Methotrexate-mediated inhibition of RAD51 expression and homologous recombination in cancer cells
Background Methotrexate is an inhibitor of folic acid metabolism. Homologous recombination is one of the most important ways to repair double-stranded breaks in DNA and influence the radio- and chemosensitivity of tumor cells. But the relationship between methotrexate and homologous recombination re...
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| Veröffentlicht in: | Journal of cancer research and clinical oncology 2012-05, Vol.138 (5), p.811-818 |
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| container_title | Journal of cancer research and clinical oncology |
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| creator | Du, Li-Qing Du, Xiao-Qing Bai, Jian-Qiang Wang, Yan Yang, Qing-Shan Wang, Xiao-Chun Zhao, Peng Wang, Hong Liu, Qiang Fan, Fei-Yue |
| description | Background
Methotrexate is an inhibitor of folic acid metabolism. Homologous recombination is one of the most important ways to repair double-stranded breaks in DNA and influence the radio- and chemosensitivity of tumor cells. But the relationship between methotrexate and homologous recombination repair has not been elucidated.
Methods
Induction of double-strand breaks by methotrexate in HOS cells is assessed by the neutral comet assay. Inhibition of subnuclear repair foci by methotrexate is measured by immunofluorescence. Western blot and quantitative real-time PCR are conducted to detect whether methotrexate affects the expression level of genes involved in homologous recombination. In addition, we used a pCMV3xnls-I-SceI construct to determine whether methotrexate directly inhibits the process of homologous recombinational repair in cells, and the sensitivity to methotrexate in the Ku80-deficient cells is detected using clonogenic survival assays.
Results
The result showed that methotrexate can regulate the repair of DNA double-strand breaks after radiation exposure, and methotrexate inhibition caused the complete inhibition of subnuclear repair foci in response to ionizing radiation. Mechanistic investigation revealed that methotrexate led to a significant reduction in the transcription of RAD51 genes. Treatment with methotrexate resulted in a decreased ability to perform homology-directed repair of I-SceI-induced chromosome breaks. In addition, enhancement of cell death was observed in Ku mutant cells compared to wild-type cells.
Conclusions
These results demonstrate that methotrexate can affect homologous recombination repair of DNA double-strand breaks by controlling the expression of homologous recombination-related genes and suppressing the proper assembly of homologous recombination–directed subnuclear foci. |
| doi_str_mv | 10.1007/s00432-011-1132-8 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_1285085141</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>2660265301</sourcerecordid><originalsourceid>FETCH-LOGICAL-c435t-beee5b1b0bf1add6d3e87dd438d53d35749f3ba661f8633ee976db4e4fbdd1923</originalsourceid><addsrcrecordid>eNp1kE1r3DAQhkVpSbZJfkAvxVAKvTjV6MOyjyHpRyClUJKzkKxxVsGWtpIXkn8fubtpSyAnSaNnRq8eQt4BPQVK1edMqeCspgA1QNm0r8gKlgpwLl-TFQUFtWTQHJK3Od_RcpaKHZBDxpgSbSNXxPzAeR3nhPdmxnpC58vqKh_W3vrZx1DFofp1diGhwvtNwpyXmgmuWscpjvE2bnOVsI-T9cH8afCh6k3oMVU9jmM-Jm8GM2Y82a9H5Obrl-vz7_XVz2-X52dXdS-4nGuLiNKCpXYA41zjOLbKOcFbJ7njUolu4NY0DQxtwzlipxpnBYrBOgcd40fk027uJsXfW8yznnxeEpiAJaQG1kraShBQ0A_P0Lu4TaGk00CBqaYTihcKdlSfYs4JB71JfjLpoUB68a93_nXxrxf_ui097_eTt7bI_NvxJLwAH_eAyb0Zh1RM-fyPk6qTnVx-w3ZcLlfhFtP_EV96_RHXeJ3C</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>1012769473</pqid></control><display><type>article</type><title>Methotrexate-mediated inhibition of RAD51 expression and homologous recombination in cancer cells</title><source>MEDLINE</source><source>SpringerLink Journals - AutoHoldings</source><creator>Du, Li-Qing ; Du, Xiao-Qing ; Bai, Jian-Qiang ; Wang, Yan ; Yang, Qing-Shan ; Wang, Xiao-Chun ; Zhao, Peng ; Wang, Hong ; Liu, Qiang ; Fan, Fei-Yue</creator><creatorcontrib>Du, Li-Qing ; Du, Xiao-Qing ; Bai, Jian-Qiang ; Wang, Yan ; Yang, Qing-Shan ; Wang, Xiao-Chun ; Zhao, Peng ; Wang, Hong ; Liu, Qiang ; Fan, Fei-Yue</creatorcontrib><description>Background
Methotrexate is an inhibitor of folic acid metabolism. Homologous recombination is one of the most important ways to repair double-stranded breaks in DNA and influence the radio- and chemosensitivity of tumor cells. But the relationship between methotrexate and homologous recombination repair has not been elucidated.
Methods
Induction of double-strand breaks by methotrexate in HOS cells is assessed by the neutral comet assay. Inhibition of subnuclear repair foci by methotrexate is measured by immunofluorescence. Western blot and quantitative real-time PCR are conducted to detect whether methotrexate affects the expression level of genes involved in homologous recombination. In addition, we used a pCMV3xnls-I-SceI construct to determine whether methotrexate directly inhibits the process of homologous recombinational repair in cells, and the sensitivity to methotrexate in the Ku80-deficient cells is detected using clonogenic survival assays.
Results
The result showed that methotrexate can regulate the repair of DNA double-strand breaks after radiation exposure, and methotrexate inhibition caused the complete inhibition of subnuclear repair foci in response to ionizing radiation. Mechanistic investigation revealed that methotrexate led to a significant reduction in the transcription of RAD51 genes. Treatment with methotrexate resulted in a decreased ability to perform homology-directed repair of I-SceI-induced chromosome breaks. In addition, enhancement of cell death was observed in Ku mutant cells compared to wild-type cells.
Conclusions
These results demonstrate that methotrexate can affect homologous recombination repair of DNA double-strand breaks by controlling the expression of homologous recombination-related genes and suppressing the proper assembly of homologous recombination–directed subnuclear foci.</description><identifier>ISSN: 0171-5216</identifier><identifier>EISSN: 1432-1335</identifier><identifier>DOI: 10.1007/s00432-011-1132-8</identifier><identifier>PMID: 22274865</identifier><identifier>CODEN: JCROD7</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer-Verlag</publisher><subject>Antimetabolites, Antineoplastic - pharmacology ; Antineoplastic agents ; Biological and medical sciences ; Cancer ; Cancer Research ; Cell death ; Cell Line, Tumor ; Cell survival ; Chromosomes ; Comet assay ; DNA damage ; DNA End-Joining Repair - drug effects ; DNA End-Joining Repair - genetics ; DNA repair ; Double-strand break repair ; Down-Regulation - drug effects ; Down-Regulation - genetics ; Folic acid ; Gene expression ; Gene Expression Regulation, Neoplastic - drug effects ; Genes, BRCA2 - drug effects ; Hematology ; homologous recombination ; Homologous Recombination - drug effects ; Homologous Recombination - genetics ; Humans ; Immunofluorescence ; Internal Medicine ; Ionizing radiation ; Medical sciences ; Medicine ; Medicine & Public Health ; Metabolism ; Methotrexate ; Methotrexate - pharmacology ; Neoplasms - genetics ; Neoplasms - pathology ; Oncology ; Original Article ; Pharmacology. Drug treatments ; Polymerase chain reaction ; Rad51 Recombinase - antagonists & inhibitors ; Rad51 Recombinase - genetics ; Rad51 Recombinase - metabolism ; Rad52 DNA Repair and Recombination Protein - genetics ; Recombinational DNA Repair - drug effects ; Recombinational DNA Repair - genetics ; RNA, Small Interfering - pharmacology ; Transcription ; Tumor cells ; Western blotting</subject><ispartof>Journal of cancer research and clinical oncology, 2012-05, Vol.138 (5), p.811-818</ispartof><rights>Springer-Verlag 2012</rights><rights>2015 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c435t-beee5b1b0bf1add6d3e87dd438d53d35749f3ba661f8633ee976db4e4fbdd1923</citedby><cites>FETCH-LOGICAL-c435t-beee5b1b0bf1add6d3e87dd438d53d35749f3ba661f8633ee976db4e4fbdd1923</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00432-011-1132-8$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00432-011-1132-8$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27924,27925,41488,42557,51319</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=25795952$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22274865$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Du, Li-Qing</creatorcontrib><creatorcontrib>Du, Xiao-Qing</creatorcontrib><creatorcontrib>Bai, Jian-Qiang</creatorcontrib><creatorcontrib>Wang, Yan</creatorcontrib><creatorcontrib>Yang, Qing-Shan</creatorcontrib><creatorcontrib>Wang, Xiao-Chun</creatorcontrib><creatorcontrib>Zhao, Peng</creatorcontrib><creatorcontrib>Wang, Hong</creatorcontrib><creatorcontrib>Liu, Qiang</creatorcontrib><creatorcontrib>Fan, Fei-Yue</creatorcontrib><title>Methotrexate-mediated inhibition of RAD51 expression and homologous recombination in cancer cells</title><title>Journal of cancer research and clinical oncology</title><addtitle>J Cancer Res Clin Oncol</addtitle><addtitle>J Cancer Res Clin Oncol</addtitle><description>Background
Methotrexate is an inhibitor of folic acid metabolism. Homologous recombination is one of the most important ways to repair double-stranded breaks in DNA and influence the radio- and chemosensitivity of tumor cells. But the relationship between methotrexate and homologous recombination repair has not been elucidated.
Methods
Induction of double-strand breaks by methotrexate in HOS cells is assessed by the neutral comet assay. Inhibition of subnuclear repair foci by methotrexate is measured by immunofluorescence. Western blot and quantitative real-time PCR are conducted to detect whether methotrexate affects the expression level of genes involved in homologous recombination. In addition, we used a pCMV3xnls-I-SceI construct to determine whether methotrexate directly inhibits the process of homologous recombinational repair in cells, and the sensitivity to methotrexate in the Ku80-deficient cells is detected using clonogenic survival assays.
Results
The result showed that methotrexate can regulate the repair of DNA double-strand breaks after radiation exposure, and methotrexate inhibition caused the complete inhibition of subnuclear repair foci in response to ionizing radiation. Mechanistic investigation revealed that methotrexate led to a significant reduction in the transcription of RAD51 genes. Treatment with methotrexate resulted in a decreased ability to perform homology-directed repair of I-SceI-induced chromosome breaks. In addition, enhancement of cell death was observed in Ku mutant cells compared to wild-type cells.
Conclusions
These results demonstrate that methotrexate can affect homologous recombination repair of DNA double-strand breaks by controlling the expression of homologous recombination-related genes and suppressing the proper assembly of homologous recombination–directed subnuclear foci.</description><subject>Antimetabolites, Antineoplastic - pharmacology</subject><subject>Antineoplastic agents</subject><subject>Biological and medical sciences</subject><subject>Cancer</subject><subject>Cancer Research</subject><subject>Cell death</subject><subject>Cell Line, Tumor</subject><subject>Cell survival</subject><subject>Chromosomes</subject><subject>Comet assay</subject><subject>DNA damage</subject><subject>DNA End-Joining Repair - drug effects</subject><subject>DNA End-Joining Repair - genetics</subject><subject>DNA repair</subject><subject>Double-strand break repair</subject><subject>Down-Regulation - drug effects</subject><subject>Down-Regulation - genetics</subject><subject>Folic acid</subject><subject>Gene expression</subject><subject>Gene Expression Regulation, Neoplastic - drug effects</subject><subject>Genes, BRCA2 - drug effects</subject><subject>Hematology</subject><subject>homologous recombination</subject><subject>Homologous Recombination - drug effects</subject><subject>Homologous Recombination - genetics</subject><subject>Humans</subject><subject>Immunofluorescence</subject><subject>Internal Medicine</subject><subject>Ionizing radiation</subject><subject>Medical sciences</subject><subject>Medicine</subject><subject>Medicine & Public Health</subject><subject>Metabolism</subject><subject>Methotrexate</subject><subject>Methotrexate - pharmacology</subject><subject>Neoplasms - genetics</subject><subject>Neoplasms - pathology</subject><subject>Oncology</subject><subject>Original Article</subject><subject>Pharmacology. Drug treatments</subject><subject>Polymerase chain reaction</subject><subject>Rad51 Recombinase - antagonists & inhibitors</subject><subject>Rad51 Recombinase - genetics</subject><subject>Rad51 Recombinase - metabolism</subject><subject>Rad52 DNA Repair and Recombination Protein - genetics</subject><subject>Recombinational DNA Repair - drug effects</subject><subject>Recombinational DNA Repair - genetics</subject><subject>RNA, Small Interfering - pharmacology</subject><subject>Transcription</subject><subject>Tumor cells</subject><subject>Western blotting</subject><issn>0171-5216</issn><issn>1432-1335</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>8G5</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNp1kE1r3DAQhkVpSbZJfkAvxVAKvTjV6MOyjyHpRyClUJKzkKxxVsGWtpIXkn8fubtpSyAnSaNnRq8eQt4BPQVK1edMqeCspgA1QNm0r8gKlgpwLl-TFQUFtWTQHJK3Od_RcpaKHZBDxpgSbSNXxPzAeR3nhPdmxnpC58vqKh_W3vrZx1DFofp1diGhwvtNwpyXmgmuWscpjvE2bnOVsI-T9cH8afCh6k3oMVU9jmM-Jm8GM2Y82a9H5Obrl-vz7_XVz2-X52dXdS-4nGuLiNKCpXYA41zjOLbKOcFbJ7njUolu4NY0DQxtwzlipxpnBYrBOgcd40fk027uJsXfW8yznnxeEpiAJaQG1kraShBQ0A_P0Lu4TaGk00CBqaYTihcKdlSfYs4JB71JfjLpoUB68a93_nXxrxf_ui097_eTt7bI_NvxJLwAH_eAyb0Zh1RM-fyPk6qTnVx-w3ZcLlfhFtP_EV96_RHXeJ3C</recordid><startdate>20120501</startdate><enddate>20120501</enddate><creator>Du, Li-Qing</creator><creator>Du, Xiao-Qing</creator><creator>Bai, Jian-Qiang</creator><creator>Wang, Yan</creator><creator>Yang, Qing-Shan</creator><creator>Wang, Xiao-Chun</creator><creator>Zhao, Peng</creator><creator>Wang, Hong</creator><creator>Liu, Qiang</creator><creator>Fan, Fei-Yue</creator><general>Springer-Verlag</general><general>Springer</general><general>Springer Nature B.V</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7TO</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>H94</scope><scope>K9.</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</scope><scope>MBDVC</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>Q9U</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope></search><sort><creationdate>20120501</creationdate><title>Methotrexate-mediated inhibition of RAD51 expression and homologous recombination in cancer cells</title><author>Du, Li-Qing ; Du, Xiao-Qing ; Bai, Jian-Qiang ; Wang, Yan ; Yang, Qing-Shan ; Wang, Xiao-Chun ; Zhao, Peng ; Wang, Hong ; Liu, Qiang ; Fan, Fei-Yue</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c435t-beee5b1b0bf1add6d3e87dd438d53d35749f3ba661f8633ee976db4e4fbdd1923</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Antimetabolites, Antineoplastic - pharmacology</topic><topic>Antineoplastic agents</topic><topic>Biological and medical sciences</topic><topic>Cancer</topic><topic>Cancer Research</topic><topic>Cell death</topic><topic>Cell Line, Tumor</topic><topic>Cell survival</topic><topic>Chromosomes</topic><topic>Comet assay</topic><topic>DNA damage</topic><topic>DNA End-Joining Repair - drug effects</topic><topic>DNA End-Joining Repair - genetics</topic><topic>DNA repair</topic><topic>Double-strand break repair</topic><topic>Down-Regulation - drug effects</topic><topic>Down-Regulation - genetics</topic><topic>Folic acid</topic><topic>Gene expression</topic><topic>Gene Expression Regulation, Neoplastic - drug effects</topic><topic>Genes, BRCA2 - drug effects</topic><topic>Hematology</topic><topic>homologous recombination</topic><topic>Homologous Recombination - drug effects</topic><topic>Homologous Recombination - genetics</topic><topic>Humans</topic><topic>Immunofluorescence</topic><topic>Internal Medicine</topic><topic>Ionizing radiation</topic><topic>Medical sciences</topic><topic>Medicine</topic><topic>Medicine & Public Health</topic><topic>Metabolism</topic><topic>Methotrexate</topic><topic>Methotrexate - pharmacology</topic><topic>Neoplasms - genetics</topic><topic>Neoplasms - pathology</topic><topic>Oncology</topic><topic>Original Article</topic><topic>Pharmacology. Drug treatments</topic><topic>Polymerase chain reaction</topic><topic>Rad51 Recombinase - antagonists & inhibitors</topic><topic>Rad51 Recombinase - genetics</topic><topic>Rad51 Recombinase - metabolism</topic><topic>Rad52 DNA Repair and Recombination Protein - genetics</topic><topic>Recombinational DNA Repair - drug effects</topic><topic>Recombinational DNA Repair - genetics</topic><topic>RNA, Small Interfering - pharmacology</topic><topic>Transcription</topic><topic>Tumor cells</topic><topic>Western blotting</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Du, Li-Qing</creatorcontrib><creatorcontrib>Du, Xiao-Qing</creatorcontrib><creatorcontrib>Bai, Jian-Qiang</creatorcontrib><creatorcontrib>Wang, Yan</creatorcontrib><creatorcontrib>Yang, Qing-Shan</creatorcontrib><creatorcontrib>Wang, Xiao-Chun</creatorcontrib><creatorcontrib>Zhao, Peng</creatorcontrib><creatorcontrib>Wang, Hong</creatorcontrib><creatorcontrib>Liu, Qiang</creatorcontrib><creatorcontrib>Fan, Fei-Yue</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Oncogenes and Growth Factors Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Public Health Database</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Research Library (Alumni Edition)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>ProQuest Central</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>Research Library Prep</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Research Library</collection><collection>Research Library (Corporate)</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>ProQuest Central Basic</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><jtitle>Journal of cancer research and clinical oncology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Du, Li-Qing</au><au>Du, Xiao-Qing</au><au>Bai, Jian-Qiang</au><au>Wang, Yan</au><au>Yang, Qing-Shan</au><au>Wang, Xiao-Chun</au><au>Zhao, Peng</au><au>Wang, Hong</au><au>Liu, Qiang</au><au>Fan, Fei-Yue</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Methotrexate-mediated inhibition of RAD51 expression and homologous recombination in cancer cells</atitle><jtitle>Journal of cancer research and clinical oncology</jtitle><stitle>J Cancer Res Clin Oncol</stitle><addtitle>J Cancer Res Clin Oncol</addtitle><date>2012-05-01</date><risdate>2012</risdate><volume>138</volume><issue>5</issue><spage>811</spage><epage>818</epage><pages>811-818</pages><issn>0171-5216</issn><eissn>1432-1335</eissn><coden>JCROD7</coden><abstract>Background
Methotrexate is an inhibitor of folic acid metabolism. Homologous recombination is one of the most important ways to repair double-stranded breaks in DNA and influence the radio- and chemosensitivity of tumor cells. But the relationship between methotrexate and homologous recombination repair has not been elucidated.
Methods
Induction of double-strand breaks by methotrexate in HOS cells is assessed by the neutral comet assay. Inhibition of subnuclear repair foci by methotrexate is measured by immunofluorescence. Western blot and quantitative real-time PCR are conducted to detect whether methotrexate affects the expression level of genes involved in homologous recombination. In addition, we used a pCMV3xnls-I-SceI construct to determine whether methotrexate directly inhibits the process of homologous recombinational repair in cells, and the sensitivity to methotrexate in the Ku80-deficient cells is detected using clonogenic survival assays.
Results
The result showed that methotrexate can regulate the repair of DNA double-strand breaks after radiation exposure, and methotrexate inhibition caused the complete inhibition of subnuclear repair foci in response to ionizing radiation. Mechanistic investigation revealed that methotrexate led to a significant reduction in the transcription of RAD51 genes. Treatment with methotrexate resulted in a decreased ability to perform homology-directed repair of I-SceI-induced chromosome breaks. In addition, enhancement of cell death was observed in Ku mutant cells compared to wild-type cells.
Conclusions
These results demonstrate that methotrexate can affect homologous recombination repair of DNA double-strand breaks by controlling the expression of homologous recombination-related genes and suppressing the proper assembly of homologous recombination–directed subnuclear foci.</abstract><cop>Berlin/Heidelberg</cop><pub>Springer-Verlag</pub><pmid>22274865</pmid><doi>10.1007/s00432-011-1132-8</doi><tpages>8</tpages></addata></record> |
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| issn | 0171-5216 1432-1335 |
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| subjects | Antimetabolites, Antineoplastic - pharmacology Antineoplastic agents Biological and medical sciences Cancer Cancer Research Cell death Cell Line, Tumor Cell survival Chromosomes Comet assay DNA damage DNA End-Joining Repair - drug effects DNA End-Joining Repair - genetics DNA repair Double-strand break repair Down-Regulation - drug effects Down-Regulation - genetics Folic acid Gene expression Gene Expression Regulation, Neoplastic - drug effects Genes, BRCA2 - drug effects Hematology homologous recombination Homologous Recombination - drug effects Homologous Recombination - genetics Humans Immunofluorescence Internal Medicine Ionizing radiation Medical sciences Medicine Medicine & Public Health Metabolism Methotrexate Methotrexate - pharmacology Neoplasms - genetics Neoplasms - pathology Oncology Original Article Pharmacology. Drug treatments Polymerase chain reaction Rad51 Recombinase - antagonists & inhibitors Rad51 Recombinase - genetics Rad51 Recombinase - metabolism Rad52 DNA Repair and Recombination Protein - genetics Recombinational DNA Repair - drug effects Recombinational DNA Repair - genetics RNA, Small Interfering - pharmacology Transcription Tumor cells Western blotting |
| title | Methotrexate-mediated inhibition of RAD51 expression and homologous recombination in cancer cells |
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