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Molecular Cloning of an Activated Human Oncogene, Homologous to V

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Molecular Cloning of an Activated Human Oncogene, Homologous to V Proc. Nati. Acad. Sci. USA Vol. 82, pp. 5641-5645, September 1985 Biochemistry Molecular cloning of an activated human oncogene, homologous to v-raf, from primary stomach cancer (transfection/human transforming gene/gastric tumor) KENJI SHIMIZU*, YOSHIMICHI NAKATSU*, MUTSUO SEKIGUCHI*t, KEIZO HOKAMURAt, KENZO TANAKAt, MASAAKI TERADA§, AND TAKASHI SUGIMURA§ *Department of Biology, Faculty of Science, and tDepartment of Pathology, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan; and §National Cancer Center Research Institute, Tokyo 104, Japan Contributed by Takashi Sugimura, May 1, 1985 ABSTRACT Transfection with high molecular weight form extraction, as described (4). Preparation of DNA from DNA from a primary stomach cancer induced foci of trans- solid tumors was as described (23). Plasmid and bacterio- formed NIH 3T3 cells, and the transformed cells were phage DNAs were prepared as described (24, 25). The tumorigenic in nude mice. By screening with a human Alu- following plasmids were generously provided [directly or family probe, we isolated the human DNA sequence from the through T. Sekiya (National Cancer Center Research Insti- secondary transformant cells. This transforming sequence tute) and M. Wigler] by the indicated scientists: pErbA, encompasses about 60 kilobase pairs and is unrelated to known pErbB, pY6271(yes), pv-myc, and pABsub3(abl) (T. human transforming genes. Examination of homologies be- Yamamoto); pHT1O(mos) (G. F. Vande Woude); pBR- tween this sequence and retroviral oncogenes revealed that the FSV(fps) (H. Hanafusa); pSRA2(src) (J. M. Bishop); pMyb human transforming sequence is closely related to the v-raf (T. Kawakami); pHB11(Ha-ras) and pKBE2(Ki-ras) (E. M. oncogene of murine transforming retrovirus 3611-MSV. Scolnick); pSSV-11(sis) (S. Aaronson); pc(hu)-fos (I. Verma); pv-fgr 1700 (K. C. Robbins); pros (L. H. Wang); Morphological transformation of NIH 3T3 cells by transfec- pRev-T3(rel) (H. M. Temin); pvski-l (E. Stavnezer); pSM- tion with genomic DNA has been used to detect dominantly FeSV(fms) (C. J. Sherr); pBB3(ets) (P. H. Duesberg); acting transforming genes in human tumors and tumor- p149(raJ) (U. R. Rapp); pMT2.5(int-1) (H. E. Varmus); derived cell lines (1-5). Most naturally occurring transform- pHuB-lym-J (G. M. Cooper); BLUR8 (C. Schmid); and ing genes thus far detected have been identified as activated pHC79 (B. Hohn). members of the ras gene family, either Ha-, Ki-, or N-ras DNA Transfection. For all DNA transfers, we used a (6-11). These activated ras genes have single point mutations modified calcium phosphate precipitation method (26) with in the ras coding regions, at either the 12th or the 61st codons NIH 3T3 cells as the recipients. Focus assays were per- (12-17). formed as described (4). Two other transforming genes, B-lym, which has been Enzymes. Restriction endonucleases were purchased from isolated from human B-cell lymphoma (18), and oncD, which Nippon Gene (Toyama, Japan) or from New England Biolabs has been identified in human colon tumor 2033 (19), do not and used according to the accompanying instructions. Esch- appear to be related to known viral oncogenes, and the erichia coli DNA polymerase I and T4 DNA ligase were from mechanisms of their activation are unclear. Another gene, New England Biolabs and Takara Shuzo (Kyoto, Japan), met, recently isolated from a human osteosarcoma-derived respectively. cell line, appears to be activated by chemical mutagenesis Southern Blot Hybridization. DNA samples were digested (20). with restriction endonucleases and subjected to agarose gel Little is known of the nature of the oncogenes activated in electrophoresis and filter-blot transfer by the method of the case of stomach cancer. Stomach cancer is the most Southern (27). Filter-blotted DNAs were hybridized with a and nick-translated 32P-labeled DNA probe, under two sets of common cancer in Japan and some European South conditions. Less stringent hybridization conditions entailed American countries. hybridization in a mixture containing 40% (vol/vol) forma- We now report detection and molecular cloning of a mide, 0.9 M NaCI, 0.09 M sodium citrate (pH 7.0), lx transforming gene from a surgically removed stomach cancer Denhardt's solution (0.02% polyvinylpyrrolidone/0.02% of a Japanese patient. DNA hybridization experiments Ficoll/0.2% bovine serum albumin), and denatured salmon showed that this gene is closely related to v-raf, the oncogene sperm DNA (20 ,ug/ml) at 42°C. More stringent hybridization of 3611-murine sarcoma virus (MSV) (21). conditions entailed hybridization in a mixture containing 50% formamide, 0.3 M NaCl, 0.03 M sodium citrate (pH 7.0), 1 x MATERIALS AND METHODS Denhardt's solution, 10 mM EDTA, and denatured salmon sperm DNA (20 ,ug/ml) at 43°C. Hybridized DNA was Cells. NIH 3T3 cells (22) obtained from M. Wigler (Cold visualized by autoradiography on Kodak XAR-5 film at Spring Harbor Laboratory) were maintained at low cell -70°C, using an intensifying screen. densities in Dulbecco's modified Eagle medium (GIBCO) Molecular Cloning. Genomic libraries of the secondary containing antibiotics (streptomycin at 100 ,ug/ml and peni- transformants were constructed in a double amber derivative cillin at 100 units/ml) and 10% calf serum (Flow Laborato- of XL47.1 (28) from DNA partially digested with either EcoRI ries). or BamHI and fractionated in a sucrose gradient, as de- Preparation of DNA. DNA was prepared from cultured scribed (4). We screened these libraries for the presence of a cells by NaDodSO4/proteinase K lysis and phenol/chloro- human sequence, using the method of Benton and Davis (29); the probe was initially BLUR8, a clone of the ubiquitous The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: kbp, kilobase pair(s). in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5641 Downloaded by guest on September 27, 2021 5642 Biochemistry: Shimizu et al. Proc. NatL Acad Sci. USA 82 (1985) repeated human Alu family sequences (30). At later stages, A B C D F F ( H we used portions of isolated clones as specific probes for isolating contiguous clones. Cosmid libraries of the same DNAs were constructed in pHC79 (31) from DNA partially digested with EcoRI and size-fractionated as above. Screening of the cosmid library was performed as described (32), with purified fragments of 4m -4 the gene from phage recombinants as probes. ..,..n RESULTS Ia t ,1 I Detection of the Transforming Sequence. High molecular weight DNAs were prepared from surgically removed human stomach cancers of three Japanese patients. These DNAs were 4.2 used to transfect mouse NIH 3T3 cells by the calcium 0.i 1 phosphate coprecipitation technique (4). One DNA prepara- tion from a Borrmann II primary cancer of a 69-year-old male 00 -44 patient gave rise to transformed foci, albeit with a low 3.1 efficiency (Table 1). DNAs of the metastasized lymph node tumor and of normal mucosa from the same patient and .-.6 DNAs from other patients did not induce foci upon transfection under the same experimental conditions. Repro- ducible occurrence of foci induction in two independent experiments suggests that the gene was already activated in the primary stomach cancer. DNAs of the primary foci thus obtained were analyzed by Southern blot hybridization, with BLUR8 as a probe for the presence ofhuman-specific repeated sequences. Seven out of ~- -1.1 seven primary transformants retained abundant amounts of human DNA sequences (Fig. 1, lane B). We observed three slightly different classes in the Southern blot hybridization FIG. 1. Presence of human sequences in NIH 3T3 transformants. Ten micrograms of each cellular DNA was profile with the DNAs from these seven primary transform- digested with EcoRI, subjected to electrophoresis in 1% agarose gel, and analyzed by blot ants arose and thereby inferred that they from at least three hybridization with BLUR8 as probe under less stringent conditions independent transforming events (data not shown). (see Materials and Methods). Lanes: A, NIH 3T3 cell DNA; B, DNA DNAs of the primary transformants were then used in the from one of the seven primary transformants; C-G, DNAs of five next round of transfection to obtain secondary transform- different secondary transformants; H, a mixture of NIH 3T3 DNA ants. Five secondary transformants were obtained indepen- (10 ,ug) and two recombinant X phage DNAs (total 200 pg) containing dently from two different primary transformants. The trans- the human c-Ki-ras-2 gene (16), used as internal size markers [indicated in kilobase pairs (kbp) at right]. Arrowheads at right formation efficiency was still very low (1 focus per 48 tkg of indicate human-specific EcoRI fragments commonly retained in DNA), whereas the positive control (an active N-ras clone) these secondary transformants. gave rise to several thousands of foci per ,tg of DNA, in the same experiment. This finding, which is in contrast to the observation that the efficiency of second-cycle transfection is transfection cycles, only a small amount of human DNA higher than that of initial transfection in many cases, suggests remains in the secondary transformants; any human DNA that this transforming sequence might be very large. fragments conserved in all secondary transformants would To characterize further the transforming sequence, we therefore be closely linked to or contained in the transforming analyzed the DNAs of the secondary transformants for the sequence. Southern blot hybridization profiles of such con- presence of human DNA sequences. After two serial served restriction fragments with human repeated sequences Table 1. Transformation of NIH 3T3 cells with DNAs from stomach cancer Primary foci Secondary foci DNA No./no. of dishes* No./no.
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