MOLECULAR AND CELLULAR BIOLOGY, Mar. 1986. p. 955-958
`0270-7306/86/030955-04$02.00/0
`Copyright © 1986, American Society for Microbiology
`
`Vol. 6, No. 3
`
`Localization of a Novel v-erbB-Related Gene, c-erbB-2, on Human
`Chromosome 17 and Its Amplification in a Gastric Cancer Cell Line
`SHIN-ICHI FUKUSHIGE,1 KEN-ICHI MATSUBARA,1 MICHIHIRO YOSHIDA,' MOTOMICHI SASAKI,2
`TOSHIMITSU SUZUKI,3 KENTARO SEMBA,4 KUMAO TOYOSHIMA,4 AND TADASHI YAMAMOTO4*
`Institute for Moleciular and Celluilar Biology, Osaka University, Suiita, Osaka 565,1 Chromosome Research Unit,
`Hokkaido University, N. 10-W.8, Sapporo 060,2 School of Medicine, Niigata University, Niigata 951,3 and The Institiute
`of Medical Science, The University of Tokyo, Minato-ku Tokyo J08,4 Japan
`Received 17 September 1985/Accepted 3 December 1985
`
`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, hybridization 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 human cancer.
`
`A number of cellular counterparts to the retroviral onco-
`genes have been identified and localized on specific chromo-
`somes. The locations of several cellular oncogenes corre-
`spond well to breakpoints of chromosomal translocations
`found in various cancers. For example, the c-myc gene on
`chromosome 8 is involved in translocations between chro-
`mosome 8 and one of the chromosomes-2, 14, or 22-that
`carries an immunoglobulin gene (5, 9, 12, 22). The resulting
`alteration in c-myc expression is suspected to be causally
`related to tumorigenesis (14).
`An avian erythroblastosis virus H strain contains an
`oncogene, v-erbB, that replaces the enm gene of an avian
`leukosis virus (25). The nucleotide sequence analysis of the
`cloned v-erbB DNA and human epidermal growth factor
`(EGF) receptor cDNA clones revealed that the v-erbB
`protein corresponds to the carboxyl half of the human EGF
`receptor, including the membrane-spanning domain (23, 24,
`26). This strongly suggests that the 3' half of the chicken
`EGF receptor gene was transduced into the H strain of avian
`erythroblastosis virus. In addition to the EGF receptor gene,
`we found another v-erbB-related gene, c-erbB-2, in the
`human genome. The c-erbB-2 gene is apparently distinct
`from the EGF receptor gene, since transcripts of the two
`genes differ from each other in length and because the amino
`acid sequence predicted from the nucleotide sequence of
`cloned c-erbB-2 gene is very similar to the corresponding
`region of the EGF receptor (17). Recently, the neu
`oncogene, active in a series of rat neuroblastoma (19), was
`found to be an erbB-related gene encoding an EGF receptor-
`like protein (C. I. Bargmann, M.-C. Huang, and R. A.
`Weinberg, Nature [London], in press). Comparison of the
`nucleotide sequences and the deduced amino acid sequences
`of human c-erbB-2 (T. Yamamoto, S. Ikawa, T. Akiyama,
`K. Semba, N. Nomura, N. Miyajima, T. Saito, and K.
`Toyoshima, Nature [London], in press) and rat neii
`(Bargmann et al., in press) revealed a strong similarity
`between the two genes, which suggests that they are in fact
`the same gene.
`Metaphase chromosomes were prepared from two cell
`lines, GM2324 and GM3197, which were provided by the
`
`* Corresponding author.
`
`Human Genetic Mutant Cell Repository and then sorted
`(Institute for Medical Research, Camden, N.J.), into nine
`fractions using a fluorescence-activated cell sorter as de-
`scribed previously (20, 28). DNA samples were prepared
`from each fraction of the sorted chromosomes (7) and
`analyzed by Southern hybridization (21) using a DNA probe
`of a 440-base-pair (bp) KpnI-XbaI restriction fragment (KX-
`DNA) generated from the c-erbB-2 genomic clone X107.
`With the GM2324 cell line, a positive signal was observed for
`a fraction that corresponded mainly to chromosomes 16, 17,
`and 18 (data not shown). Another human lymphoblast cul-
`ture, GM3197, carries the reciprocal translocation, t(17;22),
`producing derivative chromosomes (17;22 and 22q-) that are
`different in size from the normal homologs (6). Analysis of
`this cell line revealed two positive signals, one in a fraction
`that contains normal chromosome 17 and one in a fraction
`that contains the derivative chromosome 17;22 (data not
`shown). These results indicate that the c-erbB-2 gene is
`located on human chromosome 17.
`To localize the c-erbB-2 gene more precisely, we per-
`formed in situ hybridization experiments on chromosome
`spreads prepared from phytohemagglutinin-stimulated pe-
`ripheral blood cultures (3, 29). The probe used for this
`experiment was 3H-labeled pCER217 plasmid DNA, which
`is a c-erbB-2 cDNA clone containing a 2.7-kbp insert in the
`Okayama-Berg vector (15). Analysis of 85 metaphase cells
`revealed that 23.5% (20 of 85) of the silver grains were
`located on chromosome 17. Of these 20 grains, 15 (75%)
`were located on band q21-q22, and 11 grains in particular
`were in the region 17q21.1-21.3 (Fig. 1). A human version of
`the neu oncogene was recently mapped on human chromo-
`some 17 at q21 (16). Since translocation between chromo-
`somes 15 and 17, t(15;17) (q23;q21), is associated with acute
`promyelocytic leukemia (APL) (13), we examined DNA
`from seven cases of APL for the possible involvement of the
`c-erbB-2 gene in this leukemia. Using the 32P-labeled frag-
`ment prepared from pCER217 as a probe, we observed no
`sign of rearrangement of the c-erbB-2 gene by Southern
`hybridization analysis (data not shown). Recently, the p53
`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
`
`955
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`b
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`el%
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`12 0
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`II 2
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`2,1 1
`21
`212
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`000
`00000000
`0000
`71 00
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`3~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~17
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`q 22
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`0
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`FIG. 1. 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 [3H]dCTP (30 Ci/mmol) and [3H]dTTP (48 Ci/mmol). The specific activity of the probe was 3 x 107 cpm/p.g
`of DNA. The chromosomal DNA was denatured on slides in 70% formamide-2x SSC (lx 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)-105O% dextran
`sulfate-denatured salmon sperm DNA (100 pg/ml)-1 x Denhardt solution for 16 h at 40°C. After hybridization, the slides were ri,nsed 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.
`
`17
`
`with t(15;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(15;17), which include in situ hybridization on chromosome
`
`spread using c-erbB-2-specific DNA probes, is
`pated.
`Previously, we found that the c-erbB-2 gene is amplified in
`an adenocarcinoma of the salivary gland, although we could
`
`antici-
`
`a Pl.
`
`.MKN-7
`
`...
`
`kbp
`-~~~~~~~u23 _t
`L
`_ d
`*
`,15
`
`4,4
`
`1/32
`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 EcoRl. A
`nitrocellulose filter containing the EcoRI digests was probed with 32P-labeled KX-DNA (specific activity; 108 cpm/,ug of DNA). Hybridization
`was carried out in a stringent condition (17). The filter contained, in lanes from left to right, placental DNA (10 ,ug) and MKN-7 DNA (10,
`5, 2.5, 1.25, 5/8, and 5/16 jg ). (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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`PETITIONER'S EXHIBITS
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`VOL. 6, 1986
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`1 2
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`4......fffw..
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`i-4.6 kb
`
`b 1 2
`0w
`_a4.8kb
`
`b
`
`FIG. 3. Elevated expression of the c-erbB-2 gene in MKN-7
`cells. Nitrocellulose filters containing poly(A)+ RNA (2 jig) of
`MKN-7 cells (lane 2) and placental cells (lane 1) were hybridized
`with 32P-labeled KX-DNA (panel a) under stringent conditions (17)
`or with 32P-labeled v-yes probe (panel b) prepared from recombinant
`plasmid pYS2 (18) in a relaxed condition (17). DNA probes were
`labeled with [c-32P]dCTP by nick translation to a specific activity of
`1 x 101 to 2 x 108 cpm/4Lg of DNA.
`
`not examine the level of RNA transcripts due to the limited
`amount of tissue (17). To examine the possible involvement
`of the c-erbB-2 gene in other types of tumors, we examined
`the DNA of 30 human cancerous cell lines for the altered
`structures of this gene, i.e., by gene amplification or rear-
`rangement by Southern hybridization with the 32P-labeled
`KX-DNA. An MKN-7 cell line established from an adeno-
`carcinoma of the stomach was found to contain an elevated
`copy number of the c-erbB-2 gene. The degree of amplifica-
`tion was estimated by serial dilution of MKN-7 DNA to be
`about 30-fold relative to placental cell DNA (Fig. 2a). We
`also observed an additional EcoRI fragment of about 23-kbp
`that is specific to MKN-7 cells. This sequence is amplified to
`the same extent as are the other two EcoRI fragments. In
`situ hybridization analysis of MKN-7 metaphase spreads
`showed that the c-erbB-2 gene is located on at least two
`marker chromosomes other than chromosome 17 and that
`the grains were clustered (average, 4 to 5 grains) on a marker
`chromosome (Fig. 2b). Therefore, we assume that chromo-
`somal translocation in MKN-7 could lead to the fusion of a
`part of the c-erbB-2 gene to an unidentified DNA, which
`resulted in the generation of the 23-kbp EcoRI fragment.
`Poly(A)+ RNAs were prepared from MKN-7 and placental
`cells. Northern hybridization of these RNAs with 32P-labeled
`KX-DNA probe showed that an increased amount of c-erbB-
`2 mRNA (4.6 kb), in proportion to the degree of the gene
`amplification, is synthesized in MKN-7 cells relative to
`
`NOTES
`
`957
`
`placental cells, whereas the 4.8-kb yes mRNA in MKN-7
`remains at the same level as in placental cells (Fig. 3).
`There is growing evidence that gene amplification or
`elevated expression of proto-oncogenes may play a role at
`some stage during the neoplastic progress of certain tumors.
`The EGF receptor gene proto-erbB is frequently amplified
`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,
`Cancer Res., in press). Amplification of the N-myc gene
`correlates with stage III and IV neuroblastomas (1). An
`elevated copy number of the c-erbB-2 gene was found in
`MKN-7 cells, in a primary adenocarcinoma of the salivary
`gland (17), and in a primary mammary tumor (4), suggesting
`a possible role of the c-erbB-2 gene in transforming epithelial
`cells or in the malignancy of transformed epithelial cells.
`
`We thank H. Kawano for excellent technical assistance and S.
`Sasaki for help in preparing the manuscript. We also thank G.
`Merlino (National Cancer Institute) for his critical reading of the
`manuscript.
`
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