title: Wilms' tumor gene WT1 promotes homologous recombination-mediated DNA damage repair.
abstract: The Wilms' tumor gene WT1 is overexpressed in leukemia and various types of solid tumors and plays an oncogenic role in these malignancies. Alternative splicing at two sites yields four major isoforms, 17AA(+)KTS(+), 17AA(+)KTS(-), 17AA(-)KTS(+), and 17AA(-)KTS(-), and all the isoforms are expressed in the malignancies. However, among the four isoforms, function of WT1[17AA(-)KTS(+)] isoform still remains undetermined. In the present study, we showed that forced expression of WT1[17AA(-)KTS(+)] isoform significantly inhibited apoptosis by DNA-damaging agents such as Doxorubicin, Mitomycin, Camptothesisn, and Bleomycin in immortalized fibroblast MRC5SV and cervical cancer HeLa cells. Knockdown of Rad51, an essential factor for homologous recombination (HR)-mediated DNA repair canceled the resistance to Doxorubicin induced by WT1[17AA(-)KTS(+)] isoform. GFP recombination assay showed that WT1[17AA(-)KTS(+)] isoform alone promoted HR, but that three other WT1 isoforms did not. WT1[17AA(-)KTS(+)] isoform significantly upregulated the expression of HR genes, XRCC2, Rad51D, and Rad54. Knockdown of XRCC2, Rad51D, and Rad54 inhibited the HR activity and canceled resistance to Doxorubicin in MRC5SV cells with forced expression of WT1[17AA(-)KTS(+)] isoform. Furthermore, chromatin immunoprecipitation (ChIP) assay showed the binding of WT1[17AA(-)KTS(+)] isoform protein to promoters of XRCC2 and Rad51D. Immunohistochemical study showed that Rad54 and XRCC2 proteins were highly expressed in the majority of non-small-cell lung cancer (NSCLC) and gastric cancer, and that expression of these two proteins was significantly correlated with that of WT1 protein in NSCLCs. Our results presented here showed that WT1[17AA(-)KTS(+)] isoform had a function to promote HR-mediated DNA repair.
authors.affiliation: Department of Cancer Stem Cell Biology, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Oji
authors.affiliation: Department of Cancer Stem Cell Biology, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Naoya
authors.initials: N
authors.lastname: Tatsumi
authors.affiliation: Radiation Biology Center, Kyoto University, Kyoto, Japan.
authors.forename: Junya
authors.initials: J
authors.lastname: Kobayashi
authors.affiliation: Department of Functional Diagnostic Science, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Mari
authors.initials: M
authors.lastname: Fukuda
authors.affiliation: Department of Functional Diagnostic Science, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Tazu
authors.initials: T
authors.lastname: Ueda
authors.affiliation: Department of Functional Diagnostic Science, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Nakano
authors.affiliation: Department of Functional Diagnostic Science, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Chisae
authors.initials: C
authors.lastname: Saito
authors.affiliation: Department of Functional Diagnostic Science, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Syohei
authors.initials: S
authors.lastname: Shibata
authors.affiliation: Department of Functional Diagnostic Science, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Mihoko
authors.initials: M
authors.lastname: Sumikawa
authors.affiliation: Department of Functional Diagnostic Science, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Fukushima
authors.affiliation: Department of Functional Diagnostic Science, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Akari
authors.initials: A
authors.lastname: Saito
authors.affiliation: Department of Functional Diagnostic Science, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Nozomi
authors.initials: N
authors.lastname: Hojo
authors.affiliation: Department of Functional Diagnostic Science, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Miyu
authors.initials: M
authors.lastname: Suzuki
authors.affiliation: Department of Functional Diagnostic Science, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Hoshikawa
authors.affiliation: Department of Environmental Health, National Institute of Public Health, Saitama, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Shimura
authors.affiliation: Department of Pathology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
authors.forename: Eiichi
authors.initials: E
authors.lastname: Morii
authors.affiliation: Department of Cancer Immunology, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Oka
authors.affiliation: Department of Cancer Stem Cell Biology, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Hosen
authors.affiliation: Radiation Biology Center, Kyoto University, Kyoto, Japan.
authors.forename: Kenshi
authors.initials: K
authors.lastname: Komatsu
authors.affiliation: Department of Functional Diagnostic Science, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Haruo
authors.initials: H
authors.lastname: Sugiyama
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D017209:Apoptosis / Q000235:genetics; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics; D004249:DNA Damage / Q000235:genetics*; D004265:DNA Helicases / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D016162:Genes, Wilms Tumor / Q000502:physiology*; D006367:HeLa Cells; D059765:Homologous Recombination / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics; D013274:Stomach Neoplasms / Q000235:genetics; D025721:WT1 Proteins / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; C112629:RAD51D protein, human; D025721:WT1 Proteins; C583751:WT1 protein, human; C113187:XRCC2 protein, human; D004265:DNA Helicases; C103532:RAD54L protein, human
keywords: DNA repair; HR factors; WT1; chemoresistance; homologous recombination