1
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Trendel J, Trendel S, Sha S, Greulich F, Goll S, Wudy SI, Kleigrewe K, Kubicek S, Uhlenhaut NH, Kuster B. The human proteome with direct physical access to DNA. Cell 2025:S0092-8674(25)00507-0. [PMID: 40409270 DOI: 10.1016/j.cell.2025.04.037] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2024] [Revised: 01/10/2025] [Accepted: 04/27/2025] [Indexed: 05/25/2025]
Abstract
In a human cell, DNA is packed with histones, RNA, and chromatin-associated proteins, forming a cohesive gel. At any given moment, only a subset of the proteome has physical access to the DNA and organizes its structure, transcription, replication, repair, and other essential molecular functions. We have developed a "zero-distance" photo-crosslinking approach to quantify proteins in direct contact with DNA in living cells. Collecting DNA interactomes from human breast cancer cells, we present an atlas of over one thousand proteins with physical access to DNA and hundreds of peptide-nucleotide crosslinks pinpointing protein-DNA interfaces with single-amino-acid resolution. Quantitative comparisons of DNA interactomes from differentially treated cells recapitulate the recruitment of key transcription factors as well as DNA repair proteins and uncover fast-acting restrictors of chromatin accessibility on a timescale of minutes. This opens a direct way to explore genomic regulation in a hypothesis-free manner, applicable to many organisms and systems.
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Affiliation(s)
- Jakob Trendel
- Proteomics and Bioanalytics, TUM School of Life Sciences, Technical University of Munich (TUM), Freising, Germany
| | | | - Shuyao Sha
- Proteomics and Bioanalytics, TUM School of Life Sciences, Technical University of Munich (TUM), Freising, Germany
| | - Franziska Greulich
- Metabolic Programming, TUM School of Life Sciences, ZIEL-Institute for Food & Health, Technical University of Munich (TUM), Freising, Germany
| | - Sandra Goll
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
| | - Susanne I Wudy
- Bavarian Center for Biomolecular Mass Spectrometry, TUM School of Life Sciences, Technical University of Munich (TUM), Freising, Germany
| | - Karin Kleigrewe
- Bavarian Center for Biomolecular Mass Spectrometry, TUM School of Life Sciences, Technical University of Munich (TUM), Freising, Germany
| | - Stefan Kubicek
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
| | - N Henriette Uhlenhaut
- Metabolic Programming, TUM School of Life Sciences, ZIEL-Institute for Food & Health, Technical University of Munich (TUM), Freising, Germany; Institute for Diabetes and Obesity (IDO) & Institute for Diabetes and Cancer (IDC), Helmholtz Center Munich (HMGU) and German Center for Diabetes Research (DZD), Neuherberg, Germany
| | - Bernhard Kuster
- Proteomics and Bioanalytics, TUM School of Life Sciences, Technical University of Munich (TUM), Freising, Germany.
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2
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Torrecilla I, Ruggiano A, Kiianitsa K, Aljarbou F, Lascaux P, Hoslett G, Song W, Maizels N, Ramadan K. Isolation and detection of DNA-protein crosslinks in mammalian cells. Nucleic Acids Res 2024; 52:525-547. [PMID: 38084926 PMCID: PMC10810220 DOI: 10.1093/nar/gkad1178] [Citation(s) in RCA: 6] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2023] [Revised: 11/21/2023] [Accepted: 11/23/2023] [Indexed: 01/26/2024] Open
Abstract
DNA-protein crosslinks (DPCs) are toxic DNA lesions wherein a protein is covalently attached to DNA. If not rapidly repaired, DPCs create obstacles that disturb DNA replication, transcription and DNA damage repair, ultimately leading to genome instability. The persistence of DPCs is associated with premature ageing, cancer and neurodegeneration. In mammalian cells, the repair of DPCs mainly relies on the proteolytic activities of SPRTN and the 26S proteasome, complemented by other enzymes including TDP1/2 and the MRN complex, and many of the activities involved are essential, restricting genetic approaches. For many years, the study of DPC repair in mammalian cells was hindered by the lack of standardised assays, most notably assays that reliably quantified the proteins or proteolytic fragments covalently bound to DNA. Recent interest in the field has spurred the development of several biochemical methods for DPC analysis. Here, we critically analyse the latest techniques for DPC isolation and the benefits and drawbacks of each. We aim to assist researchers in selecting the most suitable isolation method for their experimental requirements and questions, and to facilitate the comparison of results across different laboratories using different approaches.
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Affiliation(s)
- Ignacio Torrecilla
- The MRC Weatherall Institute of Molecular Medicine, Department of Oncology, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DS, UK
| | - Annamaria Ruggiano
- The MRC Weatherall Institute of Molecular Medicine, Department of Oncology, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DS, UK
| | - Kostantin Kiianitsa
- Department of Immunology, University of Washington, Seattle, WA 98195-7350, USA
| | - Ftoon Aljarbou
- The MRC Weatherall Institute of Molecular Medicine, Department of Oncology, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DS, UK
| | - Pauline Lascaux
- The MRC Weatherall Institute of Molecular Medicine, Department of Oncology, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DS, UK
| | - Gwendoline Hoslett
- The MRC Weatherall Institute of Molecular Medicine, Department of Oncology, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DS, UK
| | - Wei Song
- The MRC Weatherall Institute of Molecular Medicine, Department of Oncology, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DS, UK
| | - Nancy Maizels
- Department of Immunology, University of Washington, Seattle, WA 98195-7350, USA
- Department of Biochemistry, University of Washington, Seattle, WA 98195-7350, USA
| | - Kristijan Ramadan
- The MRC Weatherall Institute of Molecular Medicine, Department of Oncology, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DS, UK
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3
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Weickert P, Li HY, Götz MJ, Dürauer S, Yaneva D, Zhao S, Cordes J, Acampora AC, Forne I, Imhof A, Stingele J. SPRTN patient variants cause global-genome DNA-protein crosslink repair defects. Nat Commun 2023; 14:352. [PMID: 36681662 PMCID: PMC9867749 DOI: 10.1038/s41467-023-35988-1] [Citation(s) in RCA: 18] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2022] [Accepted: 01/10/2023] [Indexed: 01/22/2023] Open
Abstract
DNA-protein crosslinks (DPCs) are pervasive DNA lesions that are induced by reactive metabolites and various chemotherapeutic agents. Here, we develop a technique for the Purification of x-linked Proteins (PxP), which allows identification and tracking of diverse DPCs in mammalian cells. Using PxP, we investigate DPC repair in cells genetically-engineered to express variants of the SPRTN protease that cause premature ageing and early-onset liver cancer in Ruijs-Aalfs syndrome patients. We find an unexpected role for SPRTN in global-genome DPC repair, that does not rely on replication-coupled detection of the lesion. Mechanistically, we demonstrate that replication-independent DPC cleavage by SPRTN requires SUMO-targeted ubiquitylation of the protein adduct and occurs in addition to proteasomal DPC degradation. Defective ubiquitin binding of SPRTN patient variants compromises global-genome DPC repair and causes synthetic lethality in combination with a reduction in proteasomal DPC repair capacity.
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Affiliation(s)
- Pedro Weickert
- Department of Biochemistry, Ludwig-Maximilians-University, 81377, Munich, Germany
- Gene Center, Ludwig-Maximilians-University, 81377, Munich, Germany
| | - Hao-Yi Li
- Department of Biochemistry, Ludwig-Maximilians-University, 81377, Munich, Germany
- Gene Center, Ludwig-Maximilians-University, 81377, Munich, Germany
| | - Maximilian J Götz
- Department of Biochemistry, Ludwig-Maximilians-University, 81377, Munich, Germany
- Gene Center, Ludwig-Maximilians-University, 81377, Munich, Germany
| | - Sophie Dürauer
- Department of Biochemistry, Ludwig-Maximilians-University, 81377, Munich, Germany
- Gene Center, Ludwig-Maximilians-University, 81377, Munich, Germany
| | - Denitsa Yaneva
- Department of Biochemistry, Ludwig-Maximilians-University, 81377, Munich, Germany
- Gene Center, Ludwig-Maximilians-University, 81377, Munich, Germany
| | - Shubo Zhao
- Department of Biochemistry, Ludwig-Maximilians-University, 81377, Munich, Germany
- Gene Center, Ludwig-Maximilians-University, 81377, Munich, Germany
| | - Jacqueline Cordes
- Department of Biochemistry, Ludwig-Maximilians-University, 81377, Munich, Germany
- Gene Center, Ludwig-Maximilians-University, 81377, Munich, Germany
| | - Aleida C Acampora
- Department of Biochemistry, Ludwig-Maximilians-University, 81377, Munich, Germany
- Gene Center, Ludwig-Maximilians-University, 81377, Munich, Germany
| | - Ignasi Forne
- Protein Analysis Unit (ZfP), BioMedical Center (BMC), Ludwig-Maximilians-University, 82152, Martinsried, Germany
| | - Axel Imhof
- Protein Analysis Unit (ZfP), BioMedical Center (BMC), Ludwig-Maximilians-University, 82152, Martinsried, Germany
| | - Julian Stingele
- Department of Biochemistry, Ludwig-Maximilians-University, 81377, Munich, Germany.
- Gene Center, Ludwig-Maximilians-University, 81377, Munich, Germany.
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4
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Mechanistic investigation of the deamination reaction of 6-thioguanine: a theoretical study. Struct Chem 2022. [DOI: 10.1007/s11224-022-02121-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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5
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Huo B, Zhang X, Wang C, Wang H, Zhu G, Zhu W, Zhu A, Mei H, Li L. Mechanistic Insight into the Photoinduced Damage of an Unnatural Base Pair. Chemistry 2022; 28:e202201730. [DOI: 10.1002/chem.202201730] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2022] [Indexed: 11/07/2022]
Affiliation(s)
- Bianbian Huo
- Department Henan Key Laboratory of Organic Functional Molecule and Drug Innovation Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals School of Chemistry and Chemical Engineering Key Laboratory of Green Chemical Media and Reactions Ministry of Education Henan Normal University Xinxiang Henan 453007 P. R. China
- State Key Laboratory of Cell Differentiation Regulation and Target Drug Henan Normal University Xinxiang Henan 453007 P. R. China
| | - Xiguang Zhang
- Department Henan Key Laboratory of Organic Functional Molecule and Drug Innovation Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals School of Chemistry and Chemical Engineering Key Laboratory of Green Chemical Media and Reactions Ministry of Education Henan Normal University Xinxiang Henan 453007 P. R. China
| | - Chao Wang
- Department Henan Key Laboratory of Organic Functional Molecule and Drug Innovation Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals School of Chemistry and Chemical Engineering Key Laboratory of Green Chemical Media and Reactions Ministry of Education Henan Normal University Xinxiang Henan 453007 P. R. China
| | - Honglei Wang
- Department Henan Key Laboratory of Organic Functional Molecule and Drug Innovation Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals School of Chemistry and Chemical Engineering Key Laboratory of Green Chemical Media and Reactions Ministry of Education Henan Normal University Xinxiang Henan 453007 P. R. China
- State Key Laboratory of Cell Differentiation Regulation and Target Drug Henan Normal University Xinxiang Henan 453007 P. R. China
| | - Gongming Zhu
- Department Henan Key Laboratory of Organic Functional Molecule and Drug Innovation Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals School of Chemistry and Chemical Engineering Key Laboratory of Green Chemical Media and Reactions Ministry of Education Henan Normal University Xinxiang Henan 453007 P. R. China
| | - Wuyuan Zhu
- Department Henan Key Laboratory of Organic Functional Molecule and Drug Innovation Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals School of Chemistry and Chemical Engineering Key Laboratory of Green Chemical Media and Reactions Ministry of Education Henan Normal University Xinxiang Henan 453007 P. R. China
| | - Anlian Zhu
- Department Henan Key Laboratory of Organic Functional Molecule and Drug Innovation Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals School of Chemistry and Chemical Engineering Key Laboratory of Green Chemical Media and Reactions Ministry of Education Henan Normal University Xinxiang Henan 453007 P. R. China
| | - Hui Mei
- Shenzhen Key Laboratory of Synthetic Genomics Guangdong Provincial Key Laboratory of Synthetic Genomics CAS Key Laboratory of Quantitative Engineering Biology Shenzhen Institute of Synthetic Biology Shenzhen Institutes of Advanced Technology Chinese Academy of Sciences Shenzhen 518055 P. R. China
| | - Lingjun Li
- Department Henan Key Laboratory of Organic Functional Molecule and Drug Innovation Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals School of Chemistry and Chemical Engineering Key Laboratory of Green Chemical Media and Reactions Ministry of Education Henan Normal University Xinxiang Henan 453007 P. R. China
- State Key Laboratory of Cell Differentiation Regulation and Target Drug Henan Normal University Xinxiang Henan 453007 P. R. China
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6
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Dahiya R, Hu Q, Ly P. Mechanistic origins of diverse genome rearrangements in cancer. Semin Cell Dev Biol 2022; 123:100-109. [PMID: 33824062 PMCID: PMC8487437 DOI: 10.1016/j.semcdb.2021.03.003] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Accepted: 03/08/2021] [Indexed: 12/14/2022]
Abstract
Cancer genomes frequently harbor structural chromosomal rearrangements that disrupt the linear DNA sequence order and copy number. To date, diverse classes of structural variants have been identified across multiple cancer types. These aberrations span a wide spectrum of complexity, ranging from simple translocations to intricate patterns of rearrangements involving multiple chromosomes. Although most somatic rearrangements are acquired gradually throughout tumorigenesis, recent interrogation of cancer genomes have uncovered novel categories of complex rearrangements that arises rapidly through a one-off catastrophic event, including chromothripsis and chromoplexy. Here we review the cellular and molecular mechanisms contributing to the formation of diverse structural rearrangement classes during cancer development. Genotoxic stress from a myriad of extrinsic and intrinsic sources can trigger DNA double-strand breaks that are subjected to DNA repair with potentially mutagenic outcomes. We also highlight how aberrant nuclear structures generated through mitotic cell division errors, such as rupture-prone micronuclei and chromosome bridges, can instigate massive DNA damage and the formation of complex rearrangements in cancer genomes.
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Affiliation(s)
- Rashmi Dahiya
- Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA
| | - Qing Hu
- Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA
| | - Peter Ly
- Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
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7
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Dong J, Huang C, Guo S, Xia Y, Hou Y, Yang C, Zhang X, Jie J, Zhu BZ, Su H. Free-Radical-Mediated Photoinduced Electron Transfer between 6-Thioguanine and Tryptophan Leading to DNA-Protein-Like Cross-Link. J Phys Chem B 2021; 126:14-22. [PMID: 34951313 DOI: 10.1021/acs.jpcb.1c03380] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The nucleobase analog 6-thioguanine (6-TG) has emerged as important immunosuppressant, anti-inflammatory, and anticancer drug in the past few decades, but its unique photosensitivity of absorbing strongly ultraviolet UVA light elicits photochemical hazards in many ways. The particularly intriguing yet unresolved question is whether the direct photoreaction of 6-TG can promote DNA-protein cross-links (DPCs) formation, which are large DNA adducts blocking DNA replication and physically impede DNA-related processes. Herein, by real-time observation of radical intermediates using time-resolved UV-vis absorption spectroscopy in conjunction with product analysis by HPLC-MS, we discover that UVA excitation of 6-TG triggers direct covalent cross-linking with tryptophan (TrpH) via an exquisite radical mechanism of electron transfer. The photoexcitation prepares the redox-active triplet 36-TG*, which initiates electron transfer with TrpH, creating TrpH•+ and 6-TG•- in the first step. The deprotonated Trp• undergoes radical-recombination with its geminate partner 6-TG•- and eliminates a H2S, leading to the cross-linking product 6-TG-Trp. The photoadduct structures (two chiral isomers and one constitutional isomer) are identified unambiguously, validating further the mechanism. These findings pinpoint the exact amino acid that is vulnerable to photo-cross-linking with 6-TG and establish a mechanistic framework for understanding mutagenic DPCs formation and developing photoprobes based on this new type of photo-cross-linking.
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Affiliation(s)
- Junjie Dong
- College of Chemistry, Beijing Normal University, Beijing 100875, P. R. China
| | - Chunhua Huang
- State Key Lab of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Science, Chinese Academy of Sciences, Beijing 100085, P. R. China
| | - Shaoshi Guo
- College of Chemistry, Beijing Normal University, Beijing 100875, P. R. China
| | - Ye Xia
- College of Chemistry, Beijing Normal University, Beijing 100875, P. R. China
| | - Yue Hou
- College of Chemistry, Beijing Normal University, Beijing 100875, P. R. China
| | - Chunfan Yang
- College of Chemistry, Beijing Normal University, Beijing 100875, P. R. China
| | - Xianwang Zhang
- College of Chemistry, Beijing Normal University, Beijing 100875, P. R. China
| | - Jialong Jie
- College of Chemistry, Beijing Normal University, Beijing 100875, P. R. China
| | - Ben-Zhan Zhu
- State Key Lab of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Science, Chinese Academy of Sciences, Beijing 100085, P. R. China
| | - Hongmei Su
- College of Chemistry, Beijing Normal University, Beijing 100875, P. R. China
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8
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Xie BB, Tang XF, Liu XY, Chang XP, Cui G. Mechanistic photophysics and photochemistry of unnatural bases and sunscreen molecules: insights from electronic structure calculations. Phys Chem Chem Phys 2021; 23:27124-27149. [PMID: 34849517 DOI: 10.1039/d1cp03994f] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Photophysics and photochemistry are basic subjects in the study of light-matter interactions and are ubiquitous in diverse fields such as biology, energy, materials, and environment. A full understanding of mechanistic photophysics and photochemistry underpins many recent advances and applications. This contribution first provides a short discussion on the theoretical calculation methods we have used in relevant studies, then we introduce our latest progress on the mechanistic photophysics and photochemistry of two classes of molecular systems, namely unnatural bases and sunscreens. For unnatural bases, we disclose the intrinsic driving forces for the ultrafast population to reactive triplet states, impacts of the position and degree of chalcogen substitutions, and the effects of complex environments. For sunscreen molecules, we reveal the photoprotection mechanisms that dissipate excess photon energy to the surroundings by ultrafast internal conversion to the ground state. Finally, relevant theoretical challenges and outlooks are discussed.
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Affiliation(s)
- Bin-Bin Xie
- Hangzhou Institute of Advanced Studies, Zhejiang Normal University, 1108 Gengwen Road, Hangzhou 311231, Zhejiang, P. R. China.
| | - Xiu-Fang Tang
- Hangzhou Institute of Advanced Studies, Zhejiang Normal University, 1108 Gengwen Road, Hangzhou 311231, Zhejiang, P. R. China.
| | - Xiang-Yang Liu
- College of Chemistry and Material Science, Sichuan Normal University, Chengdu, Sichuan, 610068, China
| | - Xue-Ping Chang
- College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, P. R. China
| | - Ganglong Cui
- Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education College of Chemistry, Beijing Normal University, Beijing 100875, P. R. China.
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9
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Katerji M, Duerksen-Hughes PJ. DNA damage in cancer development: special implications in viral oncogenesis. Am J Cancer Res 2021; 11:3956-3979. [PMID: 34522461 PMCID: PMC8414375] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2020] [Accepted: 02/04/2021] [Indexed: 06/13/2023] Open
Abstract
DNA lesions arise from a combination of physiological/metabolic sources and exogenous environmental influences. When left unrepaired, these alterations accumulate in the cells and can give rise to mutations that change the function of important proteins (i.e. tumor suppressors, oncoproteins), or cause chromosomal rearrangements (i.e. gene fusions) that also result in the deregulation of key cellular molecules. Progressive acquisition of such genetic changes promotes uncontrolled cell proliferation and evasion of cell death, and hence plays a key role in carcinogenesis. Another less-studied consequence of DNA damage accumulating in the host genome is the integration of oncogenic DNA viruses such as Human papillomavirus, Merkel cell polyomavirus, and Hepatitis B virus. This critical step of viral-induced carcinogenesis is thought to be particularly facilitated by DNA breaks in both viral and host genomes. Therefore, the impact of DNA damage on carcinogenesis is magnified in the case of such oncoviruses via the additional effect of increasing integration frequency. In this review, we briefly present the various endogenous and exogenous factors that cause different types of DNA damage. Next, we discuss the contribution of these lesions in cancer development. Finally, we examine the amplified effect of DNA damage in viral-induced oncogenesis and summarize the limited data existing in the literature related to DNA damage-induced viral integration. To conclude, additional research is needed to assess the DNA damage pathways involved in the transition from viral infection to cancer. Discovering that a certain DNA damaging agent increases the likelihood of viral integration will enable the development of prophylactic and therapeutic strategies designed specifically to prevent such integration, with an ultimate goal of reducing or eliminating these viral-induced malignancies.
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Affiliation(s)
- Meghri Katerji
- Department of Basic Science, Loma Linda University School of Medicine Loma Linda, CA 92354, USA
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10
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Xie BB, Cui CX. Theoretical studies on photo-induced cycloaddition and (6-4) reactions of the thymidine:4-thiothymidine dimer in a DNA duplex. Phys Chem Chem Phys 2019; 21:2006-2016. [DOI: 10.1039/c8cp07063f] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/01/2023]
Abstract
Photo-induced cycloaddition and (6-4) reactions of the thymidine:4-thiothymidine dimer in a DNA duplex.
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Affiliation(s)
- Bin-Bin Xie
- Hangzhou Institute of Advanced Studies
- Zhejiang Normal University
- Hangzhou 311231
- P. R. China
- Key Laboratory of Theoretical and Computational Photochemistry
| | - Cheng-Xing Cui
- School of Chemistry and Chemical Engineering
- Henan Institute of Science and Technology
- Xinxiang 453003
- P. R. China
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11
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Ashwood B, Pollum M, Crespo-Hernández CE. Photochemical and Photodynamical Properties of Sulfur-Substituted Nucleic Acid Bases. Photochem Photobiol 2018; 95:33-58. [PMID: 29978490 DOI: 10.1111/php.12975] [Citation(s) in RCA: 83] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2018] [Accepted: 06/28/2018] [Indexed: 12/25/2022]
Abstract
Sulfur-substituted nucleobases (a.k.a., thiobases) are among the world's leading prescriptions for chemotherapy and immunosuppression. Long-term treatment with azathioprine, 6-mercaptopurine and 6-thioguanine has been correlated with the photoinduced formation of carcinomas. Establishing an in-depth understanding of the photochemical properties of these prodrugs may provide a route to overcoming these carcinogenic side effects, or, alternatively, a basis for developing effective compounds for targeted phototherapy. In this review, a broad examination is undertaken, surveying the basic photochemical properties and excited-state dynamics of sulfur-substituted analogs of the canonical DNA and RNA nucleobases. A molecular-level understanding of how sulfur substitution so remarkably perturbs the photochemical properties of the nucleobases is presented by combining experimental results with quantum-chemical calculations. Structure-property relationships demonstrate the impact of site-specific sulfur substitution on the photochemical properties, particularly on the population of the reactive triplet state. The value of fundamental photochemical investigations for driving the development of ultraviolet-A chemotherapeutics is showcased. The most promising photodynamic agents identified thus far have been investigated in various carcinoma cell lines and shown to decrease cell proliferation upon exposure to ultraviolet-A radiation. Overarching principles have been elucidated for the impact that sulfur substitution of the carbonyl oxygen has on the photochemical properties of the nucleobases.
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Affiliation(s)
- Brennan Ashwood
- Department of Chemistry, Case Western Reserve University, Cleveland, OH
| | - Marvin Pollum
- Department of Chemistry, Case Western Reserve University, Cleveland, OH
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Arslancan S, Martínez-Fernández L, Corral I. Photophysics and Photochemistry of Canonical Nucleobases’ Thioanalogs: From Quantum Mechanical Studies to Time Resolved Experiments. Molecules 2017. [PMCID: PMC6152766 DOI: 10.3390/molecules22060998] [Citation(s) in RCA: 47] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
Interest in understanding the photophysics and photochemistry of thiated nucleobases has been awakened because of their possible involvement in primordial RNA or their potential use as photosensitizers in medicinal chemistry. The interpretation of the photodynamics of these systems, conditioned by their intricate potential energy surfaces, requires the powerful interplay between experimental measurements and state of the art molecular simulations. In this review, we provide an overview on the photophysics of natural nucleobases’ thioanalogs, which covers the last 30 years and both experimental and computational contributions. For all the canonical nucleobase’s thioanalogs, we have compiled the main steady state absorption and emission features and their interpretation in terms of theoretical calculations. Then, we revise the main topographical features, including stationary points and interstate crossings, of their potential energy surfaces based on quantum mechanical calculations and we conclude, by combining the outcome of different spectroscopic techniques and molecular dynamics simulations, with the mechanism by which these nucleobase analogs populate their triplet excited states, which are at the origin of their photosensitizing properties.
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Affiliation(s)
- Serra Arslancan
- Departamento de Química, Módulo 13, Universidad Autónoma de Madrid, Madrid 28049, Spain;
| | - Lara Martínez-Fernández
- Istituto Biostrutture e Bioimmagini-Consiglio Nazionale delle Ricerche, Via Mezzocannone 16, Napoli I-80134, Italy
- Correspondence: (L.M.-F.); (I.C.); Tel.: +34-91-497-8471 (I.C.)
| | - Inés Corral
- Departamento de Química, Módulo 13, Universidad Autónoma de Madrid, Madrid 28049, Spain;
- Institute for Advanced Research in Chemical Sciences (IADCHEM), Universidad Autónoma de Madrid, Madrid 28049, Spain
- Correspondence: (L.M.-F.); (I.C.); Tel.: +34-91-497-8471 (I.C.)
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13
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Brem R, Guven M, Karran P. Oxidatively-generated damage to DNA and proteins mediated by photosensitized UVA. Free Radic Biol Med 2017; 107:101-109. [PMID: 27989755 PMCID: PMC5462485 DOI: 10.1016/j.freeradbiomed.2016.10.488] [Citation(s) in RCA: 83] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/04/2016] [Revised: 10/19/2016] [Accepted: 10/21/2016] [Indexed: 11/19/2022]
Abstract
UVA accounts for about 95% of the solar ultraviolet (UV) radiation that reaches Earth and most likely contributes to human skin cancer risk. In contrast to UVB, which comprises the remaining 5% and is absorbed by DNA nucleobases to cause direct photodamage, UVA damages DNA indirectly. It does this largely through its interactions with cellular chromophores that act as photosensitisers to generate reactive oxygen species. Exogenously supplied chemicals, including some widely-prescribed medicines, may also act as photosensitisers and these drugs are associated with an increased risk of sun-related cancer. Because they amplify the effects of UVA on cells, they provide a means to investigate the mechanisms and effects of UVA-induced photodamage. Here, we describe some of the major lesions induced by two groups of UVA photosensitisers, the DNA thionucleotides and the fluoroquinolone antibiotics. In thionucleotides, replacement of the oxygen atoms of canonical nucleobases by sulfur converts them into strong UVA chromophores that can be incorporated into DNA. The fluoroquinolones are also UVA chromophores. They are not incorporated into DNA and induce a different range of DNA damages. We also draw attention to the potentially important contribution of photochemical protein damage to the cellular effects of photosensitised UVA. Proteins targeted for oxidation damage include DNA repair factors and we suggest that UVA-mediated protein damage may contribute to sunlight-induced cancer risk.
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Affiliation(s)
- Reto Brem
- The Francis Crick Institute, 1, Midland Road, London NW1 1AT, UK
| | - Melisa Guven
- The Francis Crick Institute, 1, Midland Road, London NW1 1AT, UK
| | - Peter Karran
- The Francis Crick Institute, 1, Midland Road, London NW1 1AT, UK.
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14
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Excited-State Dynamics of the Thiopurine Prodrug 6-Thioguanine: Can N9-Glycosylation Affect Its Phototoxic Activity? Molecules 2017; 22:molecules22030379. [PMID: 28264514 PMCID: PMC6155220 DOI: 10.3390/molecules22030379] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2017] [Revised: 02/15/2017] [Accepted: 02/24/2017] [Indexed: 12/25/2022] Open
Abstract
6-Thioguanine, an immunosuppressant and anticancer prodrug, has been shown to induce DNA damage and cell death following exposure to UVA radiation. Its metabolite, 6-thioguanosine, plays a major role in the prodrug's overall photoreactivity. However, 6-thioguanine itself has proven to be cytotoxic following UVA irradiation, warranting further investigation into its excited-state dynamics. In this contribution, the excited-state dynamics and photochemical properties of 6-thioguanine are studied in aqueous solution following UVA excitation at 345 nm in order to provide mechanistic insight regarding its photochemical reactivity and to scrutinize whether N9-glycosylation modulates its phototoxicity in solution. The experimental results are complemented with time-dependent density functional calculations that include solvent dielectric effects by means of a reaction-field solvation model. UVA excitation results in the initial population of the S₂(ππ*) state, which is followed by ultrafast internal conversion to the S₁(nπ*) state and then intersystem crossing to the triplet manifold within 560 ± 60 fs. A small fraction (ca. 25%) of the population that reaches the S₁(nπ*) state repopulates the ground state. The T₁(ππ*) state decays to the ground state in 1.4 ± 0.2 μs under N₂-purged conditions, using a 0.2 mM concentration of 6-thioguanine, or it can sensitize singlet oxygen in 0.21 ± 0.02 and 0.23 ± 0.02 yields in air- and O₂-saturated solution, respectively. This demonstrates the efficacy of 6-thioguanine to act as a Type II photosensitizer. N9-glycosylation increases the rate of intersystem crossing from the singlet to triplet manifold, as well as from the T₁(ππ*) state to the ground state, which lead to a ca. 40% decrease in the singlet oxygen yield under air-saturated conditions. Enhanced vibronic coupling between the singlet and triplet manifolds due to a higher density of vibrational states is proposed to be responsible for the observed increase in the rates of intersystem crossing in 6-thioguanine upon N9-glycosylation.
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15
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Guven M, Barnouin K, Snijders AP, Karran P. Photosensitized UVA-Induced Cross-Linking between Human DNA Repair and Replication Proteins and DNA Revealed by Proteomic Analysis. J Proteome Res 2016; 15:4612-4623. [PMID: 27654267 PMCID: PMC5154610 DOI: 10.1021/acs.jproteome.6b00717] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2016] [Indexed: 11/29/2022]
Abstract
Long wavelength ultraviolet radiation (UVA, 320-400 nm) interacts with chromophores present in human cells to induce reactive oxygen species (ROS) that damage both DNA and proteins. ROS levels are amplified, and the damaging effects of UVA are exacerbated if the cells are irradiated in the presence of UVA photosensitizers such as 6-thioguanine (6-TG), a strong UVA chromophore that is extensively incorporated into the DNA of dividing cells, or the fluoroquinolone antibiotic ciprofloxacin. Both DNA-embedded 6-TG and ciprofloxacin combine synergistically with UVA to generate high levels of ROS. Importantly, the extensive protein damage induced by these photosensitizer+UVA combinations inhibits DNA repair. DNA is maintained in intimate contact with the proteins that effect its replication, transcription, and repair, and DNA-protein cross-links (DPCs) are a recognized reaction product of ROS. Cross-linking of DNA metabolizing proteins would compromise these processes by introducing physical blocks and by depleting active proteins. We describe a sensitive and statistically rigorous method to analyze DPCs in cultured human cells. Application of this proteomics-based analysis to cells treated with 6-TG+UVA and ciprofloxacin+UVA identified proteins involved in DNA repair, replication, and gene expression among those most vulnerable to cross-linking under oxidative conditions.
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Affiliation(s)
- Melisa Guven
- The
Francis Crick Institute, Clare Hall Laboratory, South Mimms, Herts EN6
3LD, U.K.
| | - Karin Barnouin
- The
Francis Crick Institute, Clare Hall Laboratory, South Mimms, Herts EN6
3LD, U.K.
| | - Ambrosius P. Snijders
- The
Francis Crick Institute, Clare Hall Laboratory, South Mimms, Herts EN6
3LD, U.K.
| | - Peter Karran
- The
Francis Crick Institute, Clare Hall Laboratory, South Mimms, Herts EN6
3LD, U.K.
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16
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Wickramaratne S, Ji S, Mukherjee S, Su Y, Pence MG, Lior-Hoffmann L, Fu I, Broyde S, Guengerich FP, Distefano M, Schärer OD, Sham YY, Tretyakova N. Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases. J Biol Chem 2016; 291:23589-23603. [PMID: 27621316 DOI: 10.1074/jbc.m116.745257] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2016] [Indexed: 12/22/2022] Open
Abstract
DNA-protein cross-links (DPCs) are bulky DNA lesions that form both endogenously and following exposure to bis-electrophiles such as common antitumor agents. The structural and biological consequences of DPCs have not been fully elucidated due to the complexity of these adducts. The most common site of DPC formation in DNA following treatment with bis-electrophiles such as nitrogen mustards and cisplatin is the N7 position of guanine, but the resulting conjugates are hydrolytically labile and thus are not suitable for structural and biological studies. In this report, hydrolytically stable structural mimics of N7-guanine-conjugated DPCs were generated by reductive amination reactions between the Lys and Arg side chains of proteins/peptides and aldehyde groups linked to 7-deazaguanine residues in DNA. These model DPCs were subjected to in vitro replication in the presence of human translesion synthesis DNA polymerases. DPCs containing full-length proteins (11-28 kDa) or a 23-mer peptide blocked human polymerases η and κ. DPC conjugates to a 10-mer peptide were bypassed with nucleotide insertion efficiency 50-100-fold lower than for native G. Both human polymerase (hPol) κ and hPol η inserted the correct base (C) opposite the 10-mer peptide cross-link, although small amounts of T were added by hPol η. Molecular dynamics simulation of an hPol κ ternary complex containing a template-primer DNA with dCTP opposite the 10-mer peptide DPC revealed that this bulky lesion can be accommodated in the polymerase active site by aligning with the major groove of the adducted DNA within the ternary complex of polymerase and dCTP.
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Affiliation(s)
| | - Shaofei Ji
- From the Masonic Cancer Center and.,the Departments of Chemistry
| | | | - Yan Su
- Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
| | - Matthew G Pence
- Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
| | - Lee Lior-Hoffmann
- Department of Biology, New York University, New York, New York 10003-6688, and
| | - Iwen Fu
- Department of Biology, New York University, New York, New York 10003-6688, and
| | - Suse Broyde
- Department of Biology, New York University, New York, New York 10003-6688, and
| | - F Peter Guengerich
- Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
| | | | - Orlando D Schärer
- Chemistry, Stony Brook University, Stony Brook, New York 11794.,Departments of Pharmacological Sciences and
| | | | - Natalia Tretyakova
- From the Masonic Cancer Center and .,Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455
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17
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Abstract
Solar UVB is carcinogenic. Nucleotide excision repair (NER) counteracts the carcinogenicity of UVB by excising potentially mutagenic UVB-induced DNA lesions. Despite this capacity for DNA repair, non-melanoma skin cancers and apparently normal sun-exposed skin contain huge numbers of mutations that are mostly attributable to unrepaired UVB-induced DNA lesions. UVA is about 20-times more abundant than UVB in incident sunlight. It does cause some DNA damage but this does not fully account for its biological impact. The effects of solar UVA are mediated by its interactions with cellular photosensitizers that generate reactive oxygen species (ROS) and induce oxidative stress. The proteome is a significant target for damage by UVA-induced ROS. In cultured human cells, UVA-induced oxidation of DNA repair proteins inhibits DNA repair. This article addresses the possible role of oxidative stress and protein oxidation in determining DNA repair efficiency - with particular reference to NER and skin cancer risk.
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Affiliation(s)
- Peter Karran
- Francis Crick Research Institute, Clare Hall Laboratory, South Mimms, Herts. EN6 3LD, UK.
| | - Reto Brem
- Francis Crick Research Institute, Clare Hall Laboratory, South Mimms, Herts. EN6 3LD, UK
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18
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Pollum M, Ortiz-Rodríguez LA, Jockusch S, Crespo-Hernández CE. The Triplet State of 6-thio-2'-deoxyguanosine: Intrinsic Properties and Reactivity Toward Molecular Oxygen. Photochem Photobiol 2016; 92:286-292. [PMID: 26757207 DOI: 10.1111/php.12563] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2015] [Accepted: 12/19/2015] [Indexed: 12/18/2022]
Abstract
Thiopurine prodrugs are currently among the leading treatment options for leukemia, immunosuppression, and arthritis. Patients undergoing long-term thiopurine treatment are at a higher risk of developing sunlight-induced skin cancers than the general population. This side effect originates from the cellular metabolization of thiopurine prodrugs to form 6-thio-2'-deoxyguanosine, which can absorb UVA radiation, populating its reactive triplet state and leading to oxidatively generated damage. However, the photo-oxidation mechanism is not fully understood. In this contribution, the oxidation potential and the adiabatic triplet energy of 6-thio-2'-deoxyguanosine are estimated computationally, whereas the intrinsic rate of triple-state decay and the rate constant for triplet quenching by molecular oxygen are determined using time-resolved spectroscopic techniques. A singlet oxygen quantum yield of 0.24 ± 0.02 is measured in aqueous solution (0.29 ± 0.02 in acetonitrile). Its magnitude correlates with the relatively low percentage of triplet-O2 collision events that generate singlet oxygen (SΔ = 37%). This behavior is rationalized as being due to the exergonic driving force for electron transfer between the triplet state of 6-thio-2'-deoxyguanosine and molecular oxygen (ΔGET = -69.7 kJ mol-1 ), resulting in the formation of a charge-transfer complex that favors nonradiative decay to the ground state over triplet energy transfer.
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Affiliation(s)
- Marvin Pollum
- Department of Chemistry and Center for Chemical Dynamics, Case Western Reserve University, Cleveland, OH
| | - Luis A Ortiz-Rodríguez
- Department of Chemistry and Center for Chemical Dynamics, Case Western Reserve University, Cleveland, OH
| | | | - Carlos E Crespo-Hernández
- Department of Chemistry and Center for Chemical Dynamics, Case Western Reserve University, Cleveland, OH
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19
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Grasso F, Ruggieri V, De Luca G, Leopardi P, Mancuso MT, Casorelli I, Pichierri P, Karran P, Bignami M. MUTYH mediates the toxicity of combined DNA 6-thioguanine and UVA radiation. Oncotarget 2015; 6:7481-92. [PMID: 25638157 PMCID: PMC4480694 DOI: 10.18632/oncotarget.3037] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2014] [Accepted: 12/01/2014] [Indexed: 12/22/2022] Open
Abstract
The therapeutic thiopurines, including the immunosuppressant azathioprine (Aza) cause the accumulation of the UVA photosensitizer 6-thioguanine (6-TG) in the DNA of the patients' cells. DNA 6-TG and UVA are synergistically cytotoxic and their interaction causes oxidative damage. The MUTYH DNA glycosylase participates in the base excision repair of oxidized DNA bases. Using Mutyh-nullmouse fibroblasts (MEFs) we examined whether MUTYH provides protection against the lethal effects of combined DNA 6-TG/UVA. Surprisingly, Mutyh-null MEFs were more resistant than wild-type MEFs, despite accumulating higher levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG).Their enhanced 6-TG/UVA resistance reflected the absence of the MUTYH protein and MEFs expressing enzymatically-dead human variants were as sensitive as wild-type cells. Consistent with their enhanced resistance, Mutyh-null cells sustained fewer DNA strand breaks and lower levels of chromosomal damage after 6-TG/UVA. Although 6-TG/UVA treatment caused early checkpoint activation irrespective of the MUTYH status, Mutyh-null cells failed to arrest in S-phase at late time points. MUTYH-dependent toxicity was also apparent in vivo. Mutyh-/- mice survived better than wild-type during a 12-month chronicexposure to Aza/UVA treatments that significantly increased levels of skin DNA 8-oxoG. Two squamous cell skin carcinomas arose in Aza/UVA treated Mutyh-/- mice whereas similarly treated wild-type animals remained tumor-free.
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Affiliation(s)
- Francesca Grasso
- Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy
- Department of Science, University Roma Tre, Rome, Italy
| | - Vitalba Ruggieri
- Laboratory of Pre-Clinical and Translational Research, IRCCS, Referral Cancer Center of Basilicata, Rionero in Vulture, Italy
| | - Gabriele De Luca
- Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy
| | - Paola Leopardi
- Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy
| | - Maria Teresa Mancuso
- Laboratory of Radiation Biology and Biomedicine, Agenzia Nazionale per le Nuove Tecnologie, l'Energia e lo Sviluppo Economico Sostenibile (ENEA) CR-Casaccia, Rome, Italy
| | - Ida Casorelli
- Department of Immunohematology and Transfusion Unit, Azienda Ospedaliera Sant'Andrea, Rome, Italy
| | - Pietro Pichierri
- Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy
| | - Peter Karran
- Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts, UK
| | - Margherita Bignami
- Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy
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20
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Hope CM, Coates PTH, Carroll RP. Immune profiling and cancer post transplantation. World J Nephrol 2015; 4:41-56. [PMID: 25664246 PMCID: PMC4317627 DOI: 10.5527/wjn.v4.i1.41] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/11/2014] [Revised: 11/03/2014] [Accepted: 11/07/2014] [Indexed: 02/06/2023] Open
Abstract
Half of all long-term (> 10 year) australian kidney transplant recipients (KTR) will develop squamous cell carcinoma (SCC) or solid organ cancer (SOC), making cancer the leading cause of death with a functioning graft. At least 30% of KTR with a history of SCC or SOC will develop a subsequent SCC or SOC lesion. Pharmacological immunosuppression is a major contributor of the increased risk of cancer for KTR, with the cancer lesions themselves further adding to systemic immunosuppression and could explain, in part, these phenomena. Immune profiling includes; measuring immunosuppressive drug levels and pharmacokinetics, enumerating leucocytes and leucocyte subsets as well as testing leucocyte function in either an antigen specific or non-specific manner. Outputs can vary from assay to assay according to methods used. In this review we define the rationale behind post-transplant immune monitoring assays and focus on assays that associate and/or have the ability to predict cancer and rejection in the KTR. We find that immune monitoring can identify those KTR of developing multiple SCC lesions and provide evidence they may benefit from pharmacological immunosuppressive drug dose reductions. In these KTR risk of rejection needs to be assessed to determine if reduction of immunosuppression will not harm the graft.
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21
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Cadet J, Douki T, Ravanat JL. Oxidatively generated damage to cellular DNA by UVB and UVA radiation. Photochem Photobiol 2014; 91:140-55. [PMID: 25327445 DOI: 10.1111/php.12368] [Citation(s) in RCA: 226] [Impact Index Per Article: 20.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2014] [Accepted: 10/09/2014] [Indexed: 12/13/2022]
Abstract
This review article focuses on a critical survey of the main available information on the UVB and UVA oxidative reactions to cellular DNA as the result of direct interactions of UV photons, photosensitized pathways and biochemical responses including inflammation and bystander effects. UVA radiation appears to be much more efficient than UVB in inducing oxidatively generated damage to the bases and 2-deoxyribose moieties of DNA in isolated cells and skin. The UVA-induced generation of 8-oxo-7,8-dihydroguanine is mostly rationalized in terms of selective guanine oxidation by singlet oxygen generated through type II photosensitization mechanism. In addition, hydroxyl radical whose formation may be accounted for by metal-catalyzed Haber-Weiss reactions subsequent to the initial generation of superoxide anion radical contributes in a minor way to the DNA degradation. This leads to the formation of both oxidized purine and pyrimidine bases together with DNA single-strand breaks at the exclusion, however, of direct double-strand breaks. No evidence has been provided so far for the implication of delayed oxidative degradation pathways of cellular DNA. In that respect putative characteristic UVA-induced DNA damage could include single and more complex lesions arising from one-electron oxidation of the guanine base together with aldehyde adducts to amino-substituted nucleobases.
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Affiliation(s)
- Jean Cadet
- University Grenoble Alpes, INAC, Grenoble, France; CEA, INAC, Grenoble, France; Département de Médecine Nucléaire et Radiobiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada
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22
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Peacock M, Brem R, Macpherson P, Karran P. DNA repair inhibition by UVA photoactivated fluoroquinolones and vemurafenib. Nucleic Acids Res 2014; 42:13714-22. [PMID: 25414333 PMCID: PMC4267641 DOI: 10.1093/nar/gku1213] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
Cutaneous photosensitization is a common side effect of drug treatment and can be associated with an increased skin cancer risk. The immunosuppressant azathioprine, the fluoroquinolone antibiotics and vemurafenib—a BRAF inhibitor used to treat metastatic melanoma—are all recognized clinical photosensitizers. We have compared the effects of UVA radiation on cultured human cells treated with 6-thioguanine (6-TG, a DNA-embedded azathioprine surrogate), the fluoroquinolones ciprofloxacin and ofloxacin and vemurafenib. Despite widely different structures and modes of action, each of these drugs potentiated UVA cytotoxicity. UVA photoactivation of 6-TG, ciprofloxacin and ofloxacin was associated with the generation of singlet oxygen that caused extensive protein oxidation. In particular, these treatments were associated with damage to DNA repair proteins that reduced the efficiency of nucleotide excision repair. Although vemurafenib was also highly phototoxic to cultured cells, its effects were less dependent on singlet oxygen. Highly toxic combinations of vemurafenib and UVA caused little protein carbonylation but were nevertheless inhibitory to nucleotide excision repair. Thus, for three different classes of drugs, photosensitization by at least two distinct mechanisms is associated with reduced protection against potentially mutagenic and carcinogenic DNA damage.
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Affiliation(s)
- Matthew Peacock
- Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts. EN6 3LD, UK
| | - Reto Brem
- Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts. EN6 3LD, UK
| | - Peter Macpherson
- Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts. EN6 3LD, UK
| | - Peter Karran
- Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts. EN6 3LD, UK
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23
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Berra CM, de Oliveira CS, Garcia CCM, Rocha CRR, Lerner LK, Lima LCDA, Baptista MDS, Menck CFM. Nucleotide excision repair activity on DNA damage induced by photoactivated methylene blue. Free Radic Biol Med 2013; 61:343-56. [PMID: 23567189 DOI: 10.1016/j.freeradbiomed.2013.03.026] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/05/2012] [Revised: 02/20/2013] [Accepted: 03/28/2013] [Indexed: 10/27/2022]
Abstract
The nucleotide excision repair (NER) mechanism is well known to be involved in the removal of UV-induced lesions. Nevertheless, the involvement of this pathway in the repair of lesions generated after DNA oxidation remains controversial. The effects of visible-light-excited methylene blue (MB), known to generate reactive oxygen species (ROS), were examined directly in xeroderma pigmentosum (XP)-A and XP-C NER-deficient human fibroblasts. Initially, MB was confirmed as being incorporated in similar amounts by the cells and that its photoexcitation induces the generation of (1)O2 within cells. The analysis of cell survival indicated that NER-deficient cells were hypersensitive to photoactivated MB. This sensitivity was confirmed with cells silenced for the XPC gene and by host-cell reactivation (HCR) of plasmid exposed to the photosensitizing effects of photoexcited MB. The sensitivity detected by HCR was restored in complemented cells, confirming the participation of XPA and XPC proteins in the repair of DNA lesions induced by photosensitized MB. Furthermore, DNA damage (single- and double-strand breaks and alkali-sensitive sites) was observed in the nuclei of treated cells by alkaline comet assay, with higher frequency of lesions in NER-deficient than in NER-proficient cells. Likewise, NER-deficient cells also presented more γ-H2AX-stained nuclei and G2/M arrest after photoactivated MB treatment, probably as a consequence of DNA damage response. Notwithstanding, the kinetics of both alkali- and FPG-sensitive sites repair were similar among cells, thereby demonstrating not only that MB photoexcitation generates nuclear DNA damage, but also that the removal of these lesions is NER-independent. Therefore, this work provides further evidence that XPA and XPC proteins have specific roles in cell protection and repair/tolerance of ROS-induced DNA damage. Moreover, as XPC-deficient patients do not present neurodegeneration, premature aging, or developmental clinical symptoms, the results indicate that defects in the repair/tolerance of oxidatively generated DNA lesions are not sufficient to explain these severe clinical features of certain XP patients.
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Affiliation(s)
- Carolina Maria Berra
- Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP 05508-900, Brazil
| | - Carla Santos de Oliveira
- Center of Health and Biological Sciences, University of Mato Grosso do Sul, Campo Grande, MS, Brazil
| | - Camila Carrião Machado Garcia
- Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP 05508-900, Brazil
| | - Clarissa Ribeiro Reily Rocha
- Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP 05508-900, Brazil
| | - Letícia Koch Lerner
- Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP 05508-900, Brazil
| | | | - Maurício da Silva Baptista
- Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, SP 05508-900, Brazil
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24
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Zou X, Zhao H, Yu Y, Su H. Formation of Guanine-6-sulfonate from 6-Thioguanine and Singlet Oxygen: A Combined Theoretical and Experimental Study. J Am Chem Soc 2013; 135:4509-15. [DOI: 10.1021/ja400483j] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Affiliation(s)
- Xiaoran Zou
- Beijing
National Laboratory for Molecular Sciences
(BNLMS), State Key Laboratory of Molecular Reaction Dynamics, Institute
of Chemistry, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China
| | - Hongmei Zhao
- Beijing
National Laboratory for Molecular Sciences
(BNLMS), State Key Laboratory of Molecular Reaction Dynamics, Institute
of Chemistry, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China
| | - Youqing Yu
- Beijing
National Laboratory for Molecular Sciences
(BNLMS), State Key Laboratory of Molecular Reaction Dynamics, Institute
of Chemistry, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China
| | - Hongmei Su
- Beijing
National Laboratory for Molecular Sciences
(BNLMS), State Key Laboratory of Molecular Reaction Dynamics, Institute
of Chemistry, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China
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25
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Cadet J, Wagner JR. DNA base damage by reactive oxygen species, oxidizing agents, and UV radiation. Cold Spring Harb Perspect Biol 2013; 5:5/2/a012559. [PMID: 23378590 DOI: 10.1101/cshperspect.a012559] [Citation(s) in RCA: 567] [Impact Index Per Article: 47.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Emphasis has been placed in this article dedicated to DNA damage on recent aspects of the formation and measurement of oxidatively generated damage in cellular DNA in order to provide a comprehensive and updated survey. This includes single pyrimidine and purine base lesions, intrastrand cross-links, purine 5',8-cyclonucleosides, DNA-protein adducts and interstrand cross-links formed by the reactions of either the nucleobases or the 2-deoxyribose moiety with the hydroxyl radical, one-electron oxidants, singlet oxygen, and hypochlorous acid. In addition, recent information concerning the mechanisms of formation, individual measurement, and repair-rate assessment of bipyrimidine photoproducts in isolated cells and human skin upon exposure to UVB radiation, UVA photons, or solar simulated light is critically reviewed.
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Affiliation(s)
- Jean Cadet
- Direction des Sciences de la Matière, Institut Nanosciences et Cryogénie, CEA/Grenoble, 38054 Grenoble, France.
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26
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Puch CBMD, Barbier E, Sauvaigo S, Gasparutto D, Breton J. Tools and strategies for DNA damage interactome analysis. Mutat Res 2012; 752:72-83. [PMID: 23220222 DOI: 10.1016/j.mrrev.2012.11.002] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2012] [Revised: 11/01/2012] [Accepted: 11/22/2012] [Indexed: 11/26/2022]
Abstract
DNA is the target of multiple endogenous and exogenous agents generating chemical lesions on the double helix. Cellular DNA damage response pathways rely on a myriad of proteins interacting with DNA alterations. The cartography of this interactome currently includes well known actors of chromatin remodelling, DNA repair or proteins hijacked from their natural functions such as transcription factors. In order to go further into the characterisation of these protein networks, proteomics-based methods began to be used in the early 2000s. The strategies are diverse and include mainly (i) damaged DNA molecules used as targets on protein microarrays, (ii) damaged DNA probes used to trap within complex cellular extracts proteins that are then separated and identified by proteomics, (iii) identification of chromatin- bound proteins after a genotoxic stress, or (iv) identification of proteins associated with other proteins already known to be part of DNA damage interactome. All these approaches have already been performed to find new proteins recognizing oxidised bases, abasic sites, strand breaks or crosslinks generated by anticancer drugs such as nitrogen mustards and platinating agents. Identified interactions are generally confirmed using complementary methods such as electromobility shift assays or surface plasmon resonance. These strategies allowed, for example, demonstration of interactions between cisplatin-DNA crosslinks and PARP-1 or the protein complex PTW/PP. The next challenging step will be to understand the biological repercussions of these newly identified interactions which may help to unravel new mechanisms involved in genetic toxicology, discover new cellular responses to anticancer drugs or identify new biomarkers and therapeutic targets.
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Affiliation(s)
| | - Ewa Barbier
- Laboratoire Lésions des Acides Nucléiques, SCIB, UMR-E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble, F-38054, France
| | - Sylvie Sauvaigo
- Laboratoire Lésions des Acides Nucléiques, SCIB, UMR-E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble, F-38054, France
| | - Didier Gasparutto
- Laboratoire Lésions des Acides Nucléiques, SCIB, UMR-E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble, F-38054, France
| | - Jean Breton
- Laboratoire Lésions des Acides Nucléiques, SCIB, UMR-E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble, F-38054, France; UFR de Pharmacie, Université Joseph Fourier-Grenoble 1, Domaine de la Merci, La Tronche, F-38706, France.
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Benedict AL, Knatko EV, Dinkova-Kostova AT. The indirect antioxidant sulforaphane protects against thiopurine-mediated photooxidative stress. Carcinogenesis 2012; 33:2457-66. [PMID: 22983983 PMCID: PMC3510740 DOI: 10.1093/carcin/bgs293] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2012] [Revised: 08/16/2012] [Accepted: 09/05/2012] [Indexed: 01/04/2023] Open
Abstract
Long-term treatment with thiopurines, such as the widely used anticancer, immunosuppressive and anti-inflammatory agent azathioprine, combined with exposure to ultraviolet (UV) radiation is associated with increased oxidative stress, hyperphotosensitivity and high risk for development of aggressive squamous cell carcinomas of the skin. Sulforaphane, an isothiocyanate derived from broccoli, is a potent inducer of endogenous cellular defenses regulated by transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), including cytoprotective enzymes and glutathione, which in turn act as efficient indirect and direct antioxidants that have long-lasting effects. Treatment with 6-thioguanine, a surrogate for azathioprine, leads to profound sensitization to oxidative stress and glutathione depletion upon exposure to UVA radiation, the damaging effects of which are primarily mediated by generation of reactive oxygen species. The degree of sensitization is greater for irradiation exposures spanning the absorption spectrum of 6-thioguanine, and is dependent on the length of treatment and the level of guanine substitution with 6-thioguanine, suggesting that the 6-thioguanine that is incorporated in genomic DNA is largely responsible for this sensitization. Sulforaphane provides protection against UVA, but not UVB, radiation without affecting the levels of 6-thioguanine incorporation into DNA. The protective effect is lost under conditions of Nrf2 deficiency, implying that it is due to induction of Nrf2-dependent cytoprotective proteins, and that this strategy could provide protection against any potentially photosensitizing drugs that generate electrophilic or reactive oxygen species. Thus, our findings support the development of Nrf2 activators as protectors against drug-mediated photooxidative stress and encourage future clinical trials in populations at high risk for cutaneous photodamage and photocarcinogenesis.
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Affiliation(s)
- Andrea L. Benedict
- Lewis B. and Dorothy Cullman Cancer Chemoprotection Center, Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA and
| | - Elena V. Knatko
- Jacqui Wood Cancer Centre, Division of Cancer Research, Medical Research Institute, University of Dundee, Dundee DD1 9SY, Scotland, UK
| | - Albena T. Dinkova-Kostova
- Lewis B. and Dorothy Cullman Cancer Chemoprotection Center, Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA and
- Jacqui Wood Cancer Centre, Division of Cancer Research, Medical Research Institute, University of Dundee, Dundee DD1 9SY, Scotland, UK
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28
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You C, Dai X, Yuan B, Wang Y. Effects of 6-thioguanine and S6-methylthioguanine on transcription in vitro and in human cells. J Biol Chem 2012; 287:40915-23. [PMID: 23076150 DOI: 10.1074/jbc.m112.418681] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Thiopurine drugs are extensively used as chemotherapeutic agents in clinical practice, even though there is concern about the risk of therapy-related cancers. It has been previously suggested that the cytotoxicity of thiopurine drugs involves their metabolic activation, the resultant generation of 6-thioguanine ((S)G) and S(6)-methylthioguanine (S(6)mG) in DNA, and the futile mismatch repair triggered by replication-induced (S)G:T and S(6)mG:T mispairs. Disruption of transcription is known to be one of the major consequences of DNA damage induced by many antiviral and antitumor agents; however, it remains undefined how (S)G and S(6)mG compromise the efficiency and fidelity of transcription. Using our recently developed competitive transcription and adduct bypass assay, herein we examined the impact of (S)G and S(6)mG on transcription in vitro and in human cells. Our results revealed that, when situated on the transcribed strand, S(6)mG exhibited both inhibitory and mutagenic effects during transcription mediated by single-subunit T7 RNA polymerase or multisubunit human RNA polymerase II in vitro and in human cells. Moreover, we found that the impact of S(6)mG on transcriptional efficiency and fidelity is modulated by the transcription-coupled nucleotide excision repair capacity. In contrast, (S)G did not considerably compromise the efficiency or fidelity of transcription, and it was a poor substrate for NER. We propose that S(6)mG might contribute, at least in part, to thiopurine-mediated cytotoxicity through inhibition of transcription and to potential therapy-related carcinogenesis via transcriptional mutagenesis.
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Affiliation(s)
- Changjun You
- Department of Chemistry, University of California, Riverside, California 92521-0403, USA
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29
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Brem R, Karran P. Oxidation-mediated DNA cross-linking contributes to the toxicity of 6-thioguanine in human cells. Cancer Res 2012; 72:4787-95. [PMID: 22822082 DOI: 10.1158/0008-5472.can-12-1278] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
The thiopurines azathioprine and 6-mercaptopurine have been extensively prescribed as immunosuppressant and anticancer agents for several decades. A third member of the thiopurine family, 6-thioguanine (6-TG), has been used less widely. Although known to be partly dependent on DNA mismatch repair (MMR), the cytotoxicity of 6-TG remains incompletely understood. Here, we describe a novel MMR-independent pathway of 6-TG toxicity. Cell killing depended on two properties of 6-TG: its incorporation into DNA and its ability to act as a source of reactive oxygen species (ROS). ROS targeted DNA 6-TG to generate potentially lethal replication-arresting DNA lesions including interstrand cross-links. These triggered processing by the Fanconi anemia and homologous recombination DNA repair pathways. Allopurinol protected against 6-TG toxicity by acting as a ROS scavenger and preventing DNA damage. Together, our findings provide mechanistic evidence to support the proposed use of thiopurines to treat HR-defective tumors and for the coadministration of 6-TG and allopurinol as an immunomodulation strategy in inflammatory disorders.
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Affiliation(s)
- Reto Brem
- Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts, United Kingdom
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30
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Cadet J, Mouret S, Ravanat JL, Douki T. Photoinduced damage to cellular DNA: direct and photosensitized reactions. Photochem Photobiol 2012; 88:1048-65. [PMID: 22780837 DOI: 10.1111/j.1751-1097.2012.01200.x] [Citation(s) in RCA: 215] [Impact Index Per Article: 16.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
The survey focuses on recent aspects of photochemical reactions to cellular DNA that are implicated through the predominant formation of mostly bipyrimidine photoproducts in deleterious effects of human exposure to sunlight. Recent developments in analytical methods have allowed accurate and quantitative measurements of the main DNA photoproducts in cells and human skin. Highly mutagenic CC and CT bipyrimidine photoproducts, including cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are generated in low yields with respect to TT and TC photoproducts. Another striking finding deals with the formation of Dewar valence isomers, the third class of bipyrimidine photoproducts that is accounted for by UVA-mediated isomerization of initially UVB generated 6-4PPs. Cyclobutadithymine (T<>T) has been unambiguously shown to be involved in the genotoxicity of UVA radiation. Thus, T<>T is formed in UVA-irradiated cellular DNA according to a direct excitation mechanism with a higher efficiency than oxidatively generated DNA damage that arises mostly through the Type II photosensitization mechanism. C<>C and C<>T are repaired at rates intermediate between those of T<>T and 6-4TT. Evidence has been also provided for the occurrence of photosensitized reactions mediated by exogenous agents that act either in an independent way or through photodynamic effects.
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Affiliation(s)
- Jean Cadet
- Laboratoire Lésions des Acides Nucléiques, SCIB-UMR-E n°3, CEA/UJF, Institut Nanosciences et Cryogénie, CEA/Grenoble, Grenoble Cedex, France
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31
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Casorelli I, Bossa C, Bignami M. DNA damage and repair in human cancer: molecular mechanisms and contribution to therapy-related leukemias. INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH 2012; 9:2636-57. [PMID: 23066388 PMCID: PMC3447578 DOI: 10.3390/ijerph9082636] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/23/2012] [Revised: 06/12/2012] [Accepted: 07/02/2012] [Indexed: 12/12/2022]
Abstract
Most antitumour therapies damage tumour cell DNA either directly or indirectly. Without repair, damage can result in genetic instability and eventually cancer. The strong association between the lack of DNA damage repair, mutations and cancer is dramatically demonstrated by a number of cancer-prone human syndromes, such as xeroderma pigmentosum, ataxia-telangiectasia and Fanconi anemia. Notably, DNA damage responses, and particularly DNA repair, influence the outcome of therapy. Because DNA repair normally excises lethal DNA lesions, it is intuitive that efficient repair will contribute to intrinsic drug resistance. Unexpectedly, a paradoxical relationship between DNA mismatch repair and drug sensitivity has been revealed by model studies in cell lines. This suggests that connections between DNA repair mechanism efficiency and tumour therapy might be more complex. Here, we review the evidence for the contribution of carcinogenic properties of several drugs as well as of alterations in specific mechanisms involved in drug-induced DNA damage response and repair in the pathogenesis of therapy-related cancers.
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Affiliation(s)
- Ida Casorelli
- Azienda Ospedaliera Sant’Andrea, Via di Grottarossa 1035-1039, Roma 00189, Italy;
| | - Cecilia Bossa
- Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, Roma 00161, Italy;
| | - Margherita Bignami
- Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, Roma 00161, Italy;
- Author to whom correspondence should be addressed; ; Tel.: +39-6-49901-2355; Fax: +39-6-49901-3650
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32
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Kalra S, Knatko EV, Zhang Y, Honda T, Yamamoto M, Dinkova-Kostova AT. Highly potent activation of Nrf2 by topical tricyclic bis(cyano enone): implications for protection against UV radiation during thiopurine therapy. Cancer Prev Res (Phila) 2012; 5:973-81. [PMID: 22659146 DOI: 10.1158/1940-6207.capr-12-0041] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Chronic treatment with azathioprine, a highly effective anti-inflammatory and immunosuppressive agent, profoundly increases the risk for development of unusually aggressive cutaneous squamous cell carcinoma. Its ultimate metabolite, 6-thioguanine (6-TG) nucleotide, is incorporated in DNA of skin cells, and upon exposure to UVA radiation, causes oxidative stress, followed by damage of DNA and associated proteins. The acetylenic tricyclic bis(cyano enone) TBE-31 is a strong inhibitor of inflammation and a potent inducer of the Keap1/Nrf2/ARE pathway, which orchestrates the expression of a large network of cytoprotective genes. We now report that long-term (five days per week for four weeks) topical daily applications of small (200 nmol) quantities of TBE-31 cause a robust systemic induction of the Keap1/Nrf2/ARE pathway and decreases the 6-TG incorporation in DNA of skin, blood, and liver of azathioprine-treated mice, indicating extraordinary bioavailability and efficacy. In addition, TBE-31, at nanomolar concentrations, protects cells with 6-TG in their genomic DNA against oxidative stress caused by UVA radiation through induction of the Keap1/Nrf2/ARE pathway. At the same 6-TG DNA levels, Keap1-knockout cells, in which the pathway is constitutively upregulated, are highly resistant to UVA radiation-induced oxidative stress. The protective effects of both the Keap1-knockout genotype and TBE-31 are completely lost in the absence of transcription factor Nrf2. Our findings suggest that compounds of this kind are excellent candidates for mechanism-based chemoprotective agents against conditions in which oxidative stress and inflammation underlie disease pathogenesis. Moreover, their potential skin patch incorporation for transdermal delivery is an exciting possibility.
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Affiliation(s)
- Sukirti Kalra
- Division of Cancer Research, Medical Research Institute, University of Dundee, Dundee, Scotland, UK
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33
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Oxidatively generated complex DNA damage: tandem and clustered lesions. Cancer Lett 2012; 327:5-15. [PMID: 22542631 DOI: 10.1016/j.canlet.2012.04.005] [Citation(s) in RCA: 175] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2012] [Revised: 04/02/2012] [Accepted: 04/15/2012] [Indexed: 11/22/2022]
Abstract
There is an increasing interest for oxidatively generated complex lesions that are potentially more detrimental than single oxidized nucleobases. In this survey, the recently available information on the formation and processing of several classes of complex DNA damage formed upon one radical hit including mostly hydroxyl radical and one-electron oxidants is critically reviewed. The modifications include tandem base lesions, DNA-protein cross-links and intrastrand (purine 5',8-cyclonucleosides, adjacent base cross-links) and interstrand cross-links. Information is also provided on clustered lesions produced essentially by exposure of cells to ionizing radiation and high energetic heavy ions through the involvement of multiple radical events that induce several lesions DNA in a close spatial vicinity. These consist mainly of double strand breaks (DSBs) and non-DSB clustered lesions that are referred as to oxidatively generated clustered DNA lesions (OCDLs).
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34
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Cadet J, Loft S, Olinski R, Evans MD, Bialkowski K, Richard Wagner J, Dedon PC, Møller P, Greenberg MM, Cooke MS. Biologically relevant oxidants and terminology, classification and nomenclature of oxidatively generated damage to nucleobases and 2-deoxyribose in nucleic acids. Free Radic Res 2012; 46:367-81. [PMID: 22263561 DOI: 10.3109/10715762.2012.659248] [Citation(s) in RCA: 100] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
A broad scientific community is involved in investigations aimed at delineating the mechanisms of formation and cellular processing of oxidatively generated damage to nucleic acids. Perhaps as a consequence of this breadth of research expertise, there are nomenclature problems for several of the oxidized bases including 8-oxo-7,8-dihydroguanine (8-oxoGua), a ubiquitous marker of almost every type of oxidative stress in cells. Efforts to standardize the nomenclature and abbreviations of the main DNA degradation products that arise from oxidative pathways are reported. Information is also provided on the main oxidative radicals, non-radical oxygen species, one-electron agents and enzymes involved in DNA degradation pathways as well in their targets and reactivity. A brief classification of oxidatively generated damage to DNA that may involve single modifications, tandem base modifications, intrastrand and interstrand cross-links together with DNA-protein cross-links and base adducts arising from the addition of lipid peroxides breakdown products is also included.
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Affiliation(s)
- Jean Cadet
- Direction des Sciences de Matière, Institut Nanosciences et Cryogénie, CEA/Grenoble, Grenoble Cedex 9, France.
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35
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Abstract
Thiopurines are prescribed frequently as medication for cancer and for inflammatory disorders. One of them, azathioprine, has been the immunosuppressant of choice for organ transplant recipients for many years. Thiopurine use is associated with elevated sun sensitivity and skin cancer risk. Skin sensitization is selective for UVA. 6-TG integrates into DNA and unlike the canonical DNA bases, it is a strong UVA chromophore with an absorbance maximum at 342 nm. DNA 6-TG is a photosensitizer and a source of reactive oxygen species. Reactive oxygen that is generated from the photochemical activation of DNA 6-TG causes extensive damage to DNA and proteins. This damage is mutagenic and extremely toxic to cultured human cells. Here we describe some of the lesions that are known to be generated from UVA irradiation of DNA 6-TG. We discuss how this photochemical damage might contribute to the toxic effect of thiopurine/UVA treatment on cultured cells and to the high risk of skin cancer in thiopurine-treated patients.
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Affiliation(s)
- Reto Brem
- Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts, UK.
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36
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Attard NR, Karran P. UVA photosensitization of thiopurines and skin cancer in organ transplant recipients. Photochem Photobiol Sci 2012; 11:62-8. [PMID: 21860872 DOI: 10.1039/c1pp05194f] [Citation(s) in RCA: 68] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2011] [Accepted: 07/21/2011] [Indexed: 01/31/2023]
Abstract
The thiopurines azathioprine, 6-mercaptopurine and 6-thioguanine (6-TG) are important medications for cancer and inflammatory disorders. They are also widely prescribed as immunosuppressants in organ transplant patients. Their metabolism results in the incorporation of 6-TG into patients' DNA, and this increases skin sensitivity to incident UVA. Unlike the canonical DNA bases, which do not absorb UVA to a significant degree, DNA 6-TG is a strong UVA chromophore. It acts as a Type II UVA photosensitizer, and the combination of 6-TG and UVA treatment induces a synergistic toxicity in cultured human cells. Here, we review some of the damage that this interaction causes. Photochemical activation of DNA 6-TG triggers DNA and protein oxidation; it induces DNA breakage, DNA crosslinking, oxidation of DNA bases and the covalent attachment of proteins to DNA. Many of these photochemical DNA lesions are difficult for cells to deal with, and we review the evidence linking thiopurine immunosuppression with genome instability and the high incidence of skin cancer in organ transplant recipients.
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Affiliation(s)
- Natalie R Attard
- Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK
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37
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Zhang J, Bowden GT. Activation of p38 MAP kinase and JNK pathways by UVA irradiation. Photochem Photobiol Sci 2011; 11:54-61. [PMID: 21858326 DOI: 10.1039/c1pp05133d] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
There are more than two million new cases of non-melanoma skin cancers (NMSCs) diagnosed each year in the United States of America. The clear etiological factor is chronic exposure to solar radiation from the sun. The wavelengths of solar light that reach the earth's surface include UVB (280-320 nm), which accounts for 1-10%, and UVA (320-400 nm), which accounts for 90-99% of the radiation. While most published research has focused on the effects of UVB, little is known concerning UVA-mediated signal transduction pathways, and their role in skin tumor promotion and progression, giving rise to squamous cell carcinomas (SCCs). Here, we focus on UVA-mediated activation of p38 MAP kinase and c-Jun N-terminal kinase (JNK), and their roles in activator protein-1 (AP-1) mediated transcription, cyclooxygenase-2 (COX-2) and Bcl-XL expression. Since p38 MAP kinase and JNK play major roles in the expression of UVA-induced AP-1, COX-2 and Bcl-XL, pharmacological inhibitors of these kinases may be useful in the chemoprevention of SCC skin cancer.
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Affiliation(s)
- Jack Zhang
- Department of Medical Pharmacology, University of Arizona, Tucson, Arizona, USA
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38
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Brem R, Daehn I, Karran P. Efficient DNA interstrand crosslinking by 6-thioguanine and UVA radiation. DNA Repair (Amst) 2011; 10:869-76. [PMID: 21723207 DOI: 10.1016/j.dnarep.2011.05.010] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2011] [Revised: 05/24/2011] [Accepted: 05/31/2011] [Indexed: 10/18/2022]
Abstract
Patients taking the immunosuppressant and anticancer thiopurines 6-mercaptopurine, azathioprine or 6-thioguanine (6-TG), develop skin cancer at a very high frequency. Their DNA contains 6-TG which absorbs ultraviolet A (UVA) radiation, and their skin is UVA hypersensitive, consistent with the formation of DNA photodamage. Here we demonstrate that UVA irradiation of 6-TG-containing DNA causes DNA interstrand crosslinking. In synthetic duplex oligodeoxynucleotides, the interstrand crosslinks (ICLs) can form between closely opposed 6-TG bases and, in a less favoured reaction, between 6-TG and normal bases on the opposite strand. In vivo, UVA irradiation of cultured cells containing 6-TG-substituted DNA also causes ICL formation and induces the chromosome aberrations that are characteristically associated with this type of DNA lesion. 6-TG/UVA activates the Fanconi anemia (FA) pathway via monoubiquitination of the FANCD2 protein. Cells defective in the FA pathway or other factors involved in ICL processing, such as XPF and DNA Polζ, are all hypersensitive to killing by 6-TG/UVA-consistent with a significant contribution of photochemical ICLs to the cytotoxicity of this treatment. Our findings suggest that sunlight-exposed skin of thiopurine treated patients may experience chronic photochemical DNA damage that requires constant intervention of the FA pathway.
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Affiliation(s)
- Reto Brem
- Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts EN6 3LD, UK
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