MOLECULAR AND CELLULAR BIOLOGY. Mar. 1986. p. 955-958
`0270-7306/861030955-04$02. 00/0
`Copyright © 1986. American Societ y for Microbiology
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`Vol. 6. No. 3
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`Localization of a Novel v-erbB-Related Gene, c-erbB-2, on Human
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`Chromosome 17 and Its Amplification in a Gastric Cancer Cell Line
`SHIN-ICHI FUKUSHIGE.1 KEN-ICHI MATSUBARA,1 MICHIHIRO YOSHIDA,2 MOTOMICHI SASAKI.2
`TOSHIMITSU SUZUKI,3 KENT ARO SEMBA.4 KU MAO TOYOSHIMA,4 AND TAOASHI Y AMAMOT04*
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`Institute for Molecular and Cellular Biology, Osaka University, Suita, Osaka 565,1 Chromosome Research Unit,
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`Hokkaido University, N. 10-W.8, Sapporo 060,2 School of Medicine, Niigata University, Niigata 951.3 and The Institute
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`of Medical Science, The University of Tokyo, Minato-ku Tokyo 108,4 Japan
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`Received 17 September 1985/Accepted 3 December 1985
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`The c-erbB-2 gene is a v-erbB-related proto-oncogene which is distinct from the gene encoding the epidermal
`growth factor receptor. By using two independent methods, hybridizat.ion of both sorted chromosomes and
`metaphase spreads with cloned c-erbB-2 DNA, we mapped the c-erbB-2 locus on human chromosome 17 at q21,
`a specific breakpoint observed in a translocation associated with acute promyelocytic leukemia. Furthermore,
`we observed amplification and elevated expression of the c-erbB-2 gene in the MKN-7 gastric cancer cell line.
`These data suggest possible involvement of the c-erbB-2 gene in huQlan cancer.
`
`Human Genetic Mutant Cell Repository and then sorted
`A number of cellular counterparts to the retroviral onco­
`(Institute for Medical Research, Camden. N.J.). into nine
`genes have been identified and localized on specific chromo­
`fractions using a fluorescence-activated cell sorter as de­
`somes. The locations of several cellular oncogenes corre­
`scribed previously (20. 28). ONA samples were prepared
`spond well to breakpoints of chromosomal translocations
`from each fraction of the sorted chromosomes (7) and
`found in various cancers. For example, the c-myc gene on
`analyzed by Southern hybridization (21) using a DNA probe
`chromosome 8 is involved in translocations between chro­
`of a 440-base-pair (bp) Kpnl-Xbal restriction fragment (KX­
`mosome 8 and one of the chromosomes-2. 14. or 22-that
`DNA) generated from the c-erbB-2 genomic clone .U07.
`carries an immunoglobulin gene (5, 9. 12, 22). The resulting
`With the GM2324 cell line, a positive signal was observed for
`alteration in c-myc expression is suspected to be causally
`a fraction that corresponded mainly to chromosomes 16. 17.
`related to tumorigenesis (14).
`and 18 (data not shown). Another human lymphoblast cul­
`An avian erythroblastosis virus H strain contains an
`ture, GM3197, carries the reciprocal translocation, t(l7;22).
`oncogene, v-erbB, that replaces the env gene of an avian
`producing derivative chromosomes (17;22 and 22q-) that are
`leukosis virus (25). The nucleotide sequence analysis of the
`different in size from the normal homologs (6). Analysis of
`cloned v-erbB DNA and human epidermal growth factor
`this cell line revealed two positive signals, one in a fraction
`(EGF) receptor cDNA clones revealed that the v-erbB
`that contains normal chromosome 17 and one in a fraction
`protein corresponds to the carboxyl half of the human EGF
`that contains the derivative chromosome 17 ;22 (data not
`receptor, including the membrane-spanning domain (23, 24,
`shown). These results indicate that the c-erbB-2 gene is
`26). This strongly suggests that the 3' half of the chicken
`located on human chromosome 17.
`EGF receptor gene was transduced into the H strain of avian
`To localize the c-erbB-2 gene more precisely, we per­
`erythroblastosis virus. In addition to the EGF receptor gene,
`formed in situ hybridization experiments on chromosome
`we found another v-erbB-related gene. c-erbB-2. in the
`
`spreads prepared from phytohemagglutinin-stimulated pe­
`human genome. The c-erbB-2 gene is apparently distinct
`ripheral blood cultures (3, 29). The probe used for this
`from the EGF receptor gene, since transcripts of the two
`experiment was 3H-labeled pCER217 plasmid DNA, which
`genes differ from each other in length and because the amino
`is a c-erbB-2 cDNA clone containing a 2.7-kbp insert in the
`acid sequence predicted from the nucleotide sequence of
`Okayama-Berg vector (15). Analysis of 85 metaphase cells
`cloned c-erbB-2 gene is very similar to the corresponding
`revealed that 23.5% (20 of 85) of the silver grains were
`region of the EGF receptor (17). Recently, the neu
`located on chromosome 17. Of these 20 grains, 15 (75%)
`oncogene, active in a series of rat neuroblastoma (19), was
`found to be an erbB-related gene encoding an EGF receptor­
`were located on band q21-q22. and 11 grains in particular
`like protein (C. I. Bargmann, M.-C. Huang, and R. A.
`were in the region 17q21.l-21.3 (Fig. 1). A human version of
`Weinberg, Nature [London), in press). Comparison of the
`the neu oncogene was recently mapped on human chromo­
`nucleotide sequences and the deduced amino acid sequences
`some 17 at q21 (16). Since translocation between chromo­
`of human c-erbB-2 (T. Yamamoto, S. Ikawa, T. Akiyama,
`somes 15 and 17, t(l5;17) (q23;q21). is associated with acute
`K. Semba, N. Nomura, N. Miyajima. T. Saito. and K.
`promyelocytic leukemia (APL) (13). we examined DNA
`Toyoshima, Nature [London], in press) and rat neu
`from seven cases of APL for the possible involvement of the
`(Bargmann et al., in press) revealed a strong similarity
`c-erbB-2 gene in this leukemia. Using the 32P-labeled frag­
`between the two genes, which suggests that they are in fact
`ment prepared from pCER217 as a probe. we observed no
`the same gene.
`sign of rearrangement of the c-erbB-2 gene by Southern
`Metaphase chromosomes were prepared from two cell
`hybridization analysis (data not shown). Recently, the p53
`lines, GM2324 and GM3197, which were provided by the
`gene was also mapped to human chromosome 17 at bands
`17q21-q22. Although rearrangements of the p53 gene were
`not observed on Southern blotting of DNAs from APL cells
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`• Corresponding author.
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`FIG. l. Localization of the c-erbB-2 gene by in situ hybridization. (a) Photograph of a lymphocyte metaphase spread hybridized with the
`c-erbB-2 probe nick translated with (JH]dCTP (30 Ci/mmol) and [lH]dTTP (48 Ci/mmol). The specific activity of the probe was 3 x 107 cpm/µg
`of DNA. The chromosomal DNA was denatured on slides in 70% formamide-2x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)
`at 70°C for 2 min and then hybridized in a solution of 50% formamide-2x SSC-40 mM sodium phosphate (pH 7.0)-10% dextran
`sulfate-denatured salmon sperm DNA (100 µg/ml)-lx Denhardt solution for 16 h at 40°C. After hybridization. the slides were rinsed for 10
`min twice in 50% formamide-2 x SSC at 40°C and then several times in 0.2 x SSC at 37°C. Autoradiography was performed using half-strength
`Sakura NR-M2 emulsion (Konishiroku, Tokyo) for 3 weeks at 4°C. Chromosomes were Q banded using the double-staining method with
`quinacrine-mustard and Hoechst 33258 (27) and analyzed under a fluorescence microscope (left). Silver grains were detected by visible light
`and were identified on Q-banded chromosomes (right). (b) Distribution of 20 grains over chromosome 17.
`
`with t(l5;17), translocation of the p53 gene to chromosome
`15 was observed in three of three APL cases tested (8) by in
`situ hybridization. Thus, further analysis of APL cells with
`t(l5;17), which include in situ hybridization on chromosome
`
`spread using c-erbB-2-specific DNA probes, is antici­
`pated.
`Previously. we found that the c-erbB-2 gene is amplified in
`an adenocarcinoma of the salivary gland, although we could
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`a Pl. --- MKN-7
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`FIG. 2. Amplification and translocation of the c-erbB-2 gene in a gastric cancer cell line. MKN-7. (a) Amplification of the c-erbB-2 gene.
`High-molecular-weight DNAs were prepared from MKN-7 and human placental cells and digested with restriction endonuclease EcoRI. A
`nitrocellulose filter containing the EcoRI digests was probed with J?P-labeled KX-DNA (specific activity; 108 cpm/µg of DNA). Hybridization
`was carried out in a stringent condition (17). The filter contained, in lanes from left to right, placental DNA (10 µg) and MKN-7 DNA (10,
`5, 2.5, 1.25, 5/8, and 5/16 µg ). (b) Translocation of the c-erbB-2 gene. Metaphase spread was prepared from MKN-7 cells (left) and hybridized
`with 3H-labeled pCER217 DNA (right) as described in the legend to Fig. 1. Arrows indicate location of the c-erbB-2 gene on marker
`chromosomes.
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`placental cells, whereas the 4.8-kb yes mRNA in MKN-7
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`remains at the same level as in placental cells (Fig. 3).
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`There is growing evidence that gene amplification or
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`elevated expression of proto-oncogenes may play a role at
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`some stage during the neoplastic progress of certain tumors.
`The EGF receptor gene proto-erbB is frequently amplified
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`and overexpressed in glioblastomas (10, 11) and squamous
`cell carcinomas (2; T. Yamamoto, N. Kamata, H. Kawano,
`S. Shimizu, T. Kuroki, K. Toyoshima, K. Rikimaru, N.
`Nomura, R. Ishizaki, I. Pastan, S. Gamou, and N. Shimizu,
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`Cancer Res., in press). Amplification of the N-myc gene
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`correlates with stage III and IV neuroblastomas (1). An
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`elevated copy number of the c-erbB-2 gene was found in
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`MKN-7 cells, in a primary adenocarcinoma of the salivary
`gland (17), and in a primary mammary tumor (4), suggesting
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`a possible role of the c-erbB-2 gene in transforming epithelial
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`cells or in the malignancy of transformed epithelial cells.
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`We thank H. Kawano for excellent technical assistance and S.
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`Sasaki for help in preparing the manuscript. We also thank G.
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`Merlino (National Cancer Institute) for his critical reading of the
`manuscript.
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`-�4.8kb
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`LITERATURE CITED
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`2. Cowley, G., J. A. Smith, B. Gusterson, F. Hendler, and B.
`FIG. 3. Elevated expression of the c-erbB-2 gene in MKN-7
`
`Ozanne. 1984. The amount of EGF receptor is elevated on
`
`
`cells. Nitrocellulose filters containing poly(A)+ RNA (2 µ.g) of
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`p. 5-10. In J. Feramisco, B. Ozanne,
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`MKN-7 cells (lane 2) and placental cells (lane 1) were hybridized
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`with 32P-labeled KX-DNA (panel a) under stringent conditions (17)
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`
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`labeled with [a-32P)dCTP by nick translation to a specific activity of
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`
`KX-DNA. An MKN-7 cell line established from an adeno­
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`
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`
`and M. Oren. 1985. Translocation
`of the p53 gene in t(15;17)
`in
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`
`
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`
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`the grains were clustered (average, 4 to 5 grains) on a marker
`
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`
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`
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`
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`amplification, is synthesized in MKN-7 cells relative to
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