an intramolecular quadruplex, was used as one model system. Equilibrium binding constants measured by biosensor surface plasmon resonance (SPR) methods indicate a high affinity of the macrocycle for the quadruplex conformation (K > 1 x 10(7) M(-)(1)) with two equivalent binding sites. The affinity of BOQ1 for DNA duplexes is at least 1 order of magnitude lower. In addition, the macrocycle is more selective than the monomeric control compound (MOQ2), which is not able to discriminate between the two DNA structures (K(duplex) approximately K(quadruplex) approximately 10(6) M(-)(1)). Strong binding of BOQ1 to G4 DNA sequences was confirmed by fluorometric titrations with a tetraplex-forming oligonucleotide. Competition dialysis experiments with a panel of different DNA structures, from single strands to quadruplexes, clearly established the quadruplex binding specificity of BOQ1. Fluorescence resonance energy transfer (FRET) T(m) experiments with a doubly labeled oligonucleotide also revealed a strong stabilization of the G4 conformation in the presence of BOQ1 (DeltaT(m) = +28 degrees C). This DeltaT(m) value is one of the highest values measured for a G-quadruplex ligand and is significantly higher than observed for the monomer control compounds (DeltaT(m) = +10-12 degrees C). Gel mobility shift assays indicated that the macrocycle efficiently induces the formation of G-tetraplexes. Strong inhibition of telomerase was observed in the submicromolar range (IC(50) = 0.13 microM). These results indicate that macrocycles represent an exciting new development opportunity for targeting DNA quadruplexes.
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Affiliation(s)
- Marie-Paule Teulade-Fichou
- Laboratoire de Chimie des Interactions Moléculaires, Collège de France, CNRS UPR 285, 11 place Marcelin Berthelot, 75005 Paris, France
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35
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Argyle DJ, Nasir L. Telomerase: a potential diagnostic and therapeutic tool in canine oncology. Vet Pathol 2003; 40:1-7. [PMID: 12627707 DOI: 10.1354/vp.40-1-1] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
In recent years there has been considerable interest in telomerase as a target for therapeutic intervention in oncology. This largely stems from the vast number of studies that have demonstrated expression and activity of the enzyme telomerase in the majority of human cancer tissues with little or no activity detectable in normal somatic tissues. These studies have led to an interest in the role of telomerase in cancers associated with domesticated species, in particular tumors that affect dogs. This article reviews the biology of telomerase and the biological significance of telomerase activity in canine tumors and discusses the clinical implications of telomerase expression in canine cancers with regard to therapeutics and diagnostics.
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Affiliation(s)
- D J Argyle
- Department of Veterinary Clinical Studies, University of Glasgow Veterinary School, Bearsden Road, Glasgow, UK
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36
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Abstract
The continuous growth of advanced malignancies almost universally correlates with the reactivation of telomerase. While there is still a great deal of basic and applied research to be done, telomerase remains a very attractive novel target for cancer therapeutics. In this review, we will discuss the challenges and the pros and cons of the most promising antitelomerase approaches currently being investigated.
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Affiliation(s)
- Jerry W Shay
- The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75309, USA.
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37
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Fajkus J, Simícková M, Maláska J. Tiptoeing to chromosome tips: facts, promises and perils of today's human telomere biology. Philos Trans R Soc Lond B Biol Sci 2002; 357:545-62. [PMID: 12028791 PMCID: PMC1692969 DOI: 10.1098/rstb.2001.1053] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
The past decade has witnessed an explosion of knowledge concerning the structure and function of chromosome terminal structures-telomeres. Today's telomere research has advanced from a pure descriptive approach of DNA and protein components to an elementary understanding of telomere metabolism, and now to promising applications in medicine. These applications include 'passive' ones, among which the use of analysis of telomeres and telomerase (a cellular reverse transcriptase that synthesizes telomeres) for cancer diagnostics is the best known. The 'active' applications involve targeted downregulation or upregulation of telomere synthesis, either to mortalize immortal cancer cells, or to rejuvenate mortal somatic cells and tissues for cellular transplantations, respectively. This article reviews the basic data on structure and function of human telomeres and telomerase, as well as both passive and active applications of human telomere biology.
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Affiliation(s)
- J Fajkus
- Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, CZ-612 65 Brno, Czech Republic.
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38
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Olivero OA, Fernandez JJ, Antiochos BB, Wagner JL, St Claire ME, Poirier MC. Transplacental genotoxicity of combined antiretroviral nucleoside analogue therapy in Erythrocebus patas monkeys. J Acquir Immune Defic Syndr 2002; 29:323-9. [PMID: 11917235 DOI: 10.1097/00126334-200204010-00001] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Antiretroviral nucleoside analogue drugs are a major constituent of highly active antiretroviral therapy (HAART), the most advanced form of treatment for HIV-1 infection. Currently, HAART combinations that include zidovudine (ZDV) and lamivudine (3TC) are highly effective in preventing HIV-1 vertical transmission; most children are born with no evident adverse clinical effects. However, ZDV is a moderately strong transplacental carcinogen in mice, and potential long-term consequences of fetal exposure to most HAART combinations remain unknown. To model human transplacental ZDV and 3TC exposures, experiments were performed in Erythrocebus patas monkeys given human-equivalent drug exposure protocols. Pregnant monkeys were dosed with either no drug (n = 2), 40.0 mg ZDV/d (about 6 mg/kg body weight/d) for the last 50% (10 weeks) of gestation (n = 3), or with the same regimen of ZDV plus 24.0 mg 3TC/d (about 3.6 mg/kg body weight/d) for the last 20% (4 weeks) of gestation (n = 3). Multiple fetal organs were examined at term for DNA incorporation of ZDV and 3TC using two separate radioimmunoassays (RIAs). Values for ZDV-DNA incorporation were similar in fetuses exposed to ZDV alone and those exposed to ZDV plus 3TC. Values for 3TC-DNA in fetal organs were greater than or equal to values for ZDV-DNA, indicating that the total DNA damage sustained by fetuses exposed to both drugs was at least double that observed in fetuses exposed to ZDV alone. Telomere shortening, determined by Southern blot with a telomeric probe, was observed in most organs of the three animals exposed in utero to ZDV plus 3TC. No telomere shortening was evident in the unexposed fetuses, and occasional telomere shortening was found in fetuses exposed to ZDV alone. Overall, these studies demonstrate that monkey fetuses exposed in utero to the combination ZDV plus 3TC sustain a higher level of drug-DNA incorporation and show evidence of more telomere damage than monkey fetuses exposed to ZDV alone.
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Affiliation(s)
- Ofelia A Olivero
- Carcinogen-DNA Interactions Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
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39
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Søndergaard SR, Essen MV, Schjerling P, Ullum H, Pedersen BK. Proliferation and telomere length in acutely mobilized blood mononuclear cells in HIV infected patients. Clin Exp Immunol 2002; 127:499-506. [PMID: 11966767 PMCID: PMC1906322 DOI: 10.1046/j.1365-2249.2002.01790.x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The aim of the study was to investigate the mobilization of T cells in response to a stressful challenge (adrenalin stimulation), and to access T cells resided in the peripheral lymphoid organs in HIV infected patients. Seventeen patients and eight HIV seronegative controls received an adrenalin infusion for 1 h. Blood was sampled before, during and 1 h after adrenalin infusion. Proliferation and mean telomere restriction fragment length (telomeres) of blood mononuclear cells (BMNC) and purified CD8+ and CD4+ cells were investigated at all time points. In patients, the proliferation to pokeweed mitogens (PWM) was lower and decreased more during adrenalin infusion. After adrenalin infusion the proliferation to PWM was restored only in the controls. In all subjects telomeres in CD4+ cells declined during adrenalin infusion. Additionally, the patients had shortened telomeres in their CD8+ cells, and particularly HAART treated patients had shortened telomeres in all cell-subtypes. The finding that patients mobilized cells with an impaired proliferation to PWM during and after adrenalin infusion has possible clinical relevance for HIV infected patients during pathological stressful conditions, such as sepsis, surgery and burns. However, this study did not find a correlation between impaired proliferation and telomeres. It is concluded that physiological stress further aggravates the HIV-induced immune deficiency.
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Affiliation(s)
- S R Søndergaard
- Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark.
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40
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Mergny JL, Riou JF, Mailliet P, Teulade-Fichou MP, Gilson E. Natural and pharmacological regulation of telomerase. Nucleic Acids Res 2002; 30:839-65. [PMID: 11842096 PMCID: PMC100331 DOI: 10.1093/nar/30.4.839] [Citation(s) in RCA: 273] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2001] [Revised: 11/29/2001] [Accepted: 11/29/2001] [Indexed: 01/14/2023] Open
Abstract
The extremities of eukaryotic chromosomes are called telomeres. They have a structure unlike the bulk of the chromosome, which allows the cell DNA repair machinery to distinguish them from 'broken' DNA ends. But these specialised structures present a problem when it comes to replicating the DNA. Indeed, telomeric DNA progressively erodes with each round of cell division in cells that do not express telomerase, a specialised reverse transcriptase necessary to fully duplicate the telomeric DNA. Telomerase is expressed in tumour cells but not in most somatic cells and thus telomeres and telomerase may be proposed as attractive targets for the discovery of new anticancer agents.
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Affiliation(s)
- Jean-Louis Mergny
- Laboratoire de Biophysique, Muséum National d'Histoire Naturelle, INSERM U 201, CNRS UMR 8646, 43 rue Cuvier, F-75005 Paris, France.
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41
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Błasiak J, Kadłubek M, Kowalik J, Romanowicz-Makowska H, Pertyński T. Inhibition of telomerase activity in endometrial cancer cells by selenium-cisplatin conjugate despite suppression of its DNA-damaging activity by sodium ascorbate. TERATOGENESIS, CARCINOGENESIS, AND MUTAGENESIS 2002; 22:73-82. [PMID: 11754389 DOI: 10.1002/tcm.1040] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Telomerase activation can be considered as a critical step in cell immortalization. The enzyme elongates or maintains telomere length by adding to its end tandem TTAGGG repeats by using its endogenous RNA template. Telomerase is not detectable in most somatic cells but is upregulated in germ line cells and in 85-90% of human cancers, which suggests important role of telomerase in neoplastic transformation. Consequently, telomerase has been proposed as a potentially highly selective target for the development of antiproliferative agents. Platinum complexes are widely administrated in cancer therapy. A conjugate of selenite with diammineplatinum [(NH(3))(2)Pt(SeO(3))(2)] is a novel potential anticancer drug. Using alkaline single cell gel electrophoresis (comet assay), we showed that the drug at 5-30 microM induced concentration-dependent damage to DNA of endometrial cancer cells derived from tumor samples. Sodium ascorbate at 10 and 50 microM reduced the extent of the DNA damage evoked by the drug. (NH(3))(2)Pt(SeO(3)) reduced telomerase activity in the cells in a concentration-dependent manner as measured by using the telomere repeat amplification protocol (TRAP) assay. This effect was independent of sodium ascorbate. Therefore, mutagenic effects of the conjugate can be reduced by well-recognized antimutagen, sodium ascorbate, but it can still retain ability to affect neoplastic transformation. The results obtained indicate that (NH(3))(2)Pt(SeO(3)) may specifically inhibit telomerase activity in endometrial cancer cells.
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Affiliation(s)
- Janusz Błasiak
- Department of Molecular Genetics, University of Lodz, Lodz, Poland.
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42
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Abstract
Telomerase, the ribonucleoprotein enzyme that elongates chromosomal ends, or telomeres, is repressed in most normal somatic cells but reactivated in transformed cells to compensate for the progressive erosion of the telomeres during cell divisions. In accordance with this hypothesis, the presence of telomerase activity has been reported in more than 90% of human cancers, whereas most normal tissues or benign tumors contain low or undetectable telomerase activity. Reactivation of telomerase has also been widely reported in endocrine neoplasms and in hormone-related cancers. In the present study, we review the most recent publications on telomerase in these types of tumors. The hormonal regulation of telomerase activity and the possible strategies for cancer therapy based on the inhibition of telomerase has also been discussed.
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Affiliation(s)
- C Orlando
- Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, viale Pieraccini 6, 50139, Florence, Italy.
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43
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Chen Z, Smith KJ, Skelton HG, Barrett TL, Greenway HT, Lo SC. Telomerase activity in Kaposi's sarcoma, squamous cell carcinoma, and basal cell carcinoma. Exp Biol Med (Maywood) 2001; 226:753-7. [PMID: 11520941 DOI: 10.1177/153537020222600807] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Patients with acquired immune deficiency syndrome (AIDS) often develop Kaposi's sarcoma (KS), an unusual skin tumor. The malignant nature of KS has long been disputed. Telomerase activity that maintains telomere length and ensures chromosomal stability, a frequently appearing marker in human malignancies, has been proposed to play a critical role in supporting continued cell growth, hence formation of tumors. We examined telomerase activity in tissue extracts from 22 KS, 10 squamous cell carcinoma (SCC), and 22 basal cell carcinoma (BCC) using the telomeric repeat amplification protocol (TRAP). All of the tumor tissues were previously cryopreserved at -80 degrees C. In this study, all tumor samples tested were positive for telomerase activity. Consistent with the presence of the enzyme activity, the skin tumors had relatively long telomeres. Inhibitors in the tissue extracts of some samples needed to be diluted or extracted by phenol before the enzyme activity was detected in the TRAP assay. All KS as well as two other skin carcinoma samples revealed positive telomerase activity. Our finding supports telomerase's role in tumor cell immortality and suggests the true neoplastic nature of KS.
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Affiliation(s)
- Z Chen
- American Registry of Pathology, Washington, District of Columbia 20306, USA
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44
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Affiliation(s)
- CLAUDIO ORLANDO
- From the Clinical Biochemistry Unit Department of Clinical Physiopathology, University of Florence and Division of Urology, Department of Surgery, University of Pisa, Pisa, Italy
| | - STEFANIA GELMINI
- From the Clinical Biochemistry Unit Department of Clinical Physiopathology, University of Florence and Division of Urology, Department of Surgery, University of Pisa, Pisa, Italy
| | - CESARE SELLI
- From the Clinical Biochemistry Unit Department of Clinical Physiopathology, University of Florence and Division of Urology, Department of Surgery, University of Pisa, Pisa, Italy
| | - MARIO PAZZAGLI
- From the Clinical Biochemistry Unit Department of Clinical Physiopathology, University of Florence and Division of Urology, Department of Surgery, University of Pisa, Pisa, Italy
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45
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Alfonso-De Matte MY, Cheng JQ, Kruk PA. Ultraviolet irradiation- and dimethyl sulfoxide-induced telomerase activity in ovarian epithelial cell lines. Exp Cell Res 2001; 267:13-27. [PMID: 11412034 DOI: 10.1006/excr.2001.5231] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Information about telomerase regulation is incomplete, especially since various studies suggest complexity in telomerase regulation. Given the important association between telomerase and cancer, it is imperative to design and develop a model system in which telomerase activity can be regulated and studied. We employed ultraviolet (UV) radiation or dimethyl sulfoxide (DMSO) to transiently induce telomerase activity in a telomerase-positive cell line and, most importantly, in a telomerase-negative cell line. UV- or DMSO-induced telomerase activity was associated with increased hTRT, but not hTR, mRNA transcription in the telomerase-negative cells. However, no changes in hTRT or hTR mRNA transcription were noted with UV- or DMSO-induced telomerase activity in the telomerase-positive cells. Inhibition of protein synthesis or the phosphotidyl inositol 3-kinase (PI3K) pathway suppressed telomerase induction and/or activity in all cell lines examined, suggesting telomerase activity was dependent on protein synthesis and PI3K-mediated phosphorylation. Furthermore, enhanced telomerase activity was limited to UV and DMSO, since a variety of chemotherapeutic agents failed to induce telomerase activity. Therefore, our data provide a useful culture model system to study telomerase regulation in telomerase-negative and -positive cell lines and from which to obtain information about telomerase as a target for cancer intervention.
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Affiliation(s)
- M Y Alfonso-De Matte
- H. Lee Moffitt Cancer Center, University of South Florida, Tampa, Florida 33612, USA
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46
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Nakamura M, Saito H, Ebinuma H, Wakabayashi K, Saito Y, Takagi T, Nakamoto N, Ishii H. Reduction of telomerase activity in human liver cancer cells by a histone deacetylase inhibitor. J Cell Physiol 2001; 187:392-401. [PMID: 11319763 DOI: 10.1002/jcp.1087] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
The presence of telomerase has been demonstrated recently in many different malignancies. Several reports documented that in human hepatocellular carcinoma, the level of telomerase activity parallels its differentiation stage. In the present study, the effect of the differentiation-inducing agent sodium butyrate on telomerase activity in four human liver cancer cell lines was investigated using the telomeric repeat amplification protocol. We assayed telomerase activity before and after butyrate treatment and in cell cycle synchronized non-dividing quiescent cells. In addition, telomerase reverse transcriptase levels were measured at the mRNA level. All four cell lines possessed high but not identical levels of telomerase activity. Telomerase activity was significantly reduced by treatment with sodium butyrate as well as trichostatin A in a dose- and time-dependent fashion, paralleling the reduction of cell proliferation. Although methotrexate, hydroxyurea, and colchicine synchronized the cell cycle at G1, S, and G2/M, respectively, and thereby also caused proliferating cells to cease dividing and become quiescent, in this case telomerase activity remained essentially unaltered compared to the control cultures. Moreover, levels of mRNA encoding telomerase reverse transcriptase were not always significantly altered by either sodium butyrate treatment or cell cycle synchronization. These results suggest that sodium butyrate, as a histone deacetylase inhibitor, effectively reduces telomerase activity without affecting transcription levels of the reverse transcriptase component.
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Affiliation(s)
- M Nakamura
- Department of Internal Medicine, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan
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47
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Abstract
There has been a vast increase in telomerase research over the past several years, with many different pre-clinical approaches being tested for inhibiting the activity of this enzyme as a novel therapeutic modality to treat malignancy. In this review, we will provide some basic background information about telomeres and telomerase and then discuss the pros, cons and challenges of the approaches that are currently under investigation, and what we might expect in the future of this emerging field.
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Affiliation(s)
- L K White
- Depts of Internal Medicine and Cell Biology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9039, USA
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48
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Abstract
In normal somatic cells, the ends of chromosomes (the telomeres) shorten with each cell division. By contrast, in tumour cells, telomere length is maintained, generally through the reactivation of the reverse transcriptase enzyme, telomerase. At least three applications relating to telomeres and telomerase have been proposed: in cancer diagnosis and prognosis (especially through measurements of the catalytic component of telomerase, hTERT) and as a means of monitoring tumour response to therapy; as an aid to tissue engineering; and inhibition as a cancer therapeutic strategy. Mouse knockout, hTERT dominant negative, and antisense experiments suggest that telomerase inhibitors will confer anticancer activity, especially in tumours with short telomeres. Inhibitory strategies have focused on antisense molecules, inhibitors of reverse transcriptases, and small molecules able to interact with and stabilise four-stranded (G-quadruplex) structures formed by telomeres. Clinical trials involving telomerase inhibitors require careful consideration compared to those looking at conventional anticancer cytotoxic drugs.
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Affiliation(s)
- L R Kelland
- Cancer Research Campaign Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, UK.
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49
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Fletcher TM, Cathers BE, Ravikumar KS, Mamiya BM, Kerwin SM. Inhibition of human telomerase by 7-deaza-2'-deoxyguanosine nucleoside triphosphate analogs: potent inhibition by 6-thio-7-deaza-2'-deoxyguanosine 5'-triphosphate. Bioorg Chem 2001; 29:36-55. [PMID: 11300694 DOI: 10.1006/bioo.2000.1194] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
We have examined analogs of the previously reported 7-deaza-2'-deoxypurine nucleoside triphosphate series of human telomerase inhibitors. Two new telomerase-inhibiting nucleotides are reported: 6-methoxy-7-deaza-2'-deoxyguanosine 5'-triphosphate (OMDG-TP) and 6-thio-7-deaza-2'-deoxyguanosine 5'-triphosphate (TDG-TP). In particular, TDG-TP is a very potent inhibitor of human telomerase with an IC(50) of 60 nM. TDG-TP can substitute for dGTP as a substrate for telomerase, but only at relatively high concentrations. Under conditions in which TDG-TP is the only available guanosine substrate, telomerase becomes nonprocessive, synthesizing short products that appear to contain only one to three TDG residues. Similarly, the less potent telomerase inhibitor OMDG-TP gives rise to short telomerase products, but less efficiently than TDG-TP. We show here that TDG-TP, and to a lesser extent OMDG-TP, can serve as substrates for both templated (Klenow exo) and nontemplated (terminal transferase) DNA polymerases. For either polymerase, the products arising from TDG-TP are relatively short, and give rise to bands of unusual mobility under PAGE conditions. These anomalous bands revert, under treatment with DTT, to normal mobility bands, indicating that these products may contain thiol-labile disulfide linkages involving the incorporated TDG residues. This observation of potential TDG-crosslinks may have bearing on the mechanism of telomerase inhibition by this nucleotide analog.
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Affiliation(s)
- T M Fletcher
- College of Pharmacy, University of Texas, Austin, Texas 78712-1074, USA
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50
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Tejera AM, Alonso DF, Gomez DE, Olivero OA. Chronic in vitro exposure to 3'-azido-2', 3'-dideoxythymidine induces senescence and apoptosis and reduces tumorigenicity of metastatic mouse mammary tumor cells. Breast Cancer Res Treat 2001; 65:93-9. [PMID: 11261835 DOI: 10.1023/a:1006477730934] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Normal cells in culture divide a certain amount of times and undergo a process termed replicative senescence. Telomere loss is thought to control entry into senescence. Activation of telomerase in tumors bypasses cellular senescence and is thus a requirement for tumor progression. We reported previously the preferential incorporation of 3'-azido-2', 3'-dideoxythymidine (AZT) in telomeric sequences of immortalized cells in culture. In this work, we have investigated the effects of chronic in vitro AZT exposure on F3II mouse mammary carcinoma cells. We demonstrate, for the first time, that AZT-treated tumor cells have a reduced tumorigenicity in syngeneic BALB/c mice. Tumor incidence was reduced and survival was prolonged in animals inoculated with AZT-treated cells when comparing with control counterparts. The number and size of spontaneous metastases were also decreased in animals inoculated with AZT-treated cells. In addition, we present evidence of morphological and biochemical signs of senescence, as shown by the staining for senescence associated beta-galactosidase activity, and induction of programmed cell death, as demonstrated by an increase of caspase-3 activity, in tumor cells exposed to AZT. These data indicate that chronic exposure of mammary carcinoma cells to AZT may be sufficient to induce a senescent phenotype and to reduce tumorigenicity.
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Affiliation(s)
- A M Tejera
- Laboratory of Molecular Oncology Quilmes National University, Buenos Aires, Argentina
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