The EMBO Journal vol.6 no.3 pp.605-610. 1987
`~verexpression of the EGF receptor-related proto-oncogene erbB-2
`m human mammary tumor cell lines by different molecular
`mechanisms
`
`Motthios ff .Kraus, Nicholas C.Popcscu 1, Suzanne
`C.Amsbaugh1 and C.Richter King
`La~ratory of Cellular and Molecular Biology. and 1Laboratory of Biology.
`Nauonal Cancer Institute. Building 37. Room IE24, Bethesda, MD 20892,
`USA
`Communicated by J.Schlessinger
`Amplification of the erbB/EGF receptor and a structurally
`related gene, designated erbB-2, have previously been detected
`in a variety of human tumors. In a series of human mam(cid:173)
`mary tumor cell lines, analysis of transcripts of these genes
`revealed elevated levels of one or the other in more than 60%
`of tumors analyzed. Eight cell lines demonstrated erbB-2
`mRNA levels ranging from 4- to 128-fold above those of nor(cid:173)
`mal controls. erbB-2 expression was evaluated in comparison
`to the expression level of actin observed in these cell lines.
`There was no evidence of an aberrantly sized erbB-2
`transcript in any of these lines. lmmunoblot analysis indicated
`elevation in levels of the 185-kd product of the erbB-2 gene
`exp~ by these cells. In four lines erbB-2 gene amplifica(cid:173)
`tion in the absence of an apparent gene rearrangement was
`demonstrated. In a repr esentative cell line of this type, SK(cid:173)
`BR-3, the amplified erbB-2 gene copies were located in an
`at>t:rrant chromosomal location. Four additional cell lines,
`which demonstrated 4- to 8-fold overexpression of erbB-2
`mRNA, did not exhibit gene amplification. In a representative
`cell line of this type ZR-75-1, an apparently normal chromo(cid:173)
`somal location was found for the erbB-2 gene. Our findings
`indicate that overexpr~ion of the erbB-2 gene in mammary
`tumor cell lines is frequent and associated with different
`genetic abnormalities.
`Key words: erbB-2/gene amplification/growth factor receptor/
`marrunary neoplasia/ overexpression
`
`Introduction
`Cellular genes encoding effector molecules of growth regulation
`have been linked to the neoplastic process based on their
`homology to retroviral oncogenes. The c-sis proto-oncogene en(cid:173)
`codes a chain of platelet-derived growth factor (POOF) (Doo(cid:173)
`little et al., 1983; Waterfield et al., 1983), the v-erbB oncogene
`has been shown to encode a truncated form of the epidermal
`growth factor (EGF) receptor (Downward et al. , 1984), and the
`c-fms proto-oncogene product is related to the receptor for mono(cid:173)
`nuclear phagocyte growth factor (CSF-IR) (Sherr et al . • 1985).
`More recently, we and others (King et al., 1985a; Schechter et
`al., 1985; Semba et al., 1985; Coussens et al. , 1985) have iden(cid:173)
`tified a second cellular analogue of v-erbB in the human genome.
`This gene, designated erbB-2, is related to but distinct from the
`gene encoding the erbBIEGF receptor (EGFR). The predicted
`amino acid sequence of the erbB-2 gene reveals the structural
`features of a growth factor receptor molecule with close similarity
`to the EGF receptor (Coussens et al., 1985; Yamamoto et al.,
`1986a), including a cysteine-rich extracellular domain, transmem-
`
`brane region and a highly conserved tyrosine kinase domain .
`Gene alterations affecting EGFR and erbB-2 occur in tumor
`cells. A dominant cellular transforming gene activated by point
`mutation in chemically induced rat neuroblastomas (Shih et al.,
`1981), neu, is likely to be the rat homologue of human erbB-2
`based on comparative nucleotide sequence analysis and chromo(cid:173)
`somal localization (Schechter et al., 1985; Coussens et al., 1985;
`Yamamoto et al., 1986a; Bargmann et al., 1986a). In human
`glioblastoma, amplification and rearrangement of the EGFR gene
`result in extensive expression of abnormal as well as normal(cid:173)
`s.ized ~As (Libermann et al., 1985). In addition, amplifica(cid:173)
`tion without rearrangements affecting EGFR mRNA size is fre(cid:173)
`quently found in cells derived from squamous cell carcinomas
`(Yamamoto et al., 1986b) and in two distinct manunary car(cid:173)
`cinoma cell lines (King et al., 1985b; Filmus et al., 1985).
`Gene amplification of erbB-2 has been identified in a primary
`mammary adenocarcinoma (King et al., 1985a), as well as in
`a salivary gland adenocarcinoma (Semba et al., 1985). These
`~ndings have s~ggested the possibility that erbB-2 overexpres(cid:173)
`s1on may contribute to neoplastic growth (King et al. , l 985a;
`Semba et al., 1985).
`For this study, we investigated the expression of erbB-2 and
`EGFR in 16 human mammary tumor cell lines. Our results in(cid:173)
`dicate frequent overexpression of these proto-oncogenes that are
`related to growth factor receptors. Furthermore, analysis of the
`erbB-2 gene locus in these cell lines demonstrates that enhanced
`erbB-2 expression can occur in the presence or the absence of
`gene amplification, suggesting that different molecular mechan(cid:173)
`isms result in overexpression of normal size erbB-2 mRNA in
`mammary tumor cells.
`
`Results
`Isolation of erbB-2 complementary DNA
`To allow a comprehensive analysis of erbB-2 mRNA and gene
`structure we isolated cDNAs with a complexity of over 4.5 kb
`from the mRNA (Figure IA). An oligo (dT) primed normal
`human fibroblast cDNA library (Okayama and Berg, 1983) was
`screened with a 0.8 kbp Accl DNA fragment from a genomic
`clone of erbB-2 (King et al., 1985a). The largest plasmid ob(cid:173)
`tained, pMAC137, carried a 2-kbp insert comprising 1.5 kbp of
`3' coding information and 3' untranslated sequence. 11le remain(cid:173)
`ing coding information upstream was obtained from three phage
`clones, >..MAC30, AMACIO' and >..MAC14-l , identified in a ran(cid:173)
`domly primed MCF-7 cDNA library (Walter et al., 1985; Figure
`IA). Nucleotide sequence analysis and restriction mapping of
`the entire cDNA indicated that its structure was the same as an
`isolate from normal human placenta (Coussens et al., 1985).
`Overexpression of erbB-2 or EGFR proto-oncogenes in human
`mammary tumor cell lines
`To assess the role of erbB-2 in human marrunary neoplasia we
`compared the mRNA of 16 mammary tumor cell lines to nor(cid:173)
`mal human fibroblasts , M413, and a human marrunary epithelial
`cell line, HBLlOO. Increased expression of an apparently nor-
`
`605
`
`1 of 6
`
`Celltrion, Inc., Exhibit 1046
`
`

`

`M.H.Kraus el al.
`
`A
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`Fig. 1. (A) Isolation and restriction mapping of erbB-2 cDNA. Clone pMACl37 was isolated from an oligo (dn primed nom1al human fibroblast cDNA
`library (Okayama and Berg, 1983). Clones >.MAC30, >.MAC 10' and >.MAC14- l were subsequently obtained from a randomly primed MCF-7 cDNA library
`{Walter et al., 1985). Restriction sites: B = BamHI. Bil = BstEll , E = EcoRI. N = Ncol. P = Pstl. Sm = Smal. Sp = Splrl and St = Srul. (8) cDNA
`probes used in hybridization analysis.
`
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`I vc
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`of ert>B-2
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`1 128 8 128 64 64 e 4
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`13 1
`
`Fig. 2. Overexpression of erbB-2 in human mammary rumor cell lines. (A) Nonhem blot analysis. Total cellular RNA (10 µg) of mammary rumor cell lines.
`nonna.I human fibroblasts M413 and HBLIOO was hybridized with a cDNA probe derived from the 5' end of the erbB-2 coding region {Figure lB, probe a).
`M413 and HBLIOO cells contain erbB-2 specific mRNA detectable after longer autoradiographic exposures. (B) Quantitation of erbB-2 mRNA levels. Serial
`2-fold dilutions of total RNA were applied to nitrocellulose. Replicate filters were hybridized with either a erbB-2 cDNA probe (Figu re 18. probe b) or
`human 13-actin which served as control for RNA amounts present on the nitrocellulose filter. Relative amounts detected with each probe are indicated in
`comparison to the hybridization signals observed in normal human fibroblasts M413.
`606
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`2 of 6
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`Celltrion, Inc., Exhibit 1046
`
`

`

`Overexpr~ion of the erbB-2 gene in mammary tumor cells
`
`Table I. Overexpression of erbB-2 and EGFR proto-oncogenes in human
`mammary neoplasia
`
`EGFR
`erbB-2
`Overexpression Gene
`Overexpression Gene
`amplification
`ampl ification of mRNA3
`of mRNA•
`
`A
`
`(')
`
`(')
`I
`CI:
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`<
`Cl)
`+ - +
`
`MW x 10- 3
`
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`
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`I
`I
`,._
`CI:
`II)
`I
`CD
`u.
`I u
`I
`~ CI:
`Cl) N ~
`
`M41 3
`HBLIOO
`MCF-7
`SK-BR-3
`BT474
`MDA-MB361
`MDA-MB453
`ZR-75-1
`ZR-75-30
`MDA· MB1 75
`BT483
`BT20
`MDA·MB468
`
`I
`128
`128
`64
`64
`8
`4
`8
`8
`
`I
`4-8
`4-8
`2-4
`2
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`32
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`32
`
`'Overexpression above nonnal fibroblast and HBLIOO.
`
`ma! size 5-kb transcript was detected in 8 of 16 tumor cell Jines,
`when total cellular RNA was subjected to Northern blot analysis.
`Figure 2A shows the results using a cDNA probe comprising
`the coding sequences of the amino-terminal extracellular domain
`of erbB-2 (Figure 18, probe a). These results of overexpression
`of normal-sized mRNA were confirmed by hybridization of po(cid:173)
`ly(A) + selected RNA using several erbB-2-specitic probes com(cid:173)
`prising coding information for the transmembrane and tyrosine
`kinase domains. An aberrantly sized erbB-2 mRNA was not
`detected in any of the cell lines analyzed.
`To quantitate more precisely the amount of erbB-2 transcript
`in eight mammary tumor cell lines which overexpress erbB-2,
`serial 2-fold dilutions of total cellular RNA were subjected to
`dot blot analysis using human (3 actin as a control for the amount
`of RNA applied to the nitrocellulose filters. As shown in Figure
`2B, the highest levels of erbB-2 mRNA, which ranged from 64-
`to 128-fold over that of our controls, were observed in the cell
`lines MDA-MB453, SK-BR-3, MDA-MB361 and BT474. More(cid:173)
`over, erbB-2 mRNA levels were increased 4- to 8-fold in four
`cell lines including BT483, MDA-MB175, ZR-75-30 and
`ZR-75-1 (Table I) .
`To determine if the overexpression of erbB-2 mRNA resulted
`in a steady state increase of its encoded gene product, we
`developed a specific immunoblot assay. Antisera were raised
`against a synthetic peptide whose sequence corresponded to a
`portion of the putative erbB-2 tyrosine kinase domain. As this
`region is partially conserved between the encoded proteins of
`the EGFR and erbB-2 genes, we tested its specificity using A43 l
`and SK-BR-3 cell lines which overexpress EGFR or erbB-2
`mRNA, respectively . As shown in Figure 3A, a specific band
`of - 185 kd was detected in ex.tracts of SK-BR-3 but not in A431
`cells. This band was not detected when the antibody was pre(cid:173)
`incubated with the synthetic peptide corresponding to its antigen.
`The human erbB-2 and rat neu products have been reported to
`be glycoproteins of 185 kd (Akiyama et al. , 1986; Stem et al. ,
`1986).
`To estimate the relative amounts of erbB-2 protein in different
`mammary tumor cell lines, immunoblot analysis was conducted
`using equivalent amounts of total cellular protein. As shown in
`Figure 3B, an intense band of protein was detected in extracts
`of SK-BR-3 and a less intense but readily detectable band in ex(cid:173)
`tracts of ZR-75-1. No erbB-2 protein was detected in extracts
`
`-
`
`- 200
`
`- 97
`
`MWX10 - J
`
`- 200
`
`- 97
`
`Fig. 3. Elevated erbB-2 protein levels in mammary tumor cell lines. 40 µg
`total cellular protein was separated by electrophoresis and transferred to
`nitrocellulose filters. The erbB-2 protein was detected with an antipeplide
`antibody coupled to t2S1 protein A. The specificity of antibody detection was
`determined by pre-incubation of the antibody with excess amounts of peptide
`prior to immunodetection. { +) preincubation with peptide. {-) no peptide.
`In panel B, nonspecific bands a1 110 kd are observed in longer exposures of
`peptide-blocked immunoblots (panel A).
`
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`Fag. 4. Gene amplification of erbB-2 in mammary tumor ceU lines. {A)
`Southern blot analysis. For each lane 10 µg genomic DNA were restricted
`with Xbal and hybridized with a probe comprising the entire coding region
`of erbB-2. HindIII restriction fragments of lambda DNA seived as mol. wt
`standards. (8) DNA dot-blot analysis. Genomic DNA (10 µg) digested with
`EcoRI was applied in serial 2-fold dilutions to nitrocellulose filters. Filters
`were hybridized either with erbB-2 {Figure I 8 , probe b) or mos, which
`served as a control for DNA amounts applied to replicate nitrocellulose
`filters. Gene copy numbers of erbB-2 relative to M41 3 indicate the minimal
`extent of gene amplification detected in DNA from mammary tumor cell
`lines.
`of MCF-7 , a mammary tumor cell line, that tlid not display
`overexpression of erbB-2 mRNA. We interpret these results to
`indicate that substantially more erbB-2 protein is found in both
`SK-BR-3 and Z R-75- 1 than in MCF-7 cells where the amount
`of protein escapes the sensitivity of the assay. Dilution ex(cid:173)
`periments suggest that SK-BR-3 contains between 5- and IO-fold
`more erbB-2 protein than does ZR-75-1 (data not shown).
`We also analyzed total cellular RN As of the same mammary
`tumor cell lines for evidence of EGFR receptor mRNA overex(cid:173)
`pression. Increased amounts of an apparently normal size EGFR
`607
`
`3 of 6
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`Celltrion, Inc., Exhibit 1046
`
`

`

`M.H.Kraus tt al.
`
`.1, ..
`
`,
`
`Fig. S. Representative metaphase from the SK-BR-3 carcinoma cell line
`after in situ hybridization with an erbB-2 cDNA probe, autoradiography and
`G·banding. (A) A chromosome spread exhibiting silver grains on a
`distinctive, highly rcamnged chromosome (arrow). (B) The same spread
`after trypsin/EDT A treatment to produce G·bands. Detailed G·banding
`analysis did not indicate that the site of hybridization contains a
`homogeneously stained region (HSR).
`
`mRNA were observed in BT20 and MDA-MB468. These two
`cell lines have previously been shown to contain amplified EGFR
`genes. EGFR transcripts were elevated 16-fold in BT20 and
`32-fold in MDA-MB468 above the level seen in nonnal human
`fibroblasts as detennined by RNA dot-blot analysis (Table I).
`Genetic abnormalities associated with elevated erbB-2 expression
`To investigate alterations of the erbB-2 gene associated with
`mRNA overexpression, we examined Xbal-restricted high mol.
`wt DNA by Southern blot analysis using a probe comprising the
`entire coding sequence of erbB-2 (Figure IB, probe c). The nor(cid:173)
`mal restriction pattern of Xbal fragmentc; was dete:eted in all DNA
`samples analyzed indicating that gene rearrangements in prox(cid:173)
`imity of the erbB-2 coding region did not occur in these cell lines.
`When compared with nonnal human fibroblast DNA (Figure 4A,
`lane I) the erbB-2-specific Xbal restriction fragments appeared
`clearly amplified in the cell lines SK-BR-3, BT474 and MDA(cid:173)
`MB361 (Figure 4A, lanes 2-4). Similar results were obtained
`with restriction enzymes EcoRJ and Sad (data not shown).
`Quantitation of erbB-2 gene copy number was accomplished
`using DNA dot-blot analysis. These studies revealed a 4- to 8-fold
`
`608
`
`erbB-2 gene amplification in SK-BR-3 and BT474 relative to nor(cid:173)
`mal human DNA and a 2- to 4-fold erbB-2 gene amplification
`in MDA-MB361. In addition, a 2-fold erbB-2 gene amplifica(cid:173)
`tion was identified for the cell line MDA-MB453 by DNA dot(cid:173)
`blot analysis. Thus, gene amplification was associated with
`overexpression in the four tumor cell lines with the highest levels
`of erbB-2 mRNA (Table I). In contrast, gene amplification could
`not be detected by Southern blot analysis or DNA dot-blot
`analysis in the four tumor cell lines with erbB-2 transcript in(cid:173)
`creased to intennediate levels.
`To examine the nature of any chromosomal abnonnalities
`associated with overexpression we used in situ hybridization to
`localize the erbB-2 gene in two cell lines which either do or do
`not contain amplified gene copies. The erbB-2 gene has been
`mapped in nonnal human cells on chromosome 17ql 1.2-22
`(Schechter et al., 1985; Coussens et al., 1985; Fukushige et al.,
`1986). Mammary tumor cell line SK-BR-3 contains 4- to 8-fold
`gene amplification and 128-fold overexpression of erbB-2
`mRNA. In SK-BR-3, G-banding showed no copies of normal
`chromsome 17. In situ hybridization of the erbB-2 gene to these
`cells revealed accumulations of grains on two large abnonnal
`marker chromosomes derived from complex rearrangements in(cid:173)
`volving at least three chromosomes. An average of three grains
`was observed at each labelled site (Figure 5). These results in(cid:173)
`dicate that the amplification of the erbB-2 gene occurs in an ab(cid:173)
`nonnal chromosomal location and is not associated with either
`a homogeneously stained region or double minute chromosomes,
`abnormalities diagnostic for gene amplification (Biedler and
`Spengler, 1976; Levan et al., 1977). Mammary tumor cell line
`ZR-75-1 showed no evidence of gene amplification and an 8-fold
`overexpression of mRNA (Table I). Chromosome 17 was pre(cid:173)
`sent in one or two copies per cell. Analysis of 50 cells after in
`situ hybridization with a erbB-2 cDNA probe revealed the largest
`accumulation of grains on chromosome 17 with 85 % of these
`clustered on chromosome bands 17ql 1.2-21, the nonnal loca(cid:173)
`tion of the erbB-2 gene. This indicates that overexpression of
`the erbB-2 gene can occur in the absence of detectable structural
`abnormalities of chromosome 17.
`Mammary tumor cell lines overexpressing erbB-2 do not con(cid:173)
`tain readily detectable transforming genes
`In chemically induced rat neuroblastomas, a point mutation within
`the transmembranous domain activates the rat homologue of
`erbB-2, neu, to transforming activity readily detectable in the
`NIH/3T3 transfection assay (Bargmann et al., 1986b). Previous
`transfection analysis of21 mammary tumors and tumor cell lines
`did not reveal activation of erbB-2 as a transforming gene in
`human mammary neoplasia (Kraus et al., 1984). To investigate
`whether an activating lesion similar to the rat neu gene was
`associated with the overexpression of erbB-2 in human mam(cid:173)
`mary tumor cell lines, we transfected genomic DNA of these
`cell lines into mouse Nlli/31'3 cells. Under conditions where high
`mol. wt DNA from the cell line T24 which is known to contain
`an activated H -ra.~ oncogene induced 4-8 foci/plate, genomic
`DNA from eight mammary tumor cell lines which overexpress
`erbB-2 did not induce detectable morphological transfonnation
`(Table II).
`
`Disc~ion
`Analysis of 16 human mammary tumor cell lines for the EGF
`receptor and the related erbB-2 gene revealed frequently increased
`transcript levels of either member of this family of growth fac(cid:173)
`tor receptor genes. In BT20 and MDA-MB468 overexpression
`
`4 of 6
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`Celltrion, Inc., Exhibit 1046
`
`

`

`Table D. DNA transfeclion of human mammary rumor cell lines
`
`Source of genomic DNA
`
`FFU/plate•
`
`T24 prep I
`T24 prep 2
`T24 prep 3
`SK-BR-3
`BT474
`MDA-MB361
`MDA-MB453
`ZR-75-1
`ZR-75-30
`BT483
`MDA-MBl75
`
`32/4
`19/4
`16/4
`0112
`0112
`012
`0/4
`0/8
`0/4
`014
`0/8
`
`"FFU/plate = focus-forming units/number of 1ransfected plates. 30 µg high
`mol. wt DNA were coprecipitated with calcium phosphate and transfected
`onto NDi/3T3 fibroblasts as previously described (Wigler et al., 1977).
`Genomic T24 DNA served as positive control for each assay.
`
`of normal size EGF receptor gene transcripts is associated with
`gene amplification (King et al., I 985b; Filmus er al., 1985). In(cid:173)
`creased transcript levels of erbB-2 were detected in the presence
`and absence of gene amplification, indicating that erbB-2 over(cid:173)
`expression in human mammary tumor cell lines can be caused
`by different molecular mechanisms. The absence of aberrantly
`sized erbB-2 mRNA in Northern blot analysis suggests that a
`nonnal size mRNA is overexpressed in mammary tumor cell lines
`rather than a rearranged form. Moreover, the fact that genomic
`DNA from these cell lines lacked the ability to transform
`NIH/3T3 cells by transfection indicates that point mutations
`similar to those that can activate the rat neu gene did not occur.
`These observations provide evidence that a structurally normal
`coding sequence of erbB-2 is overexpressed as mRNA in human
`mammary tumor cell lines. Protein analysis of representative
`samples with erbB-2 overexpression suggests that elevated erbB-2
`transcript levels are translated into erbB-2 proteins.
`Several lines of evidence link the overexpression of proto(cid:173)
`oncogenes to the neoplastic process. The increased transcription
`of nonnal coding sequences of either the human c-sis/PDGF gene
`or the H-ras gene using a viral long terminal repeat promoter
`induces transformation of NIH/3T3 cells in culture (Chang er
`al., 1982; Gazit er al., 1984). Moreover, in human tumors myc
`or N-myc amplification correlates with increased malignancy
`(Kohl er al., 1983; Schwab er al., 1983; Nau er al., 1984). Our
`results link erbB-2 overexpression to the neoplastic growth of
`mammary tumor cells.
`Gene amplification of multidrug resistance genes is observed
`in cells selected for the ability to grow in media containing cer(cid:173)
`tian metabolic inhibitors (AJt er al., 1978). There is evidence
`that overexpression of these genes can precede gene amplifica(cid:173)
`tion in the development of multidrug resistance (Shen er al. ,
`1986). Our observation of elevated erbB-2 transcript levels in
`the presence and absence of gene amplification may reflect a
`similar pathway. Overexpression of erbB-2 may confer an in(cid:173)
`itial selective growth advantage to the tumor cell and subsequent
`gene amplification cause a further step where the selective growth
`advantage is enhanced and stabilized. erbB-2 overexpression is
`consistently higher in those samples with gene amplification when
`compared with cell lines lacking gene amplification. Interestingly,
`the amount of overexpression per gene copy is approximately
`constant in the cell lines that overexpress erbB-2 (Table I). This
`suggests that deregulated erbB-2 expression due to different
`molecular mechanisms in mammary tumor cell lines may be part
`
`Overexp~on of the erbB-2 gene in mammary tumor cells
`
`of a multistep process which confers selective growth advantage
`to a mammary tumor cell.
`In all cell lines examined we detected no abnormalities of
`erbB-2 gene structure by Southern blot hybridization. Moreover,
`in ZR-75-1, in which overexpression occurs without gene
`amplification, the erbB-2 gene was located at its normal site on
`chromosome 17. These results indicate that mechanisms of
`deregulation are unlikely to involve the type of rearrangements
`responsible for activation of the myc gene in Burkitt's lymphoma
`(Taub er al., 1982; Dalla-Favera er al., 1983). Deregulation of
`erbB-2 gene expression may therefore involve subtle changes in
`cis-acting control sequences, changes involving transacting
`regulatory elements, or changes which enhance mRNA stab~ity.
`Abnormalities of erbB-2 and EGFR genes are not restncted
`to mammary tumor cells in culture, as we and others have iden(cid:173)
`tified erbB-2 or EGFR gene amplification in several samples of
`mammary tumor tissue (King er al. , l 985a; Yokota et al. , 1986,
`and unpublished observation). The precise action of growth fac(cid:173)
`tor receptor gene overexpression in the neoplastic process of
`mammary tumor cells has yet to be established. However, since
`tumorigenicity of mammary tumor cells can be enhanced by
`steroid hormones or polypeptide growth factors (Kasid er al. ,
`1985; Dickson et al., 1986), it is tempting to hypothesize that
`the overexpression of polypeptide growth factor receptors in(cid:173)
`creases tumorigenicity by stimulation of the same or similar
`growth signalling pathways. Forty percent of mammary tumor
`cell lines analyzed did not exhibit overexpression of erbB-2 or
`EGFR. Heterogeneity of steroid receptor expression is of con(cid:173)
`siderable therapeutic and prognostic value in human mammary
`neoplasia (McClelland er al., 1986; DeSombre er al., 1986). Our
`results strongly suggest an additional level of heterogeneity defin(cid:173)
`ed by overexpression of the erbB-2 or EGFR gene.
`In the cell line SK-BR-3, where we identified an amplified and
`overexpressed erbB-2 gene, a myc gene amplification with over(cid:173)
`expression has been previously reported (Kozbor and Croce,
`1984). The myc gene has been shown to cooperate with a ras
`gene in the transformation of primary fibroblasts (Land er al. ,
`1983). It will be of interest to determine whether overexpres(cid:173)
`sion of erbB-2 and myc can complement each other in promoting
`the growth of mammary tumor cells. In addition, systematic
`analysis of mammary tumors will determine whether overexpres(cid:173)
`sion of erbB-2 or the EGFR gene can be associated with a par(cid:173)
`ticular clinical manifestation of mammary carcinoma.
`
`Materials and methods
`
`Tumor cell lines
`Human mammary rumor cell lines were obtained from ATCC (American Type
`Culture Collection) and cultured according to the supplier's recommendation.
`
`RNA blorting
`Total cellular RNAs were isolated by CsCl-gradient centrifugation (Chirgwin et
`al., 1979). For Northern blot analysis 10 µg tOOll RNA were denarured and electro(cid:173)
`phoresed in I% formaldehyde gels (Lehrach et al., 1977). Following mild alkali
`degradation (50 mM NaOH at 24°C for 30 min) RNA wa~ transfen-ecl to
`nitrocellulose filters using I M ammonium acetate as a convectant. RNA dot(cid:173)
`blot analysis was conducted by applying serial 2-fold dilutions of total RNA in
`I M ammonium acetate to replicate nitrocellulose filters in a dot-blot manifold.
`
`DNA blotting
`For Southern blot analysis DNA samples (10 µg} were restricted with Xbal and
`subjected to electrophoresis in 0.8% agarosc gels. DNAs were transferred to ni~
`cellulose filters by the method of Southern (Southern, 1975). For dot-blot analysis
`DNAs were cleaved with EcoRI, denatured with 0.1 M NaOH and nwtraliz.cd
`with I M ammonium acetate. Serial 2-fold dilutions of each sample were ap(cid:173)
`plied to replicate nitrocellulose filters using a dot-blot manifold.
`
`609
`
`5 of 6
`
`Celltrion, Inc., Exhibit 1046
`
`

`

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`Received on January 5, 1987
`
`Note added in proof
`
`After submission of this manuscript, a srudy by Slamon,0. et al., ((1987) Science,
`235, 177 - 182] indicated a correlation of erbB-2 gene amplification and reduced
`disease-free survival in breast cancer patients.
`
`M.H.Kraus el al.
`
`Hybridi(.Qtion
`Hybridization was conducted at 42°C in 750 mM NaCl, 75 mM Na citrate and
`50% formamide. The filters were washed under stringency conditions of 15 mM
`NaCl and 1.5 mM Na citrate at 50°C.
`lmmunoblotting
`One confluent 100 mm plate of each cell line was lysed in I % Triton, 0 .1 % SOS.
`0 .5% deoxycholate, 10 mM sodium phosphate pH 7.4, 0.1 M sodium chloride,
`I mM phenylrnethyl sulfonyl fluoride, and centrifuged at 12 000 g for 10 min
`at 4 °C. The supernatant was tested for protein concentration using the method
`of Lowry (Lowry et al., 1951) and adjusted to I% SOS, 50 mM Tris-HCI pH
`6 .8, 0.1 % (3 mercaptoethanol, 5% glycerol and 0.01 % bromophenol blue. After
`heat treatment (100°C for 5 min), 40 µg of each sample was subjected to poly(cid:173)
`acrylamide gel (7 % ) electrophoresis and transferred to nitrocellulose. The nitro(cid:173)
`cellulose was treated for 1 h in Blotto solution (2% nonfat dry milk, 1 % Triton,
`50 mM Tris- HCI pH 7.4, JO mM EOTA). The lgG fraction of an antipeptide
`antibody directed against amino acids 866-880 of the erbB-2 protein was con(cid:173)
`ducted in Blotto for 2 h. Blots were washed three times with Blotto solution for
`JO min each. Reaction with 2 x lOS c.p.m./ml of 1125 protein A was conducted
`for 20 min at room temperarure. The blots were then washed three times for
`10 min with Blotto solution and 20 times with water. Detection was accomplish(cid:173)
`ed by autoradiography at - 70°C.
`In situ hybridiwtion
`Exponentially growing culrures of ZR-75-1 and SK-BR-3 were exposed to col(cid:173)
`cemid (0.05 µg/ml) and ethidium bromide (7 .5 µglml) for 4 and 2 h, respective(cid:173)
`ly, before harvesting for chromosome preparation. In situ hybridization was canied
`out under stringent conditions as previously described (Harper and Saunders, 1981;
`Popescu et al., 1985a) using 3H-labelled erbB-2 cDNA as probe. The hybridiz(cid:173)
`ed slides were coated with nuclear track emulsion NTB-2, diluted 1: 1 with H20
`and sto