`
`5E0 E
`
`l
`5;
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`BIOEPIS EX. 1046
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`BIOEPIS EX. 1046
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`BIOEPIS EX. 1046
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`

`The EMBO Journal
`Contents
`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 GABAA/benzodiazepine receptor is a heterotetramer
`of homologous a and 6 subunits
`
`Differentially expressed bovine cytokeratin genes.
`Analysis of gene linkage and evolutionary conservation of
`5'-upstream sequences
`
`J .Verver, R.Goldbach, J .A.Garcia and P.Vos
`
`V.Herzog, W.Neumiiller and B.Holzmann
`
`C.Mama1aki, F.A.Stephenson and E.A.Bamard
`
`M.Blessing, H.Zentgraf and J .L.Jorcan0
`
`Irnmunoglobulin heavy chain switch region recombination
`within a retroviral vector in murine pre-B cells
`
`D.E.Ott, F.W.Alt and K.B.Marcu
`
`Superinduction of the human gene encoding immune
`interferon
`
`M.A.Lebendiker, C.Ta1, D.Sayar, S.Pi10, 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-B promoter
`
`H.Dinter and H.Hauser
`
`Overexpression of the EGF receptor-related proto-
`oncogene erbB-2 in human mammary tumor cell 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 mouse cells
`
`M.Tsuchiya, H.Nomura, S.Asano, YKaziro and
`S.Nagata
`
`Close genetic and physical linkage between the murine
`haemopoietic growth factor genes GM-CSF and Multi-
`CSF (1L3)
`
`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 retinal-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.Sch1'itz,
`R.Schl‘ile and R.Renkawitz
`
`V.Colantuoni, AAPirozzi, C.Blance and R.Cortese
`
`Molecular cloning of cDNA coding for rat proliferating
`cell nuclear antigen (PCNA)/cyclin
`
`K.Matsurnoto, T.Moriuchi, T.Koji and P.K.Nakane
`
`Molecular cloning of the B-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.My11y1éi, M.—L.Huhtala, J .Koivu and
`K.I.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.Wi11iams
`
`549
`
`555
`
`561
`
`567
`
`577 '
`
`585
`
`591
`
`599
`
`605
`
`611
`
`617
`
`625
`
`631
`
`637
`
`643
`
`651
`
`
`
`BIOEPIS EX. 1046
`
`Page 3
`
`BIOEPIS EX. 1046
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`
`

`

`r
`
`The EMBO Journal
`Contents (comm)
`Volume 6 number 3 March 1987
`
`
`Enzymatic activation of Fujinami sarcoma vims gag—fps
`
`fitransforming 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 DNA in a hepatoma cell 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
`
`p activity
`
`Import of frog prepropeptide GLa into microsomes
`
`inquires ATP but does not involve docking protein or
`
`{ribosomes
`
`Independent mutations at the amino terminus of a
`j protein act as surrogate signals for mitochondrial import
`
`
`, Three suppressor mutations which cure a mitochondrial
`
`. RNA maturase deficiency occur at the same codon in the
`
`' open reading frame of the nuclear NAMZ gene
`
`
`-A yeast mutant lacking mitochondrial porin is
`
`.mpiratory-deficient, but can recover respiration with
`
`:simultaneous accumulation of an 86—kd
`
`extramitochondrial protein
`
`
`.
`1' The product of the mei3+ gene, expressed under control
`3 of the mating-type locus, induces meiosis and sporulation
`
`lln fission yeast
`
`
`: Yeast DNA polymerase—DNA primase complex: cloning
`
`‘ of PRI 1, a single essential gene related to DNA primase
`
`activity
`
`
`EStructural transition in inactive Balbiani ring chromatin
`in! 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
`
`gum
`
`
`Developmental and molecular analysis of Deformed; a
`f homeotic gene controlling Drosophila head development
`
`
`'7 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.L0, T.-s.Su, L.—
`P.Ting, C.-K.Chou, S.—h.Han, E.Pfaff, J.Salfeld
`and H.Schaller
`
`R.Cattaneo, GiRebmann, A.Schmid, K.Baczk0,
`V.ter Meulen and M.A.Billeter
`
`M.G.Katze, D.DeCorato, B.Safer, J.Ga]abru and
`A.G.Hovanessian
`
`G.Sch]enstedt and R.Zimmermann
`
`A.Vassar0tti, R.Str0ud and M.Douglas
`
`M.Lab0uesse, 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.Véissin and J.A.Campos—Ortega
`
`M.Regu1ski, 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
`
`
`
`BIOEPIS EX. 1046
`
`Page 4
`
`BIOEPIS EX. 1046
`Page 4
`
`

`

`I‘ Ol’ltCIltS (continued)
`
`The EMBO Journal
`Volume 6 number 3 March 1987
`
`"I cture and sequence of the Drosophila zeste gene
`
`V.Pirrotta, E.Manet, E.Hard0n, S.E.Bickel and
`M.Benson
`
`e-specific transcription of fs(1)K10: a Drasophila
`t.
`w affecting dorsal—ventral developmental polarity
`
`M.Haenlin, C.Roos, A.Cassab and E.Mohier
`
`_
`
`| regulation of bacteriophage P2 repressor
`
`S.Saha, B.Lundqvist and E.Haggéird—Ljungquist
`
`‘I tacts between 76 resolvase and the 745 res site
`
`E.Falvey and N.D.F.Grindley
`
`811"- peptide amino acid sequences in Escherichia coli
`it :'| information related to final protein localization.
`multivariate data analysis
`
`M.Sj6strom, S.Wold, A.Wieslander and L.Rilfors
`
`t
`
`that index
`
`uuct news
`
`.1‘
`
`tions vacant/announcements
`
`791
`
`801
`
`809
`
`815
`
`823
`
`833
`
`.5
`Vver illustration: The cover shows the anterior end of a germ band stage Drosophila embryo, viewed from the ventral side. At this stage the
`my enta] organization of the head and thoracic regions is most evident. The embryo shown is a null mutant for the homeotic gene, Deformed,
`:iu exhibits abnormal development of two head segments. For more details, see pages 767—777 and pages 779—789 in this issue.
`
`PUBLISHERS ANNOUNCEMENT
`
`Submission of manuscripts on floppy disks
`
`(0865) 882283.
`
`'y Photosetting directly from floppy disks facilitates faster
`production of proofs and eliminates the opportunity for
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`, currently able to process 5% inch floppy disks in IBM
`PC/XT/AT (and compatibles) format (either 360 kbyte
`‘ double sided, 180 kbyte single sided or 1.2 Mbyte AT).
`'
`The format used must be clearly marked on the disk. In
`.
`addition, the publishers prefer that the material be keyed
`
`:
`
`using WordStar. Alternatively, if this is not possible, the
`material should be in standard ASCII files (i.e.
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`It is important to note that material submitted to the
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`Eynsham, Oxford 0X8 1].], UK. Tel.
`
`BIOEPIS EX. 1046
`
`Page 5
`
`BIOEPIS EX. 1046
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`
`

`

`The EMBO Journal vo|.6 no.3 pp.(>05—610, 1987
`
`Overexpression of the EGF receptor-related prom—oncogene erbB-2
`in human mammary tumor cell lines by different molecular
`mechanisms
`
`Matthias I'I.Kraus, Nicholas C.I’opescu', Suzanne
`(LAmsbaughl and C.Richter King
`
`Laboratory of Cellular and Molecular Biology, and lLaboratory of Biology,
`National Cancer Institute. Building 37, Room 1124, Bethesda. MD 20892.
`USA
`
`Communicated by J.Schlessinger
`
`Amplification of the erbB/EGF receptor and a structurally
`related gene, designated erbB-Z, have previously been detected
`in a variety of human tumors. In a series of human mam-
`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 crbB-Z
`mRNA levels ranging from 4- to 128—1'old above those of nor-
`mal controls. erbB-Z 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—Z
`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-Z 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 erbB-Z gene copies were located in an
`aberrant chromosomal location. Four additional cell lines,
`which demonstrated 4- to 8-fold overexpression of erbB-Z
`mRNA, did not exhibit gene amplification. In a representative
`cell line of this type 'l.R—75-l , an apparently normal chromo-
`somal location was found for 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: ('rbB—Z/gcne amp]itication/growth factor receptor/
`mammary neoplasiii/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 of platelet—derived growth factor (PDGF) (D00-
`little cl (:1. , 1983; Waterfield e101,, 1983), the v-crbB oncogene
`has been shown to encode a truncated form of the epidermal
`growth factor (EGF) receptor (Downward (’t (:1. , 1984), and the
`c-jim' prom-oncogene product is related to the receptor for mono-
`nuclear phagocyte growth factor (CSF—IR) (Sherr at (11. , 1985).
`More recently, we and others (King er (11., 198521; Schcchter er
`(1]., 1985; Semba er ((1., 1985; Coussens er ((1., 1985) have iden-
`tified a second cellular analogue of v~erbB in the human genome.
`This gene, designated crbB-2, is related to but distinct from the
`gene encoding the erbB/EGF receptor (EGFR). The predicted
`amino acid sequence of the (Ir/)B—Z gene reveals the structural
`features of a growth factor receptor molecule with close similarity
`to the EGF receptor (Coussens cl (1]., 1985; Yamamoto et (11.,
`1986a), including a cysteine—rich extracellular domain, transmem—
`
`brane region and a highly conserved tyrosine kinase domain.
`Gene alterations affecting EGFR and erbB-Z occur in tumor
`cells. A dominant cellular transforming gene activated by point
`mutation in chemically induced rat neuroblastomas (Shih et a1. ,
`1981), neu, is likely to be the rat homologue of human er-bB-Z
`based on comparative nucleotide sequence analysis and chromo-
`somal localization (Seheehter et a1. , 1985; Coussens et (11., 1985',
`Yamamoto et (11., 1986a; Bargmann at (11., 1986a). In human
`glioblastoma, amplification and rearrangement of the EGFR gene
`result in extensive expression of abnormal as well as normal~
`sized mRNAs (Libermann et (11., 1985). In addition, amplifica-
`tion without rearrangements affecting EGFR mRNA size is fre—
`quently found in cells derived from squamous cell carcinomas
`(Yamamoto et (11., 1986b) and in two distinct mammary car—
`cinoma cell lines (King (31(11, 1985b; Filmus er ((1., 1985).
`Gene amplification of crbB-Z has been identified in a primary
`mammary adenocarcinoma (King et (11., 1985a), as well as in
`a salivary gland adenocarcinoma (Semba et (11., 1985). These
`findings have suggested the possibility that erbB-2 overexpres—
`sion may contribute to neoplastic growth (King et (11., 1985a;
`Semba er (1]., 1985).
`For this study, we investigated the expression of erbB—Z and
`EGFR in 16 human mammary tumor cell lines. Our results in-
`dicate frequent overexpression of these prom-oncogenes that are
`related to growth factor receptors. Furthermore, analysis of the
`erbB-2 gene locus in these cell lines demonstrates that enhanced
`erbB—Z expression can occur in the presence or the absence of
`gene amplification, suggesting that different molecular mechan—
`isms result in overexpression of normal size erbB—Z mRNA in
`mammary tumor cells.
`
`Results
`
`[solution of erbB-Z complementaly DNA
`To allow a comprehensive analysis of crbB-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
`human fibroblast cDNA library (Okayama and Berg, 1983) was
`screened with a 0.8 kbp Ach DNA fragment from a genomic
`clone of crbB—Z (King et (11., 1985a). The largest plasmid ob-
`tained, pMACl37, carried a 2-kbp insert comprising 1.5 kbp of
`3’ coding information and 3’ untrzmslated sequence. The remain-
`ing coding information upstream was obtained from three phage
`clones, )\MAC30, )xMAClO’ and )xMACl4-1, identified in a ran-
`domly primed MCF—7 cDNA library (Walter et (11., 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 at (11., 1985).
`Overexpresxion of erbB—2 or EGFR prom-oncogenes in human
`mammary tumor cell lines
`To assess the role of crbB-2 in human mammary neoplasia we
`compared the mRNA of 16 mammary tumor cell lines to nor—
`mal human fibroblasts, M413, and a human mammary epithelial
`cell line, HBLlOO. Increased expression of an apparently nor—
`
`l'his material wascflpied
`at: the N LM 3 1'le may 1312
`Subject Uiflopyright Laws
`
`605
`
`BIOEPIS EX. 1046
`
`Page 6
`
`BIOEPIS EX. 1046
`Page 6
`
`

`

`MJLKraus et all
`
`NN
`31
`all
`BIIE
`BSmEKSmBIIP
`PP SpBPBB
`Si
`\
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`
`% )‘.MACSO
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`
`L‘lfi—i‘fi—a—hfiw
`0
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`2
`3
`4
`5
`
`kbp
`
`l: E probes
`
`luulmn Illnnblai r_*l>l\"\
`ll-‘Vlllml
`Fig. l. (A) Isolation and restriction mappingY ol' (‘rbUiZ EDNA, (‘lonu pMMfl £7 wzr. wilaiutl from an ulisro til ll I‘lllll‘,‘,l
`
`
`library (Okayama and Ber”
`1983), Clones XMAS“), AMAC [0’ and hM/V'liirl wen; sub» quantiv illllnillytl
`lmm ll
`isizitlnml’» [Hunt-l \'l"l
`7 HUNA lIl'VIJL
`.b‘lllrll‘
`‘s'p
`(Walter L'/ ul., 1985). Rustriction silcs: B : [fa/NHL Bll
`7’
`It’s/Ell. Ii
`. lz'le, N
`le. l’
`.I‘Sll‘
`is'm
`Nil/ll anxl
`‘s'l
`5/111
`(Ii) UNIV"
`probes used in hybridization analysis.
`
`A
`
`B
`
`"minon,“
`('3ka
`m
`C)
`IT gag?
`m$szii.;‘j$o
`~||v<<(,v:
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`relative amount
`
`l
`
`”4 1/2
`
`1/2
`
`il? ‘/l
`
`‘12
`
`ovarexpression
`0t a_vbB—2
`
`1 Q8 8 128 64 64
`
`3
`
`1
`
`4
`
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`
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`
`,M_
`
`lmcs.
`'I‘oial cullular RNA (1!) lug) ot HHIHHIILII'} 11mm «.‘L‘ll
`(A) Northcrn blot analysis,
`Fig. 2. Ovurcxprcssion ol (Ir/)BQ in human mammary tumor cell Imus.
`normal human fibroblasts M413 and HBl.l()0 was liybridizctl with a CDNA probc {icerL‘Ll Iron) mt» ‘3’ cml ol’ [lit (7)!” 2 LULllllL’
`[Hymn il'iytm-
`lll. pmlwv;
`in
`M413 and HBLIOO cells contain ('rbBiE specific mRNA dctcctahlc al'tcr longcr auloiutliogiaphic cxpusum, (B) Qiiamnatimi o1 WWI)“ l ian\/\ lL'\/.'l'\. Scial
`Z—lbld dilutions ol
`total RNA wen: applied to niuocullulosc. chliuatc filters wurc hybridized with Clil'lL‘l' a wr/rli 2 EDNA pmbt- ll‘lL’llIL’
`Ill
`pmbv: b; or
`human [ivactin which served as control
`lor RNA amounts present on the nutmu-llulosc liltcr. Relative amounts (lCiL‘L‘lL’Ll Wllll L'iltll morn:
`lllL‘ imlitaml
`in
`comparison to the hybridization signals observed in normal human fibroblasts M413.
`This material was [apiad
`atthe NLM andmavba
`Subject U5 Capilrright Laws
`
`606
`
`BIOEPIS EX. 1046
`
`Page7
`
`BIOEPIS EX. 1046
`Page 7
`
`

`

`'l'al)le l. f)ver'esprcssion ot' t’r‘l/ll l and liGl’R protooncogenes in humatt
`marmnary rrcoplasia
`
`A
`
`
`latil'Rr
`7
`cr/ll} _" 7
`f)vcrcsr)r'cssron (jenc
`(lverexpressron (lene
`
`ot’ n'rRNA"
`am rlification ot mRNA"
`atrrplifieation
`
`’r
`
`Ml! l
`lllll,llltl
`.\l(.‘l‘ 7
`5K llls’.
`ll'l'rl’rl-t
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`/.l’- 7‘3 l
`/.l1. 7:.
`to
`.‘\'ll)A.\1l3]"/‘S
`ll] JX‘
`11'] It)
`.‘v‘lllA Mil-lot“;
`
`l
`I
`l
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`I
`
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`1‘4
`1‘2
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`4
`t4
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`
`
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`
`()verexpression of the erbB-2 gene in mammary tumor cells
`
`c?
`0:
`z
`‘0
`<
`+ - + - MWxto'3
`
`.. ~200
`
`.—97
`
`B
`
`m .—
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`
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`
`i
`Mero 3
`
`*200
`
`.-97
`
`lines. 40 pg
`Fig. 3, Elevated ('r/JBQ protein levels in mammary tumor cell
`total cellttlar protein was separated by electrophoresis and translerrcd to
`nitrocellulose filters. The t'r‘IIBQ protein was detected with an antipeptide
`antibody coupled to 13“] protein A. The specificity 01‘ antibody detection was
`determined by pre—incubatron ot' the antibody with excess amounts of peptide
`prior to irnmurtodetectton, H J prerneubatron with peptide. (7) no peptide.
`ln panel B. nonspecific bands at “0 kd are observed irt longer exposures of
`pepttdeeblocked immtmoblots (panel A).
`
`
`
`'t)verr_~-'.prr_".ston ahotc rror'riial fibroblast and Illll,
`
`lltt
`
`rtral size 52kt: transcript was detected in 8 o!" lo ttrrttor cell lines.
`when total cellular RNA was subjected to .\ortl1ern blot analysis.
`Figure _A shows tlte resttlts usingy a cl)N/\ probe comprising
`the coding sequences ot' the amino—terminal extracellular domain
`at z'r/JllQ (Figure lli. probe a). These results ot overexpression
`oi normal sized mRNA were cortfirrtted by hybridization of po—
`lytA) ' selected RNA using several ('rbBQispecilic probes eorw
`prising coding inl'orrnation tor the transmembrane and tyrosine
`lginase dornairts. An aberrantly sized (Ir/Ilia? mRNA was not
`«,letected in arty ol' the cell
`lines analyzed.
`'l'o quantitate more precisely the amottnt ot' «MB—2 transcript
`in eight matrrmary tumor cell
`litres which overexpress «MB—2.
`serial Zil‘old dilutions ot' total cellular RNA were subjected to
`tlot blot artalysis using human I)" actin as a control for the amount
`<>l RNA applied to the nitrocellulose filters, As sltown in lir‘igure
`EB. the highest levels ot'erbBQ mRNA. which ranged 11-011th
`to lZX-t‘old over that ol' our controls. were observed iii the cell
`lines MDA»MB453. SK—BR—l MDAVMBJOI arid B’l‘474. Morei
`over. ('11de mRNA levels were incr‘ased 47 to 8—fold in four
`cell
`litres
`includingr
`[311183. Ml)A—MB|75.
`[RI/5730 arid
`list—75v]
`t'l‘able I).
`To determine it the overexpression ol‘erbB—Z ntRNA resulted
`in a steady state increase ol‘
`its encoded gene product. we
`developed a specilic immttnoblot assay. Antisera were raised
`against a synthetic peptide whose sequence corresponded to a
`portion ol' the putative (Ir/287:2 tyrosine kinase domain. As this
`region is partially conserved betweert the encoded proteins of
`the EUI'R and (Ir/)B—Z genes. we tested its specificity using A431
`and SK~BR5§ cell
`lines which overexpress EGFR or erbB~2
`rnRNA. respectively. As shown in Figure 3A. a specific band
`rrl
`-~ 185 kd was detected in extracts oli SK—BRJ but not in A431
`cells. This band was not detected when the antibody was pre4
`incubated with the synthetic peptide corresponding to its antigen.
`'l‘he human orbli—Z and rat um products have been reported to
`be glycoproteins ol' 185 ltd (Akiyama ('1 1d,. 1986; Stern 0111],.
`”86).
`the assay. Dilution ex—
`of protein escapes the sensitivity ol'
`To estimate the relative amounts ol‘ (Ar/)BQ protein indifferent
`periments suggest that SK-BRS contains between 5 and 107t‘old
`mammary tttrnor cell lines, irttrnuttoblot analysis was condttcted
`more w‘bB—Z protein than does ZR—75—l (data not shown).
`usingr equivalent amounts ol total cellular protein. As shown in
`We also analyzed total cellular RNAs ol' the same mammary
`l‘r'gurc 58. an intense bartd ol‘ protein was detected itt extracts
`tumor cell lines for evidence of EGFR receptor mRNA over-ere
`ol SKillRV-Ii arid a less intense httt readily detectable band in ex;
`pression. Increased amounts of an apparently normal size EGFR
`tracts ot' ZR—75 I. No (WM! 2 protein was detected itt extracts
`This material was cupied
`atthe NLM andmavbe
`Subjett US Cupyright Laws
`
`er,
`1mg
`5"
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`relative amount
`1
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`
`relative-gene copy
`1
`4
`t
`1
`2
`t”
`I
`2
`try
`manner at area 7 2
`
`Fig. 4. Gene amplification ot' crbBQ in mammary tumor cell lines. (A)
`Southern blot analysis. For each latte 10 a}; genomic DNA were restricted
`wrth X/ml and hybridized with a probe cttrtipr‘isirtg the entire coding region
`at (”1,3,3 Hr'ndlll restriction fragments of lambda DNA served as mol. wt
`standards.
`(I!) DNA dot—blot analysis. Genomic DNA (l0 pg) digested with
`Ii‘t'oRl was applied in serial 27fold dilutions to nitrocellulose filters. Filters
`were hybridized either with whlSQ (Figure ”3. probe b) or mm, which
`served as a control
`l'or DNA amounts applied to replicate nitrocellulose
`filters. Gene copy numbers of crbB—2 relative to Mill?» indicate the minimal
`extent of gene amplification detected itt DNA from mammary tumor cell
`lines,
`
`that did not display
`line,
`of MCF-7, a mammary tumor cell
`overexpression of erbB~2 mRNA. We interpret these results to
`indicate that substantially more ()rbB-2 protein is found in both
`SKVBR-3 and ZRA75—l than in MCF—7 cells where the amount
`
`607
`
`BIOEPIS EX. 1046
`
`Page 8
`
`BIOEPIS EX. 1046
`Page 8
`
`

`

`M.H.Kraus et a].
`
`l.
`
`
`
`I
`
`A.
`
`,1-
`.
`s
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`
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`M.
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`.r
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`
`line
`Fig. 5. Representative nietaphtisc l'rom tbc SKiliR—Ti carcinoma cell
`after in .ri'tu hybridization with an cr/JBQ cDNA probe, autoradiopraphy and
`GAbanding. (A) A chromosome spread exhibiting silver grains on a
`distinctive, highly rearranged chromosome (arrow).
`(H) The same spicad
`alter trypsin/EDTA treatment to produce G—bantls. Detailed (irhandiiig
`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 normal human
`fibroblasts as determined by RNA dot—blot analysis (Table 1).
`Genetic abnormalities associated with elevated (#17872 express/(in
`To investigate alterations of the erbBQ gene associated with
`mRNA overexpression, we examined Xbalirestrictcd high mol.
`Wt DNA by Southern blot analysis using a probe comprising the
`entire coding sequence of erbB 2 (Figure 18, probe c). The nor;
`mal restriction pattern of Xbal fragments was detected in all DNA
`samples analyzed indicating that gene rearrangements in prox-
`imity of the erbB-2 coding region did not occur in these cell lines.
`When compared with normal human fibroblast DNA (Figure 4A,
`lane 1) the crbB—Z—specific XbaI restriction fragments appeared
`clearly amplified in the cell lines SK—BR-3, BT474 and MDA7
`M8361 (Figure 4A, lanes 2‘4). Similar results were obtained
`with restriction enzymes EcoRI and Said (data not shown).
`Quantitation oi er/zB-2 gene copy number was accomplished
`using DNA dot—blot analysis. These studies revealed 1147 to 87lold
`
`608
`
`(’I‘/)B-3 gene amplification in SK lilt 3 and H1474 icl:lll\c_tdniflil
`mal human DNA and a 27 to 4 told wr/ili 3 PM“ innplilicatlti'
`in MM Masai. in addition, a 27min vrblilfiwc ”mimmi‘.
`non was identified im- the cell tint- MDA MBJJ.’ WPM dig
`blot analysis.
`'lhus‘ gene amplification was associated it'll“:
`ovcrcxprcssion in the tour tumor ccll lines With lllc“ lilgllgxl knit
`ol'crl)BrZ mRNA (Table I). In contrast, ccnc amplillczillon L‘Ulll i
`not be detected by Southern blot analysb 01' DNA 9?“) L1:
`analysis in the tour tumor ccil
`lincs with cr/iliQ [Hm-Still“ ”
`creased to intermediate levels.
`_
`.
`To examine the nature of any chromosomal abnormaliti:H
`associated with overcxprcss‘ion we used in \i/u hybridization t
`i()CLtil'/,L' the cr/ilifl acne in two cell liiicW Wh'd' unlit-“l d.” m it]
`not contain amplified gcnc coplcfi. “W "”l’lg’z Elwyn“ bi;
`”WPPCd in normal human cells on clllwnimfimg I
`(II I" i—
`(Schcchtcr «In/H WXS: ('ous‘scns ('I (1/.‘ “)8”: I'lil‘l‘bhlgwfiililti
`1986i. Mammary tumor ccll linc SK BR 3 CUHlil‘m 4730,6112;j
`gene amplification and
`llxilold ()VCIVCXPIICSMU.“ ”I. H J H]
`mRNA.
`in SKiliRfi. (iibanding showed no copies oi mull;
`chronisonie 17. In .i'im hybridization ol'thc (Ir/2112 gcnc to Ill“ IL.
`cells revealed accumulations of grains on two large abnoil‘i‘ti.
`marker chromosomes dcrivcd l'roin complex rearrangements lll'
`volvitig at least three chromosomes. An avcraglc ”‘ thrcc 14W“
`was observed at each labelled sitc (l’igurc 5)‘ ilillcwd'c‘ullb'lg:
`dicate that the amplification ot' the (WM?2 E'U'lCI‘K'Clmm ‘Hi ‘1‘,
`normal chromosomal location and is iioi associated Wllil c1111LI
`a homogeneously stained region or double minute chromosome:
`abnormalities diagnostic for gene amplification (Bicdlci
`im:
`Spengler. W76; chan (‘I ul., W77). Mammary Illm‘” H” _““1
`741(775 1 showed no evidence ol'gciic amplification and an X-tolt
`overcxprcssion ol' mRNA (Table l)’ Vhr“”"“f’”” 17 was In
`sent in one or two copies per cclli Analysb 0' 5” “CH“ ”It“: \Hi
`silu hybridization with a midi—2 cl)N/\ probc i‘c‘vcalcdpthc laigf \.
`accumulation ol' grains on chromosome 17 W11“ 85f ”I Ih‘f
`clustered on chromosome bands l7ql l.2»21t tilt? ”0””“1 I‘M"
`tioii ol' the (Ir/>82 gene, This indicates that
`(iVCl‘CXPrL‘S'H‘m f):
`the crbliQ gene can occur in the absence ol‘dctcctablc Nlltic‘ltll-h
`abnormalities of chromosome l7.
`
`i’Vlunmiwjy Iiiiiim' ('c/l Iiiim oi'(‘t‘tatyirt's's'iiii! ""I’B'g d“ W” (WP
`Iai/i readily (/clt’t'luhlc II'mi.\_'/in‘niiii,:: miter
`_
`_
`_
`lti chemically induced rat iicuroblastomas. a point mutation with”!
`the transincmbranous domain activates the rat
`lionioldguC m
`(Jr/)BrZ,
`III’H,
`to traiisloriiiing activity readily detectable intlic
`NIH/3T3 transl'cction assay (Bargniann c/ (11.. NRbhi Pl'm’lmi“
`translcclion analysis ol’21 mammary tumors and tumor cell lincs
`did not reveal activation ol
`(‘I‘lIii—z as a ti‘aiisloll‘mlig‘ gut” ”1
`human mammary iicoplasia (Klaus cl u/rt 1984) I“ iiivestigtlls‘
`whether an activating lesion similar to the rat m’" £40113 “‘15
`associated with the ovcrexpression ol’ w'I)BA2‘m human imam:
`mary tumor cell
`lines‘ we traiisl'cctcd genomicl)NA ol
`the.»
`cell lines into mouse NIH/3T3 cclls. Under conditions where high
`mol. wt DNA from the cell line 124 which is known to contain
`an activated H—ms oncogene iiidticcd 4 ”8 locil/Plillu genomic
`DNA from eight mammary tumor cell lines which chI'CM’lF”
`(Ir/)BQ did not induce detectable morphological translormatiori
`(Table ll).
`
`Discussion
`
`lines lUl' thc 1‘11};
`Analysis of lo human mammary tumor cell
`receptor and the related (Ir/mil gene revealed lrcqucntly incrcasc‘t.
`transcript levels ol'either mcmbcr of this lamily of growth lac
`tor receptor genes.
`in BT20 and Ml)/\7Mli-’1t’18 ovcrcxprcssion
`
`BIOEPIS EX. 1046
`
`Page 9
`
`BIOEPIS EX. 1046
`Page 9
`
`

`

`'lablc ll. DNA trarr».tcctioii ol human mammary tttiiior cell
`"s‘iiiiru:
`til er'iioiiiit DNA
`
`lznes
`
`liliU tplate"
`
`'13,} prep 1
`1171 prep 1
`11-1 prep '4
`“$15.
`111’.
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`n] H»)
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`titlitt
`\tl‘m Mltl fit
`
`12/4
`19/4
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`(1/12
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`0/2
`0/4
`WK
`0/4
`0/4
`0/8
`
`1t)t.'|.t‘~. ltttlltlllt’ tinits’iitliiilwr o1 transt'ected plates. 30 ng high
`‘1-1-1?,/1il:iii.-
`iiiol wt 1)ts.-\ were eripieeipitatetl
`ivith calcitiiii phosphate and transfeeted
`tllll) Nllli ’51}.
`ltl)t'ttl)l;i:~,ts a. picwiotisly described (Wigler e/ ul.. 1977).
`{iciioiiiic lil DNA net wet] as positive control
`tor each assay
`
`ol normal size litil‘ receptor gene transcripts is associated with
`gene amplification (King ('I (1].. 1985b; Filmus or ill. . 1985). hi—
`creased transcript levels ofcr/iBQ were detected iii the presence
`and absence of gene amplification. indicating that (’I’ltB-2 over»
`expression in litiiiiaii iiiaiiiriiary tumor cell
`lines can be caused
`by ditlerent niolecttlar mechanisms. 'l‘lie absence of aberraritly
`.i/ed (#21172 mRNA in Northern blot analysis suggests that a
`normal si/e mRNA is overexpressed iii mammary tumor cell lines
`rather than a rearranged form. Moreover. the fact that genomic
`DNA lroiii
`these cell
`lines lacked the ability to transform
`Nlll/fl'l'fl cells by transfection indicates that point mutations
`similar to those that can activate the rat new gene did not occtir.
`'1 lie-re observations provide evidence that a structurally normal
`it Mine sequence of erbBQ is overexpressed as mRNA in human
`mammary tumor cell
`lines. Protein analysis of representative
`i2t111ple‘f‘t with (Ir/)BQ ovcrcxpression suggests that elevated erbB—Z
`lt'ttlt.’~.Ct‘l[)l
`levels are translated into (Jr/I132 proteins.
`Several
`lines of evidence link the overexl‘iression of pron»
`oncogenes to the neoplastic process. The increased transcription
`of normal ctxling sequences ol‘eitlier the littiiiaii c-.\i.\'/l’l)Gll gene
`or the H ms gene using a viral
`long terminal repeat promoter
`induces transloriiiation ol Nllrl/Ll'lfll cells in culture (Chang ct
`(//.. 1982‘. (la/it (’I (1/.. 1984). Moreover. in human tumors More
`or Nairn‘ amplification correlates with increased iiialignaticy
`(Kohl (’/(1/.. 1983'. Schwab e/ (l/.. 1983'. Nati ct (I/.. 1984). Our
`results link ('r/Jlifl overexpression to the neoplastic growth of
`mammary tumor cells.
`(ieiic amplilieation of itiultidrug resistance genes is observed
`in cell». selected for the ability to grow in media containing cer-
`tian metabolic inhibitors (All at (1].. 1978). There is evidence
`that overexpression of these genes can precede gene amplificte
`rioii
`in the development of iiiultidrug resistance (Sheri cl al..
`19546). Our observation of elevated ”/7112 transcript levels in
`the presence and absence ol gene ariiplificatiori may rellect a
`mimilar pathway. ()verexpression of (Ir/)BQ riiay confer art
`inv
`ll ial selective growth advantage to the tumor cell and subsequent
`gene aiiiplilicatioit cause a [rather step where the selective growth
`advantage is enhanced and stabilized. (Ir/)BQ overexpressioti is
`consistently higher in those samples with gene aiiiplilication when
`',tititt)ttt'e(l with cell lines lacking gene aiiiplilication. lnterestingly.
`the amount of overexpressioii per gene copy is approximately
`corritant iii the cell lines that overexpress ('rhlt—Q t'l'able 1). This
`llgge’d‘s that deregulated er/IIL'Z expression dtie to different
`molecular mechanisms in mammary tumor cell lilies may be part
`
`()vercxprcssion of the crbB—Z gene in mammary tumor cells
`
`ofa multistcp process which confers selective growth advantage
`to a mammary tumor cell,
`In all cell
`lines examined we detected no abnormalities of
`(Ir/J82 gene structure by Southern blot hybridization. Moreover.
`iii ZR—75—l.
`in which ovcrcxpression occurs without gene
`amplification. the crbB—Z gene was located at its normal site on
`chromosome 17. These results indi‘ate that mechanisms of
`deregulation are unlikely to involve the type of rearrangements
`responsible for activation of the myc gene in Burkitt‘s lymphoma
`(Tatib (it (11.. 1982; Dalla—Favera (it (11.. 1983). Deregulation of
`erbB-Z gene expression may therefore involve subtle changes in
`cisuacting control
`sequences. changes involving transacting
`regulatory elements. or changes which enhance mRNA stability.
`Abnormalities of erbB—Z and EGFR genes are not restricted
`to mammary tumor cells in culture, as we and others have iden—
`lifted (’I‘IJB-2 or EGFR gene amplification in several samples of
`mammary tumor tissue (King 6’] (11.. 1985a; Yokota (It (1].. 1986.
`and unpublished observation). The precise action of growth fac—
`tor receptor gene overexpression in the neoplastic process of
`mammary tumor cells has yet to