OCTOBER 1987
`VOLUME 84
`NUMBER 20
`
`I~
`
`Proceedings
`
`OF THE
`
`National Academy
`of Sciences
`
`BIOEPIS EX. 1047
`Page 1
`
`

`

`Proceedings
`OF THE
`National Academy
`of Sciences
`OF THE UNITED STATES OF AMERICA
`
`Officers
`oftlze
`Academy
`
`Editorial Board
`of the
`Proceedings
`
`FRANK PRESS, President
`JAMES D. EBERT, Vice President
`PETER H. RAVEN, 1/ome Secretary
`WILLIAM E. GoRDON, Foreign Secretary
`ELKAN R. BLOUT, Treasurer
`
`ROBERT H. ABELES
`GORDON A. BAYM
`WILLIAM F. BRACE
`RONALD BRESLOW
`MICHAEL J. CHAMBERLIN
`
`MAXINE F. SINGER, Chairman
`MARY-DELL CIIIL TON
`EDWARD E. DAVID, JR.
`STUART A. KORNFELD
`DANIEL E. KOSIILAND, JR.
`PETER D. LAX
`DANIEL NATIIANS
`
`HERBERT E. SCARF
`SoLoMON H. SNYDER
`HAROLD V ARM US
`THOMAS A. WALDMANN
`FRANK H. WESTIIEIMER
`
`Managing Editor: FRANCES R. ZWANZIG
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`
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`
`BIOEPIS EX. 1047
`Page 2
`
`

`

`Proc. Na rl . A cad. Sci. USA
`Vo l. 84. pp. 71 59 -7 163, Oc tobe r 1987
`Cell Biology
`
`Increased expression of the putative growth factor receptor
`pl85HER2 causes transformation and tumorigenesis of NIH 3T3 cells
`
`R OBE RT M. HUD Z IAK *, JOSE PH S C HL ESS INGER T, AND AX E L ULLRI C H *
`
`*Department of Deve lopme ntal lliology. Genen tech, Inc .. 460 Point Sa n Bruno Boulevard. South Sa n Francisco, CA 94080; and t Biotechnology Research
`Ce nt er. Meloy Laboratories. 4 Resea rch Court. Rockville. MD 20850
`·
`
`Co mmunicar ed by 1/i/ary Koprou ·ski. July 13. ! 987
`
`The HER2 gene encodes a cell-surface glyco(cid:173)
`ABSTRACT
`protein with extensive homology to the epidermal growth factor
`receptor. Recently it was found to be amplified in about 30 %
`of primary human breast malignancies. In experiments de(cid:173)
`signed to assess the role of the HER2 gene in oncogenesis, we
`found that overexpression of unaltered HER2 coding sequences
`in NIH 3T3 cells resulted in cellular transformation and
`tumorigenesis.
`
`The IIER2 ge ne e ncodes a transmembrane glycoprotein with
`ex ten s ive stru ct ural homology to the human e pidermal
`growth factor (EGF·) receptor and the chicken oncogene
`v-erhB (1-3) . Chro moso ma l mapping and sequence compar(cid:173)
`iso n stro ngly sugge st that th e IIER2 gene product and the
`eth ylnitrosourea-ac ti vated , rat neuroblastoma oncogene neu
`represe nt spec ies variants of the same polypeptide (4) . The
`ne11 o ncoge ne e ncodes a 185-k Da cell- surface glycoprotein
`that po ssesses intrin sic tyros ine-specific kinase activity that
`is like ly to be activ ated by an as yet unidentified ligand (5 , 6).
`Co mpariso n of th e transfo rming ne11 oncogene sequence with
`its norma l rat protooncogcne counte rpa rt suggested that a
`point mut ati o n in th e tra nsme mbra ne domain resulting in
`substitution o f a va lin e res idue by gluta mate unmasked the
`tra ns forming potential of thi s put ative growth factor receptor
`(7) . Anal ogously , stru ctural alterati ons have converted nor(cid:173)
`mal ge nes coding for the receptors for macrophage colony(cid:173)
`stimul ating fac tor type 1 a nd EGF into v ~fins (8) and v-erhB
`(9) o ncoge nes, respec tiv e ly.
`So uth e rn a nalysis of primary human tumors and estab(cid:173)
`li shed tumor-de ri ved ce ll lines revealed a mplification a nd in
`some cases rearrangement of the EGF rece ptor gene . Am(cid:173)
`plification was pa rti cu la rl y appare nt in squamous carcinomas
`00, 11) a nd gli ob lastomas (12) . The HER2 gene was also
`found to be amplified in a human salivary gland ade nocarci(cid:173)
`noma (3), a ma mma ry gland carcinoma (2), and a gastric
`ca nce r cell line 03). Rece ntl y, Slamon et a/ . (14) de mon(cid:173)
`strated that abo ut 30% of primary hum an breast carcinoma
`tumors cont a in ed a n a mplifi ed HER2 ge ne. Although a few
`sequ e nce rea rra nge me nt s were detected , in most tumors
`there we re no obvious differences between amplified and
`no rm a l HER2 ge nes. Fu rthermore , a mplificatio n of the
`Hl:-'1?2 gene corre la ted signifi ca ntl y with the prognosis of the
`di sease a nd th e proba bility of re lapse .
`To investi gate th e significa nce of the co rrelation betwee n
`overexpression a nd ce llul a r transformation as it has been
`observed for prot ooncogcnes c-mos (1 5) and c- Ha-ras / (16),
`we e mplo yed a IIER2 expression vector and a selection
`sc he me th at pe rmitted seq ue nce amplification aft er transfec(cid:173)
`ti o n of mo use NIH 3T3 ce ll s. We report here that amplifi(cid:173)
`ca tio n of th e una ltered HER2 ge ne in NIH 3T3 cell s leads to
`ove rex prcssio n of p185 1 tE Rl as well as cellular transform ation
`and tum o r formation in ath ymic mi ce. These findings, in
`
`combination wit~ the_ re sul~ s of Slamon et a/. (14) , suggest
`that mere amplificatiOn of the HER2 gene and resulting
`overexpression of its product may play a crucial role in the
`genesi s and development of some types of huma n cancer.
`
`MATERIALS AND METHODS
`
`Expression Plasmids. The mammalian expression vector
`CVN (17) contained expression unit s for mou se dihydrofolate
`reductase (DHFR) eDNA (18) and the bacterial neomycin
`phosphotransfera se (neo) gene (19) , both under simian virus
`40 early promoter control. Transcription of a 4.4-kilobase(cid:173)
`pair Sal l-Dra I HER2 fragment containing the full-length
`l-IER2 coding region (1) was driven by the Rou s sarcoma
`virus (RSY) long terminal repeat promoter (L TR) . The
`poly(A) site was provided by the 3' untran slated sequence of
`the hepatitis B virus surface antigen gene (20). The control
`CVN plasmid was identical but lacked eDNA sequences
`downstream from the RSY L TR.
`Cell Culture. NIH 3T3 cell s were cultured in a 1:1 mixture
`of Dulbecco's modified Eagle's medium and Ham' s nutrient
`mi xture F-12 supplemented with glutamine (2 mM) , pe nicillin
`(100 units/ml), streptomycin (100 ~-tg/ml) , and 10% HyCione
`(Logan , Utah) calf serum in a humidified incubator under 5%
`C0 2 in air atmosphe re .
`Transfections and Amplification. Plasmid DNA was intro(cid:173)
`duced into mammalian cells by the calcium phosphate
`coprecipitation method (21). Half-confluent plates of cells (60
`mm) were exposed to 5 ILg of plasmid DNA in 1 ml of
`precipitate for 6-8 hr . After a 20% (vol/vol) glycerol shock
`(22), the cells were fed with nonselective medium . Two days
`later, they we re passaged into selective medium containing
`Geneticin (G418) at 400 ~-tg/ ml.
`Clones were picked using glass cloning cylinders with
`petroleum jelly for the bottom seal. Colonies ari~,; 1g from
`transfected cells selected for growth in G418 were picked ,
`expanded, and subcultured into medium containing 7%
`dialy zed fetal bovine serum in place of 10% calf serum and the
`appropriate concentration of me thotrexate for plasmid am(cid:173)
`plification (23). The dialy sis step removes trace amounts of
`purines and pyrimidines present in serum that decrease the
`efficiency of the methotrexate se lection . To apply selective
`pressure, stepwise increasing concentrations of methotrex(cid:173)
`ate were used with a final concentration of 400 nM . To avoid
`enriching for sponta neously transformed cells, cells were
`kept subcontluent. An additional control was to amplify the
`CYN neo-DHFR vector without the HER2 eDNA in sert in
`the NIH 3T3 recipie nt cell line.
`Immunoprccipitations and Labeling. The G-H2CT17 anti(cid:173)
`bod y recogni zing the C-terminall7 amino acids of H ER2 was
`prepared in rabbits using a synthetic peptide conjugated with
`soy bean tryp sin inhibitor.
`
`Th e pub li ca tion costs oft hi ~ arti c le we re defra yed in part by page charge
`pa yment. Thi s arti c le mu st thc rcl'o re be hereb y marked " adverrisemenr"
`in accorda nce with 18 U. S.C. ~ 1734 solely to indi ca te thi s fact.
`
`Abbre viations: DH F R , dih ydrofolat e red ucta se; 1\ res istance; EGF,
`e ptde rmal growth factor; RSY, Rou s sarcoma viru s; L TR, long
`terminal repeal.
`
`7159
`
`BIOEPIS EX. 1047
`Page 3
`
`

`

`7160
`
`Ce ll Bio logy : Hud z ia k eta/.
`
`Pmc. N ot/. A('(/(/. Sc i. U.S'A 84 ( 1987)
`
`Tab le I . A s~ a y fo r grow th in >Oft aga r a nd I 'Jr, ca lf serum of
`H ER2 prim<t ry a nd amrlilied ce ll lines
`
`Ce ll line
`
`II LR2 g.: nc cop ies
`per hap loid
`ge nome. no.
`
`Soft aga r
`colo ni es.
`no.
`
`Pl ating
`effi cie ncy
`in 1 'It calf
`-;c rum . 1/r
`
`Ce ll s we re ha rvested by try ps ini zati o n a nd count ed by
`Coulter Counte r, a nd 1. 5 x 101' ce ll s were pl a ted pe r 60- mrn
`c ulture di s h . Aft er 36 hr , th e ce ll s we re lysed a t 4°C wi th 0.4
`ml ofHNEG buffe r pe r pla te (50 mM H e pes, pH 7.5/ 150 mM
`NaC I/1 mM EGTA / 10% glyce rol) containing 1.0% Trit o n
`X-100 de te rgen t a nd 1 mM ph e nylmet h yls ulfo nyl flu o rid e.
`Afte r 10 min , 0 .8 m l o f lys is dilution buffe r iHN EG bulle r
`with 1% bovine se rum a lbumin a nd 0.1 % Triton X- 100) wa s
`added to eac h plat e and th e ex trac ts were pe ll e te d a t 12 ,000
`x g for 5 min.
`HER2 a ntibody was added to the ce ll ex trac ts, w hi c h we re
`th e n in c ubated a t 4°C fo r 2 hr ; thi s wa s followed by in c uba ti o n
`with protein A-Sepha rose bead s for 20 min a nd three wa shes
`with 1 ml o f HN EG buffe r w ith 0. 1% Triton X- 100 .
`A utoph osphoryl a tion reac tion s we re ca rri e d o ut a t 4°C in 50
`p.l of HNEG wash b uffe r with 5 mM MnCI 2 and 3 p.C i o f
`[y_32 P]ATP (Am e rs ha m , 5000 C i/ mm o l; 1 C i = 37 GBq) fo r
`20 min . Pro te in s were se pa rat ed o n 7. 5% NaDodS04/ po ly(cid:173)
`acryla mid e ge ls and a nal yzed by a uto ra di ography.
`Transformation Assays . Th e e ffi c ie ncy of colo ny fo rma ti o n
`in soft agar (24) was de te rmin ed by plating 25 ,000 ce ll s in 3
`ml of0.2% agar (Oifco, "p urifie d ") ove r 4 ml of0.4 % agar in
`a 60- mm di sh . After 2- 4 week s, colo nies of abo ut 100 ce ll s o r
`more we re co unt ed.
`The plating e ffici e ncy of cell line s (25) in l % ca lf se rum wa s
`dete rmin e d by pl a ting equal numbe rs of ce ll s into 100-mm
`pla tes with e ith e r 10% o r 1% ca lf se rum . Afte r 2-3 week s , th e
`plates we re stained with crysta l vio le t a nd colo ni es we re
`counted .
`Mouse Tumorigenicity Assays . A th ym ic (nu /n u ) mi ce were
`obtained from C ha rl es Riv e r Bree ding La bora to ries. Contro l
`NIH 3T3 and NIH 3T3/CY N ce ll s and ex pe rim e nt a l H E R2-
`34oo ce ll s were ha rves ted by trypsini zation and count e d w ith
`a Co ulter Co unte r . They were th e n coll ected by low- s peed
`centrifugation a nd res us pe nde d in ice-co ld phos ph a te-buff(cid:173)
`e red salin e to e ith e r 2.5 x 106 , 5. 0 x 106 , or 1.0 x 107 ce ll s
`per mi. A nim a ls we re injec ted s ubc utaneou s ly w ith 0 . 1-ml
`vo lume of th e ce ll sus pe ns io ns . Tumor occ urrence a nd s ize
`we re mo nitored twice week ly.
`
`RESULTS
`For ex press io n of H ER2 sequences inN IH 3T 3 ce ll s, a e DNA
`codi ng for th e e ntire 1255 -amino acid polypeptid e (1) wa s
`pl aced under tra nsc ripti o na l co ntro l of the RSV LTR . Tran (cid:173)
`scripti o na l termination s igna ls a nd a poly(A) site were pro(cid:173)
`vid e d by 3' seq ue nces of the he patiti s viru s surface a ntigen
`ge ne (20). In additio n , the exp ressio n vector contained th e
`neo re sistance ( neo R) gene , w hich confers ce llul ar res is tan ce
`to the ami noglycos id e a ntibiotic G418 (18) a nd th e re fore
`a ll ows se lect ion of primary trans fectant s , as we ll as the
`DHFR gene for methotrexate res ista nce, which was used to
`a mplify transfec ted DNA sequences under selective pres (cid:173)
`sure . Both drug res ista nce genes we re und e r s imian vi ru s 40
`early promote r t ra nscriptional co ntro l. Bacterial plasm id
`sequence s, in c luding an o rigin of replica tion a nd the ge ne for
`ampici llin re sista nce , a ll owed re pli ca ti o n of th e e ntire ex pres(cid:173)
`sion plasmid in Eschl'richia coli.
`Th e tran sforming ac tivity of HER2 sequ ences wa s initi a ll y
`tes te d using a conventiona l NIH 3T3 ce ll focu s-form a ti on
`assay. Und e r co nditi o ns that resulted in a bout 104 foci pe r p.g
`of a v)ins vi ra l co nstruct , we were unab le to de tect any
`H£R2-tran sformin g activit y. Beca use of the rece ntly re port(cid:173)
`ed finding th at abo ut 30% of mammary carc ino ma s con ta in
`amplifi e d f-IER2 ge ne sequences without a pparent sequence
`rea rrange ment s (14), we in vestiga ted whether a mplifi catio n
`of a n unalte red H ER2 ge ne could tran s form mou se fibro(cid:173)
`bla sts . NIH 3T3 cell s we re tran s fecte d with the pC YN / H E R2
`cons tru c t. An identical plas mid mi ss ing the HER2 exp ress ion
`modu le was used a s a co ntro l. Four ind ependent prim a r y
`
`()
`
`()
`3
`60
`2
`55
`4
`<)()
`I
`131
`
`0
`0
`
`0.27
`0
`3
`38
`1.4
`11.7
`0. 2
`49
`0.6
`50.2
`
`NIH 3T3/CVN
`NIH 3 T3/ C VN .I(~'
`H ER2- l
`H ER2-14 ,.,
`424
`0
`H E R2-3
`X36
`H ER2-3.11 "
`0
`H E R2-4
`376
`H E R2 - 4•1~,
`HER 2-I:D
`{)
`H ER2-B3 4.,1
`373
`T wo co ntrol lin es we re used . The firs t o ne was a NIH 3T3 line
`tran sfected with" pla smid co ntaining o nl y the neo a nd /) 1/FR ge nes.
`The seco nd co ntro l lin e co nt ain ed th e n<.:o-DHFR plasmid a nd was
`amplifi ed to re si-; tance to 400 mM me th o trexa te. II LR2 ge ne copy
`num be rs we re determined u' ing a hum a n DNA standa rd and
`den sit o mete r -;cann ing of So uthe rn hyb ridi zatio n autorad iogr<llll '>.
`Equ al ce ll numbe rs (25, 000) we re plat ed in soft aga r and colonic '
`we re count ed aft e r 2- 4 week , . Th e pla ting cfli cie ncy in 1'/r ca ll'
`se rum is rela ti ve to th e num be r of colo nic-; ari, ing when an equal
`aliquo t was simultaneou sly pla ted in medium conta ining 10'/t ca lf
`se rum .
`
`G4 18- res is ta nt c lo nes (HER 2- l. H E R2-3. H E R2-4. H E R2-
`B3) we re iso la te d. Ce ll
`lin es co ntaining a mp lifi ed H ER2
`coding sequ e nce s we re ge ne ra te d from th e se pare nt a l c lo nes
`by c ulturing th e ce ll s in g radu a ll y in c rea s ing co nce ntra ti o ns
`of me th o trexa te up to 400 nM ( H E R2-l.1ot>- H ER2-3.H~I ·
`H E R2-4 41Mh H E R2-3B.11 w1) . So uth e rn h y bridi za ti o n a na lys is of
`pa re ntal a nd a mplifi e d ce ll lines de mo ns tra ted tha t th e !IL'R2
`cON A copy numbe r in c rea sed fro m l- 4 to 55-13 1 pe r haplo id
`ge no me (Tab le 1) .
`T o tes t w he th e r ge ne a m plifi ca ti o n re s ult e d in ovc rcxpre s(cid:173)
`sio n of th e I/LR2 ge ne prod uc t , ce ll
`lysa tcs we re im (cid:173)
`mun o prec ipit a te d w ith a n a nti bod y aga in s t th e C- te rmin a1 17
`amin o ac id s o f th e /I LR2 sequence. As s how n in F ig . 1.
`substa nti a ll y in c rea sed leve ls o f th e pl 85 11 10 R ~ ge ne produ c t
`were fo und in a mplifi e d ce ll line s re lati ve to the ir parenta l
`G418R tran sfectant s . Th e parenta l ce ll s had a no rm a l mor(cid:173)
`phol ogy that wa s indi s ting ui s hab le fro m NIH 3T 3 cell s.
`Howeve r, amp lifi e d ce ll s ha d th e typica l re fractil e, spin dl e-
`
`1 23456789
`
`200 -
`
`11 6 -
`
`FIG. l. Qua nti tation ofpUl511"K" in fo ur primary , unamp lilled cell
`lin es a nd lines de rived from them by a mplifica ti o n to res istance to 400
`nM methotrexate. Lan e 1, nco- DH 1-'R control; la nes 2 and 3. H ER2 -l
`parent a nd a mp lifi ed lines ; la nes 4 a nd 5. H E R2-3 pa rent and
`a mplified lin es ; lanes 6 and 7, H ER2-4 pare nt a nd a rn plificd line s:
`Janes 8 a nd 9. H ER2-l:l3 pa re nt a nd amp lili ed lines. Positio ns of th e
`siLe marke rs myos in a nd f:l-ga lactosidasc a re indi cated in kDa.
`
`BIOEPIS EX. 1047
`Page 4
`
`

`

`Ce ll Biology: Hud ziak et a/.
`
`Proc. Nat/. A c ad. Sc i. USA 84 ( 1987)
`
`7161
`
`F IG. 2. Morphology of NIH 3T3 cell s transfected with HER2
`ex pre~s ion co nstru ct. ( /) N IH 3T3 cd ls transfected with th e control
`neo- DHFR plasmid. (2) NIH 3T3 cell s wi th th e neo-D H FR plasmid
`amp lified to rc~i stance to 400 mM met hotrexate. (J) Primary G4J8R
`ce ll line 1-1 ER2- l , an unamplified c lone exp ressi ng low levels ofpl85.
`(4) The same c lone amp lified to 400 mM methotrexate resistance.
`( Y50.)
`
`shaped appearance of transform ed ce ll s and grew in irregular,
`piled -up c lumps (Fig. 2).
`NIH 3T3 cell lin es with an a mplified J-IER2 gene and high
`levels of 111.:-R2 gene ex pression also di splayed other char(cid:173)
`acteristics assoc iated w ith a tran sformed phenotype . As
`shown in Table l and Fig. 3, th ese cell s all form ed colonies
`in soft agar a nd were able to grow at low den sity at low serum
`conce ntrat io n. In co ntras t. primary transfecta nt s did not
`grow under these co nditi o ns . The primary transfectant s did,
`howeve r, grow to a hi ghe r saturati on density than the
`pa rental NIH 3T 3 cell s .
`The co rrespond ence betwee n a tra nsformed phe notype
`a nd HLU2 ge ne amplification a nd overexpress ion was inde(cid:173)
`pendently co nfirmed by directl y selecting for tran sform ed
`ce ll s and I he n a na lyzi ng th e resulting clones. Fo r thi s purpose
`the parental cell lin e H E R2-3, whic h cont ains about two
`cop ies o f the IIER2 express ion co nstru ct , was cu ltured in
`medium co ntaining a low conce ntrati o n of fetal calf serum
`<0. 5%); con trol cell s cont a ining the express ion vector without
`fii.:-R2 coding sequences (pCYN) were cu ltured in parallel.
`Afte r 5 weeks, a few colo ni es appea red in the control culture
`and roughly 10-fold more coloni es appeared in di shes con(cid:173)
`ta ining th e H ER2-3 cell line. T hese colo ni es appeared to be
`morphologically tra nsform ed and we re subsequ ently ana(cid:173)
`lyzed fo r IIJ:.R 2 overexp ress ion. As shown in Fig. 4, three
`indi vidu a l c lo nes as we ll as a pool of th e re maining colonies
`had e levated leve ls o f pl85 11 ER 2 co mpared with the original
`parental G4 18-res ista nl H E R2-3 ce ll line. In add ition , th ere
`
`FIG. 3. Ancho rage-indepe nd ent growth of H£R2-transformed
`cell s in soft agar. Cells were plated in 0 .2% soft aga r over a 0.4% agar
`lower la ye r. After 3 weeks th e plates were photographed at 40 x
`magnification using a Nikon mic roscope with phase-contrast optics.
`(Upper) Control untransform e d NIH 3T3 cells. (Lower) Anchorage(cid:173)
`indepe ndent growth of th e cell line HER2-34,XI containing the HER2
`ex pression plasmid and amplified to resistance to 400 mM methotrex(cid:173)
`ate. (x25 .)
`
`was a 26-fold increase in the number of cells plating in 100 nM
`methotrexate in the selected cells compared with the parental
`cells, implying that the unselected but linked DHFR gene had
`also been coamplified.
`The tumorigenicity of cell lines with a high HER2 eDNA
`copy number was tested in nude mice by subcutaneou s
`
`2 3 4 5 6
`
`•
`
`- 185
`
`F iG. 4. Quantitation of pl85 in the prima ry line 1-1 ER2-3 and cell
`lines selec ted for growth in 0 .5% calf serum. HER2-encoded protein
`was immunoprecipitated a nd labe led. Lanes 1-3, three colonies
`picked and expanded from plates after selecti o n fo r growth in 0.5%
`se rum ; lane 4. the starting cell line, H E R2-3; lane 5, a pool of0 .5%
`serum -selec ted colonies of H ER2-3; lane 6. a clone de riv ed fro m a
`G418H co ntro l line selecte d for growth in 0. 5% calf serum . The
`position ex pected for a protein of ap parent molec ul ar mass of 185
`kDa is indicated.
`
`BIOEPIS EX. 1047
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`7162
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`Ce ll Biology: Hudzi ak et a!.
`
`Proc. Nat !. Aunt. Sci. USA 84 ( 1987)
`
`T ab le 2. Tumorige ni c it y tes ting of cell lines
`
`Cell line
`
`Cell s injec ted , no .
`
`Mice with tumors/
`mi ce injec ted
`
`N IH 3T3
`
`N IH 3T3/CYN
`
`H E R2-3 4,l()
`
`0.25 X 10''
`0.5 X 10''
`1.0 X 10''
`0.25 X 10"
`0.5 X 106
`1.0 X 101'
`0. 25 X 10''
`0. 5 X 10"
`1.0 X 106
`
`0/6
`0/6
`0/ 6
`0/6
`0/6
`0/6
`5/5
`6/ 6
`5/5
`
`The cell line H E R2 -3 a nd two contro l lines. NIH 3T3 cells a nd a
`N IH 3T3 line co nta ining the co ntrol plas mid , were injected subc u(cid:173)
`taneo usly at three d iffe re nt dosages into nud e mi ce. The tim e afte r
`injection before tum o rs beca me vi sible was dose rela ted: 22 days
`(ave rage) fo r 1 x 10" ce ll s, 2!! da ys (ave rage) for 0.5 x 10" ce lls. a nd
`34 days (average) for 0. 25 x 10" ce ll s.
`
`inj ect ion of three different ce ll numbers pe r an im al. As s hown
`lin e HER2-34<~l was
`in Ta ble 2, th e ov e rex press ing cell
`s trongly tumorigenic at all dosages te sted , whereas th e
`contro l cell
`lin es, NIH 3T3 ce ll s and NIH 3T3 ce ll s
`tran sfected with CVN vector wi th o ut the HER2 in se rt , were
`both negative unde r the same conditions. N ecrops y of three
`mice wit h we ll -establi shed tumo rs fail ed to id e ntify a ny
`met astasis. Ce ll lines reesta bli s hed from th ree excised tu(cid:173)
`mors still expre ssed the G418R phenotype, we re resis ta nt to
`methotrexate , and expressed high levels of p185H ERZ (not
`s hown).
`
`DISCUSSION
`Amplifi cation of th e HER2 ge ne has been re ported in a few
`prima ry human tumors (2 , 3) a nd tumor-d e rived ce ll lin es 0 3,
`26). Rece ntly , S lamon et al. (14) fou nd th at th e J-/ER2 gene
`is a mplified in 30% of prima ry breast tumors, a commo n
`human ma lignancy tha t affect s a bout 7% of all America n
`femal es. Notably , o nl y 3/189 tumors s urveyed s howed a ny
`gross rea rrange me nt of the HER2 gene. To assess th e rol e of
`the HER2 ge ne in th e neoplas tic process, we c haract erized
`the tran sforming potential of the HER2 gene in an in vitro ce ll
`cu lture system .
`Express ion of the full-length cON A in NIH 3T3 ce ll s did
`not lead to tran sform a tion as determined by a standard
`focu s-forming tra nsfection assay. Howe ver, HER2 overex(cid:173)
`press io n caused by gene ampli fi cation tra nsform ed th ese
`cell s. Colonies that survived methotrexate se lection we re
`morphol ogically tran sformed a nd exhib ited loss of contact
`inhibiti on. Such ce ll s a lso grew in soft aga r and wou ld grow
`in 1% calf se rum . F urthe rmore , cell s tran sform ed by /-1 ER2
`were tumorigenic in a thymic mice .
`Selection of ce ll s tran sfected with the /-IER2 eDNA for
`growth in low serum provided independe nt ev idence that
`high- level expression of an una ltered HER2 ge ne product
`cau sed cellul a r tran sformation . Since DNA introduced into
`mammali an ce ll s by tran sfection is more labi le than genom ic
`DNA (23, 27), we reasoned that selection for a prope rty
`demon strated by pharmacologicall y amplified H ER2 lines(cid:173)
`name ly , growth in low serum-mig ht lead to amplification or
`other c hanges resulting in overexpress io n of p1 85 HER2.
`Clones derived from a n unamplified HER21ine by thi s selection
`procedure appeared morphologically tra nsformed and ex hibit(cid:173)
`ed ele vated levels of p185HER2_
`Ta ken toge ther , the c haracteri stic morphologica l cha nges ,
`res ults of in vitro and in vivo tran sformation and tumorigenic(cid:173)
`ity assays, a nd th e e levated leve ls of pl85 HER Z in ce ll s
`se lected for a transformed phenotype imply that high-leve l
`express ion of HER2 re sult s in tra nsformation of NIH 3T3
`
`ce ll s . Ano th e r me mber o ft he ty ros ine kinase ge ne family was
`rece ntl y fo und to be a mplifi ed 4- to ~-fo ld in s pontaneou s ly
`a ri s ing foci of NIH 3T3 ce ll s (28). A mplifi ca ti o n of th e m et
`ge ne a ppears to be a frequent eve nt a nd . s imil a r to H£ 1?2
`a mplifi cati o n in mamm a ry carcinomas, is rarely accompa(cid:173)
`ni ed by gross rearra nge me nt s. These findin gs and th e di s(cid:173)
`co ve ry o f a mplifi ed EG F rece pt or ge nes in primary human
`tum o rs (10 - 12) s ugges t th a t overexpre ss io n of other grow th
`fac to r rece pt o r ge nes w ill a lso lea d to tran sfo rm a ti on a nd
`tum o rige nes is. Wh e th e r s usce ptibilit y to s po nt a neous a mpli (cid:173)
`fi cation is ca used by c ha rac teris ti cs of th e ge ne ti c loc i for
`HI:.R2, m e t and EGF receptor, o r cell-s pec ifi c selecti ve
`advantages ca used by rece ptor ove rexpress io n re ma in s to be
`inves tiga ted.
`It is no t ye t c lea r w he th e r tra ns fo rm a ti o n of ce ll s
`ove rexpress ing a grow th fac tor rece pt o r ge ne is de pe nde nt o n
`pa rac rine o r autoc rin e stimul a ti o n by th e a ppropria te li ga nd .
`For III:.R2. thi s qu esti o n ca nn o t ye t be ad dressed sin ce it s
`liga nd has not bee n id e ntifi ed . In th e case of th e EGF
`rece pt o r , howeve r, we we re a bl e to de mo ns tra te th a t primary
`hum a n tumors and tum o r-d e ri ved ce ll lin es frequ e ntl y ex(cid:173)
`press mRN As fo r bo th th e rece ptor a nd tra nsfo rming growth
`l~tctor type a liga nd (29). Th e findin g th a t me re ovc rexp rcs(cid:173)
`s io n of a n int ac t g rowt h facto r rece ptor may sub ve rt norma l
`cellul a r g row th co ntro l mec ha ni s ms a nd lead to tumo rige ni c
`growth provides new pot e ntia l fo r diagno-; ti c a pproach es a nd
`the rapeutic s tra teg ies fo r trea tm e nt o f human malig nancies.
`
`·u pe rb tec hni ca l assi\ tanc.: or
`We ex press app rec iatio n fo r th e
`Thomas Dull in co n ~ tru c tin g th e H ER2 ex press io n plas m ids and llill
`Lagrimas for he lp wi th th e mo use tum o ri ge ni c it y assays. We a re
`gra teful to Jea nn e Arc h for her patience a nd s kill in ty ping thi s
`ma nu sc ript and to Or. S uza nn e Pfeffe r fo r he r va lua ble editorial
`co mme nt \.
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`BIOEPIS EX. 1047
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`Ce ll Biology : Hud zia k et a/.
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`Proc. Nat/. A cad. S ci. USA 84 ( /987)
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`7163
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`BIOEPIS EX. 1047
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