Proc. Nati. Acad. Sci. USA
`Vol. 82, pp. 6497-6501, October 1985
`Biochemistry
`
`A v-erbB-related protooncogene, c-erbB-2, is distinct from the
`c-erbB-1/epidermal growth factor-receptor gene and is
`amplified in a human salivary gland adenocarcinoma
`(src gene family/kinase family/gene amplification/cancer)
`
`KENTARO SEMBA*, NOBUYUKI KAMATA*t, KUMAO TOYOSHIMA*, AND TADASHI YAMAMOTO*
`*The Institute of Medical Science, The University of Tokyo 4-61, Shirokanedai, Minato-ku, Tokyo 108, Japan; and tTokyo Medical and Dental University,
`Faculty of Dentistry 1-545, Yushima, Bunkyo-ku, Tokyo 113, Japan
`Communicated by Takashi Sugimura, June 10, 1985
`
`From a human genomic library, we obtained
`ABSTRACT
`six v-erbB-related DNA clones. A DNA probe prepared from
`one of the clones, X107, hybridized to EcoRI fragments of 6.4
`and 13 kilobase pairs of human DNA. Neither of these
`fragments was amplified in A431 vulva carcinoma cells, in
`which the gene encoding the epidermal growth factor receptor
`is amplified. In addition, the probe from X107 hybridized with
`a single, 4.8-kilobase poly(A)+ RNA species and did not react
`with EGF receptor mRNA. Thus, we conclude that clone X107
`represents a v-erbB-related gene (c-erbB-2) that is distinct from
`the EGF receptor gene. In contrast, the other five clones were
`shown to represent the EGF receptor gene (c-erbB-1). Partial
`nucleotide sequence analysis of the X107 insert showed that this
`clone contained at least seven putative exons and that six of
`them could encode the kinase domain characteristic of protein
`products of the src oncogene family. Southern blot analysis
`showed close similarity of the restriction patterns of the rat
`c-erbB-2 gene and the rat neu oncogene, suggesting possible
`involvement of c-erbB-2 in human cancer. In fact, =30-fold
`amplification of c-erbB-2 was observed in a human adenocar-
`cinoma of the salivary gland.
`
`At least 19 genes have been identified as retroviral oncogenes
`that are responsible for inducing tumors in vivo and trans-
`forming cells in vitro (1). Ten of them apparently encode
`transforming proteins that share a kinase domain homologous
`to that of pp60src, a tyrosine-specific protein kinase. The
`cellular cognate, encoded by the c-src gene, also exhibits
`tyrosine-specific kinase activity. Of particular interest is the
`fact that tyrosine-specific kinases are also encoded by the
`genes for several receptors for polypeptide growth factors,
`including the receptors for epidermal growth factor (EGF)
`(2), platelet-derived growth factor (PDGF) (3), insulin (4), and
`insulin-like growth factor I (5). This implies a possible link
`between the action of the growth factor-receptor complex
`and the oncogene product with tyrosine-specific kinase
`activity. In fact, recent analysis of the v-erbB gene and the
`EGF receptor gene indicated that the v-erbB gene is a part of
`the EGF receptor gene and codes for the internal domain and
`transmembrane portion ofthe receptor (6-8). These findings,
`together with the extensive identity of the amino acid
`sequences of the v-sis protein and platelet-derived growth
`factor (9, 10), suggest that some viral oncogene products
`mimic the action of the polypeptide growth factor-receptor
`complex in activating a cellular pathway involved in cell
`proliferation.
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertisement"
`in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`We examined details of the relation between the v-erbB
`gene and the EGF receptor gene and the possible involve-
`ment of this gene in human cancer. During this study we
`identified two v-erbB-related genes, c-erbB-1 and c-erbB-2, in
`the human genome.
`
`MATERIALS AND METHODS
`Cells and Tissues. A431 vulva carcinoma cells and human
`embryo fibroblasts were maintained in Dulbecco's modified
`Eagle's medium with 10% fetal calf serum. K562 chronic
`myelogenous leukemia cells and MT2 adult T-cell leukemia
`cells were maintained in RPMI 1640 medium with 10% fetal
`calf serum. Mouse FM3A cells were from M. C. Yoshida
`(Hokkaido University) and primary tumors were provided by
`K. Rikimaru (Tokyo Medical and Dental University).
`Isolation of Clones. A human genomic library was con-
`structed from placental DNA as described (11). The library
`was screened for the v-erbB-related sequence by plaque-
`hybridization as described (12) in 30% (vol/vol) formamide/
`4x NaCl/Cit (lx is 0.15 M NaCI/15 mM sodium citrate)/50
`mM Hepes, pH 7.0/denatured salmon sperm DNA (20
`,ug/ml)/10x Denhardt's solution (lx is 0.02% polyvinyl-
`pyrrolidone/0.02% Ficoll/0.02% bovine serum albumin) at
`42°C for 16 hr. After hybridization, the filters were washed
`with 2x NaCl/Cit/0.1% NaDodSO4 at room temperature and
`then with 0.6x NaClI/Cit/0.1% NaDodSO4 at 50°C. Plaques
`were purified by successive plaque-hybridization. The probe
`used was a 1.7-kilobase-pair (kbp) Sst I-Stu I DNA fragment
`that represents the v-erbB gene of avian erythroblastosis
`virus strain H (6); it was labeled with [a-32P]dCTP by
`nick-translation (13) to a specific activity of 2 x 108 cpm/,g
`of DNA. Phage DNAs were prepared as described (14).
`Nucleotide Sequence Analysis. The nucleotide sequence
`was determined by the procedure of Maxam and Gilbert (15)
`and the dideoxy chain-termination method (16, 17) in con-
`junction with bacteriophage M13 mpl9 (18).
`Blot-Hybridization Analysis of DNA and RNA. High mo-
`lecular weight DNAs were prepared from chicken blood,
`human embryo fibroblasts, mouse FM3A cells, rat spleen,
`and primary tumors. The DNAs (10 jig per lane) were
`digested with restriction endonucleases under the conditions
`recommended by suppliers (Takara Shuzo) and fractionated
`by electrophoresis in 1% agarose gels. The fragments were
`subjected to Southern blot hybridization (19) at 42°C for 16 hr
`in 4x NaCI/Cit/SO mM Hepes, pH 7.0/lOx Denhardt's
`solution/denatured salmon sperm DNA (20 gg/ml)/30%o
`(relaxed conditions) or 50% (stringent conditions) formam-
`ide. After hybridization, the filters were washed with 2x
`
`Abbreviations: EGF, epidermal growth factor; kb, kilobase(s); kbp,
`kilobase pair(s).
`
`6497
`
`PETITIONER'S EXHIBITS
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`Exhibit 1042 Page 1 of 5
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`

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`6498
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`Biochemistry: Semba et al.
`
`Proc. Natl. Acad. Sci. USA 82 (1985)
`
`'It
`
`1
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`
`X E K
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`
`S S S
`I
`
`x
`
`X107
`
`Identification and cloning of the v-erbB-related sequence that is distinct from the EGF receptor gene. (a) Hybridization with the
`FIG. 1.
`v-erbB probe. DNAs prepared from A431 cells (lanes 1, 3, and 5) and placenta (lanes 2, 4, and 6) were digested with EcoRI (lanes 1 and 2), Sac
`I (lanes 3 and 4), or Pvu II (lanes 5 and 6). Because the placental DNA was contaminated with RNA, samples in lanes 2, 4, and 6 contained
`less DNA than those in lanes 1, 3, and 5. The digests were subjected to Southern blot hybridization using a v-erbB probe under the relaxed
`conditions described in Materials and Methods. Autoradiographs obtained by longer exposures of lanes 2, 4, and 6 are shown to the right of
`the respective lanes. Arrowheads indicate positions of fragments of HindIII-digested X DNA used as standards. Stars indicate bands that were
`not amplified in the A431 samples. (b) Restriction map of the X107 insert. E, K, S, and X represent restriction sites of EcoRI, Kpn I, Sac I,
`and Xba I, respectively. The thick line indicates the Kpn I-Xba I fragment (KX fragment) used as a specific probe of the c-erbB-2 gene. (c)
`Hybridization with the KX fragment. The same filter represented in a was washed and then hybridized with the KX probe under the stringent
`conditions described in Materials and Methods.
`
`NaCI/Cit/0.1% NaDodSO4 and then with either 0.2x
`NaCl/Cit/0.1% NaDodSO4 (stringent) or 0.6x NaCl/Cit/
`0.1% NaDodSO4 (relaxed). RNAs were prepared by the
`guanidinium isothiocyanate/cesium chloride method (20).
`Poly(A)+ RNA selected by oligo(dT)-cellulose (P-L Biochem-
`icals type 7) column chromatography was denatured with
`50% formamide/2.2 M formaldehyde and 5 ,ug of each RNA
`sample was subjected to electrophoresis in 1% agarose gel
`containing 2.2 M formaldehyde (21). RNAs on the gel were
`transferred directly to a nitrocellulose filter and subjected to
`blot hybridization under stringent conditions, as described
`(22). The DNA probes used for blot hybridization were
`v-erbB DNA (described above), an EGF receptor cDNA
`clone (pE7) (22), and c-erbB-2-specific DNA (described
`below).
`
`RESULTS
`Isolation of a Human v-erbB-Related Sequence Distinct from
`the EGF Receptor Gene. High molecular weight DNAs
`prepared from A431 cells and human placenta were digested
`with either EcoRI, Sac I, or Pvu II, and the digests were
`analyzed by Southern blot hybridization under relaxed con-
`ditions with the v-erbB probe, which covers almost 90% of
`the v-erbB gene. Most of the v-erbB-related sequences were
`amplified in A431 cells at least 20-fold compared with those
`in placenta (Fig. la). These sequences were observed as 8.1-,
`5.9-, 5.4-, and 3.5-kbp EcoRI fragments; 20-, 4.7-, 3.3-, 1.7-,
`0.9-, and 0.5-kbp Sac I fragments; and 6.6-, 3.3-, 2.9-, 2.3-,
`1.9-, and 0.9-kbp Pvu II fragments. The amplified sequences
`were of the EGF receptor gene as reported (8, 23). However,
`we observed several restriction fragments that hybridized to
`the v-erbB probe but that were not amplified in A431 cells (13-
`and 6.4-kbp EcoRI fragment, a 6.9-kbp Sac I fragment, and
`a 5.1-kbp Pvu II fragment, marked with stars in Fig. la). This
`suggested that besides the EGF receptor gene, there may be
`
`another v-erbB-related gene, from which the unamplified
`restriction fragments are generated.
`We searched a human genomic library for v-erbB-related
`sequences, using the v-erbB probe, under the relaxed hy-
`bridization and washing conditions, and isolated six indepen-
`dent clones. Restriction map analysis showed that all the
`cloned inserts except the X107 insert represented one gene,
`c-erbB-1. In these clones, we identified four EcoRI fragments
`as exon-containing sequences by the criterion that they
`hybridized to the v-erbB probe. All these fragments were
`
`1
`
`2
`
`3
`
`4
`
`12341
`
`-
`
`4.8 kb
`
`U
`
`kI-)
`-1().()
`- 5.6
`
`_a-- 2.9
`
`Expression of the EGF receptor gene and the c-erbB-2
`FIG. 2.
`gene in human cells. Duplicate samples of poly(A)+ RNA were
`subjected to blot hybridization with the 32P-labeled KX fragment (a)
`or with the 32P-labeled EGF receptor cDNA clone pE7 (22)(b). Sizes
`of RNA species that hybridize with the probes are given. Chicken
`ribosomal RNAs (28S, about 4.8 kb, and 18S, about 2.0 kb) and Rous
`sarcoma virus RNA (39S, about 10.0 kb) served as size standards.
`RNAs were isolated from human placenta (lanes 1), A431 cells (lanes
`2), human embryo fibroblasts (lanes 3), MT2 cells (lanes 4), and K562
`cells (lanes 5).
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1042 Page 2 of 5
`
`

`
`Biochemistry: Semba et al.
`
`Proc. Natl. Acad. Sci. USA 82 (1985)
`
`6499
`
`E
`
`5,1
`
`li U*
`
`I
`
`I
`
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`
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`
`c-erbB-2
`EGFR
`
`c-erbB-2
`EGFR
`
`c-erbB-2
`EGFR
`
`c-erbB-2
`EFGR
`
`c-erbB-2
`EGFR
`
`c-erbB-2
`EGFR
`
`c-erbB-2
`EGFR
`
`c-erbB-2
`EGFR
`
`c-erbB-2
`EGFR
`
`c-erbB-2
`EGFR
`
`c-erbB-2
`EGFR
`
`30
`1
`60
`GlyIleTrpIleProAspGlyGluAsnValLysIleProValAlaIleLysValLeuArgGluAsnThrSerProLysAlaAsnLys
`agGGCATCTGGATCCCTGATGGGGAGAATGTGAAAATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACCTCCCCCAAAGCCAACAAA
`GCA A T
`G
`G
`G AA
`CC T
`A
`A
`A T
`AC
`A A T
`90
`120
`150
`GluAlaTyrValMetAlaGlyValGlySerProTyrValSerArgLeuLeuGlyIleCysLeuThrSer
`GluIleLeuAsp
`GAAATCTTAGACgt-----agGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCTCCCGCCTTCTGGGCATCTGCCTGACATCC
`CA2%O
`CC1W0
`CC C G G
`C
`C
`A AA
`C
`G 240
`ThrValGlnLeuValThrGlnLeuMetProTyrGlyCysLeuLeuAspHisValArgGluAsnArgGlyArgLeuGlySerGlnAspLeu
`ACGGTGCAGCTGGTGACACAGCTTATGCCCTATGGCTGCCTCTTAGACCATGTCCGGGAAAACCGCGGACGCCTGGGCTCCCAGGACCTG
`TC
`C G
`T
`C
`AAA ACAATA T
`C
`T
`A CA C
`G
`C
`300
`330
`270
`GlyMetSerTyrLeuGluAspValArgLeuValHisArgAspLeuAlaAlaArg
`LeuAsnTrpCysMetGlnl eAlaLys
`CTGAACTGGTGTATGCAGATTGCCAAGgt-----agGGGATGAGCTACCTGGAGGATGTGCGGCTCGTACACAGGGACTTGGCCGCTCGG
`C
`A
`T
`CCGT CT G
`G
`C C
`C
`A
`C A
`CA
`G
`C
`390
`360
`420
`AsnValLeuValLysSerProAsnHisValLysIleThrAspPheGlyLeuAlaArgLeuLeuAspIleAspGluThrGluTyrHisAla
`AACGTGCTGGTCAAGAGTCCCAACCATGTCAAAATTACAGACTTCGGGCTGGCTCGGCTGCTGGACATTGACGAGACAGAGTACCATGCA
`C
`T
`T
`CAAA
`GTGCG
`A
`A
`A
`G A CA GC G
`G
`A
`
`480
`510
`450
`ValProIleLysTrpMetAlaLeuGluSerIleLeuArgArgArgPheThrHisGlnSerAspValTrp
`AspGlyGlyLys
`GATGGGGGCAAGgt-----agGTGCCCATCAAGTGGATGGCGCTGGAGTCCATTCTCCGCCGGCGGTTCACCCACCAGAGTGATGTGTGG
`AT
`A
`A
`T A A A AATC AT
`C
`T
`A
`A
`A
`5
`lyValThrValTrpGluLeuMetThrPheGlyAlaLysProTyrAspGlyIleProAlaArgGluIleProAsp
`SerTyrG
`AGTTATGgt-----agGTGTGACTGTGTGGGAGCTGATGACTTTTGGGGCCAAACCTTACGATGGGATCCCAGCCCGGGAGATCCCTGAC
`G
`C T
`T
`C
`AT
`G A
`T
`C
`A
`T
`A C
`T CTC
`C
`C
`
`630
`600
`660
`LeuLeuGluLysGlyGluArgLeuProGlnProProIleCysThrIleAspValTyrMetIleMetValLysC
`ysTrpMet
`CTGCTGGAAAAGGGGGAGCGGCTGCCCCAGCCCCCCATCTGCACCATTGATGTCTACATGATCATGGTCAAATgt-----agGTTGGATG
`G
`G A A A C C T
`A T
`C
`C
`A
`A C
`750*
`690
`750
`IleAspSerGluCysArgProArgPheArgGluLeuValSerGluPheSerArgMetAlaArgAspProGlnArgPheValValIleGln
`ATTGACTCTGAATGTCGGCCAAGATTCCGGGAGTTGGTGTCTGAATTCTCCCGCATGGCCAGGGACCCCCAGCGCTTTGTGGTCATCCAG
`AG 780T
`CA
`A CATC
`AAA
`ACC T
`T
`C
`TA
`G A
`A
`~~~~~~~~~~~~~840
`810
`780
`AsnGluAspLeuGlyProAlaSerProLeuAspSerThrPheTyrArgSerLeuLeuGluAspAspAspMetGlyAspLeu
`gt-----agAATGAGGACTTGGGCCCAGCCAGTCCCTTGGACAGCACCTTCTACCGCTCACTGCTGGAGGACGATGACATGGGGGACCTG
`CATTTGC A
`TACA
`TC
`A
`TG C
`A
`T A
`A
`AC
`AAGAA
`G
`GGGG
`870
`ValAspAlaGluGluTyrLeuValProGlnGlnGlyPhePhe
`GTGGATGCTGAGGAGTATCTGGTACCCCAGCAGGGCTTCTTCT--
`C
`CA C
`A
`A
`C
`C
`
`Nucleotide sequence of the exons for the kinase domain ofc-erbB-2. (a) Putative exons in the X107 clone are indicated by thick vertical
`FIG. 3.
`bars. The two EcoRI (E) sites are shown by vertical lines. (b) Nucleotide sequence of the putative exons. Restriction fragments that hybridized
`to v-erbB DNA were sequenced. The exons were defined by the splicing consensus sequence and by the high homology of the deduced amino
`acid sequences to those of the EGF receptor gene EGFR. The nucleotide sequence of putative exons of c-erbB-2 (in capital letters) is compared
`with that of the EGF receptor gene (8). Only nonidentical nucleotides are shown for the EGF receptor gene. Intron sequences at the splicing
`sites that flank the exons are shown in lowercase letters. The splicing donor site of the last exon was not identified. The predicted amino acid
`sequence is shown above the nucleotide sequence.
`
`amplified in A431 cells (data not shown), suggesting that the
`c-erbB-1 gene is the same as the EGF receptor gene.
`A restriction map of the X107 DNA insert is shown in Fig.
`lb. The 440-bp Kpn I-Xba I fragment (KX fragment) was
`used as a probe in the following hybridization experiments.
`This fragment did not contain human repetitive sequences
`and hybridizes with the v-erbB probe under the relaxed
`conditions. The v-erbB probe on the filter represented in Fig.
`la was washed off and the filter was rehybridized with the
`32P-labeled KX fragment. Fig. lc shows that the KX probe
`reacted with the restriction fragments (13- and 6.4-kbp EcoRI
`fragments, 6.9-kbp Sac I fragment, and 5.1-kbp Pvu II
`fragment) that were not amplified in A431 cells, suggesting
`that the X107 insert does not represent the EGF receptor
`gene. Therefore, we tentatively concluded that the X107
`insert represented a v-erbB-related gene (c-erbB-2) that
`differs from the EGF receptor gene.
`Then we examined the expression of the v-erbB-related
`genes. RNAs were prepared from human cell lines (K562,
`MT2, and A431 cells), human embryo fibroblasts, and human
`placenta. Hybridization was carried out under stringent
`conditions (Fig. 2 a and b). Poly(A)+ RNA samples were
`subjected to blot-hybridization analysis. As reported previ-
`
`ously, most transcripts detected with the EGF receptor
`cDNA probe were of 10 kilobases (kb) and 5.6 kb and were
`apparently overproduced in A431 cells (Fig. 2b and refs. 8
`and 23). In addition, variant 2.9-kb mRNA was detected in
`A431 cells, as reported previously (8, 23). On the other hand,
`the mRNA that hybridized with the KX probe was a single
`species of about 4.8 kb (Fig. 2a). The level of expression of
`this mRNA was virtually the same in A431 cells as in placenta
`and was slightly higher in human embryo fibroblasts than in
`other cells. Interestingly, transcription of the EGF receptor
`gene and c-erbB-2 genes was not detected in the leukemic cell
`lines K562 and MT2. The above results strongly indicate that
`the human genome contains two v-erbB-related genes, the
`c-erbB-I/EGF receptor gene and the c-erbB-2 gene.
`c-erbB-2 Encodes a Protein with a Kinase Domain. We
`determined the nucleotide sequence of the X107 insert that
`hybridized with the v-erbB DNA and identified seven puta-
`tive exons flanked by a consensus sequence of splicing
`junctions (data not shown). Fig. 3 shows that the nucleotide
`sequences of all seven exons (total, 885 bp) were highly
`homologous with the corresponding regions of the cDNA
`clone for the EGF receptor (74%). Only one reading frame
`deduced from putative exons was not interrupted by termi-
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1042 Page 3 of 5
`
`

`
`6500
`
`Biochemistry: Semba et al.
`
`Proc. Natl. Acad. Sci. USA 82 (1985)
`
`c-erbB-2
`EGFR
`
`GIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQL-60
`S DN H C
`E
`E
`K
`L
`A
`
`c-erbB- 2
`EGFR
`
`VTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSP- 120
`T
`HKDNI
`Y
`N
`Y
`I
`F
`R
`V
`
`c-erbB-2
`EGFR
`
`c-erbB-2
`EGFR
`
`c-erbB-2
`EGFR
`
`*
`
`***
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`NHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELM-180
`H IY
`GAE K
`E
`Q
`K
`
`****
`
`*
`
`*
`
`*
`
`**
`
`TFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEF-240
`II
`S
`ADS
`S
`SSI
`K
`
`*
`
`*
`
`*
`
`** *
`
`SRMARDPQRFVVIQ-NEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFF
`D
`YL
`I
`N
`D V
`T
`A MDEE
`GD RMHLP
`K
`
`FIG. 4.
`Comparison of the deduced amino acid sequences of c-erbB-2 and the EGF receptor (EGFR). The standard one-letter abbreviations
`are used. Only nonmatching amino acids are shown for the EGF receptor. The kinase domain of EGF receptor is shown as the amino acid
`sequence between positions 705 and 937 (8). Asterisks indicate residues common to the protein products of the v-abl, v-erbB, v-fgr, v-fms, v-fps,
`v-ros, v-src, and v-yes genes.
`
`expression of the c-erbB-2 gene in a cell line (MKN-7)
`established from human gastric cancer (data not shown).
`
`nation codons. The amino acid sequence deduced from this
`open-reading frame suggested that these exons in c-erbB-2
`could encode a polypeptide characteristic of the kinase
`domain (residues 1-233 in Fig. 4) that showed high homology
`DISCUSSION
`with the EGF receptor kinase domain (82%). The amino acid
`sequence of the kinase domain of the c-erbB-2 product is
`There are two v-erbB-related genes in the human genome: the
`highly homologous to that ofthe v-erbB product and distantly
`c-erbB-J/EGF receptor gene and the c-erbB-2 gene. The
`related to those of the protein products of other members of
`c-erbB-2 gene is apparently not a pseudogene of the EGF
`the src gene family (Table 1).
`receptor gene because it consists of both exons and introns
`Conservation of the c-erbB-2 Gene in Vertebrates. High
`and is transcribed in a cell-type-specific manner. We could
`molecular weight DNAs were prepared from chicken blood,
`not find any termination codon interrupting the reading frame
`mouse FM3A cells, and rat spleen. The DNAs were digested
`of the putative kinase domain coded by c-erbB-2. In addition,
`with EcoRI and subjected to Southern hybridization with the
`we have shown that the c-erbB-2 gene is conserved in
`32P-labeled KX probe. Since the hybridization conditions
`chickens, mice, rats, and humans, indicating that this gene is
`were stringent enough to avoid detection ofthe EGF receptor
`not confined to the human genome and probably fulfills an
`gene with the KX probe (see Fig. 5a, lane 1), all the fragments
`indispensable function. Obviously, efforts to identify a pro-
`shown in Fig. 5 are specific to the c-erbB-2 gene. Thus, we
`tein product of the c-erbB-2 gene are required.
`conclude that c-erbB-2 is conserved in vertebrates.
`The amino acid sequence deduced from the nucleotide
`The possible relation between c-erbB-2 and the recently
`sequence ofc-erbB-2 exons in A107 suggests that the c-erbB-2
`gene may code for a tyrosine-specific protein kinase. The
`discovered neu oncogene was examined by Southern blot
`putative kinase domain encoded by the c-erbB-2 gene shows
`hybridization. The 32P-labeled KX probe hybridized with a
`82% homology with that of the EGF receptor and the v-erbB
`rat EcoRI fragment of more than 23 kbp and with a 4.4-kbp
`protein. The eight protein products of the src family show
`fragment generated by digestions ofthe DNA with EcoRI and
`tyrosine kinase activity in vitro. All the amino acid residues
`BamHI (Fig. 5). These fragments are the same sizes as the
`that are common to these eight proteins are also conserved in
`respective restriction fragments of the neu oncogene (24).
`the c-erbB-2 gene product, strongly supporting the idea that
`Association of Amplification of the c-erbB-2 Gene with a
`this gene could encode a protein kinase. Our nucleotide
`Primary Human Tumor. Next we examined whether the
`sequence data further show that the c-erbB-2 protein carries
`c-erbB-2 gene is amplified in human cancers. High molecular
`an amino acid sequence (residues 234-295) that is 65%
`weight DNAs were isolated from human placenta, A431 cells,
`homologous to the corresponding portion of the EGF recep-
`and several primary tumors (one neuroblastoma, two
`tor. Since the deduced amino acid sequence did not contain
`epidermoid cell carcinomas, and one adenocarcinoma of the
`a putative transmembrane portion, it is not known whether
`salivary gland). These DNAs were digested with EcoRI and
`c-erbB-2 encodes a receptor-like protein or only a polypep-
`analyzed by Southern blot hybridization with the KX probe.
`tide similar to the intracellular portion of the EGF receptor.
`Two EcoRI fragments (13 and 6.4 kbp) were identified in all
`Analysis of the amino-terminal portion of the protein is
`the DNA samples and were amplified about 30-fold in the
`necessary to determine whether it has a transmembrane
`adenocarcinoma of the salivary gland (UY adenocarcinoma),
`sequence and a domain that recognizes some growth fac-
`indicating possible involvement of c-erbB-2 gene expression
`tor(s).
`in this cancer (Fig. 6a). We do not know whether the entire
`Recently, the neu oncogene was identified, by gene-
`transfer techniques, in the genomes of four neuro/glioblas-
`c-erbB-2 gene is amplified in this tumor, since the KX probe
`toma cell lines derived from chemically induced rat tumors
`detects only part of the c-erbB-2 gene. When the same filters
`were hybridized with the EGF receptor cDNA probe, am-
`(24). The neu gene is related to the v-erbB gene and encodes
`plification of the EGF receptor gene was observed in A431
`a tumor antigen with a molecular weight of 185,000 (p185).
`p185 is serologically related to, but distinct from, the EGF
`DNA, as has been reported (8, 23), but not in the UY
`receptor (25). Our data show that the rat c-erbB-2 gene yields
`adenocarcinoma. Owing to the limited amount of tissue
`restriction fragments of the same sizes as does the rat neu
`available, RNA of the UY adenocarcinoma could not be
`oncogene when digested with EcoRI or EcoRl plus BamHI.
`analyzed. We have detected amplification and enhanced
`Table 1. Amino acid sequence homology between the c-erbB-2-encoded kinase domain and those
`encoded by various viral oncogenes
`Viral oncogene:
`erbB
`src
`% homology:
`82
`43
`
`abI
`42
`
`yes
`42
`
`fgr
`41
`
`ros
`38
`
`fps
`37
`
`mil
`28
`
`fMs
`27
`
`mos/rel
`<25
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1042 Page 4 of 5
`
`

`
`Biochemistry: Semba et al.
`
`Proc. Natl. Acad. Sci. USA 82 (1985)
`
`6501
`
`w_r__,~~~~~~312, 513-516.
`
`3.
`
`4.
`
`5.
`
`8.
`
`9.
`
`14.
`
`18.
`19.
`20.
`
`21.
`22.
`
`23.
`
`28.
`
`31.
`
`32.
`33.
`
`34.
`
`Conservation of the c-erbB-,
`2 gene in vertebrates. (a)
`FIG. 5.
`Conserved c-erbB-2 sequences in chicken
`Iand mouse. Nitrocellulose
`from human placenta (lane
`filters containing EcoRI digests of DNA I
`icken blood (lane 3) were
`1), mouse FM3A cells (lane 2), and chi
`probed with the KX DNA. (b) Close simi
`ilarity of the c-erbB-2 gene
`ted with either EcoRI (lane
`to the neu oncogene. Rat DNA was cleav
`digests were analyzed by
`1) or EcoRI plus BamHI (lane 2). The
`)robe. Arrowheads indicate
`Southern blot hybridization with the KX p
`positions of fragments from HindIll dig
`gestion of bacteriophage x
`DNA (see Fig. la).
`
`might function in establishing and maintaining the cancerous
`state.
`Note Added in Proof. King et al. (34) have independently isolated an
`erbB-2 gene amplified in a human mammary carcinoma.
`We thank Y. Miura and H. kawano for excellent technical
`assistance, M. Nishizawa and N. Miyajima for helpful discussions,
`and S. Sasaki for typing this manuscript. We also wish to thank N.
`Nomura for DNA from a human primary neuroblastoma and I.
`Pastan for recombinant plasmid pE7. This work was supported in
`part by a Grant-in-Aid for Special Project Research Cancer-Biosci-
`ence from the Ministry of Education, Science, and Culture of Japan.
`Land, H., Parada, L. F. & Weinberg, R. A. (1983) Science 222, 771-778.
`1.
`Cohen, S., Carpenter, G. & King, L. (1980) J. Biol. Chem. 255,
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`Kasuga, M., Zick, Y., Blithe, D. L., Crettaz, M. & Kahn, C. R. (1982)
`Nature (London) 298, 667-669.
`Rubin, J. B., Shia, M. A. & Pilch, P. F. (1983) Nature (London) 305,
`438-440.
`6. Yamamoto, T., Nishida, T., Miyajima, N., Kawai, S., Ooi, T. &
`Toyoshima, K. (1983) Cell 35, 71-78.
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`Nature (London) 307, 521-527.
`Ullrich, A., Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A., Tam,
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`ward, J., Mayes, E. L. V., Whittle, N., Waterfield, M. D. & Seeburg,
`P. H. (1984) Nature (London) 309, 418-425.
`Waterfield, M. D., Scrace, G. T., Whittle, N., Stroobant, P., Johnsson,
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`e that there is a close
`Deuel, T. F. (1983) Nature (London) 304, 35-39.
`These findings lead us to speculate
`Ic. Doolittle, R. F., Hunkapiller, M. W., Hood, L. E., Devare, S. G.,
`relation between c-erbB-2 and neu.
`Robbins, K. C., Aaronson, S. A. & Antoniades, H. N. (1983) Science
`such as myc and ras has
`Amplification of protooncogenes E
`221, 275-277.
`various human cancers
`Maniatis, T., Hardison, R. C., Lacy, E., Lauer, J., O'Connel, C., Quon,
`been reported to be associated with
`11.
`(26-30). This amplification suggests t
`that the protein products
`p2. Benton, W. D. & Davis, R. W. (1977) Science 196, 180-182.
`D., Sim, G. K. & Efstratiadis, A. (1978) Cell 15, 687-701.
`involved in some stage
`of the amplified protooncogenes are
`Maniatis, T., Jeffrey, A. & Kleid, D. G. (1975) Proc. Natl. Acad. Sci.
`13.
`st is the role(s) ofgrowth
`USA 72, 1184-1188.
`oftumorigenesis. Ofparticular intere
`factor receptors in tumorigenesis, wl
`Davis, R. W., Botstein, D. & Roth, J. R. (1982) Advanced Bacterial
`liich is inferred from the
`Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY),
`are expressed in large
`i0-ui.
`p
`observations that EGF receptors
`epidermoid carcinomas
`numbers on the surface of cells of
`15. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 499-560.
`Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci.
`(31-33). Since the c-erbB-2 gene c(
`ould code for a protein
`16.
`USA 74, 5463-5467.
`similar to the EGF receptor or at leas
`it its intracellular do'main
`17.
`Messing, J., Crea, R. & Seeburg, P. H. (1981) Nucleic Acids Res. 9,
`cinoma of the salivary
`and was amplified in an adenocar
`309-321.
`gland, the gene products expressed a
`abnormally in this tumor
`Messing, J. & Vieira, J. (1982) Gene 19, 269-276.
`Southern, E. M. (1975) J. Mol. Biol. 98, 503-517.
`Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Ruffer, W. J.
`(1979) Biochemistry 18, 5294-5299.
`Lehrach, H., Diamond, K., Wozney, J. M. & Boedtker, H. (1977)
`Biochemistry 16, 4743-4751.
`Xu, Y.-H., Ishii, S., Clark, A. J. L., Sullivan, M., Wilson, R. K., Ma,
`D. P., Roe, B. A., Merlino, G. T. & Pastan, I. (1984) Nature (London)
`309, 806-810.
`Merlino, G. T., Xu, Y.-H., Ishii, S., Clark, A. J. L., Semba, K.,
`Toyoshima, K., Yamamoto, T. & Pastan, I. (1984) Science 224, 417-419.
`Schechter, A. L., Stern, D. F., Vaidyanathan, L., Decker, S. J.,
`24.
`,.ww Drebin, J. A., Greene, M. I. & Weinberg, R. A. (1984) Nature (London)
`Derbin, J. A., Stern, D. F., Link, V. C., Weinberg, R. A. & Green,
`25.
`_ S_ _
`26.Collins, S. J. & GrouIne,M.(1984) Nature (London)
`312, 545-548.
`.
`
`i.1i~iw_i. .26. Collins, S. J. & Groudine, M. (1982) Nature(London) 298, 679-681.
`Dalla-Favera, R., Wong-Staal, F. & Gallo, R. C. (1982) Nature (London)
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`Alitaro, K., Schwab, M., Lin, C. C., Varmus, H. E. & Bishop, J. M.
`(1983) Proc. Natl. Acad. Sci. USA 80, 1707-1711.
`29. McCoy, M. S., Toole, J. J., Cunningham, J. M., Chang, E. H., Lowy,
`D. R. & Weinberg, R. A. (1983) Nature (London) 302, 79-81.
`30.
`Schwab, M., Alitalo, K., Varmus, H. E., Bishop, J. M. & George, D.
`(1983) Nature (London) 303, 497-501.
`Carpenter, G., King, L. E. & Cohen, S. (1979) J. Biol. Chem. 254,
`4884-4891.
`Fabricant, R., Delarco, J. E. & Todaro, G. J. (1977) Proc. Nat!. Acad.
`Sci. USA 74, 565-569.
`Cowly, J., Gutason, B., Smith, J., Hendlor, F. & Ozanne, B. (1984) in
`Cancer Cells, eds. Levine, A. J., Vande Woude, G. F., Topp, W. C. &
`Watson, J. D. (Cold Spring Harbor Laboratory, Cold Spring Harbor,
`NY), Vol. 1, pp. 5-10.
`King, C. R., Kraus, M. H. & Aaronson, S. A. (1985) Science, in press.
`
`a
`
`1
`
`2
`
`3
`
`---P-MM-l
`
`b
`
`II--
`
`Do-
`
`9
`
`a
`
`5 6
`
`1
`
`2
`
`b
`
`I
`
`IiA
`
`I
`.1
`4
`
`a
`
`I
`
`lr.l
`.,;
`VIL4,
`
`.,!-
`
`.:
`
`'..,
`
`a
`
`3
`
`4
`
`*
`
`>
`
`6
`
`t
`
`'-2 gene in human primary
`Amplification of the c-erbB
`FIG. 6.
`iing EcoRI digests of tumor
`tumors. (a) Nitrocellulose filters contain
`:cific KX DNA. (b) After
`DNAs were probed with c-erbB-2-spe
`re then hybridized with the
`removal of the first probe, the filters we
`tion was carried out under
`EGF receptor cDNA probe. Hybridizat
`tdifromgand (laneur2stwom
`ted from a neuroblastoma
`stringent conditions. DNAs were isola
`lary gld(lane 2), two
`(lane 1), an adenocarcinoma of the sa
`I), placenta (lane 5), and the
`squamous cell carcinomas (lanes 3 and 4
`a a and b indicate positions
`A431 cell line (lane 6). Markers at left ir
`DNA.
`of fragments from HindIII digestion of )
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1042 Page 5 of 5