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A New Human Gene KCNRG Encoding Potassium Channel Regulating Protein Is a Cancer Suppressor Gene Candidate Located in 13Q14.3

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A New Human Gene KCNRG Encoding Potassium Channel Regulating Protein Is a Cancer Suppressor Gene Candidate Located in 13Q14.3 FEBS Letters 539 (2003) 156^160 FEBS 27082 View metadata, citation and similar papers at core.ac.uk brought to you by CORE A new human gene KCNRG encoding potassium channel regulatingprovided by Elsevier - Publisher Connector protein is a cancer suppressor gene candidate located in 13q14.3 D.V. Ivanova;b, T.V. Tyazhelovaa, L. Lemonnierc, N. Kononenkoc, A.A. Pestovaa, E.A. Nikitind, N. Prevarskayac, R. Skrymac, Y.V. Panchinc;e;f , N.K. Yankovskya, A.V. Baranovaa;b;Ã aVavilov Institute of General Genetics, 3 Gubkina Str., 119991 Moscow, Russia bCenter for Biomedical Genomic and Informatics, Biology Department, George Mason University, 10910 University Blvd., MSN 4E3, Manassas, VA 20110 USA cLaboratoire de Physiologie Cellulaire, INSERM EMI 0228, Ba“timent SN3, USTL, 59655 Villeneuve d’Ascq, France dHematology Research Center of Russia, Novozykovski pr.4a, 125167 Moscow, Russia eInstitute for Information Transmission Problems, 19 Bolshoi Karetny, 101447 Moscow GSP-4, Russia f A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia Received 13 January 2003; revised 14 February 2003; accepted 20 February 2003 First published online 10 March 2003 Edited by Maurice Montal tations in any of these genes ([4] and our unpublished data on Abstract We report the primary characterization of a new gene KCNRG mapped at chromosome band 13q14.3. This C13ORF1, and RFP2). These data suggest the existence of a gene includes three exons and has two alternatively spliced iso- di¡erent tumor suppressor gene, located in the area of inter- forms that are expressed in normal tissues and in some tumor est. cell lines. Protein KCNRG has high homology to tetrameriza- In several cell types, enhanced Kþ channel gene expression tion domain of voltage-gated K+ channels. Using the patch- or increased Kþ channel activity has been found to be asso- clamp technique we determined that KCNRG suppresses K+ ciated with cell division [10]. Important role of voltage-gated channel activity in human prostate cell line LNCaP. It is known Kþ channels in mitogenic growth control has been shown for + that selective blockers of K channels suppress lymphocyte and human lymphocytes [11]. A high expression of EAG channels LNCaP cell line proliferation. We suggest that KCNRG is a inhibited by physiological [Ca2þ]i has been demonstrated in candidate for a B-cell chronic lymphocytic leukemia and pros- several human melanoma [12] and somatic cancer cell lines tate cancer tumor suppressor gene. þ ß 2003 Published by Elsevier Science B.V. on behalf of the [13]. In human prostate cancer, LNCaP cells, the K channels 2þ Federation of European Biochemical Societies. controlling proliferation are also inhibited by [Ca ]i [14].It has been suggested that TEA [15^19] and verapamil [20^23] Key words: Tumor suppressor gene; inhibit the cancer cell proliferation by suppressing Kþ channel B-cell chronic lymphocytic leukemia; Prostate cancer; activity. We have previously demonstrated that in the andro- 13q14.3; Kþ channel regulating protein gen-sensitive prostate cancer cell line LNCaP the rate of the mitoses is decreased when voltage-activated Kþ channels are blocked implying that Kþ channels are involved in prostate 1. Introduction cell proliferation [24,25]. Here we report genomic organization and tissue distribu- Deletions in chromosomal band 13q14.3 have been previ- tion of the new human gene KCNRG located between ously associated with several di¡erent tumor types, including D13S1168 and D13S25. KCNRG encodes a soluble protein B-cell chronic lymphocytic leukemia (BCLL) [1] and prostate that shows strong homology to the cytoplasmic tetrameriza- cancer [2], indicating importance of this region for suppres- tion domain (T1) of voltage-gated Kþ channels. We suggest sion of a tumor development. The well-known tumor suppres- that such protein might interfere with the normal assembly of sor gene RB1 located at chromosome 13q has been excluded the Kþ channel protein by binding to their tetramerization as potential candidate for these diseases [3], implying the ex- domain causing the suppression of the Kþ channels. We istence of one or more tumor-suppressor genes in this region. also examine the suppressor e¡ect of KCNRG expression on Previously it was demonstrated that the region bordered by the whole-cell potassium currents in human prostate cell line STS markers D13S1168 and D13S25 is frequently deleted in LNCaP by the patch-clamp method. The results of our re- tumor cells form patients with BCLL [4,5]. The indicated area search suggest that KCNRG is a strong candidate for a is immersed in a wider region that is deleted in prostate cancer long-searched BCLL and prostate cancer tumor suppressor samples [6,7]. Four candidate genes belonging to this region, gene in human 13q14.3. namely DLEU1 and DLEU2 [4], RFP2 [8], C13ORF1 [9] were cloned. All candidates have been extensively checked for mu- tations by SSCP analysis and have failed to detect small mu- 2. Materials and methods 2.1. Molecular cloning of a new gene KCNRG To isolate a new gene KCNRG we have performed a BLASTN *Corresponding author. Fax: (1)-703-993 4293. search of the expressed sequence tag (EST) database of GenBank E-mail address: [email protected] (A.V. Baranova). with genomic sequence accession number AF440619 as a bait. We 0014-5793 / 03 / $22.00 ß 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/S0014-5793(03)00211-4 FEBS 27082 12-3-03 Cyaan Magenta Geel Zwart D.V. Ivanov et al./FEBS Letters 539 (2003) 156^160 157 Fig. 1. A: Position of genes in the BCLL critical region in 13q14.3; B: genomic structure of KCNRG. retrieved two expressed human sequences with the accession numbers priate. Each experiment was repeated several times. Student’s t-test BI770863 and BG537776. Corresponding cloned cDNAs IMAGE: was used for statistical comparison among means, and di¡erences 5207702 and IMAGE: 4690662 were obtained from ATCC USA. with P 6 0.05 were considered signi¢cant. The plasmid cDNAs were isolated and completely sequenced using the following primers: NGf: 5P-GGTAGCCTCTAGTTTGAAGTG- 2.5. Computer analysis 3P; NGr: 5P-GAGGCAGTGGAAGTAAGGTC-3P; NGf871: 5P- Nucleic acid and protein sequences were compared with sequences GAAATGAAGTTGTCTATCCTC-3P; NGr871: 5P-GAGGATAGA- in the GenBank databases using program BLAST. The ‘Search the CAACTTCATTTC-3P; NGr110: 5P-GGAAAGATGTATTGAAGG- Conserved Domain Database using RPS-BLAST’ (http://www.ncbi. TAG-3P and standard M13-rev and M13-forv sequencing primers. nlm.nih.gov/Structure/cdd/wrpsb.cgi) program has been used for search of conservative domains. The sequences of the open reading 2.2. RT-PCR analysis frames of the KCNRG and KCNRG-like genes were aligned, using the A number of human poly(A) RNAs samples (Clontech, Palo Alto, ClustalW program (www2.ebi.ac.uk/ClustalW/). CA, USA) were reverse transcribed to generate ¢rst-strand cDNAs with Advantage RT-for-PCR kit (Clontech, USA). PCR was carried out with these ¢rst-strand cDNAs, corresponding to 5 ng poly(A) 3. Results and discussion RNA, with GenePakTM PCR Universal 100 (IsoGene, Russia) using the following primer pair NGf and NGr110. PCR was performed for 3.1. Molecular cloning of a novel putative tumor suppressor 35 and 39 cycles at 94‡C for 1 min, 58‡C for 1 min and 72‡C for 1 min. gene KCNRG The ampli¢ed fragments were sequenced on both strands. As a con- trol, GAPDH was ampli¢ed using speci¢c primers (Clontech, USA). To identify the candidate for tumor suppressor genes for BCLL and prostate cancer locus at 13q14.3, an integrated 2.3. Plasmid construction genomic contig [8] and corresponding transcript map [9] KCNRG cDNA encompassing the entire coding region was synthe- were constructed. The new gene KCNRG was found via sized by PCR ampli¢cation of cDNA insert from clone IMAGE: BLASTN search of the EST database of GenBank seeded 4690662 using two gene-speci¢c primers. A sense primer, containing with a genomic sequence (AF440619). As a result we retrieved a XhoI site at its 5P-end (NgreF: 5P-TATCTCGAGATGAGTAGT- CAGGAACTGGT-3P) and an antisense primer, containing a BamHI two expressed human sequences with the accession numbers site at its 5P-end (NgreR: 5P-TATGGATCCATACCTGTAATCC- BI770863 and BG537776. Corresponding cloned cDNAs IM- CAGCTA-3P) were used. 500 ng of the pEGFP-N1 vector (Clontech, AGE: 5207702 and IMAGE: 4690662 were completely se- USA) and PCR product were digested by XhoI and BamHI. The quenced (mRNAs AY129654 and AY129653) and found to digested vector and the PCR products were gel puri¢ed, ligated to- gether and cloned. The cDNA insert was sequenced to verify its iden- contain two full-length coding sequences corresponding to tity and absence of mutation. di¡erent mRNA isoforms that belongs to the same gene. Sequence alignment of the genomic locus AF440619, which 2.4. Electrophysiological studies consists of 350 000 bases with the KCNRG mRNAs LNCaP cells from the ATCC were grown and prepared for electro- AY129654 and AY129653 revealed genomic structure of physiological experiments as described previously [14,25]. The whole- cell modes of the patch-clamp technique were used. This technique 5460 bp KCNRG. mRNA AY129654 consists of two exons has been described in detail in previous publications [14,25]. (612 nt and 709 nt in size) divided in genome by single intron The extracellular solution contained (in mM): 140 NaCl, 5 KCl, 4139 nt in size, mRNA AY129653 consists of three exons 2 CaCl2, 2 MgCl2, 0.3 Na2HPO4, 0.4 KH2PO4, 4 NaHCO3, 5 glucose (612, 95 and 709 nt in size) divided by two introns (2750 and 10 HEPES (N-2-hydroxyethylpiperazine-NP-2-ethano-sulfonic acid).
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