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`Growth Regulation of Human Breast and Ovarian Tumor Cells by Heregulin:
`
`.
`
`Evidence for the Requirement of ErbBZ as a Critical Component in
`
`Mediating Heregulin Responsiveness
`
`Gall DuLevils'. Julie A. Infgren, Array E. McMurtrey. Andrew Nuijetts, Brian M. Fendly. Kenneth D. Bauer. and
`massacres:
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`biologiulresponaestoHRG werealsoeontpared to EGFandtothe
`growth-Inhibitory anti-Erbflz antibody. 405. In most cases. HBG stimu-
`lation of DNA synthesis undated with positive efl'ectl on cell cycle
`pmgruton and cell number and wlth enhancement of colony formatlorr
`www.mumdrummmrmcmuummgmmm
`from EGI‘ and IDS. Our [ladlngs Indicate that HRG Induces cell prolif-
`eration In a number oftuntor cell Ilnu. In addition, we show that methods
`for measuring cell proliferation. as ed! as experimental condlllons. are
`critical for determining l-Ille effect on tumor cell youth in vino.
`
`INTRODUCTION
`
`’ Clinical interest in the Etbfi family of receptor tyrosine kinases
`stems from the observations that these receptors are frequently over-
`ettpressed in a number of human cancers (1—5). Five different gene
`products are known to activate the prototypical rrternherof this family.
`EGFR.‘ In several different tumor types. the coeaprcssion of com
`with one of its cognate ligands. transforming growth factor or. has
`been correlated with greater metastatic potential (6. “ll. A second
`group of ligands. which collectively have been termed neuregulins.
`are known to arise from alternative splicing of a single gene mapped
`to human chromosome Spitz-pl! (8. 9). This family of proteins which
`includes HRG (I0). NDF (ll. [2). 55:30 (13.
`Ill). aeotylcholine
`receptor-iirdueing cetivity (15}. glial growth factor (16]. and sensory
`and motor neuron-derived factor (I?) are ligands for ErbBJ and
`Btblld (18). One hallmark of this class of receptors is their propensity
`for heteroditnertration. which upon ligand stimulation can lead to
`transphosphorylation and ultimately u-ansactlvation (19). To date. no
`ligands have been chateau-iced. to the extent that their cDNA clones
`have been obtained. that specifically interact with ErbBZ. Ed)!!! is.
`however. frequently maneuvered by either EGFR ligands or neu-
`
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`regulins. and in several instances. the activation of 1&sz has been
`shown to be essential for the generation of an active receptor signaling
`complex (20—22).
`The Importance of Ebb: as a negative prognostic indicator in
`breast (‘23) and ovarian (24) cancer is now well established (25). Since
`l-[RG (IO) and NDF (ll) were originally identified based‘on their
`ability to activate Erbnz. it is of interest to dcten'nlne the biological
`outcome of this activation on the growth of human breast and ovarian
`tumor cells. To address this question. we have undutalten a system.
`atic surdy of HRG responsiveness using a panel of human breast and
`ovarian armor cell lines with known BrbBZ levels. These cell litres
`
`were thenchancterized widttegardtoboththeiraffinitlesandcapcc-
`itiet to birrd HRG. Antibodies directed against 81132 were used to
`determine if ErbBZ was essential in mediating HRG interactions with
`Eth3 or ErbB4. To detemtine whether HRG treatment of these tumor
`cell lines resulted in cell proliferation (ID) or growth inhibition (12.
`26). we preformed these assays under a series of well-defined caper—
`imerrtal conditions and using a number of different assay formats. In
`addition. HRG responses were compared to EGF and a cytostatic
`monoclonal antibody directed against ErbBZ. 405 (21. 28}. Our
`conclusions from these studies are that ErbBE plays a critical role in
`mediating HRG responsiveness over a wide range of EthZvespres-
`siort levels. Cellular responses to erogenous HRG under carefully
`conu'olled experimental conditions are cell specific but, in general.
`result in the proliferation of human breast and ovarian tumor cells.
`These studies suggest that development of HRS antagonists or com-
`pounds that target HRG receptors may find clinical utility in the
`treatment of a number of important human cancers.
`-
`
`MATERIALS AND METHODS
`
`'
`
`Cells and Cell Cullul'e. The following cell lines were obtained from the
`American Type Culture Collctlon: MCI-‘1. .SK-BR-Il. MDA—MB-l'lS-Vll.
`MBA-M5131. ”DAME—36!. WNW-453. MBA-M3463. 37-474.
`trauma. Taro. arr-co. H483. Grov3. and sxova. For or experiments.
`eellaaereutedhermpuugudurd l5. nitrates wctetnaintaincdio Ham's
`BIEDNIEM (50:50) supplemented with 10% heat-inactivated FBS and 1 out
`u-gluumine.
`ERG Binding Assays. Tumor cells were plated in 24-well plates at [0’
`eellsr'well'ta FIUDMEM containing lO‘lr F'BS. After 48 h. the cultures were
`washed two times With FIZfDMEM. Cells were placed on ice and briefly
`incubated with binding bulTer (0.1!: BSA In FI'IJ'DMEM).
`rut-labeled
`“OHM.” (5 pH to! ml was then added to each well. Binding reactions
`were performed for It It on ice. Kinetic studies showed that equilibrium was
`obtained at d h. and no significant strange: in binding constants were observed
`lfthemetiontwereconductcdupto I6 Ir.Cellt weretlven washed twice with
`0.5 ml binding buffer. and burnt! radioactivity was determined afla solubili-
`tation with (MI: 508 in (ll ti NaOlt or with l M urea in 3 M acetic laid.
`Scatcluttd analysis was performed as described previously (lo. 29}. The VIII-WI
`reported in Table l for then binding elpet'irnmu are the average of three
`different binding eaperimentt. each of much emsisted of triplicate imitations
`for each concentration of '"l-lahelcd rr-raopr mm. turns. Error mm for
`both K, and titeafoell area on the order of 10% or less for each bindinl
`"Mm"
`Genentech 2063
`I457
`
`Hospira v. Genentech
`|PR2017-00737
`
`Genentech 2063
`Hospira v. Genentech
`IPR2017-00737
`
`

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`
`36m Tum Colony image Analysis System (longing Ptoducn bites-national.
`Inc.)-
`
`Inhibition ofHRG binding to muesli lionsby antl‘EabBZ antibodies was:
`mwtumdonicwithmclayuamwesiaaflwell-plflelm
`Anti-MRI mlaaalsnfibndics‘ueadtbdtoeacbwllandincubndfw
`30 min. ml-labeied rl-lROflImM ('35 pit) was added. end the incubation
`wuoontimled I'tl'dto Ibhfimuflcflluminmbflionsmlflfm
`twdhdaeebmsnbubafiunnndrreadtanpummdrediaeeiis
`urchin from the plate.
`Mmmmflmmflsmfisfldbuwfl
`mumumtwmommuw antibod-
`htoErblzwneaddedtneaebwellandinmbatedior-Jllniaatmnr
`reruperanueuitendeahmwasaddedtoeaehwefl fwafiatl noses»
`hundOJmmdlhtincubaticnwasmtlmedthmmMediawas
`caehdlysrpiratodlromuehwdtandraacdomwcrcneppedbydreaddifioa
`d implofSDSuntpleb'uifcruiSDS.BanIUfi.and15muTris-HCL
`pHfiBLFach-rnplemuliwelemnflnuednoad-IZSMmgel
`Gloves) afl‘dnr eluttropboreeluily nurtured to polyvinylideoe difluuide
`MW(¢GIO.MUBLMII I misalfimmu-
`WMWNRWHMWMRMH
`H, atom was quantified by refleclame densltomeo-y. as described previ-
`ously (to. at
`Cell Proliferation hunted by l’ll'rlbysnldlne Inmr'poration Into
`DHLCeann-eaneddintoWlplatuusdeuiryole‘Neflfw
`nunneanuptoSdaysaaIi-S X ro’mu fthyhmbafimFollwring
`anhvuttigbtitlubadontoallowfaceli adbumhmditoawasrupnved
`andreplmdafithmedltnncnnuiningm‘lflStogrowlbmtbecelb.
`AflaaMirurbaticmlhls Indium wureplaod with orediumeoatairiing
`o.t. LO. or tots fits and attract (5 put-l0 m). 3 net cor (Sigma (licorice!
`Cu). or llllI laghni [65 nit) of lhe anti-Elba: transitional antibody 4DS (17. 28.
`30}. The cells we then incubated for 0.5. I. 3. or 6 days. pulsed with l
`timbre“ l’i'nlhyrnidine (An-realism) for 4 h. and harvested onto Unifilter
`GFIC plates (Packard Instrument Co.) using a Packard Fiherrrtalc l96 har-
`vestcr Alter allowing the filter plates to dry. MicroScint 10 liquid scintillation
`cocktail (Packard) was added to each well; thenthe plateswere heatsealed and
`counted in a Packard Tnpcount
`CCII Cycle Analysts. Cells were plated at I density of 2 X if cellsfrlish in
`60 x lsvrnm culture dishes and allowed to adhere overnight. The monolayers
`were then washed With PBS and incubated With medium containing 0.”: FBS
`for 48 It to arrest cell growth. The medium was removed and replaced with
`medium supplemented with Ill. 1.0. or NH: PBS alone or containing 0.3 rttt
`rHRGflI. 3 rat EOF. or 65 run of”. After a I-day incubation. the cells were
`uypsiniend. washed with PBS. fixed in ice-cold methanol. and stored at
`-20“C.1heftaedoelismdtenwashedtwicem988ufliucubatedwith
`"in M11 RHAse (Worthington Biochemical) for 10 min st 31'C.'lhc RNAsc
`was rumved by centrifuging the cells. and the pellet was incubated with 50
`mini propidium iodide (Molecular Probes. Inc.) in PBS for DNA staining.
`‘The samples were then incubated overnight at 4'1: and analynnd on an Eples
`FJite flow cytorneter (Coultrr Corporation} using Modftt LT software (Verity
`Software Haste).
`Ceilhollfaratlno Amy wiibCrystalVlolet. 'l‘umcellswueplatedat
`adensityol’! it lo'lwcllinchll piatesin media cordsirriogQI‘lrFBS and
`allowedtoadhereforlit Mooneionalarttibodiaflflntnnrnudiaalooem
`ddsd.andlbeeellswueioeubstedfor2hat31'€.rflRGfll(OJoat)waslheo
`added. arrdtilcells were imbued (widen. Monolayerswethcnwashed
`with PBSsndliaedistained witboj‘lreryetaiviotrs. Hemmfldflfiure
`dye was elutul withOJ It sodiumcitl'lic (pH 4.2) in ethanol (50:30).“ ill
`absorbence was read at 540 run to determine cell viability (30).
`Determination of Cell Number. Cells was plated as described for the
`[’Hlthymidine assays. Mia each incubation. the molayers were washed
`out: withBs.eellswetedelachedwith u-ypsioandyisbleeellswerecourued
`by “791" blue dye eaclusion.
`Quanlil'rution at Cell Proliferation by Anchorage-independent Growth
`in Salinger. Cells were seeded in 60 x lS-rnrndisltuontoabonom layer
`of 0.5% Bane-agar (Ditto) at the folieuring densities: ltl'idish for SK—OV-S'.
`2 x lo‘tdish for MC“. SK-BR-J. MDA-Mfl-nl. 31-414. and REL-KO:
`and IO‘idish for MBA-M3461. MBA-M3451. and MBA-M3463. A top
`layer of 0.!5‘lr'agar in medium containing in cat rl-IRGBI or 3 tllrl EGF or
`medium alone (as aconuol) was added to each dish. Afton-d weeks. colonies
`were stained with 250 nudish 3—[IiJ-dimcthylthiaml-2---yi]-1.5-diphenyltetn—
`I It. w. Altita. N. carol. t. ans. 6. Stilts-rice. n. M. Ferdly. and M. x. Sllwtowsbi.
`Minnie limitation of hetegtdin binding to troll. manuscript in preparation.
`aoliurn bromide (Sigma (mercies! Co.) and enunciated using an Omnicoo
`1453
`
`RESULTS
`
`Determination of HRG Binding. Using '"l-labelod rHRGiil m.
`eutoperi’orrrtdiroctbindingeaperimentt. wedctcrrrtioedboihihe
`aifinityandlhenurnberoflmarecnptnrsbyScucltardanalysison 13
`differentcelllimcfbreastandomianmigin. A utmrnaryofllrcse
`dataisahowtrioTeblel. Forenchnftlflecelllimthcbindingdsu
`could be fit to a single class of high affinity binding sites. Only the
`intern-tallied human mammary epithelial cell line. HBLIUJ. lacked
`detectable HRG binding. -Very low (410.000 receptorskell) ERG
`biedingceuldbcdctmtedon theovariartcell line SK-OV-Sand the
`breast armor cell lines MDA—MB-ITS-Vn. MBA-M3468. and 31'.
`20. The following breast cancer cell lines. MCF'i. MDA-MB-Sbl.
`TdTD. and BT483. and the ovarian cancer cell line Canvii were found
`to have between lam-21M receptorflcell. and than cell lines
`were arbitrarily characterized as containing Intermediate levels of
`ERG binding sites. Cell lines having high numbers of ERG binding
`- sites (in. 3-25.!!!) receptor-aloe") wen SK-BR—3. MBA-HMS.
`and BT-II‘M. heviottsly. we have determined that in the absence of
`Erbflz. ErbBZl binds HRG with a binding affinity of 0.9-4 use (3]).
`When 0057 cells or mum cells cocspress ErbBZ with ErbBB. a
`higher affinity binding site of ~20 phi is observed (29. 32}. Using a
`similar COS? cell system. Tether er til. (33) have reported recently
`that NDF binds to Erde and ErbB3 with It‘s of LS and B nu.
`respectively. These authors speculated that the difference in the bind-
`ing constants may be related to cell-specific determinants or are due
`to the preparation or radiolabcling of the recombinant ligands. Re-
`cently. we have determined that Erde or Erbli3 binds HRG with
`similar affinity. if either is expressed singly in cells of hematopoietic
`origin in the absence of any other Erbli receptors.’ 111: average K. for
`the l2 cell lines shown in Table I that exhibit HRG binding is 99 : I4 .
`phi. The binding constants shown in Table l are at least an order of
`magnitude higher in affinity than what has been reported for film! or
`ErbB‘l alone. These data suggest that other cornportertts besides ErbBB
`or 511334 are contributing to the formation of a high affinity HRG
`binding site on these cells.
`Monoclonal Antibodies to Erbflz Block HRG Binding to Breast
`_ and Ovarian Tumor Cells. Based on our previous observations with
`C051i cells expressing Erblili and Ersz. as well as our experience
`with human Schwann cells. we hypothesized that the high-affinity
`HRG receptors present or: these tumor cells were the result of het-
`crodirner formation of Erbli} with FabBZ or possibly ErbBIl with
`ErbBiZ (2|. 29). To evaluate whether ErbBZ contributes to the fame-
`tion of this high-affinity HRG binding site on human tumor cells. We
`surveyed a panel of well-duracterized anti-Erb32 antibodies (28) for
`their ability to inhibit ERG—stimulated tyrosine phosphorylaiioo of
`proteins in the M, 180.“ range from whole-cell Iysates of MCFT
`cells. MCI-7 cells are reported to express all known FAbB martinis.
`but at relatively low levels (34). Since ErbBZ. ErbBS. and Erde have
`nearly identical molecular sizes (35. 36). it is not possible to discern
`which protein is becoming tyrosine phosphorylated when whole-cell
`lysates are evaluated by Western blot analysis. However. these cells
`are ideal for HRG tyrosine phosphorylation assays because under the
`assay conditions used. in the absence of cangcrtously added HRG.
`they exhibit low to undetectable levels of tyrosine phosphorylation
`proteins in the M, [80.0w range. As shown in Fig. M. several 0‘
`these antibodies. including 201. TF3. and dDS. significantly inhibited
`
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`esatnple. BT-ZO. MCFT. and W3 express - IO‘ firth! WON
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`cell (30).Giveotlrewiderangeof£tbm expression intheaeerllsartd
`thedatainfig.1.ltwasoonoludedutatdeintuaodonborwunmgz
`and mm or ErbB'ti. was itself a high-afiudty interaction that takes
`plsoeontltesttrfaoe ofthe plasma membrane. Almilismleu
`whether these oomplesea involving Ersz-Erbnli or ErbM-ErbBtt
`are preexisting in the absence of ERG or whether the association with
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`the generation of a HRS-induced tyrosine phosphorylation signal at
`M, tendon. in the absence of Him. none of these antibodies were
`able to stimulate tyrosine phosphorylation of proteins in the M.
`180.0!!! range (data not shown). Also. these antibodies do not cross-
`reset with MFR (28). ErbBii. or Ethll.‘ Antibodies 20! and 7B
`significantly inhibited HRG stimulation of plat“) tyrosine phosphoiyl-
`ation to <25!) of control. However. 405 was sble to block HRG
`stimulation of tyrosine phosphorylalion by ~50!» Previously. 201
`and 7H were assigned the same 6:sz epitope as reported in the
`original characterization or this antibody panel. and this epitope is
`dirrercttt than that recognized by 405 as). Fig.
`IB tttcws date.
`. response curves for 20% or TF3 inhibition of HRG stimulation of 9180
`tyrosine phosphorylation as determined by reflectance densitometry.
`Evaluation of these inhibition curves using a 4-parameaer fit yielded
`an IC” of 2.8 1: 0.? net and 29.0 x 4.]
`It." for 264 and Eli-‘3.
`respectively. Varying concentrations of 20% or TF3 were inathaled
`with MCI-‘7 cells in the presence of mll-lttlr'eltrd rHRCiBI. and the
`inhibition curves are shown in Fig. lC. Analysis of these data yielded
`an [Cm of 1d 1 0.3 mi and 19.0 1- 1.3 rot [or 2C4 and TF3.
`respectively. A maximum inhibition of ~14% for 201 and “it"! are in
`agreement with the tyrosine phosphorylation data We concluded from
`time espen'rnents that in MCF7 cells. EsbBZ was critical for ERG
`stimulation of tyrosine phownorylation and was an important eons-
`portent for high-affinity HRG binding
`It was oi interest to determine whether the effect of the anti-Brita:
`antibodies observed on MCF'l was a general phenomenon. To address
`this question. human tumor cell lines were incubated with 10'! or 7F3
`and the degree of specific "’l-labeled instant binding was deter-
`mined. The results from this study are shown in fig. 2. Binding oi
`'“t-tahcted rl-IRGBI ceutd be significantly inhibited by eitttcr 2C4 or
`TFSinalloelllines. withdteertoeptionol'thebt'essteanorsoell line
`MBA-M3468. which has been reported to upsets little (3?) or no
`ErbBZ (30. 3B). The remaining cell
`lines are reported to express
`FJIJBI. with the level of E6332 expression varying widely among
`these eell lines {30. 38). in fact. the range of ErbBZ expression in the
`cell
`lines tested varies by more titan 2 orders of magnitude. For
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`there was no effect in 0.1% FBS. [’Hlthyrnidine incorporation in-
`creased in cells exposed to rl-lltGBl in l or 10% FBS.
`Comparison ol' the ERG Response to EGF and 4135. Since
`responsiveness of a number of tumor cell lines to either EGF or the
`anti-ErbBZ monoclonal antibody 4135 has been extensively studied.
`we compared treatment of these cell lines with either EGF or 405
`under identical conditions as a comparison to rl-IRGBl. In vitro. EGF
`inhibits the growth of tumor cell lines such as MDA'MB-dfia {43) and
`Mill (4d. 45). both of which overespress EGHt. Conversely. cells
`that express nomutlflow or moderate levels of EGFR are frequently
`grow-stimulated by er'togenous EGF (39). Inhibition of cell growth
`by 4D5 is dependent on the expression level of ErbBZ (30}. i.e.. 405
`is cytostatic only for cells that over-express ErbBZ. Using a 3«day
`assay. the data in Fig. 4 show that the changes in [’Hlthyrnidine
`incorporation in these cell
`lines were distinct. depending on the
`ueaunent auditions. rHRGBl stimulated thyrniditte incorporation in
`MCI-"l. _SK-BR-3. BT474. MBA-M3453. MDA-MB-Jfil. T471).
`and BT-Zt'l cells at all serum concentrations tested. However the
`
`min is driven by the formation of HRG-Erblil or Has-mar
`complexes.
`Stimulation .of Mltogenesls by [“16 In Human Tumor Cells.
`We initially reported that HRG stimulated the grovnh ol' SK—BR-S
`breast tumor cells as measured by crystal violet staining (l0). Having
`determined the levels of HRG receptors in a number of different
`human tumor cell lines. we examined the mitogenic response of these
`cells to rHRGBl. Dose-response curves consisting of 5 pint-10 I'lM
`rHRGfil in dill'erent serum concentrations were generated for all of
`the cell lines listed in Table l. Prior to treaunent with rHRGBl. cells
`were growl! arrested by incubation in medium containing 0.1% [-153
`for #8 h. Fig. 3 shows typical curves [or three breast tumor cell lines
`after treatment for 3 days. MCFJr responsiveness to rHRGpI was
`dependent on serum concentration in the assay medium. When its-
`sayed in lll'l; FBS. MCF! cells showed a 4-5-fold stimulation of
`['Hllhymidine incorporation after treatment for
`l day (data not
`shewn] and 3 days (Fig. 30). will) the rrtaairttal response counting
`between 0.1 and I out rHRGBl. consistent with the affinity constant
`shown in Table l. rl-[ltGBl also stimulated DN A synthesis Lid-told
`magnitude of the response varied. depending on the serum concen—
`in ii: HS but had no efiect in NH: F‘B-S (Fig. 3a). A likely
`trations present
`in the assay. In contrast. EGF {3 tut) stimulated
`explanation for this effect is that MCF‘l cells contain high levels of
`[’Hlthymidine locum-nation in HBLrli'Xl. “mp. 31-20. and SK-
`atrogen receptors (39) and. in the absence oi other eaogeoously
`OV-3 cells under all conditions tested. Both rHRGfll and EGF
`added growth factors. requires estrogen for growth (#0). Alternatively.
`stimulated MCI-’1 cells in both OJ!» and lit “8. although the
`the presence of peptide grail-1h factors such as insulin-like growth
`magnitude ofthe response was less with EGF than with rHRGflI. 4D5
`factor-l may reduce cellular responses to HRt'i (all. Sufficient quan-
`had no eflect on this cell line. as reported previously (30). DNA
`tities of these [actors are present when cells are cultured in [0%
`synthesis was inhibited by EGF and. more potently. by 405 in
`serum: her-item. 0.l% serum is not sufficient. In low serum concen-
`SK-BR-B cells at all serum concentrations tested- Similarly. [’Hlthy—
`trations. HRG activation of ErbB receptor pathways is then sufficient
`midine incorporation in BT4'M. MBA-M3453. and MBA-M366]
`for stimulating rnitogen'tc activity. These data are consistent with
`breast tumor cells was also inhibited by 405 since these cell lines
`those reported recently by Pietras rt cl. (42]. which demonstrate a
`overespress ErbBZ. rHRGflI and 405 did not affect [’Hlthymldine
`direct interaction between HRG-mcdiatcd activation of ErbB path-
`incorporation in the MDA-MB-23l and MDA-MB-463 breast tumor
`ways and cross-tall: with estrogen receptor pathways. In contrast. the
`lines. Consistent with previous reports (43). EGF was inhibitory l‘or
`ErbB! overespi'cssing breast tumor cell lines. SK-BR-J and MBA-
`MBA-M8468 cells. No significant changes in MDA-MB-Ziil cells
`MB-453. showed enhanced incorporation of [’l-llthynridine in a se-
`were detected with either r'HRGBI. EGF. or 405 under the conditions
`rum-dependent manner alter 1 day of treatment with rHItGfll (data
`tested.
`not shown) and after 3 days of treatment (Fig. 3. a and c). Although
`I460
`
`

`

`~..l
`
`IMGMWWTESIG’IM
`
`+0.15!» FBS +19% FBS +10% FBS
`
`A
`
`no MOP?
`g-r no
`3% an
`2%f:
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`
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`
`0.1
`
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`
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`
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`
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`i;
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`5.6
`
`E g
`
`”
`
`s.
`
`250 MDA-MB-453
`
`Sphagnumiediealerofpmlifuafivemnmwdm -30h
`following manna. For both MCF! and SIC-3R4 eells. the change;
`in a; pea-mug: «IS-phase cells afiu each Wm correlates well
`With the effects m [’Hlthymidiue hum-amino (F13. 3). tHRGfll
`unsedaLfoldkmueintheMgeofMCfieellsinS-phag
`in 0.1% I-BS. ESP mullet-It induced e smalls proportion of eells to
`enter 5 phase in 0.] and I‘ll FBS (1.6- ml 13-fold, mpeetively),
`e
`
`I "HO
`
`U EOF
`
`‘0'
`
` 'H-IdlIIonaof
`com!+I-me.)
`
`9
`
`
`
`'l-I-ThymldlneIncorporation(9;olcontrol+!-3.0.)
`
`len
`
`(96ofeemrel+1-3.9.)
`
`
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`
`o
`
`0.0!
`
`0.1
`Human
`
`l
`
`10
`
`F131. suuuuulenel om lynllu'uby IHRGBI. HGT (A). Sit-Bil»: {8). led
`m-MASSIQmmmmMulmdlfl'odthl
`mmmmummmmimmmmwum
`hmnflimtfldfififlflop—nmhufinflluflhlfiemfiwm
`mmummmuaeflmmdmcpmm
`ens-elemental will-OJ {0). 1 Me: In F35 (A) Inlays. AI undated:
`Whaflsmlfluw rill: I pCMll’HlflIymilfiml'ntdILm
`erduphufaefimllmmheMmW-hm
`crude-swieeu:kmsl
`
`Cell Cycle Changes Induced by rHRGBI. EGF. 1mg Murine
`Monoclonal Antibody 4D5. To fnnhet ehmclcriu the effects of
`rHRGfll. EGF. and 4D5 on tumor cell growth. analyses of eell cycle
`phase fraction distillation: wese performed. MCF‘l. SIC-BIL}. and
`MBA-M3453 breast tumor cells were serum slanted and then treated
`
`Hetmwflmefllumpemotdiflmmwwefinwufl ileum
`I'I'IRGBI. EFF. ml ‘1'”. Milnpni: nun. we flaw “bullied in Fig. 1C¢11l
`resume! will: 0.31MIHR051J mW.“66nniDShmodlumeuminluU-15
`(A), I'lawlw lflll'lCl F35 fuldlyl.mmnelpusedufl|tm°[
`for I day with 0.3 mt rHRGBl. 3 me EOEWGG mu 4D5 in OJ. I, or
`['l‘llurrmlflim Wine (nun cl 4-! malieuu: Mn. SE) n unwed lo tune-Id
`enlwd eelll.
`10% FBS. Fig. 5 shows the mulls presented as Ihe fraction of cells in
`I46I
`
`

`

`.
`
`.
`
`'
`
`.
`
`mumsmmmfinm
`
`while 4DS bad no effect. With SK-BR‘S cells». rl-IRGBI stimulation of
`the percuttageofcells inSphasewascohar-tcedwithincreasingactum
`ccneermation (Is-lj-lold). BGF had an inhibitory effect in OJ and
`lit Fns.andasespocted. 405deoeasedtlteperoeatageof S—phate
`cells at all concentrations of serum MC?! and sx-on—a cells were
`also treated for 3 days with rHRGBI with similar results. In contrasl.
`thefractionofoellsinSphaselntheMDA-hm-dflcelllioeupon
`espoauretorHRGfll didnotdilferfromconn'olsin thethroesertrrn
`mnafiooamsteduwhneseresultsaredifiaentfrpmdtedata
`shown in Fig. 4. where rilRGfll
`increased the incorporation of
`[’l-mnymidineintheMDA-MB-tlflcellsio I and I0%FBS.Onthe
`other hand. EGF and 4D5 have similar effects on both [’l-llthymidine
`inoupuationemioellcyoleprogreuionAsMEGF-oeatcd
`WA-MB-dfl cells were not different from control. since this cell
`lineupreases littleornodctcctableEGFRflO.38).whereasneat—
`merit with 4D5 reduced the numb of Sv-phase cells. In contrast to
`previous reports (ll. 16. 46). upon HRG treatment. there was no
`mggudonofaGrMy-omharrestnorwasthereevidenoefor
`induction of a ploidy abnormality. Explanations for moire-span—
`ciescanlikelyboamibtttedtcdifierenoeslntheptoitycfdteNDF
`motionlortheparticularoelllinesbeinguscdinthcseearticr
`studies.
`
`Eflect of rflRGfll on Cell Number. Because of the discrepancy
`between the NBA-M3453 [‘Hlthyrnidine incorporation and chl
`cycle eaperirnents. it was necessary to determine actual cell number
`after rl-lRGBl treatment. Fig. ti shows that the effects of rliRGfll on
`MCF'? and SK-BR-3 cell number are in agreement with the data in
`Figs. 3 and 5. The MCF’l breast tumor cells show the greatest increase
`in cell number to rl-lRGBl after 3 days of treatment in 0.!‘11 PBS.
`while proliferation of SK-BR-S cells is increased in higher serum
`canoentrations. 'I‘l'tet't: was no change in MBA-M3453 cell number
`after exposure to 0.3 tort rHRGfll in 0.1. I. or It“: F35. Therefore.
`it appears that the incorporation of [‘Hlthyrnidine in this cell line is
`not an indicator of DNA synthesis and cell proliferation. These results
`are in agreement with those reported earlier for the response of
`MBA—M3453 cells to concentrated conditioned medium containing
`recombinant NDF (25). Our data for the responsiveness of MBA-MB-
`453 and SK-Bl‘t-3 cells differ from those reported earlier for NDFcr
`(12) or purified natural gp30. which has been shown to be related to
`HRGINDF {14. 26}.
`
`sscoo
`
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`
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`-
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`:IE
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`:3.
`1'.
`0.1 1010.0
`0.1 1.0 10.0
`0.1 1.0 10.0
`
`Serum Commutation {as}
`mtmeuolrifllfifll onMCFT.SK-IR-3.IIIIMDA-MB-45! oellnttrnher.Cella
`Humanist-1g. Leaccptatdteccdddtelocuhuionthsmrolayuawe
`mmmummmmnmmmm
`
`HRG Stimulation of Tumor Cell Growth Is Inhibited by Antl-
`ErbBZ Monoclonal Antibodies 101 and TF3. Since 2124 and “ll-21
`are cytostatic for cells that overeapress ErbB! (30). we could not test
`the antagonist effect of 1C4 or 7F! on HRS-induced growth of cells
`that overeapress ErbBI. The effect of 2C4 or 1F! on three cell lines
`that express lowlnormal levels of E11332 is shown in Fig. 'l. MCF‘l and
`Td‘l'D are breast cancer cell lines that are known to express low to
`intermediate levels of all E11313 receptors {34. 36. 39). Although 51'de
`is not expressed in the ovarian cancer cell line Caov3 (35). other ErbB
`receptors are present 130) (data not shown]. The effect of 201 or TF3
`on rl-lROlll stimulation of cell growth using a 3-day crystal violet
`assay format is shown in Fig. 1. In each case. rHRGBl-mcdiated
`growth was inhibited by 201 or 7F} to levels close to those observed
`without HRG treatment. The results observed with 201 or TF3 are
`similar to those reported recently by Gratis-Porto er cl. (22). using a
`version of Td‘lD cells engineered to sequester 5:082 in the endoplas-
`mic reticulum with a single-chain anti-Elba! antibody. These data are
`also similar to those obtained when rHRGflI was tested in combina-
`tion with 2C4 on human Schwann cells (2”.
`Anchorage-independent Growth in Soft Agar. For a final deter-
`mination of the effects of rl-lltOlll on cell proliferation. we studied
`the anchorage—independent growth of different breast and ovarian
`armor cell lines in soft agar and compared the response with EGF. To
`allow for enhanced proliferation of colonies in soft agar. cells were
`seeded at densities resulting in minimal colony formation in untreated
`cells. and all erperiments had to be perfumed in 10% serum. As
`shown in Fig. 8. MCI-“l. T410. Sit-BIL}. BT474. and SK-OV-S cells
`showed a greater than 2-fold increase in colony formation in response
`to treatment with 0.3 not rHltGfll. Growth in soft agar of the MBA-
`lle-468 cell line was also slightly enhanced. Since this cell line does
`not express Erbln (30. 38) and presonurbly low or undetectable levels
`of Erde but does express ErbBB. this response might be mediated by
`ErbBJ. Since ErbBS is an impaired kinase (4?. 118}. these data suggest
`that E11133 may be heterodimerizing with another ErbB family mem-
`ber other than Erbllz. One plausible candidate for this active signaling
`complex may be EGFlt-ErbBZl since MBA-M8468 cells ovaespress
`EGFR (-191. Colony formation by MDAaMBASJ and MDA-MBJGI
`cells was inhibited by HRS treatment relative to control plates. In
`comparison. EGF had little effect on MCI-‘l. MDA-MB-dfl. MBA-
`MB-Sdl. or MDA-MB-Zill cells. Growth in soft agar was stimulated
`by EGF in Sit-0V») cells and was slightly enhanced in arcane and
`BTd'M cells. Treatment of MDA-MB-463 cells With EGF completely
`abolished colony formation.
`1462
`
` 7
`
`‘05
`
`SK-BFl-a
`
`a Control I HRG El cor
`
`
`
`S-phasoCells(at) 8
`
`
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`
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`
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`
`
`
`0.1 1.0 10
`0.1 1.01
`
`0.1 1.O 10
`
`Scrum Concentration (‘36)
`fig. 3. Effects of ducal. EGF. and £05 on cell cycle progression in HEY-1'.
`stt-tllt-J. oat MBA-n54!) breast month. not ttuurut was dose in triplicate. at
`dcuihdh‘MuuiahudMnlnda'flcdmahowmpohdfr-unlluutofw
`mundmhrasfi.
`
`

`

`mummmmsmm
`
`suggesting that the font-tattoo oi this high-sliittity binding site was
`likely the result of heterodimeritation of mm with EthJ or with
`Eb“. In. addition. the binding affinity of HBO for E11354 appears to
`be considerably lower than the values determined here for this pane]
`of human tttntor‘cell lines (33). Although ErbM is uprated in some
`cities: cell lines (34. 35). our analysis ofErbB¢ expression in thee:
`cells using a panel of PAIN-specific monoclonal antibodies indicate;
`that the 15le expression level is significantly lower than BOP-1t.
`131sz or F333." 5:de is a fully functional l-lltG receptor (35. SO),
`anditltuboe