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`e
`TAN
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`BIOEPIS EX. 1046
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`yreunolOPW|
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`BIOEPIS EX. 1046
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`Page 2
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`BIOEPIS EX. 1046
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`BIOEPIS EX. 1046
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`Contents
`
`
`
`The EMBO Journal
`Volume 6 number 3. March 1987
`
`
`In vitro expression of a full-length DNA copy of cowpea
`mosaic virus B RNA:identification of the B RNA
`encoded 24-kd protein as a viral protease
`
`Thyroglobulin, the major and obligatory exportable
`protein of thyroid follicle cells, carries the lysosomal
`recognition marker mannose-6-phosphate
`
`The GABA,g/benzodiazepine receptor is a heterotetramer
`of homologous a and £ subunits
`
`Differentially expressed bovine cytokeratin genes.
`Analysis of gene linkage and evolutionary conservation of
`5’-upstream sequences
`
`Immunoglobulin heavy chain switch region recombination
`within a retroviral vector in murine pre-B cells
`
`J.Verver, R.Goldbach, J.A.Garcia and P.Vos
`
`V.Herzog, W.Neumiiller and B.Holzmann
`
`C.Mamalaki, F.A.Stephenson and E.A. Barnard
`
`M.Blessing, H.Zentgraf and J.L.Jorcano
`
`D.E.Ott, F.W.Alt and K.B.Marcu
`
`Superinduction of the human gene encoding immune
`interferon
`
`M.A.Lebendiker, C.Tal, D.Sayar, S.Pilo, A.Eilon,
`Y.Banai and R.Kaempfer
`
`Single amino acid changes that render human IFN-a2
`biologically active on mouse cells
`
`H.Weber, D.Valenzuela, G.Lujber, M.Gubler and
`C.Weissmann
`
`Superinduction of the human interferon-6 promoter
`
`H.Dinter and H.Hauser
`
`Overexpression of the EGF receptor-related proto-
`oncogene erbB-2 in human mammary tumorcell lines by
`different molecular mechanisms
`
`M.H.Kraus, N.C.Popescu, S.C.Amsbaugh and
`C.R.King
`
`Characterization of recombinant human granulocyte-
`colony-stimulating factor produced in mousecells
`
`M.Tsuchiya, H.Nomura, S.Asano, Y.Kaziro and
`S.Nagata
`
`Close genetic and physical linkage between the murine
`haemopoietic growth factor genes GM-CSF and Multi-
`CSF (IL3)
`
`Glucocorticoid induction of the rat tryptophan oxygenase
`gene is mediated by two widely separated glucocorticoid-
`responsive elements
`
`Negative control of liver-specific gene expression: cloned
`human retinol-binding protein gene is repressed in HeLa
`cells
`
`D.P.Barlow, M.Buéan, H.Lehrach, B.L.M.Hogan
`and N.M.Gough
`
`U.Danesch, B.Gloss, W.Schmid, G.Schiitz,
`R.Schiile and R.Renkawitz
`
`V.Colantuoni, A.Pirozzi, C.Blance and R.Cortese
`
`Molecular cloning of cDNA coding for rat proliferating
`cell nuclear antigen (PCNA)/cyclin
`
`K.Matsumoto, T.Moriuchi, T.Koji and P.K.Nakane
`
`Molecular cloning of the 8-subunit of human prolyl
`4-hydroxylase. This subunit and protein disulphide
`isomerase are products of the same gene
`
`T.Pihlajaniemi, T.Helaakoski, K.Tasanen,
`R.Myllyla, M.-L.Huhtala, J.Koivu and
`K.1.Kivirikko
`
`Characterization and expression of a murine gene
`homologous to human EPA/TIMP: a virus-induced gene
`in the mouse
`
`D.R.Gewert, B.Coulombe, M.Castelino, D.Skup
`and B.R.G. Williams
`
`549
`
`555
`
`561
`
`567
`
`S77
`
`585
`
`591
`
`599
`
`605
`
`611
`
`617
`
`625
`
`631
`
`637
`
`643
`
`651
`
`
`
`BIOEPIS EX. 1046
`Page 3
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`BIOEPIS EX. 1046
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`

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`
`Enzymatic activation of Fujinami sarcoma virus gag—fps
`
`transforming proteins by autophosphorylation at tyrosine
`
`
`_ Xenopus cytoskeletal actin and human c-fos gene
`
`promoters share a conserved protein-binding site
`
`
`Production of hepatitis B virus in vitro by transient
`
`expression of cloned HBV DNAin a hepatomacell line
`
`
`
`Altered transcription of a defective measles virus genome
`derived from a diseased human brain
`
`
`Adenovirus VAI RNA complexes with the 68 000 M,
`protein kinase to regulate its autophosphorylation and
`
`activity
`
`
`‘Import of frog prepropeptide GLa into microsomes
`
`requires ATP but does not involve docking protein or
`
`ribosomes
`
`
`; Independent mutations at the amino terminus of a
`_protein act as surrogate signals for mitochondrial import
`
`
`- Three suppressor mutations which cure a mitochondrial
`
`_RNAmaturasedeficiency occur at the same codon in the
`
`_ open reading frame of the nuclear NAM2 gene
`
`
`_A yeast mutant lacking mitochondrial porin is
`
`_respiratory-deficient, but can recover respiration with
`
`simultaneous accumulation of an 86-kd
`
`_extramitochondrial protein
`
`
`: The product of the mei3+ gene, expressed under control
`_of the mating-type locus, induces meiosis and sporulation
`
`in fission yeast
`
`
`Yeast DNA polymerase—DNA primase complex: cloning
`
`of PRI 1, a single essential gene related to DNA primase
`
`activity
`
`
`Structural transition in inactive Balbiani ring chromatin
`of Chironomus during micrococcus nuclease digestion
`
`
`Characterization and localization of the even-skipped
`protein of Drosophila
`
`
`EGF homologous sequences encoded in the genome of
`Drosophila melanogaster, and their relation to neurogenic
`
`: genes
`
`
`_ Developmental and molecular analysis of Deformed; a
`_homeotic gene controlling Drosophila head development
`
`
`Temporal and spatial distribution of transcripts from the
`_ Deformed gene of Drosophila
`
`
`K.Meckling-Hansen, R.Nelson, P.Branton and
`T.Pawson
`
`T.Mohun, N.Garrett and R.Treisman
`
`C.Chang, K.-s.Jeng, C.-p.Hu, S.J.Lo, T.-s.Su, L.-
`P.Ting, C.-K.Chou, S.-h.Han, E.Pfaff, J.Salfeld
`and H.Schaller
`
`R.Cattaneo, G.Rebmann, A.Schmid, K.Baczko,
`V.ter Meulen and M.A.Billeter
`
`M.G.Katze, D.DeCorato, B.Safer, J.Galabru and
`A.G.Hovanessian
`
`G.Schlenstedt and R.Zimmermann
`
`A. Vassarotti, R.Stroud and M.Douglas
`
`M.Labouesse, C.J.Herbert, G.Dujardin and
`P.P.Slonimski
`
`M.Dihanich, K.Suda and G.Schatz
`
`M.McLeod, M.Stein and D.Beach
`
`G.Lucchini, S.Francesconi, M.Foiani, G.Badaracco
`and P.Plevani
`
`R.M.Widmer, M.Lezzi and Th.Koller
`
`M.Frasch, T.Hoey, C.Rushlow, H.Doyle and
`M.Levine
`
`E.Knust, U.Dietrich, U.Tepass, K.A.Bremer,
`D.Weigel, H.Vassin and J.A.Campos-Ortega
`
`M.Regulski, N.McGinnis, R.Chadwick and
`W.McGinnis
`
`R.Chadwick and W.McGinnis
`
`659
`
`667
`
`675
`
`681
`
`689
`
`699
`
`705
`
`713
`
`723
`
`729
`
`737
`
`743
`
`749
`
`761
`
`767
`
`779
`
`
`
`Contents continues
`
`The EMBO Journal
`Volume 6 number 3. March 1987
`
`
`
`
`BIOEPIS EX. 1046
`Page 4
`
`BIOEPIS EX. 1046
`Page 4
`
`

`

`r
`X ontents (continued)
`
`The EMBO Journal
`Volume 6 number 3 March 1987
`
`structure and sequence of the Drosophila zeste gene
`
`V.Pirrotta, E.Manet, E.Hardon, S.E.Bickel and
`M.Benson
`
`ocyte-specific transcription of fs(1)K10: a Drosophila
`ene affecting dorsal—ventral developmental polarity
`
`M.Haenlin, C.Roos, A.Cassab and E.Mohier
`
`\utoregulation of bacteriophage P2 repressor
`
`S.Saha, B.Lundqvist and E.Haggard-Ljungquist
`
`Contacts between +6 resolvase and the 6 res site
`
`E.Falvey and N.D.F.Grindley
`
`Signa peptide amino acid sequences in Escherichia coli
`ontain information related to final protein localization.
`multivariate data analysis
`
`M.Sjéstrém, S.Wold, A.Wieslander and L.Rilfors
`
`Author index
`
`oduct news
`
`Situations vacant/announcements
`
`791
`
`801
`
`809
`
`815
`
`823
`
`833
`
`R
`overillustration: The cover showsthe anterior end of a germ band stage Drosophila embryo, viewed from the ventral side. At this stage the
`segmental organization of the head and thoracic regions is most evident. The embryo shownis a null mutant for the homeotic gene, Deformed,
`and exhibits abnormal development of two head segments. For more details, see pages 767—777 and pages 779—789 in this issue.
`
`PUBLISHERS ANNOUNCEMENT
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`
`BIOEPIS EX. 1046
`Page 5
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`BIOEPIS EX. 1046
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`

`

`The EMBO Journal vol.6 no.3 pp.605—-610, 1987
`
`Overexpression of the EGF receptor-related proto-oncogene erbB-2
`in human mammary tumorcell lines by different molecular
`mechanisms
`
`Matthias H.Kraus, Nicholas C.Popescu!, Suzanne
`C.Amsbaugh! and C.Richter King
`Laboratory of Cellular and Molecular Biology, and 'Laboratory ofBiology,
`National Cancer Institute, Building 37, Room 1E24, Bethesda, MD 20892,
`USA
`
`Communicated by Schlessinger
`
`Amplification of the erbB/EGFreceptor and a structurally
`related gene, designated erbB-2, have previously been detected
`in a variety of human tumors. In a series of human mam-
`mary tumorcell lines, analysis of transcripts of these genes
`revealedelevatedlevels 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-
`mal controls. evbB-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. Immunoblot analysis indicated
`elevation in levels of the 185-kd product of the erbB-2 gene
`expressed by these cells. In four lines erbB-2 gene amplifica-
`tion in the absence of an apparent gene rearrangement was
`demonstrated. In a representative cell line of this type, SK-
`BR-3, the amplified evbB-2 gene copies were located in an
`aberrant chromosomal location, Four additional cell lines,
`which demonstrated 4- to 8-fold overexpression of erbB-2
`mRNA,did not exhibit pene amplification. Ina representative
`cell line of this type 7R-75-1, an apparently normal chromo-
`somallocation was foundfor the erbB-2 gene. Our findings
`indicate that overexpression of the erbB-2 gene in mammary
`tumor cell
`lines is frequent and associated with different
`genetic abnormalities.
`Key words: crbB-2/gene amplification/growth factor receptor/
`mammary 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-
`codes a chain ofplatelet-derived growth factor (PDGF) (Doo-
`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 ef al. , 1984), and the
`c-fins proto-oncogene productis related to the receptor for mono-
`nuclear phagocyte growth factor (CSF-1,) (Sherr ez al., 1985).
`More recently, we and others (King ef a/., 1985a; Schechter er
`al., 1985; Sembaet al., 1985; Coussenset al. , 1985) have iden-
`tified a second cellular analogue of v-erB in the human genome.
`This gence, designated erbB-2, is related to but distinct from the
`gene encoding the erbB/EGF 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 er al., 1985; Yamamotoer al.,
`1986a), including a cysteine-rich extracellular domain, transmem-
`
`brane region and a highly conserved tyrosine kinase domain.
`Genealterations 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-
`somallocalization (Schechter et al., 1985; Coussens er al., 1985;
`Yamamoto et al., 1986a; Bargmanner al., 1986a). In human
`glioblastoma, amplification and rearrangement of the EGFR gene
`result in extensive expression of abnormal as well as normal-
`sized mRNAs(Libermannet al., 1985). In addition, amplifica-
`tion without rearrangements affecting EGFR mRNAsizeis fre-
`quently found in cells derived from squamous cell carcinomas
`(Yamamoto ef al., 1986b) and in two distinct mammary car-
`cinomacell 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 (Sembae¢ al., 1985). These
`findings have suggested the possibility that erbB-2 overexpres-
`sion may contribute to neoplastic growth (King ef al., 1985a;
`Sembaet al., 1985).
`For this study, we investigated the expression of erbB-2 and
`EGFRin 16 human mammary tumorcell lines. Our results in-
`dicate frequent overexpression of these proto-oncogenesthat arc
`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
`gence amplification, suggesting that different molecular mechan-
`isms result in overexpression of normal size erbB-2 mRNA in
`mammary tumorcells.
`
`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 1A). An oligo (dT) primed normal
`humanfibroblast cDNAlibrary (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-
`tained, pMAC 137, carried a 2-kbp insert comprising 1.5 kbp of
`3’ coding information and 3’ untranslated sequence. The remain-
`ing coding information upstream was obtained from three phage
`clones, MMAC30, AMAC10’ and MAC 14-1, identified in a ran-
`domly primed MCF-7 cDNAlibrary (Walter er al. , 1985; Figure
`1A). 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 ef al., 1985).
`Overexpression of erbB-2 or EGFR proto-oncogenes in human
`mammary tumorcell lines
`Toassess the role of erbB-2 in human mammary neoplasia we
`compared the mRNA of 16 mammary tumorcell lines to nor-
`mal humanfibroblasts, M413, and a human mammaryepithelial
`cell line, HBL100. Increased expression of an apparently nor-
`
`This material was copied
`atthe NLM and maybe
`Subject US Copyright Laws
`
`605
`
`BIOEPIS EX. 1046
`Page 6
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`BIOEPIS EX. 1046
`Page 6
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`

`

`M.H.Kraus et al.
`
`NN
`
`St
`
`Bll
`
`BIE
`
`PP SpBPBB
`\
`
`BSMEKSmBIIP
`\
`
`St
`
`5
`
`3
`
`E
`aEotSe pMAC137
`E
`E
`eaeel|»hMAC30
`
`Ep
`
`
`
`aeee AMAC10°
`
`——————
`
`AMAC14~1
`
`9
`
`1
`
`2
`
`3
`
`4
`
`5
`
`kbp
`
`Ca)aa|probes
`
`Fig. 1. (A) Isolation and restriction mapping of erbB-2 cDNA. Clone pMAC137 was isolated from an oligo (dT) primed normal human fibroblast CDNA
`
`
`library (Okayama and Berg,
`1983). Clones AMAC30, AMAC10" and AMAC 14-1 were sub: quently obtained from a randomly primed MCBE-7 CDNATibran
`
`(Walter er al., 1985). Restriction sites: B = BamHI, BIL = BstEll, E = EcoRI, N=Neol, P Psil, Sm Smal, Sp Sphl and St Sad. (B) cDNA
`
`
`
`
`probesused in hybridization analysis.
`
`A
`
`B
`
`ew,on 2
`o
`ot ~ 9
`oO
`rhe BEE?
`o8 2ft 1
`+
`© S&S
`so a a. Se.
`tear adogera
`zHnNwmotzFERPR OS
`
`—_
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`& 8
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`=f
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`*
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`erbB-2
`a
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`®,
`+
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`fe
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`ey
`s
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`28s +
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`185 ~
`
`ose relative amount
`
`4
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`
`4
`
`64 326 4
`32 4
`1
`seossesees
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`@eeeeeeoe@
`actin @@ @©6@806080
`@®vreee@eres®e- 6
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`ie
`e
`
`relative amount
`
`1 V4 v2 V2 V2 V4 W2
`
`4
`
`va
`
`1
`
`overexpression
`of erbB-2
`
`178 8 2864 64
`
`8
`
`4 6 1
`
`lines.
`(A) Northern blot analysis. Total cellular RNA (10 pe) of Mammary tumor cell
`lines.
`Fig. 2. Overexpression of erbB-2 in human mammary tumor cell
`normal human fibroblasts M413 and HBLIOO was hybridized with a cDNA probe derived from the 5‘ end ofthe erbB-2 coding region (Figure 1B, probe a)
`M413 and HBL100cells contain erbB-2 specific MRNA detectable after longer autoradiographic exposures. (B) Quantitation of crbB-2 mRNAlevels. Serial
`2-fold dilutions of total RNA were appliedto nitrocellulose. Replicate filters were hybridized with either a erhB-2 cDNA probe (Figure 1B. probe b) or
`human f-actin which served as control for RNA amounts present on the nitrocellulose filter, Relative amounts detected with cach probe are indicated in
`comparison to the hybridization signals observed in normal human fibroblasts M413.
`This material was copied
`606
`atthe NLM and may be
`Subject US Copyright Laws
`
`BIOEPIS EX. 1046
`Page 7
`
`BIOEPIS EX. 1046
`Page 7
`
`

`

`Overexpression of the erbB-2 gene in mammarytumorcells
`
`;
`Mw x10 °
`~— 200
`
`
`
`B
`
`Oo -
`aw
`iL Be
`ped
`oN S
`
`;
`
`|h
`
`
`
`°
`c
`|
`3
`=a
`+- + - mwxto®
`@ ~ 200
`
`
`
`Fig. 3. Elevated erbB-2 protein levels in mammary tumorcell lines, 40 pg
`total cellular protein was separated by electrophoresis and transferred to
`nitrocellulose filters. The erbB-2 protein was detected with an antipeptide
`antibody coupled to 125] 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 at 110 kd are observed in longer exposures of
`peptide-blocked immunoblots (panel A).
`
`Table [. Overexpression of erbB-2 and EGFR proto-oncogenes in human
`mammary neoplasia
`
`A
`
`
`EGER |
`erbBe
`Overexpression Gene
`Overexpression Gene
`of mRNA"
`amplification of mRNA"
`amplification
`
`
`
`
`I
`|
`M413
`I
`|
`HBL 100
`1
`|
`MCF-7
`I
`4-8
`128
`SK-BR-3
`e
`l
`8
`128
`BT474
`
`MDA-MB301—64 2-4 ! - 97 - - 97
`
`MDA MB453
`64
`2
`<
`1
`ZR-75-1
`&
`|
`l
`ZR-75-30
`4
`|
`al
`MDA-MB1I75
`8
`|
`:
`BT483
`8
`I
`<
`BT20
`|
`16
`+—$
`32
`32
`MDA-MB-+408
`|
`
`
`|
`
`|
`
`<
`
`‘Overexpression above normal fibroblast and HBL1OO
`
` =
`
`2
`Sok»
`eo oof ¢
`gi 2.2
` k
`fn t Zn FB wy
`LKR tt 8
`ononrnr
`oot * Spt ats
`~_oer Oe GOE
`ONO EN S53 w
`=
`
`erpB-
`
`2
`
`9e*e@eon+8
`*
`@ees
`*
`
`mos
`
`- 0
`an
`o
`OT &
`\
`ao? 7
`m
`£y FSPon wo
`om On '$ 1s & @
`to
`@ © Hite
`s
`Zve-rp QaOan ace
`>oo SSN N
`
`i
`
`23 -
`
`relative amount
`To.
`ove
`21
`1
`2
`
`4
`2% 1
`2
`0
`telalive gene copy 14 1
`numper of erbB-2
`
`Fig. 4. Gene amplification of erbB-2 in mammary tumorcell lines. (A)
`Southern blot analysis. For each lane 10 pg 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 served as mol. wt
`standards.
`(B) DNA dot-blot analysis. Genomic DNA (10 pg) digested with
`EcoRI was applied in serial 2-fold dilutions to nitrocellulose filters. Filters
`were hybridizedeither with erbB-2 (Figure 1B, 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 M413 indicate the minimal
`extent of gene amplification detected in DNA from mammary tumorcell
`lines.
`
`607
`
`BIOEPIS EX. 1046
`Page 8
`
`mal size 5-kb transcript was detectedin 8 of 16 tumorcell lines,
`when total cellular RNA was subjected to Northernblot analysis.
`Figure 2A shows the results using a cDNA probe comprising
`the coding sequences of the amino-terminal extracellular domain
`of erbB-2 (Figure 1B, probe a). These results of overexpression
`of normal-sized mRNAwere confirmedby hybridization ofpo-
`kbp
`ly(A)* selected RNA using several erbB-2-specific probes com-
`prising codinginformation for the transmembrane and tyrosine
`2a- -
`—
`kinase domains. An aberrantly sized erbB-2 mRNA was not
`we — —
`9.4
`
`
`6.6 - ooGeen - -
`detected in any of the cell
`lines analyzed.
`relative amount 147 641 121
`‘To quantitate more precisely the amount of erbB-2 transcript
`A.3 -
`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 appliedto the nitrocellulose filters, As shownin Figure
`2B, the highest levels of erbB-2 mRNA, which ranged from 64-
`to 128-fold overthat of our controls, were observed in the cell
`lines MDA-MB453, SK-BR-3, MDA-MB361 and BT474. More-
`over, erbB-2 mRNA levels were increased 4- to 8-fold in four
`cell
`lines
`including BT483, MDA-MBI75, ZR-75-30. and
`ZR-75-1 (Table 1).
`To determineif the overexpression oferbB-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 er/B-2 tyrosine kinase domain. As this
`region is partially conserved between the encodedproteins of
`the EGFRand erhB-2 genes, wetestedits specificity using A431
`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 wasdetectedin extracts of SK-BR-3 but not in A431
`cells. This band was not detected when the antibody was pre-
`that did not display
`of MCF-7, a mammary tumorcell line,
`incubated with the synthetic peptide corresponding toits antigen.
`overexpression of erbB-2 mRNA. Weinterpret these results to
`The human erbB-2 andrat new products have been reported to
`indicate that substantially more erbB-2 protein is found in both
`be glycoproteins of 185 kd (Akiyamaer al., 1986; Sternef al.,
`SK-BR-3 and ZR-75-1 than in MCF-7 cells where the amount
`1986).
`of protein escapes the sensitivity of the assay. Dilution ex-
`To estimate the relative amounts of evhB-2 protein in different
`periments suggest that Sk-BR-3 contains between 5- and 10-fold
`mammary tumorcell lines, immunoblot analysis was conducted
`more erbB-2 protein than does ZR-75-1 (data not shown).
`using equivalent amounts of total cellular protein. As shown in
`We also analyzed total cellular RNAs of the same mammary
`Figure 3B, an intense band of protein was detected in extracts
`tumorcell lines for evidence of EGFR receptorMRNA overex-
`of SK-BR-3 anda less intense but readily detectable bandin ex-
`pression. Increased amounts of an apparently normal size EGFR
`tracts of ZR-75-1. No erbB-2 protein was detected in extracts
`This material was copied
`atthe NLM and may be
`Subject US Copyright Laws
`
`BIOEPIS EX. 1046
`Page 8
`
`

`

`erbB-2 gene amplification in SK-BR-3 and BT 474 relauNG te ae
`mal human DNA and a 2- to 4-fold erbB-2 gene amplificatio
`in MDA MB361.
`In addition, a 2-fold erbB-2 gene amplifice
`tion was identified for the cell line MDA-MB453 byDNA fo
`blot analysis. Thus, gene amplification was nsseriaid -
`overexpression in the four tumorcell lines with the: highest Sa
`of erbB-2 MRNA(Table I). In contrast, gene amplification ae "
`not be detected by Southern blot analysis or PIA ene “
`analysis in the four tumorcell lines with erbB-2 Wanseript!
`creased to intermediate levels.
`malities
`To examine the nature of any chromosomal Ss
`associated with overexpression we usedin sift hyoriciabon 105
`localize the erhB-2 genein two cell lines which either door ¢
`:
`The erbB-2 gene has been4
`not contain amplified gene copies.
`. Wal l.22
`mapped in normal human cells on chromosome
`qii.e
`(Schechterer al. , 1985; Coussens eral. , 1985; Fukushige Hen
`1986). Mammarytumorcell line SK-BR-3 contains 4- SF 32
`gene amplification and
`128-fold overexpression of er :
`‘al
`mRNA.
`In SK-BR-3, G-banding showed no copics of norn a
`-
`watts
`.
`-bB-2 gene to these
`chromsome 17. /n situ hybridization of the erbB-2 ge
`ae
`cells revealed accumulations of grains on two large abnorma’
`marker chromosomes derived from complex rearrangements In-
`volving at least three chromosomes. An average Of three grains
`was observedat each labelled site (Figure 5). Seca
`dicate that the amplification of the erbB-2 gene occurs Inan : 7
`normal chromosomal location and is not associated with eit a
`a homogeneously stained region or double minute eeia
`abnormalities diagnostic for gene amplification Biedier ea
`Spengler, 1976; Levaner al., 1977). Mammary tumor cell nul
`ZR-75-1 showed no evidence of gene amplificauon and an o-fok
`overexpression of mRNA (Table I). Chromosome 17 an nel
`sent in one or two copies percell. Analysis of 50cells on i
`situ hybridization with a erbB-2 cDNA probe revealed!the large a
`accumulation of grains on chromosome 17 with 85% of these
`clustered on chromosome bands 17q11.2-21,
`the normal ine
`tion of the erbB-2 gene. This indicates that aveTe x pressuon ee
`the erbB-2 gene can occurin the absence of detectable structura!
`abnormalities of chromosome 17.
`Mammary tumorcell lines overexpressing erbB-2 do not con-
`tain readily detectable transforming genes
`In chemically induced rat neuroblastomas, @ point mutation within
`the transmembranous domain activates the rat homologue of
`erbB-2, neu,
`to transforming activity readily detectable in the
`NIH/373 transfection assay (Bargmannef al, 1986b). ou
`transfection analysis of 21 mammary tumors and tumor cell lines
`did not reveal activation of erbB-2 as a transforming gene mn
`human mammary neoplasia (Kraus ef al., 1984). To nVestients
`whether an activating lesion similar to the rat new gene was
`associated with the overexpression of erbB-2 in human roan
`mary tumor cell
`lines, we transfected genomic DNA ol
`these
`cell lines into mouse NIH/313cells. Under conditions where high
`mol. wt DNAfrom the cell line T24 which is knownto contain
`an activated H-ras oncogene induced 4—8 foci/plate, genomic
`DNA from eight mammary tumorcell lines which overexpress
`erbB-2 did not induce detectable morphological transformation
`(Table I).
`
`Discussion
`
`Analysis of 16 human mammary tumorcell lines for the EGE
`receptor andthe related erbB-2 gene revealed frequently increased
`transcript levels of cither member ofthis family of growth fac
`tor receptor genes.
`In BT20 and MDA-MB468 overexpression
`
`BIOEPIS EX. 1046
`Page 9
`
`M.H.Krausef al.
`
`‘
`
`¥
`¥ ¥
`; a Pa Te
`9
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`ue
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`1.
`
`9
`
`e
`s” 4
`*
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`Ss”
`
`4
`
`’
`
`Fig. 5. Representative metaphase from the SK-BR-3 carcinomacell 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 rearranged chromosome (arrow),
`(B) The same spread
`after trypsin/EDTA 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 shownto contain amplified EGFR
`genes. EGFR transcripts were elevated 16-fold in BT20 and
`32-fold in MDA-MB468 abovethe level seen in normal human
`fibroblasts as determined by RNAdot-blot analysis (Table I).
`Genetic abnormalities associated with elevated erbB-2 expression
`To investigate alterations of the erbB-2 gene associated with
`mRNAoverexpression, we examined Xbal-restricted high mol.
`wt DNAby Southernblotanalysis using a probe comprising the
`entire coding sequence oferbB-2 (Figure 1B, probe c). The nor-
`mal restriction pattern of Xbal fragments was detected in all DNA
`samples analyzed indicating that gene rearrangements in prox-
`imity ofthe erbB-2 coding regiondid not occurin these cell lines.
`When compared with normal humanfibroblast DNA (Figure 4A,
`lane 1) the erbB-2-specific Xbalrestriction fragments appeared
`clearly amplified in the cell lines SK-BR-3, BT474 and MDA-
`MB36] (Figure 4A, lanes 2—4). Similar results were obtained
`with restriction enzymes EcoRI and Sacl (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
`
`BIOEPIS EX. 1046
`Page 9
`
`

`

`Overexpression of the erbB-2 gene in mammary tumorcells
`
`Table I. DNA transfection of human mammary tumor cell
`Source of genomic DNA
`
`lines
`
`FFU/plate"
`
`of a multistep process which confers selective growth advantage
`to a mammary tumorcell.
`In all cell
`lines examined we detected no abnormalities of
`erbB-2 gene structure by Southern blot hybridization. Moreover,
`32/4
`T24 prep |
`in ZR-75-1,
`in which overexpression occurs without gene
`19/4
`124 prep 2
`amplification, the erbB-2 gene waslocated at its normal site on
`16/4
`[24 prep 3
`chromosome 17. These results indicate that mechanisms of
`0/12
`SK-BR-3
`O/12
`BI474
`deregulation are unlikely to involve the type of rearrangements
`0/2
`MDA MB361
`responsible for activation of the myc gene in Burkitt’s lymphoma
`0/4
`MDA-MB453
`(Taub ef al., 1982; Dalla-Favera ef al., 1983). Deregulation of
`0/8
`7R-T5-1
`erbB-2 gene expression may therefore involve subtle changesin
`0/4
`7R-75-30
`
`cis-acting control sequences, changes involving§transacting
`BT483
`0/4
`regulatory elements, or changes which enhance mRNA stability.
`MDA-MBI75
`O/8
`Abnormalitics of erbB-2 and EGFR genes are not restricted
`to mammary tumor cells in culture, as we and others have iden-
`tified erbB-2 or EGFR gene amplification in several samples of
`mammary tumortissue (King er al., 1985a; Yokota er al., 1986,
`and unpublished observation). The precise action of growth fac-
`tor receptor gene overexpression in the neoplastic process of
`mammary tumorcells has yet to be established. However, since
`tumorigenicity of mammary tumorcells can be enhanced by
`steroid hormones or polypeptide growth factors (Kasid et al.,
`1985; Dickson et al., 1986),
`it is tempting to hypothesize that
`the overexpression of polypeptide growth factor receptors in-
`creases tumorigenicity by stimulation of the same orsimilar
`growthsignalling pathways. Forty percent of mammary tumor
`cell lines analyzed did not exhibit overexpression of erbB-2 or
`EGER. Heterogeneity of steroid receptor expression is of con-
`siderable therapeutic and prognostic value in human mammary
`neoplasia (McClelland ef al., 1986; DeSombreer al., 1986). Our
`results strongly suggest an additional level of heterogeneity defin-
`ed by overexpression of the erbB-2 or EGFR gence.
`In the cell line SK-BR-3, where weidentified an amplified and
`overexpressed erbB-2 gene, a myc gene amplification with over-
`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 (Lander al.,
`1983).
`It will be ofinterest to determine whether overexpres-
`sion of erbB-2 and myc can complement each other in promoting
`the growth of mammary tumorcells. In addition, systematic
`analysis of mammary tumors will determine whether overexpres-
`sion of erbB-2 or the EGFR gene can be associated with a par-
`ticular clinical manifestation of mammary carcinoma.
`
`transfected plates. 30 pg high
`focus-forming units/number of
`‘BE U/plate
`mol. wt DNA were coprecipitated with calcium phosphate and transfected
`mito NIH/313 fibroblasts as previously described (Wigler er al., 1977).
`Genomic T24 DNA served as positive control for each assay
`
`of normal size EGFreceptor gene transcripts is associated with
`gene amplification (Kingef al., 1985b; Filmus ef al., 1985). In-
`creased transcript levels of erbB-2 were detected in the presence
`and absence of gene amplification, indicating that erbB-2 over-
`expression in human mammary tumorcell lines can be caused
`by different molecular mechanisms. The absence of aberrantly
`sized erbB-2 mRNA in Northern blot analysis suggests that a
`normal size MRNA is overexpressed in mammary tumorcell lines
`rather than a rearranged form. Moreover, the fact that genomic
`DNA from these cell
`lines lacked the ability to transform
`NIH/31T3cells by transfection indicates that point mutations
`similar to those that can activate the rat new gene did not occur.
`These observations provide evidence that a structurally normal
`coding sequence of erbB-2 is overexpressed as MRNA in human
`mammary tumorceil lines. Protein analysis of representative
`amples 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-
`oncogenesto the neoplastic process. The increased transcription
`of normal 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/31T3cells in culture (Chang ef
`al., 1982; Gazit eral., 1984). Moreover, in human tumors nye
`or N-mye amplification correlates with increased malignancy
`(Kohl et al., 1983; Schwab ef al., 1983; Nauer al., 1984). Our
`results link erbB-2 overexpression to the neoplastic growth of
`mammary tumorcells.
`Gene amplification of multidrug resistance genes is observed
`in cells selected for the ability to grow in media containing cer-
`tian metabolic inhibitors (Alt et a/., 1978). There is evidence
`that overexpression of these genes can precede gene amplifica-
`uion in the development of multidrug resistance (Shen ef al.
`1986). Our observation of elevated er/B-2 transcript levels in
`the presence and absence of gene amplification may reflect a
`similar pathway. Overexpression of erbB-2 may confer an in-
`itial selective growth advantage to the tumorcell and subsequent
`gene amplification cause a further step where the selective growth
`advantage is e