Proc. Natl. Acad. Sci. USA Vol. 90, pp. 10280-10284, November 1993 Medical Sciences An ets-related gene, ERG, is rearranged in human myeloid leukemia with t(16;21) chromosomal translocation KIMIKO SHIMIZU*t, HITOSHI ICHIKAWA*t, ARINOBU Tojo§, YASUHIKO KANEKO¶, NOBUO MASEKIII, YASUHIDE HAYASHI**, MIKI OHIRA*t, SHIGETAKA ASANO§tt, AND MISAO OHKI*t *Department of Immunology and Virology, Saitama Cancer Center Research Institute, and lDepartment of Laboratory Medicine and IlHematology Clinic, Saitama Cancer Center Hospital, Ina, Saitama 362, Japan; §Department of Hematology and Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108, Japan; and **Department of Pediatrics and ttFourth Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Communicated by Takashi Sugimura, July 19, 1993 ABSTRACT The t(16;21)(pll;q22) translocation is a non- MATERIALS AND METHODS random chromosomal abnormality found in several types of myeloid leukemia, which show variable cytomorphological Leukemia Samples, Cel Lines, and DNA Markers. Periph- features. We constructed rodent-human somatic cell eral blood or bone marrow cells were obtained from five hybrids patients with t(16;2l)(pll;q22) at diagnosis or in relapse. The containing the der(16) chromosome from leukemic cells of a patients were diagnosed as having AML, Ml subtype (patient patient with t(16;21). Using these hybrids, we mapped the 1); AML, M2 subtype (patient 2); AML, Ml or M7 subtype translocation breakpoint on the Not I restriction map of (patient 3); or MDS that had evolved to AML (patients 4 and chromosome 21 which we had previously constructed. The 5). result showed the proximity ofthe breakpoint to theERG gene, The adenine phosphoribosyltransferase (APRT)-deficient a member of the ets oncogene superfamily. Polymerase chain rodent cell lines used for construction of somatic cell hybrids reaction and Southern blot analyses of genomic DNA from the were a derivative ofthe mouse mammary carcinoma cell line hybrids and from peripheral blood cells and bone marrow cells FM3A (Cior5) (9) and a derivative of the Chinese hamster of patients with t(16;21) showed that the breakpoints were ovary cell line CHO (D118) and were obtained from H. clustered within a single intron in the coding region oftheERG Koyama (Kihara Institute, Yokohama, Japan). Three human gene. This finding and the results obtained by Northern blot hematopoietic cell lines, Namalwa (Burkitt lymphoma), Ka- analysis suggested the formation of a chimeric product(s) by sumi-1 (AML) (10), and MOLT-4 (T-cell leukemia), were fusion of the ERG gene and an unknown counterpart gene on used as controls. The human-mouse somatic cell hybrid chromosome 16. WAV17, which contains human chromosome 21, and its mouse parent cell line A9 were also used. Chromosomal translocations are implicated as important The chromosome 21-specific Not I linking clones have events in the pathogenesis ofhematopoietic malignancies. By been described (11). These clones were divided by Not I analysis of translocation breakpoints, many protooncogenes digestion into two half linking fragments. The DNA markers involved in neoplastic processes have been isolated (1, 2). pVK20B (D16S96) (12), pCJ52.196Ml (D16S160) (13), SF21 The translocation is a nonrandom chromo- (D21S41), and SF85 (D21S46) (14) were also used. t(16;21)(pll;q22) Construction ofSomatic Cell Hybrids. Bone marrow cells (5 somal abnormality found in human myeloid leukemia. This x 106) from patient 1 were fused with Cior5 or D118 cells (2 translocation was first identified in acute nonlymphocytic x 106) by the use of polyethylene glycol. Hybrids were leukemia (3-5), and its association with chronic myeloid selected as APRT+ cells growing in ES medium (Nissui leukemia in the blast crisis was also reported (6). In addition, Seiyaku, Tokyo) containing 10 ,uM azaserine and 100 ,uM myelodysplastic syndrome (MDS) evolved to acute myeloid adenine and were cloned after 20 days of culture. Residual leukemia (AML) with the same translocation (Y.H., unpub- human chromosomes in these clones were examined by lished data). AML with t(16;21) has been classified into Southern blot analysis. various subtypes in the French-American-British classifica- Southern Blot Analysis. Genomic DNA was digested with tion because it shows diverse cytomorphological features. appropriate restriction enzymes, and the restriction frag- This diversity raises a possibility that the breakpoints in each ments were separated by electrophoresis and transferred to leukemia may be inconsistent. These breakpoints on chro- a nylon membrane (Hybond-N; Amersham) by standard mosome 21 are cytogenetically indistinguishable (3) from methods. For pulsed-field gel electrophoresis (PFGE), ge- those ofthe t(8;21)(q22;q22) translocation, which is one ofthe nomic DNA was prepared and digested in agarose and then most common abnormalities in AML. Previously, we isolated fractionated with a Bio-Rad apparatus (CHEF Mapper). a protooncogene, AMLI, which is located across the t(8;21) Hybridization and autoradiography were performed as de- breakpoint on chromosome 21 (7). However, the AMLI gene scribed (15). is not rearranged in the leukemic cells with t(16;21) (8). PCR Amplfication and Primers. PCR amplification from 1 To determine the exact location of the t(16;21) transloca- Mg of genomic DNA was carried out with 0.2 nmol of tion breakpoint and the gene(s) involved in the neoplastic oligonucleotide primers in 100 ,ul of the reaction mixture [10 process of the leukemia, we mapped and analyzed the mM Tris'HCl, pH 7.5/50 mM KCI/1.5 mM MgCl2/0.01% breakpoint region on chromosome 21. Here, we report that gelatine/0.2 mM 4 dNTPs containing 2 units of DNA poly- the ERG gene, a member ofthe ets oncogene superfamily, on chromosome 21, was rearranged by this translocation. Abbreviations: AML, acute myeloid leukemia; APRT, adenine phos- phoribosyltransferase; MDS, myelodysplastic syndrome; PFGE, pulsed-field gel electrophoresis. The publication costs ofthis article were defrayed in part by page charge tPresent address: Radiobiology Division, National Cancer Center payment. This article must therefore be hereby marked "advertisement" Research Institute, Chuo-ku, Tokyo 104, Japan. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be sent at the present address. 10280 Downloaded by guest on October 2, 2021 Medical Sciences: Shimizu et al. Proc. Natl. Acad. Sci. USA 90 (1993) 10281 merase (AmpliTaq; Perkin-Elmer)] by 30 cycles of denatur- cell hybrids between rodent cells and leukemic cells. Mouse ation at 95°C for 1 min, annealing at 60°C for 2 min, and APRT- cells (FM3A CiorS) were fused with bone marrow extension at 72°C for 3 min with a Perkin-Elmer thermal cells of one patient (patient 1) and selected in medium cycler. For reverse transcription-PCR, cDNA was synthe- containing azaserine and adenine. This selection resulted in sized from 1 ug of poly(A)+ RNA in 20 Al of the reaction enrichment ofhybrid cells retaining a normal chromosome 16 mixture [10 mM Tris HCl, pH 8.3/75 mM KCl/3 mM or der(16) chromosome as APRT+ clones, since the human MgCl2/1 mM dithiothreitol/l mM each dNTP containing 10 APRT gene is located at 16q24. Residual human chromo- units of RNasin (Promega), 25 pmol of random hexamer somes in the resulting eight APRT+ clones were examined by (Pharmacia), and 200 units of reverse transcriptase (Super- Southern blot analysis using chromosome 16 or 21-specific Script RNase H-; GIBCO/BRL)] at room temperature for 10 probes for D16S160 (16pl3), D16S96 (16q21-q22), D21S46 min and then at 37°C for 1 hr. One-tenth ofthe cDNA product (21qll-q21), and D21S41 (21q22.3) loci. Two of the eight was used for PCR amplification by 30 cycles of denaturation APRT+ clones retained D16S96 and D21S41 loci but had lost at 95°C for 20 sec, annealing at 60°C for 30 sec, and extension D16S160 and D21S46 loci, indicating that they harbored the at 72°C for 1 min. The amplified products were analyzed by der(16) chromosome but not normal chromosome 16, normal electrophoresis in 3% NuSieve 3:1 agarose (FMC Bioprod- chromosome 21, or der(21) chromosome. These two clones ucts) gel. For use as hybridization probes, the PCR products (NSC532 and NSC533) were used for subsequent analyses. were purified from the agarose gel. By using Chinese hamster APRT- cells (CHO D118), two The primers for the D2JS55 locus (pPW518-1R) were more somatic cell hybrids (NSD122 and NSD130), which also described by Tanzi et al. (16). The sequences of the primers harbored only the der(16) chromosome among the four rel- for the ETS2 and ERG genes were designed according to the evant chromosomes, were constructed similarly. known sequences (17, 18) as follows: ETS2F, 5'-TTCT- Mapping of the t(16;21) Translocation Breakpoint on Chro- CAGGGCTGTGCAAGTA-3'; ETS2R, 5'-CATGGTGAC- mosome 21. We have constructed a complete Not I restriction TCTAGGTCTAG-3'; ERGa, 5'-GGCCAAAGGCGGGAA- map of the long arm of human chromosome 21 by using Not GATGG-3'; ERGb, 5'-AGCGTAGGATCTGCTGGCAC-3'; I linking clones (11). Use of these Not I linking clones ERGc, 5'-CATGCATTAACCGTGGAGAG-3'; ERGd, 5'- together with the somatic cell hybrids provides a simple CCATCAGCGTGCATTCACCA-3'; ERGe, 5'-GCAT- system for mapping the t(16;21) translocation breakpoint. It GAACCCTCGAGTCTCC-3'; ERGf, 5'-CTGCAGTTA- can be mapped on the Not I restriction map by Southern blot GAGGCTCACTG-3'; ERGg, 5'-CAGCTCCTGAGCTG- analysis using the Not I linking clones as probes to examine TAGCCA-3'. the presence or absence of Not I sites in genomic DNA from Northern Blot Analysis. Total RNA of cultured cells and these hybrids. This analysis showed that the Not I sites of peripheral blood cells was isolated by the acid guanidinium linking clones LL46LP, LJ105, and LL42SP were present on thiocyanate/phenol/chloroform method (19).