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`MOLECULAR AND CELLULAR BIOLOGY
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`VOLUME 6 • MARCH 1986 • NUMBER 3
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`PFIZER EX. 1044
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`

`MOLECULAR AND CELLULAR BIOLOGY. Mar. 1986. p. 955-958
`0270..73061861030955-04$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
`S HIN-IC HI FUKUSH IGE,1 KEN-ICH I MATSUBARA} MICH IH IRO YOSHIDA.2 MOTOMIC HI SASAKI.2
`TOSHIMITSU SUZUKI ,3 KENTARO SEMBA,4 KUMAO TOYOSHIMA,4 AND TADASH I YAMAM0T04*
`Institute for Molecular and Cellular Biology, Osaka Unit•ersity, Suiw, Osaka 565, 1 Chromosome Research Unit.
`Hokkaido University, N. 10-W.8, Sapporo 060.2 School of Medicine. Niigata University, Niigata 95/ ,3 and The l nstifllte
`of Medical Science, The University of Tokyo, Minato-ku Tokyo /08,4 Japan
`
`Received 17 September 1985/Acccpted 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 1ocus 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 .
`-- - - - - - - - - --'~--
`Human Genetic Ylutant Cell Repository and then sorted
`A number of cellular counterparts to the retroviral onco(cid:173)
`genes have been identified and localized on specifi c chromo(cid:173)
`(Institute for Medical Research, Camden. N.J .). into nine
`somes. The locations of several cellular oncogenes corre(cid:173)
`fractions using a fluorescence-activated cell sorter as de(cid:173)
`scribed previously (20. 28). DNA samples were prepared
`spond well to breakpoints of chromosomal translocations
`found in various cancers. For example, the c-myc gene on
`from each fraction of the sorted chro mosomes (7) and
`chromosome 8 is involved in translocations between chro(cid:173)
`a nalyzed by Southern hybridization (21) using a DNA probe
`of a 440-base-pair (bp) Kpn l-Xbal restriction fragme nt (KX(cid:173)
`mosome 8 and one of the chromosomes-2. 14, or 22-that
`DNA) generated from Lhe ~.:-erbB-2 genomic clone H07 .
`carries an immunoglobulin gene (5, 9, 12, 22). The resulting
`alteration in c-myc expression is suspected to be causally
`With the GM2324 cell line. a positive signal was observed for
`related to tumorigenesis (14).
`a fraction that corresponded mainly to chromosomes 16. l 7,
`and 18 (data not shown). Another human lymphoblast cul(cid:173)
`An avian erythroblastosis virus H stn1in contains a n
`ture , GM3197, carries the reciprocal translocation, t(17;22).
`oncogene, v-erbB , that replaces the env gene of an avian
`producing derivative chromosomes (17;22 and 22q- ) that are
`leukosis virus (25). T he nucleotide sequence analysis of the
`clo ned v-erbB DNA and human epidermal growth factor
`different in size from the no rmal homolog (6). Analysis of
`(EGF) receptor eDNA clones revealed that the v-erbB
`this cell line revealed two positive signa ls, one in a fraction
`that contains normal chromosome 17 a nd one in a fraction
`protein corresponds to the carboxyl half of the human EGF
`receptor, including the membrane-spanning domain (23, 24,
`that contains the derivative c hromosome 17:22 (data not
`shown). These results indicate that the c-erb B-2 gene is
`26). This strongly suggests that the 3' half of the chicken
`EGF receptor gene was transduced into the H strain of avian
`located on huma n chro mosome 17.
`To localize the c-erbB-2 gene more precisely, we per(cid:173)
`erythroblastosis virus. In addition to the EGF receptor gene,
`we found another v-erbS-related gene, c-erbB-2. in the
`formed in situ hybridization experiments on chromosome
`spreads prepared from phytohemagglutinin-stimulated pe(cid:173)
`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
`experime nt was 3H-Iabeled pCER217 plasmid DNA. which
`genes differ from each other in length and because the amino
`is a c-erbB-2 eDNA 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
`region o f the EGF receptor (17). Recently , the neu
`revealed that 23.5% (20 of 85) of the silver grains were
`oncogene, active in a series of rat neuroblastoma (19). was
`located on chromosome 17. Of these 20 grains, 15 (75% )
`found to be an erbB-related gene encoding an EGF receptor(cid:173)
`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 17q2l.l-2l.3 (Fig. 1). A huma n version of
`Weinberg, Nature [London]. in press). Comparison of the
`the neu oncogene was recently mapped on human chromo(cid:173)
`nucleotide sequences and the deduced a mino acid sequences
`some 17 at q21 (16). Since translocation between chromo(cid:173)
`of human c-erbB-2 (T . Yamamoto, S. lkawa. T . Akiyama,
`somes 15 and 17. t(15 ;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-Iabeled frag(cid:173)
`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 anal ysis (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
`
`• Corresponding author.
`
`955
`
`PFIZER EX. 1044
`Page 3
`
`

`

`956
`
`NOTES
`
`MOL. CELL. BtOL.
`
`b
`
`0
`
`000
`
`00000000
`0000
`00
`
`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 [lH]dCTP (30 Ci/mmol) and ['H]dTTP (48 Cilmmol). The specific activity of the probe was 3 x 107 cpm 118
`of D A. The chromosomal 0 A was denatured on slides in 70% formamide-2x SSC (I X SSC is 0.15 M aCI plus 0.015 M sodium citrate)
`at 1o•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-<lenatured salmon perm DNA (100 11g/ml)-1 x Denhardt ~olution for 16 h at 40•c. Arter hybridization. the slides were rinsed for 10
`min twice in 50% formamide- 2X SSC at40°C and then several times in 0.2X SSC at37•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 o f the p53 gene to chromosome
`15 was observed in three of three APL cases tested (8) by in
`situ hybridization. Thus, further anal ysis o f APL cells with
`t(l5 ;17), w hic h include in situ hybridization o n chromosome
`
`spread using c-erbB-2-specific DNA probes,
`pated.
`Previously, we found t hat the c-erbB -2 gene i~ amplilit:u in
`an adenocarcinoma of t he salivary gla nd, although we could
`
`is antici(cid:173)
`
`a Pl. - MKN-7 -
`
`.. 6.4
`
`FIG. 2. Amplification and translocation of the c-erbB-2 gene in a gastric cancer cell line. M KN-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 £coR I digests was probed with n p.Jabelcd KX-DNA (specific activity; 108 cpm/11g of DNA). Hybridization
`was carried out in a stringent condition (17). The filter contained, in lanes from left to right. placental DNA (10 118) and MKN-7 DNA (10,
`5, 2.5. 1.25, 5/8. and 5/16 11g ). (b) Translocation of the c-erbB· 2 gene. Metaphase spread was prepared from MK N-7 cells (left) and hybridized
`with 3H-Iabeled pCER217 DNA (right) as described in the legend to Fig. 1. Arrows indicate location of the c-erbB-2 gene on marker
`chromosomes.
`
`PFIZER EX. 1044
`Page 4
`
`

`

`VOL. 6. 1986
`
`NOTES
`
`957
`
`kb
`
`b
`1 2
`•
`
`-
`
`.-4.8kb
`
`FIG. 3. Elevated expression of the c-erbB-2 gene in MKN-7
`cells. Nitrocellulose filters containing poly(A)+ RNA (2 11-g) of
`MKN-7 cells (lane 2) and placental cells (lane 1) were hybridized
`with 32P-labcled KX-DNA (panel a) under stringent conditions (17)
`or with 32P-Iabeled v-yes probe (panel b) prepared from recombinant
`plasmid pYS2 (18) in a relaxed condition (17). DNA probes were
`labeled with ln-32P]dCTP by nick translation to a specific activity of
`1 x lOS to 2 x 108 cpm/11-g 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 alte red
`struc tures of this gene, i.e. , by gene a mplificatio n or rear(cid:173)
`rangement by Southern hybridization with the 32P-Iabeled
`KX-DNA. An MKN-7 cell line established from an adeno(cid:173)
`carcinoma of the stomach was found to contain a n elevated
`copy number of the c-erbB-2 gene. The degree of amplifica(cid:173)
`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 hybridizatio n a nalysis of MKN-7 me taphase spreads
`showed that the c-erbB-2 gene is located on a t least two
`marker c hromosomes other tha n chromosome 17 and that
`the grains were clustered (average, 4 to 5 grains) o n a marker
`chromosome (Fig. 2b). Therefore, we assume that chromo(cid:173)
`somal translocation in MKN-7 could lead to the fusion of a
`pa rt of the c-erbB-2 gene to a n unidentified DNA, which
`resulted in the generatio n of the 23-kbp EcoRI fragment.
`Poly(A)+ RNAs were prepared from MKN-7 a nd placental
`cells. Northern hybridizatio n of these RNAs with 32P-labeled
`KX-DNA probe s howed that a n inc reased amount of c-erbB-
`2 mRNA (4.6 kb), in proportion to the degree of the gene
`a mplificatio n, is synthesized in MKN-7 cells relative to
`
`placental cells, whereas the 4.8-kb yes mRNA in MKN-7
`re mains 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 a t
`some stage during the neoplastic progress of certain tumors.
`The EGF receptor gene proto-erbB is frequently a mplified
`a nd overexpressed in glioblastomas (10, 11) and squamous
`cell carcinomas (2; T. Ya mamoto, N. Kamata, H. Kawano,
`S. Shimizu, T. Kuroki , K . Toyoshima, K . Rikima ru, N.
`Nomura. R. lshizaki, I. Pastan, S. Gamou, a nd N. S himizu ,
`Cancer Res., in press). Amplification of the N -myc gene
`correlates with s tage Ill a n.d IV ne uro blastomas (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), a nd in a primary mammary tumor (4), s uggesting
`a possible role of the c-erbB-2 gene in transforming e pithelial
`cells o r 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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`NOTES
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