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(LIMD1), a Tumor Suppressor Encoded at Chromosome 3P21.3, Binds Prb and Represses E2F-Driven Transcription

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(LIMD1), a Tumor Suppressor Encoded at Chromosome 3P21.3, Binds Prb and Represses E2F-Driven Transcription LIM domains-containing protein 1 (LIMD1), a tumor suppressor encoded at chromosome 3p21.3, binds pRB and represses E2F-driven transcription Tyson V. Sharp*†, Fernando Munoz*, Dimitra Bourboulia*, Nadege Presneau*, Eva Darai‡, Hsei-Wei Wang*, Mark Cannon*, David N. Butcher§, Andrew G. Nicholson§, George Klein‡, Stephan Imreh‡, and Chris Boshoff*† *Cancer Research UK, Viral Oncology Group, Wolfson Institute for Biomedical Research, Cruciform Building, University College London, London WC1E 6BT, United Kingdom; ‡Microbiology and Tumor Biology Center, Karolinska Institute, S-171 77 Stockholm, Sweden; and §Department of Histopathology, Royal Brompton Hospital, London SW3 6NP, United Kingdom Contributed by George Klein, September 29, 2004 LIM domains-containing protein 1 (LIMD1) is encoded at chromo- FuGene (Roche Diagnostics) per the manufacturer’s instructions. some 3p21.3, a region commonly deleted in many solid malignan- The reporter plasmid pGL2–3xE2F-luciferase contains three cop- cies. However, the function of LIMD1 is unknown. Here we show ies of the E2F DNA-binding site derived from the adenovirus E2A that LIMD1 specifically interacts with retinoblastoma protein (pRB), gene promoter and has been described (11). Luciferase activity in inhibits E2F-mediated transcription, and suppresses the expression cell lysates was normalized to cotransfected ␤-galactosidase values of the majority of genes with E2F1-responsive elements. LIMD1 [relative E2F-luciferase (E2F-luc) activity]. blocks tumor growth in vitro and in vivo and is down-regulated in the majority of human lung cancer samples tested. Our data Plasmids. To create N-terminal hemagglutinin (HA)-tagged indicate that LIMD1 is a tumor-suppressor gene, the protein prod- LIMD1, we performed PCR amplification on human LIMD1 uct of which functionally interacts with pRB and the loss of which cDNA (kindly provided by S.I.) by using the 5Ј-HA-START promotes lung carcinogenesis. primer (5Ј-GCAGCATGTACCCATACGATGTTCCAGA- TTACGCTATGGATAAGTATGACGACCTGGGCCTGGA- lung cancer ͉ retinoblastoma GGCC-3Ј) and the 3Ј-STOP primer (5Ј-GAAGTTGTGCGT- GTGAAGGGCTGTAGATGAGGGTCT-3Ј). The PCR prod- he archetypal tumor-suppressor gene (TSG) is represented by uct was TA cloned into pcDNA3.1 Topo (Invitrogen) and Tthe retinoblastoma 1 (RB1) gene (1, 2). Loss of its function sequence-verified. results in susceptibility to retinoblastoma, a sporadic or hereditary pHR-CMV-HA-LIMD1-IRES-GFP was produced as follows: pediatric neoplasm arising from retinal cells harboring either pcDNA3.1-HA-LIMD1 was cut with HindIII, treated with the deleted or mutated RB alleles (1, 3, 4). To identify binding partners Klenow fragment of DNA polymerase to produce blunt ends, of retinoblastoma protein (pRB), we performed a yeast two-hybrid purified and cut with XhoI, and then ligated directionally into the screen using full-length pRB fused to the GAL4 DNA-binding SmaI͞XhoI-cut pHR-CMV-IRES-GFP plasmid downstream of the domain as bait. We identified the LIM domains-containing protein cytomegalovirus (CMV) promoter and upstream of the internal 1 (LIMD1) sequence, which is localized within the common elim- ribosome entry site (IRES)-GFP. inated region 1 (C3CER1; also called CER1) on chromosome Xpress (Invitrogen)-tagged LIMD1 and deletion mutant plas- 3p21.3 (5, 6). C3CER1 is one of the putative tumor-suppressor mids were created as follows. LIMD1 cDNA was used as a template regions identified by the ‘‘elimination test,’’ a functional test system for PCR to create consecutive deletions in a C- to N-terminal that identifies regularly lost (eliminated) chromosome segments in direction. PCR amplification was performed by using the 5Ј- microcell hybrid-derived severe combined immunodeficiency LIMD1-START primer (5Ј-GAGATCGAATTCGCAATG- (SCID) tumors (7). Within chromosome 3p are several TSG GATAAGTATGACGACCTGGGCCTG-3Ј) in combination with regions. Nine homozygous deletion regions have been described, the 3Ј primer, which introduced the indicated C-terminal deletion four of which were found in lung tumors. C3CER1 (megabases followed by a stop codon. Consecutive LIMD1 deletions in an N- to 43.32–45.74) is located between two such regions, AP20 and LUCA C-terminal direction were achieved by using the 3Ј-LIMD1-STOP (8, 9). Deletions in this chromosome 3p region are a common event primer (5Ј-CTCTGCAGGTCGACCTAGAAGTGGTGCT- in solid malignancies including breast, gastric, colorectal, ovarian, GGTGAAGGGCTGTA-3Ј) in combination with the indicated 5Ј and renal (6). Furthermore, C3CER1 loss of heterozygosity (LOH) primer, which annealed at the indicated amino acid and introduced exceeds 90% in lung tumors compared to the putative TSG FHIT an ATG start codon before this residue. All PCR products pro- (65%) and the TSG VHL (72%) (6), which map at 3p14.2 and duced by this PCR deletion mutagenesis method were gel-purified 3p25.3, respectively. Therefore, because deletion in this region is a and TOPO cloned into pcDNA4͞HisMax (Invitrogen), which common event in lung tumors, chromosome 3p is under intense resulted in an N-terminal Xpress-tagged LIMD1 (when using just study for the identification of putative TSGs (10). the 5Ј-START and 3Ј-STOP primers) or the indicated deletion Here, we confirm the specific binding of LIMD1 to pRB and mutants. All PCR full-length and deletion mutants of LIMD1 were its ability to repress E2F-driven transcription and cell prolifer- sequence-verified. ation. Furthermore, we demonstrate that LIMD1 inhibits tumor pGEX6P-1-LIMD1 plasmid was constructed as follows. Hu- growth in both in vitro and in vivo models. Finally, we show that man LIMD1 cDNA was PCR-amplified by using primers de- LIMD1 expression is down-regulated in lung cancer. Materials and Methods Abbreviations: LIMD1, LIM domains-containing protein 1; pRB, retinoblastoma protein; TSG, tumor suppressor gene; C3CER1, common eliminated region 1; LOH, loss of heterozy- Cell Culture, Transfection, and Reporter Assays. All cell lines were gosity; HA, hemagglutinin; VO, vector only; qRT-PCR, quantitative RT-PCR; LMB, leptomy- cultured in DMEM (GIBCO͞BRL) supplemented with 10% FCS, cin B; NES, nuclear export signal. 2 mM glutamine, 50 units͞ml penicillin, and 50 ␮g͞ml streptomy- †To whom correspondence may be addressed. E-mail: [email protected] or cin. For transient transfections, 8 ϫ 105 adherent cells were plated [email protected]. CELL BIOLOGY in a 25-cm2 flask and the following day were transfected by using © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0407123101 PNAS ͉ November 23, 2004 ͉ vol. 101 ͉ no. 47 ͉ 16531–16536 Downloaded by guest on October 1, 2021 scribed above to incorporate an EcoRI and SalI site. The was used for transfections (16). According to standard protocols, resulting PCR product was restriction enzyme-digested with 50–70% confluent monolayer A9 cells were transfected with a EcoRI and SalI and ligated into similarly cut pGEX6P-1 vector mixture of 1–2 ␮g of PAC DNA and 10 ␮l of Lipofectamine (Life (Amersham Pharmacia Biotech). The plasmid with the correct Technologies, Rockville, MD) per well, in six-well plates. The insert was sequence-verified on both DNA strands. transfectants were selected on blasticidin (1–2 ␮g͞ml). Two positive pHR-CMV-E7-IRES-GFP plasmid was constructed by BamHI clones were chosen and expanded in vitro for inoculation into SCID digestion of pLXSN-E7 (a gift from David Beach, Queen Mary’s mice. All transfectants were analyzed by fluorescent in situ hybrid- School of Medicine and Dentistry, London) to excise the E7 cDNA, ization (FISH) and PCR. which was then ligated into similarly cut pHR-CMV-IRES-GFP. One million A9 and A9-LIMD1-PAC transfectants were inocu- lated s.c. into 6-week-old SCID mice in three sites, with a fourth site Construction, Production, and Infection of Lentivirus Expressing used for A9 controls. The mice were observed for tumor formation LIMD1. To obtain pseudotyped lentivirus [recombinant HIV-1 with once a week up to 6 weeks. The two positive LIMD1-PAC clones vesicular stomatitis virus G (VSV-G) envelope protein], which were inoculated in two series into four mice per clone and three expresses HA-tagged LIMD1 or E7 protein, we used the gene inoculation sites per mouse. A9 cells were inoculated as controls at delivery and production system developed by Naldini et al. (12). one site per mouse. Tumor growth was monitored once a week for 6 weeks. Yeast Two-Hybrid Screen. The cloning of full-length pRB into the pAS2.1 bait vector has been described (13). The resulting plasmid BrdUrd Cell Proliferation Assay. The Cell Proliferation ELISA, BrdU (pAS2.1-pRB) was pretransformed into the Saccharomyces cerevi- (Colorimetric) (Roche Applied Science) immunoassay for the siae PJ69-4a (MATa trp1-90 leu2-3,112 ura3-52 his3-200 quantification of cell proliferation, based on the measurement of gal4⌬gal80⌬LYS2ϻGAL1-HIS3 GAL2-ADE2 met2ϻGAL7-lacZ) BrdUrd incorporation during DNA synthesis, was used per the (14) reporter strain by using a modified lithium acetate protocol manufacturer’s instructions. (see Yeast Protocols Handbook, BD Biosciences Clontech). Subse- quently, this strain was cotransformed with the BC3 cDNA library Microarray Processing and Analysis. RNA preparation, microarray (produced by using the Stratagene HybriZAP cDNA library kit) in processing, and data analysis of the MDA-MB435 cell line (here- the GAL4 DNA activation domain (GAL4AD) fusion ‘‘prey’’ after referred to as MB435) were performed as described by Wang vector. Selection for positive colonies and cDNA clone isolation et al. (17). Affymetrix (Santa Clara, CA) Hg-U133 Plus 2.0 chips were as previously described (15). were used in this analysis. The human orthologs for the E2F1- responsive genes were selected by using lists previously obtained by Generation of Recombinant GST–LIMD1 Fusion Protein. Full-length Ma et al. (18). The false discovery rate q of 0.05 assures that Ͻ5% LIMD1 was expressed as a recombinant GST fusion protein from of genes with q values Ͻ0.05 can be expected to be false positives. the pGEX6P-1-LIMD1 plasmid.
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