`
`5E0 E
`
`l
`5;
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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
`
`
`
`PFIZER EX. 1546
`
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`
`

`

`r
`
`The EMBO Journal
`Contents (comma)
`Volume 6 number 3 March 1987
`
`
`Enzymatic activation of Fujinami sarcoma vims gag—fps
`transforming proteins by autophosphorylation at tyrosine
`
`’ Xenopus cytoskeletal actin and human c-fos gene
`promoters share a conserved protein-binding site
`
`A 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
`
`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, GrRebmann, 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
`
`
`
`_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
`
`7 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
`respiratory-deficient, but can recover respiration with
`:simultaneous accumulation of an 86—kd
`extramitochondrial protein
`
`.
`
`it 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
`i9! 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
`f homeotic gene controlling Drosophila head development
`
`'7 Temporal and spatial distribution of transcripts from the
`-_ Deformed gene of Drosophila
`
`
`
`PFIZER EX. 1546
`
`Page 4
`
`PFIZER EX. 1546
`Page 4
`
`

`

`I‘
`
`(continued)
`
`The EMBO Journal
`Volume 6 number 3 March 1987
`
`"I cture and sequence of the Drosophila zeste gene
`
`V.Pirrotta, E.Manet, E.Hardon, S.E.Bickel and
`M.Benson
`
`e-specific transcription of fs(1)K10: in Drosophila
`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 EiHaggiird—Ljungquist
`
`‘I tacts between 76 resolvase and the 745 res site
`
`E.Falvey and N.D.F.Grindley
`
`1,1“: peptide amino acid sequences in Escherichia coli
`it 5| information related to final protein localization.
`multivariate data analysis
`
`M.Sj6strom, S.Wold, A.Wieslander and L.Rilfors
`
`1'
`
`that index
`
`nuct 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 Drosaphila 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
`the introduction of typographic errors. The publishers are
`, 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 A7)
`'
`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.
`those that
`are readable when TYPEd on to screen or printer).
`It is important to note that material submitted to the
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`copy. Queries regarding formats should be addressed to
`the Production Editor, IRL Press Ltd, PO Box 1,
`Eynsham, Oxford 0X8 1].], UK. Tel.
`
`PFIZER EX. 1546
`
`Page 5
`
`PFIZER EX. 1546
`Page 5
`
`

`

`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
`
`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 al. ,
`1981), neu, is likely to be the rat homologue of human crbB-Z
`based on comparative nucleotide sequence analysis and chromo-
`somal localization (Schechter et a1. , 1985; Coussens et al. , 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 al., 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 at (11., 1985b; Filmus et 01., 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 al, 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.
`
`Subject Uiflepyright Laws
`
`Matthias I'I.Kraus, Nicholas C.I’opescu', Suzanne
`(LAmslmughl and C.Richter King
`
`Laboratory of Cellular and Molecular Biology. and lLaboratory of Biology,
`National Cancer Institute, Building 37, Room 1E24, Bethesda. MD 20892.
`USA
`
`Communicated by J.Schlessinger
`
`Amplification of the erbB/EGF receptor and a structurally
`related gene, designated crbB-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 aetin 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: crbB—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—oncogenc en—
`codes a chain of platelet—derived growth factor (PDGF) (D00-
`little cl ul. , 1983; Waterficld et al. , 1983), the v-crbB oncogene
`has been shown to encode a truncated form of the epidermal
`growth factor (EGF) receptor (Downward at u]. , 1984), and the
`c-jim' prom-oncogene product is related to the receptor for mono-
`nuclear phagocytc growth factor (CSF—IR) (Sherr at (11., 1985).
`More recently, we and others (King er (1]., 198521; Schcchter er
`(1]., 1985; Semba er al. , 1985; Coussens er al., 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—
`
`Results
`
`[solution of erbB-Z complementary 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 al, 1985a). The largest plasmid 0b-
`taincd, 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 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 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, HBLIOO. Increased expression of an apparently nor—
`
`l'his material wascflpied
`at: the N LM 3 1'in may be
`
`PFIZER EX. 1546
`Page 6
`
`

`

`MJLKraus et all
`
`all
`BIIE
`BSmEKSmBIIP
`PP SDBPBB
`,
`\
`_
`‘
`5'_JL_____L_____L_____LL___li__l_lLLi———_———l—l—Jl—J—i—————J-—-—————- 3
`E
`
`——i—__h___ pMAC137
`
`% ).MACSO
`
`AMAClO'
`
`AMACM *1
`
`iwlzuml than an Mlls'n M ii inmml
`Fig. l. (A) Isolation and rcxtrictiun mappingY ol' (‘rbUiZ EDNA, (‘lmiu pMMfl i7 wzr.
`“brier “)ki'ym'm and Berg. 1983} ClOHC‘x XMAS“), AMAC [0’ and hM/V'liirl “wen:
`‘yill'r'uljtlll‘QIJilV whimlml
`lmm 1|
`is:
`(Wither L'l u/.,
`I985). Rmtrictiun silt»: B : [fa/NHL Bll
`7’ [is/Ell. Ii
`1 lz'le, N
`le. l’
`.I‘kll‘ 3m
`.‘mml‘
`‘x'p
`probcx mud in hybridization analysis.
`
`luulmii Illvmblai r_*l>l\"\
`ll-‘Vlllml
`luml’» l"“”“>'*] “H 7 "DNA hm’n'
`Sir/1| awl
`‘w’l
`5/111
`in) EDNA
`
`A
`
`v—
`"I
`w mw vm m
`
`2 Il I
`
`< <C! Li
`I I
`
`MBA-M8361
`
`MDA‘MB453
`
`-30
`
`ZFl~T
`
`erbB' 2
`
`I
`
`o.
`
`00.
`
`:0.
`
`g.MBA—mews
`
`OBT4B’3
`
`relative amoun’l
`
`1
`
`3.2
`
`4
`
`64 3215
`
`a
`
`a
`
`4
`
`I
`
`Subject U5 Capilrright Laws
`
`lmcm
`'I‘mal cyllulnr RNA (1!) W) Ul mmnmm‘; 111mm «.‘L‘ll
`(A) Nul'lllcrn l)l(![ anulyxi»,
`Fig. 2. Ovurcxprcsxion oi (Ir/)BQ in human mammary Iumnr cull Imus.
`nUrmal human fibroblasts M413 and HELIOO was hybridile with a CDNA pmhc dcrivul l’mm mg ‘3’ mid ul’ lllL‘ (Ir/1H 2 LULllllL’
`IL'leHIi tl'iyuu-
`lll. pmiiv;
`in
`M413 and HBLIOO cells contain ('rbBiE specific mRNA dctcctahlc allcr lnngcr auluiauliugiaphic prlinurfl’», (B) Qiiumnaimn n1 4’71)“ 2 le\A luwlx. Scull
`Z—lkild dilutions 0|
`total RNA wen: applied to niuucullulusc. chliuaic fillers, wurc hyliridi/ud with cilnrr :1 (MB 2 EDNA pmlu- (l‘wmr:
`Ill pmlw in nr
`human [ivuclin which served as conlml
`lkir RNA amnums prosecnl on Ihu nullitylluluxc lilicr. RclllllVU anmunia downed wuh L'iltll nml'u: 2m- imlnnml
`in
`comparison to the liyhridizaliun signals Observed in annual human lihi‘ohlasl‘. M413.
`This material was [apiad
`atthe NLM antimavi
`
`“W38?
`
`0
`.00...
`000000000.
`MnOooooooooO
`.0...Q..'.
`0 v.1
`0
`
`.v
`
`relative amounl
`
`ovarexpression
`of a_vbB—2
`
`l
`
`1
`
`1/4
`
`1/2
`
`1/2
`
`ll? ‘ll
`
`‘12
`
`‘28 8 QB 64 64
`
`3
`
`1
`
`4
`
`1/4
`
`)6
`
`I
`
`1
`
`606
`
`PFIZER EX. 1546
`Page 7
`
`

`

`'l'al)le l. f)ver'espression ot' t’r‘l/ll l and liGl’R protooncogenes in humatt
`mammary rrcoplasia
`cr/rll."
`
`rztrr'rc
`()verexpressron (lene
`f)vcrcsr)r'cssron (.lenc
`ot’ n'rRNA"
`am rlification ot ntRNA"
`atrrplifieation
`
`()verexpressien of the erbB-2 gene in mammary tumor cells
`
`A
`
`B
`
`‘N'
`I'
`
`"97
`
`lines. 40 pg
`lt‘ig. 3, Elevated ('r/JBQ protein levels in mammary tumor cell
`total cellttlar protein was separated by electrophoresis and translerrcd to
`nitrocellulose filters. The ('r‘IIBQ protein was detected with an antipeptitle
`antibody coupled to ‘3”! protein A. The specificity 01‘ antibody detection was
`determined by pre—incubatron ot' the antibody with excess amounts of peptide
`prior to immunodetectton, t) J prerncubatron with peptide. (7) no peptide.
`ln panel B. nonspecific bands at “0 kd are observed irt longer exposures ot’
`pepttdeiblocked immunoblols (panel A).
`
`l
`l
`1
`l
`l
`l
`l
`l
`
`Ill42
`
`it
`
`R
`
`MDAMBEGt
`
`MDAAMBTTS
`
`MDA<MBQSJ
`
`.
`
`,,
`
`r/
`lt’r
`12
`
`
`
`’r
`
`Mill
`lllll,llX)
`.\l(.‘lr 7
`5K llii
`ll'l'rlrl-l
`.\1]).-\ Mllittl
`MDA Mll-if’it
`/.l’. 7‘3 l
`/.l’-. 7::
`it)
`.\'ll)Ar.\llll"/5
`ll'lJXl
`11'] It)
`.‘v‘ll)/\ Mil-lot“:
`
`1
`I
`l
`lll‘l
`11*?
`(rl
`or
`b’
`rl
`s
`H
`l
`
`
`
`.
`
`"
`
`'t)verr_~-;prr_".ston ahotc rror'rtial fibroblast and Illll, UH
`
`Subjett US Cupyright Laws
`
`rtral size 52kt: transcript was detected in 8 of lo ttrrttor cell lines.
`when total cellular RNA was subjected to .\orthern blot analysis.
`Figure _A shows tlte resttlts usiany a cl)N/\ probe comprising
`the coding sequences ot' the amino—terminal extracellular domain
`at z'r/JllQ (Figure lli. probe a). These results ol overexpression
`oi normal sized mRNA were cortfirrned by hybridization of po—
`lytA) ' selected RNA using several ('rbBQispecilic probes eorw
`prising coding inl'orrnation tor the transmembrane and tyrosine
`lginase dotnaitts. An aberrarttly sized (Ir/Ilia? mRNA was not
`detected in any ol' the cell
`lines analyzed.
`'l'o quantitate more precisely the amottnt ot' «MB—2 transcript
`in eight matrrmary tumor cell lines which overexpress w'bB—L’.
`serial Zil‘old dilutions ot' total cellular RNA were subjected to
`tlot blot artalysis using human I)" actin as a control for the amount
`oi RNA applied to the nitrocellulose filters, As sltown in lir‘igure
`EB. the highest levels ot'crbBQ mRNA. which ranged from ()4;
`to lZX-t‘old over that ol' our controls. were observed iii the cell
`lines MDA»MB453. SK—BR-B. MDAVMBJOI arid B’l‘474. More?
`over. ('11de mRNA levels were incr‘ased 47 to 8—fold in four
`cell
`litres
`includianr
`[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 imntttnoblot 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 between 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 irt extracts oli SK—BRJ bttt not in A431
`cells. This band was not detected when the antibody was pro;
`incubated with the synthetic peptide corresponding to its antigen.
`'l‘he human erbli—Z and rat um products ltave been reported to
`be glycoproteins ol' 185 ltd (Akiyarna ('1 ul,. 1986; Stern ('t 11],.
`HMO).
`of protein escapes the sensitivity of the assay. Dilution ex—
`To estimate the relative amounts ol‘ crbliQ protein indifferent
`periments suggest that SK-BRS contains between 5 and 107t‘old
`mammary tttrnor cell lines, irttrnunoblot analysis was conducted
`tnore w‘bB—Z protein than does 7..R—75—l (data not shown).
`using, equivalent amounts ol total cellular protein. As shown in
`We also analyzed total cellular RNAs ol' the same mammary
`l‘r'gurc )8. an intense bartd ol‘ protein was detected in extracts
`tumor cell lines for evidence of EGFR receptor mRNA over-ere
`oi SKiliRr-Ii arid a less intense httt readily detectable band in ex;
`pression. lrtcreased amounts of an apparently normal size EGFR
`tracts ot' ZR—75 I. No w'lrli 2 protein was detected itt extracts
`This material was cupied
`atthe NLM andmavbe
`
`MDA—MBS"r
`
`MDAJvthSZ}
`
`relative amount
`
`1
`
`4
`
`Va
`
`8
`
`4
`
`t
`
`1
`
`2
`
`t
`
`000......
`0.0-
`o
`‘
`
`relative amount
`reldttve gene copy
`ntrmnnr 0t erbaig
`
`Fig. 4. Gene atnplifieation ot' erbBQ in mammary tumor cell lines. (A)
`Southern blot analysis. For each latte 10 a}; genomic DNA were restricted
`wrtlt X/ml and hybridized with a probe comprising the entire coding region
`at (Ir/)BVZ, Hindlll restriction fragments of lambda DNA served as mol. wt
`standards.
`(I!) DNA dot—blot analysis. Genomic DNA (l0 pg) digested with
`Ii‘t'okl was applied irt serial 2it‘old dilutions to nitrocellulose filters. Filters
`were hybridized either with whlSQ (Figure lB. 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 M4l3 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
`
`PFIZER EX. 1546
`Page 8
`
`

`

`M.H.Kraus et al.
`
`whit-2 gene amplification iii SK lilt 3 and H1474 iclull\c_ttlniflil
`trial human DNA and a 27 to 4 told wr/ili 3 PM“ “mplmcgilii'
`in MDA Masai. In addition, a 27min obligfiwc “mlmmi‘.
`tion was identified lor the cell imt- MDA MBJJ.’ "Hm" all;
`blot analysis.
`'llius‘ gene atnplification was associated pvt“:
`ovcrcxprcssion in the tour tumor ccll lines With lllc“ lily,th lt‘~l‘w
`ol'crbBQ ttiRNA (Table l). [ti contrast, gcnc ainplillcztllttn L‘Ulll
`not bc detected by Southern blot analysb 01' DNA fllll’h L1:
`analysis iii the tour tumor ccll
`lines with cr/iliQ [Hm-Still“ ”
`creased to intermediate levels.
`_
`.
`To examine the nature of any chromosomal
`illi‘litillli‘clllllzy
`associated with overexprcss‘ion we used in win hybridization
`lt)CLtll'/,L' thc cr/ilifl gene in two cell lincW ‘Vh'd' cum} (1.00Ifxtl
`not contain amplified gene coplcfi. “W "MHZ Elwyn“ bi;
`“lul’PCd in normal human cells on clll'mnimfimfii
`I
`(II I" T—
`(Schcchtcr (’IH/H IQXS: ('ous‘scns ('I (1/.‘ “)8”: I‘lil‘ibhlgcfllililrl
`1986). Mammary tumor cell line SK BR 3 Clllllil‘m 47 tuba-l i5;j
`gene amplification and
`llxitold iivcrcxlwwwin m. H H]
`niRNA.
`ln SKiliRd. (iibanding showed no copies ol mull;
`chromsomc 17. [H mm hybridization ol'thc (Ir/2112 gcnc to Ill“ IL.
`cells revealed accumulations of grains on two large ltlHIUHli‘iL
`marker chromosomes derivcd l'roin complex rearrangcincins in
`volving at least three chromosomes. An avcraglc “l mm“ will!“
`was observed at each labelled sitc (l’igurc 5t ilillcwd'c‘lllbllg:
`dicate that the amplification ot' the (WM?2 E'U'WI‘WCLHUH ‘Hl ‘1‘,
`tiortiial chromosomal location and is iioi associated Wllll cillltLI
`a homogeneously stained region or double minute L‘lll'ttliltts‘tilllti
`abnormalities diagnostic for gene amplification (litcdlct
`Spengler. W76; chan (‘I ul., W77). Mammary Illm‘” Ls” ll:
`741(775 1 showed no evidence ol'gcnc amplification and an X-tolt
`overcxprcssion oi mRNA (Table l)’ (lllrt"l"“l’lllc l7 ‘Wufi
`sent in one or two copies per ccllr Analysb 0' 5” CCU“ “Hf” 12'
`still hybridization with a «Hill—2 cl)N/\ probc l‘DVCillULlJllL‘ laigg
`accumulation ol' grains on chromosome 17 W11“ 85": “l [mi
`clustered on chromosome bands l7ql I 2»th Ills? “0””“1 I‘M"
`tioti ol' the (Ir/>82 gene, This indicates that
`(tVCl‘CXPrL‘S'HOn
`the (Ir/)BQ gene can occur in the abscttce ol‘dctcctablc Niltic‘ltll-h
`abnormalities of chromosome l7.
`
`In BT20 and Ml)/\*Mli¥lt’18 over-expression
`
`Munniiwjv tumor cell lines ot'(‘Hatyirt's's'i'lii! ("i/’11“? d“ W” a)”
`min readily (/(’l(’('l((/)/('
`II'(III.\_'/i)l‘lllill,t,’ critter
`_
`_
`_
`ln chemically induced rat nctiroblastomas. a point mutation Within
`the transinembranous domain activates tltc rat
`ll(llll(ll0guc m
`(Jr/Mia,
`III’H,
`to translitrniing activity readily detectable ititlic
`NIH/3T3 ti‘anlection assay (Bargmann (’I (11.. 1985'” Pl'cvlml“
`translcction analysis ol21 mammary tumors and tumor cell lincs
`did not reveal activation ol
`(Ir/Ili—Z as a transloflimig‘ gc'ic 1“
`human mammary itcoplasia (Klaus oi (i/tt 1984) l“ “lvcmgllw
`whether an activating lcsion similar to the rat m’" £40113 “‘15
`associated with the ovcrexprcssion ol’ w'I)BA2‘m human imam:
`mary tumor cell
`lines‘ we transt'ccted genomicl)NA ot
`ihcsu
`cell lines into mouse NIH/3T3 cells. Under conditions where high
`mol. wt DNA from the cell litre 124 which is known to contain
`an activated H—ms oncogene induced 4 fits locil/Plillu 11C"““““
`DNA from eight mammary tumor cell lines wlncli chI'CM’lF”
`crbBQ did not induce detectable morphological translormatiori
`(Table ll).
`
`Discussion
`
`lines lor the 1‘11};
`Analysis of lo human mammary tumor cell
`receptor and the related (Ir/mil gctic revealed lrcqucntly incrcasc‘t.
`transcript levels ol'eithcr member of this l'amily ol growth lacr
`tor receptor genes.
`
`line
`Fig. 5. Representative rnctaphasc l'rom the SKABR—3 carcinoma cell
`after ill
`.ri'tu hybridization with an cr/JBQ cDNA probe, autoradiogiaphy and
`Gabanding. (A) A chromosome spread exhibiting silver grains oti a
`distinctive, highly rearranged chromosome (arrow).
`(B) The same spread
`alter trypsin/EDTA treatment to produce G—bands. Detailed (irbanding
`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
`CCll 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/(iii
`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—specifie 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 Sac-l (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
`
`PFIZER EX. 1546
`Page 9
`
`

`

`trarist'ecied plates. 30 )1g high
`1t)t.'|.t‘~. toimnie units’riiiiiilm' ol
`‘1-1-1?/p11ne
`niol wt 1)1s.-’\ were eripieeir‘iitated tvith ctilcitiiii phosphate and transfeeted
`tllll) NHL ’51}. 111)l'ltl)l;12»l's a.
`t)tc‘\lt)tt’sl)’ described (Wigler e/ ul.. 1077).
`{icrioiiiic 1L1 DNA sci wet] as positive control
`tor each assay
`
`()vcrcxprcssion of the crbB—Z gene in mammary tumor cells
`
`ofa multisth 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.
`in ZR—75—1.
`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
`(Truth or al.. 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 er‘bB—Z and EGFR genes are not restricted
`to mammary tumor cells in culture, as we and others have iden—
`lifted arbB-Q or EGFR gene amplification in several samples of
`mammary ttimor tissue (King 6’] (1].. 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 be established. However, since
`tumorigenicity of mammary tumor cells can be enhanced by
`steroid hormones or polypeptide growth factors (Kasid cf (11.,
`1985: Dickson ()7 (11., 1986),
`it is tempting to hypothesize that
`the ()VCI'CXpI‘CSSlOn of polypeptide growth factor receptors iri-
`creascs ttiiiiorigenicity by stimulation of the same or similar
`growth signalling pathways. Forty percent of mammary tumor
`cell lines analyzed did not exhibit overexpression of ('rbB—2 or
`EGFR. Heterogeneity of steroid receptor expression is of con—
`siderable therapetiti ‘ and prognostic value in human mammary
`neoplasia (McClelland (It (1]., 1986; DcSombre cl al., 1986). Otir
`results strongly suggest art additional level of heterogeneity defin»
`ed by overexpression of the (Ir/)Bi2 or EGFR gene.
`In the cell line SK-BR-B. where we identified an amplified and
`overexpresscd crbBQ gene. a nryc gene amplification with over-
`expression has been previously reported (Kozbor and Croce,
`1984). The mvc gene has been shown to cooperate with a ms
`gene in the transtormation of primary fibroblasts (Land 6! (11..
`1983).
`It will be of interest to determine whether overexpres<
`sion of (’l‘bB‘Z 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-
`sion of (’I‘hB-2 or the EGFR gene can be associated with a par-
`ticttlar clinical manifestation of mammary carcinoma.
`
`Materials and methods
`
`'llmmr cell llmnr
`
`Human mammary tumor cell lines were obtained from ATCC tAmcriean Type.
`Culture Collection) and cultured according to the supplier‘s recommendation.
`
`RNA blotting
`'l'otal cellular RNA,» were isolated by CsCligradient ccntriftigation (Chirgwin or
`(i/.. 1079). lror Northern blot analysis 10 [lg total RNA were denatured and electrtv
`plltri‘csctl in 1% lornitildehydc gels (Lehrticli 0111].. 1977). Followrng mild alkali
`degradation (50 niM NaOH at 24"(‘ for 30 min) RNA was transferred to
`nitrocellulose filters using 1 M ammonium acetate as a convcetant. RNA doe
`blot analysis was conducted by applying serial 2 told dilutions of total RNA in
`1 M ammonium acetate to replicate nitrocellulose filters in a dot-blot manifold.
`
`DNA blotting
`For Southern blot analysis DNA samples t 10 ltg) were restricted with lel and
`subjected to electrophoresis in 0.8% agai'osc gels. DNAs were tr‘tlrislei’i‘etl to nitro—
`cellulose filters by the method o1 Southern (Southern. 1975).
`l"ordot—b1ot analysis
`l)N/\s were cleaved with licoRl. denatured with 0.1 M NaOH and neutralized
`with 1 M ammonium acetate. Serial 3~lold dilutions of each sample were ap—
`plied to replicate nitrocellulose filters using ti dot—blot manifold.
`
`Th'
`'
`|
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`is materia was copra
`atthe NLM and may be
`
`609
`
`'lablc ll. DNA traristccriori o1 human mammary tttiiior cell 12nes
`"s‘rniru:
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`o1 norri‘ial si/c litil‘ receptor gene transcripts is associated with
`gene amplification (King ('I (1].. 1985b; Filmus of NI. . 1985).
`In—
`creased transcript levels ofcr/iBQ were detected iii the presence
`and absence of gene ariiplificatiori. indicating that (’I’llB-2 over»
`expression in litiiiiaii iiiaiiiriiary tumor cell
`lines can be caused
`by ditterent molecular mechanisms. 'l‘lie absence of aberraiitly
`.i/ed (#2112 mRNA in Northern blot analysis suggests that a
`normal si/c mRNA is overexpressed iti mammary tumor cell lines
`rather than a rearranged form. Moreover. the fact that genomic
`DNA 1roiii
`these cell
`lines lacked the ability to transform
`Nlll/fl'l'fl cells by translcction indicates that point mutations
`similar to those that can activate the rat m'u gene did not occtir.
`'1 liesc observations provide evidence that a structur