[CANCER RESEARCH 59, 342–346, January 15, 1999] Advances in Brief Transcription Factor Y-Box Binding Protein 1 Binds Preferentially to Cisplatin- modified DNA and Interacts with Proliferating Cell Nuclear Antigen 1 Tomoko Ise, Gunji Nagatani, Toshihiro Imamura, Ken Kato, Hiroshi Takano, Minoru Nomoto, Hiroto Izumi, Haruki Ohmori, Tatsuro Okamoto, Takefumi Ohga, Takeshi Uchiumi, Michihiko Kuwano, and Kimitoshi Kohno 2 Department of Molecular Biology, University of Occupational and Environmental Health, Japan School of Medicine, Kitakyushu Fukuoka 807-8555 [T. Is., G. N., T. Im., K. Ka., H. T., M. N., H. I., H. O., K. Ko.]; and Department of Biochemistry, Kyushu University School of Medicine, Fukuoka 812-8582 [T. Ok., T. Oh., T. U., M. K.], Japan Abstract The Y-box binding protein (YB-1) binds to inverted CCAAT box sequences that are present in the promoter region of many genes. We previously showed that YB-1 is overexpressed in human cancer cell lines that are resistant to cisplatin and that the depletion of YB-1 by transfec- tion of a vector expressing YB-1 antisense RNA increases the sensitivity of human cancer cells to cisplatin. To determine whether YB-1 can bind to cisplatin-modified DNA, we fused YB-1 cDNA to glutathione S-transferase (GST) cDNA and purified the resulting GST fusion protein. When we tested the fusion protein with unmodified or cisplatin-modified oligonu- cleotides, we found that GST-YB-1 bound more strongly to cisplatin- modified oligonucleotides, as did GST fusion proteins of high mobility group 1 (HMG1), HMG2, and xeroderma pigmentosum group A protein. When we assayed the ability of proliferating cell nuclear antigen (PCNA) to interact with the GST fusion proteins, we observed binding to YB-1 but not to HMG1, HMG2, or xeroderma pigmentosum group A. Subsequent experiments demonstrated that YB-1 and PCNA interact directly via the COOH-terminal region of YB-1. Using immunochemical coprecipitation methods, we observed binding of YB-1 and PCNA in vivo. These results suggest that YB-1 can function as a recognition protein for cisplatin- damaged DNA and that it may be important in DNA repair or in directing the cellular response to DNA damage. Introduction The protein YB-1, 3 which was first identified by its ability to bind to the Y-box (i.e., the inverted CCAAT box) of the MHC class II promoter (1), has been shown to regulate gene expression (2). Since then, several additional eukaryotic genes, including thymidine kinase, cyclin-dependent kinase 1, DNA topoisomerase II, and MDR1 genes, have been found to contain a Y-box in their promoter region (3). Interestingly, the family of Y-box binding proteins has been shown to contain a unique DNA-binding domain, the cold shock domain, which is highly conserved from prokaryote to eukaryote (2). In previous reports from our laboratory, we demonstrated that MDR1 promoter activity increases in response to various environ- mental stresses in a manner that is dependent on both the inverted CCAAT box and YB-1 (4). We have also shown that YB-1, which is expressed ubiquitously in human tissues, is overexpressed in human cancer cell lines that are resistant to cisplatin (5). In addition, we have found that transfection of cells with a mammalian expression vector expressing YB-1 antisense RNA leads to increased drug sensitivity to cisplatin (5). Taken together, these findings suggest that YB-1 may recognize regions of DNA modified by cisplatin and that it may participate in DNA repair processes. Cisplatin is a widely used anticancer agent; its therapeutic efficacy is believed to result from its interaction with DNA (6, 7). Cisplatin has been shown to cause the formation of intrastrand cross-links between adjacent purines in genomic DNA. The major cisplatin DNA cross- links are intrastrand 1, 2-d (GpG) and d (ApG), whereas the minor cross-links include intrastrand 1,3-d (GpNpG) (7). Because major cross-links are not formed by transplatin, the inactive isomer of cisplatin, attention has focused on the major cross-links and the cellular proteins that specifically recognize these cross-links (6). Cellular proteins that recognize these cross-links and that bind preferentially to cisplatin-modified DNA include HMG1, HMG2 (8), and XPA (9); several mechanisms have been suggested for their activity (10, 11). Both HMG1 and HMG2 are members of the abun- dant HMG of nonhistone chromosomal proteins, and both have been shown to contain two internal repeat HMG boxes that mediate DNA binding (12). Both are implicated in various cellular processes, including tran- scription and DNA repair (8, 12). XPA is a zinc finger DNA-binding protein that is altered in group A xeroderma pigmentosum cells, and it has been shown to be involved in the damage recognition steps of the NER processes (11, 13). Although our previous results have suggested indirectly that YB-1 is associated with cisplatin resistance, there has been no direct dem- onstration that YB-1 binds to cisplatin-modified DNA. Furthermore, although both HMG1 and HMG2 are known to function as class II transcription factors (8), the ability of a sequence-specific transcrip- tion factor to preferentially recognize cisplatin-modified DNA has not been shown. Here, we demonstrate that YB-1 binds to cisplatin- modified DNA and that it interacts with PCNA, an essential protein in DNA repair. Materials and Methods Antibodies. Antibodies to XPA, PCNA, GST, and TRX were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antiserum to YB-1 was generated as described previously (5). Antibody to HMG1 was generated from the synthetic peptide KGETKKKFKDPNAP (K plus amino acids 83–95), and antibody to HMG2 was generated from the peptide KSEAGKKGPGRPTG (K plus amino acids 168 –180). Preparation of Cisplatin-modified Oligonucleotides. Twenty-base-long oligonucleotides were annealed with the complementary strands. The double- stranded oligonucleotides were end-labeled with [- 32 P]ATP (Amersham) using polynucleotide kinase (Takara Suzo, Kyoto, Japan), and half of each was treated with cisplatin (Sigma Chemical Co., St. Louis, MO; Ref. 11). Expression of Fusion Protein. Total RNA was prepared from KB cells, and full-length cDNAs for human YB-1, HMG1, HMG2, XPA, and PCNA were amplified by reverse transcription-PCR using the following primer Received 10/22/98; accepted 12/1/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by a grant-in aid for cancer research from the Ministry of Education, Science, Sports and Culture of Japan, and from the Fukuoka Anticancer Research Fund (Fukuoka, Japan). 2 To whom requests for reprints should be addressed, at Department of Molecular Biology, University of Occupational and Environmental Health, School of Medicine, Yahatanishi-ku, Kitakyushu 807-8555, Japan. Phone: 81-93-691-7423; Fax: 81-93-692- 2766; E-mail: k-kohno@med.uoeh-u.ac.jp. 3 The abbreviations used are: YB-1, Y-box binding protein 1; MDR1, multidrug resistance 1; HMG, high mobility group; XPA, xeroderma pigmentosum group A; NER, nucleotide excision repair; PCNA, proliferating cell nuclear antigen; GST, glutathione S-transferase; TRX, thioredoxin. 342 on July 15, 2015. © 1999 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from