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Functional Characterization of the Candidate Tumor Suppressor Gene NPRL2/G21 Located in 3P21.3C

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Functional Characterization of the Candidate Tumor Suppressor Gene NPRL2/G21 Located in 3P21.3C [CANCER RESEARCH 64, 6438–6443, September 15, 2004] Functional Characterization of the Candidate Tumor Suppressor Gene NPRL2/G21 Located in 3p21.3C Jingfeng Li,1 Fuli Wang,1 Klas Haraldson,1 Alexey Protopopov,1 Fuh-Mei Duh,2,3 Laura Geil,2,3 Igor Kuzmin,2,3 John D. Minna,4 Eric Stanbridge,5 Eleonora Braga,6 Vladimir I. Kashuba,1,7 George Klein,1 Michael I. Lerman,2 and Eugene R. Zabarovsky1,8 1Microbiology and Tumor Biology Center, Center for Genomics and Bioinformatics, Karolinska Institute, Stockholm, Sweden; 2Cancer-Causing Genes Section, Laboratory of Immunobiology, Center for Cancer Research, National Cancer Institute, Frederick, Maryland; 3Basic Research Program, SAIC-Frederick, Inc., Frederick, Maryland; 4Hamon Center for Therapeutic Oncology, Research, University of Texas Southwestern Medical Center, Dallas, Texas; 5Department of Microbiology and Molecular Genetics, College of Medicine, University of California at Irvine, Irvine, California; 6Russian State Genetics Center, Moscow, Russia; 7Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kiev, Ukraine; and 8Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia ABSTRACT accompanied by chromosome 3p homozygous deletions, is a charac- teristic feature of most major epithelial carcinomas, such as lung, Initial analysis identified the NPRL2/G21 gene located in 3p21.3C, the breast, cervical, oral cavity, ovary, and kidney (2, 3). These changes lung cancer region, as a strong candidate tumor suppressor gene. Here we indicate the involvement of multiple tumor suppressor genes. provide additional evidence of the tumor suppressor function of NPRL2/ G21. The gene has highly conserved homologs/orthologs ranging from We have performed a comprehensive deletion survey of 3p on more yeast to humans. The yeast ortholog, NPR2, shows three highly conserved than 400 major epithelial carcinoma cases (4–8), using a defined set regions with 32 to 36% identity over the whole length. By sequence of markers, combining conventional loss of heterozygosity with quan- analysis, the main product of NPRL2/G21 encodes a soluble protein that titative real-time PCR, and comparative genomic and Southern hy- has a bipartite nuclear localization signal, a protein-binding domain, bridizations. We identified two most frequently affected 3p21.3 re- similarity to the MutS core domain, and a newly identified nitrogen gions, LUCA at the centromeric and AP20 at the telomeric border of permease regulator 2 domain with unknown function. The gene is highly 3p21.3. Aberrations of either region were detected in more than 90% expressed in many tissues. of the studied tumors. These 3p21.3 aberrations were complex and, in We report inactivating mutations in a variety of tumors and cancer cell addition to deletions, could involve gene amplifications as well. lines, growth suppression of tumor cells with tet-controlled NPRL2/G21 Homozygous deletions were detected in 10 to 18% of all of the tumors transgenes on plastic Petri dishes, and suppression of tumor formation in SCID mice. Screening of 7 renal, 5 lung, and 7 cervical carcinoma cell lines in both the LUCA and AP20 sites. The frequent chromosome aber- showed homozygous deletions in the 3؅ end of NPRL2 in 2 renal, 3 lung, rations in these regions suggest that they harbor multiple tumor .(and 1 cervical (HeLa) cell line. Deletions in the 3؅ part of NPRL2 could suppressor genes (2, 3 result in improper splicing, leading to the loss of the 1.8 kb functional Analysis of 15 homozygous deletions in the LUCA region permit- NPRL2 mRNA. We speculate that the NPRL2/G21 nuclear protein may be ted us to establish the smallest homozygously deleted region in involved in mismatch repair, cell cycle checkpoint signaling, and activa- 3p21.3C, located between D3S1568 (CACNA2D2 gene) and D3S4604 tion of apoptotic pathway(s). The yeast NPR2 was shown to be a target of (SEMA3F gene). It contains 17 genes that were defined previously as cisplatin, suggesting that the human NPRL2/G21 may play a similar role. lung cancer candidate tumor suppressor genes. Mapping of 19 ho- At least two homozygous deletions of NPRL2/G21 were detected in 6 mozygous deletions in the 3p21.3T/AP20 region resulted in localiza- tumor biopsies from various locations and with microsatellite instability. tion of its smallest homozygous deletion to the region flanked by This study, together with previously obtained results, indicates that NPRL2 is a multiple tumor suppressor gene. D3S1298 and D3S3623. It contains 4 genes, namely APRG1, ITGA9, RBSP3/HYA22, and VILL, which need to be analyzed (9). In this study, we report the functional characterization of the INTRODUCTION NPRL2/G21 gene from the LUCA region and its tumor suppressor Epithelial tumors are the most prevalent and lethal cancers in the genes-like features. world. For example, lung cancer alone kills Ͼ150,000 patients each year in the United States and more than a million around the world (1). MATERIALS AND METHODS Loss of heterozygosity involving several chromosome 3p regions, Cell Lines and General Methods. Small cell lung carcinoma cell line U2020 was described earlier (10). The ACC-LC5 small cell lung carcinoma Received 12/10/03; revised 6/1/04; accepted 7/9/04. Grant support: the Swedish Cancer Society, the Swedish Research Council, the cell line that carried a deletion in 3p21.3 (11) was kindly provided by Dr. Swedish Foundation for International Cooperation in Research and Higher Education Yusuke Nakamura (University of Tokyo, Tokyo, Japan), and the GLC20 small (STINT), the Swedish Institute, the Royal Swedish Academy of Sciences, INTAS, cell lung carcinoma cell line with homozygous deletion in LUCA region was Karolinska Institute, The Program of Russian Academy of Science Basic Research - to obtained from Dr. Charles H. Buys (12). A498, Caki1, and Caki2 renal clear Medicine, the Russian Ministry of Science and Technology, the Russian Foundation for Basic Research Grant 01-04-48028 (E. Braga), the National Cancer Institute, NIH contract cell carcinoma lines, 7 cervical carcinoma cell lines (HTB, C33A, HeLa, no. NO1-CO-56000 (M. Lerman), NIH contract no. NO1-CO-12400 (F. Duh, L. Geil, and CaSki, C4–1, MS-75, and SiH2), and the N417 small cell lung carcinoma cell I. Kuzmin), Lung Cancer SPORE P50 CA70907 and CA71618 (J. Minna), and the Avon line were purchased from the American Type Culture Collection (Manassas, Foundation (E. Stanbridge). MD). Lymphoblastoid cell line CBMI-Ral-STO, osteosarcoma cell line 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 Saos-2, non-small cell lung carcinoma line A549, and renal clear cell carci- 18 U.S.C. Section 1734 solely to indicate this fact. noma lines KRC/Y, ACHN, TK164, HN4, TK10, and KH-39 were obtained Note: The content of this publication does not necessarily reflect the views or policies from the MTC-KI (Stockholm, Sweden) cell lines collection (6). of the Department of Health and Human Services, nor does mention of trade names, KRC/Y, Caki 1, Caki 2, ACHN, TK10, and TK164 cell lines were karyo- commercial products, or organizations imply endorsement by the United States Govern- ment; J. Li and F. Wang contributed equally to this work; J. Li is currently in the National typed using trypsin-Giemsa banding, and different cell clones were found (data Cancer Institute, Frederick, Maryland; A. Protopopov is currently in the Dana-Farber not shown). Heterogeneity of the small cell lung carcinoma U2020 and renal Cancer Institute, Boston, Massachusetts. cell carcinoma KH39 cell lines was shown previously (10, 13). Requests for reprints: Eugene R. Zabarovsky, Microbiology and Tumor Biology Molecular cloning of the human NPRL2/G21 gene was described previously Center, Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden. Phone: 46-8-728- 67-50; Fax: 46-8-31-94-70; E-mail: [email protected]. (ref. 14; accession no. AF040707). The structures of NPRL2 and PCR primers ©2004 American Association for Cancer Research. used in the study are shown in Fig. 1. 6438 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2004 American Association for Cancer Research. NPRL2 IS A MULTIPLE TUMOR SUPPRESSOR Fig. 1. Genomic structure of NPRL2 (A) and primers (B) used in the study. Accession no. for NPRL2 is AF040707; accession no. for BLU is NM_015896; and accession no. for RASSF1A is NM_007182. Noncoding sequences are shown in black. Thick horizontal arrows indicate the orien- tation of transcription. Position of the F2a primer on the RASSF1A gene transcript is 97-116nt from the 5Ј end. Small arrowheads indicate position of PCR primers. (ex, exon) For amplification of RASSF1A gene, the following primers were used: RESULTS AND DISCUSSION forward, F2a 5Ј-GCCCAAAGCC AGCGAAGCAC-3Ј; and reverse, F2int2 5Ј-ACCCAGGCAG CCCTCGAGAA-3Ј (Fig. 1). The PCR primers were Specific Aims and Experimental Design. The NPRL2 gene was purchased from Invitrogen (Carlsbad, CA). The PCR was performed as de- one of the promising tumor suppressor gene candidates that we scribed earlier (4). However, we used only 28 cycles for detection of homozy- analyzed previously (14). Forced, uncontrolled expression of wild- gous deletions in NPRL2 gene in both cancer cell lines and tumor biopsies. type NPRL2/G21 transgenes from adenovirus vectors inhibited tumor This was done to reduce the effect of normal cell contamination in the case of cell growth by inducing apoptosis and cell cycle arrest of non-small biopsies and heterogeneity in the case of cancer cell lines. cell lung carcinoma line cell lines H1299 and A549. Also, intratu- PCR products were cloned, using the TOPO TA cloning kit for sequencing moral injection of a NPRL2 expressing adenovirus vector or systemic (Invitrogen). All molecular biology and microbiology procedures were performed as administration of protamine-complexed vectors significantly sup- described previously (15, 16).
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