Proc. Natl. Acad. Sci. USA
`Vol. 84, pp. 7159-7163, October 1987
`Cell Biology
`
`of the putative growth factor receptor
`Increased expression
`p185HER2 causes transformation
`of NIH 3T3 cells
`and tumorigenesis
`ROBERT M. HUDZIAK*, JOSEPH SCHLESSINGERt, AND AXEL ULLRICH*
`
`CA 94080; and tBiotechnology
`Inc .• 460 Point San Bruno Boulevard.
`Research
`South San Francisco.
`Genentech.
`Biology.
`of Developmental
`•Department
`MD 20850
`4 Research Court. Rockville.
`Center, Meloy Laboratories.
`
`Communicated by Hilary Koprowski, July 13, 1987
`
`MATERIALS AND METHODS
`
`with the results of Slarnon et al. (14), suggest
`combination
`ABSTRACT The HER2 gene encodes a cell-surface glyco·
`that mere amplification
`of the HER2 gene and resulting
`protein with extensive homology to the epidermal
`growth factor
`of its product may play a crucial role in the
`overexpression
`Recently it was found to be amplified in about 30%
`receptor.
`of some types of human cancer.
`genesis and development
`In experiments de­
`of primary human breast malignancies.
`signed to assess the role of the HER2 gene in oncogeoesis, we
`HER2 coding sequences
`of unaltered
`found that overexpression
`in cellular
`transformation and
`in NIH 3T3 cells resulted
`tumorigenesis.
`
`vector
`The mammalian expression
`Expression Plasmids.
`units for mouse dihydrofolate
`CVN (17) contained expression
`neomycin
`(DHFR) cDNA (18) and the bacterial
`reductase
`glycoprotein
`with
`The HER2 gene encodes a transmembrane
`(neo) gene (19), both under simian virus
`phosphotransferase
`homology to the human epidermal
`extensive structural
`of a 4.4-kilobase­
`Transcription
`40 early promoter control.
`growth factor (EGF) receptor and the chicken oncogene
`pair Sal I-Dra I HER2 fragment containing
`the full-length
`v-erbB (1-3). Chromosomal mapping and sequence compar­
`HER2 coding region (1) was driven by the Rous sarcoma
`ison strongly suggest that the HER2 gene product and the
`virus (RSV) long terminal repeat promoter (L TR). The
`oncogene neu
`rat neuroblastoma
`ethylnitrosourea-activated,
`sequence of
`poly(A) site was provided by the 3' untranslated
`(4). The
`of the same polypeptide
`species variants
`represent
`B virus surface antigen gene (20). The control
`the hepatitis
`neu oncogene encodes a 185-kDa cell-surface
`glycoprotein
`but lacked cDNA sequences
`CVN plasmid was identical
`that possesses intrinsic tyrosine-specific
`that
`kinase activity
`downstream from the RSV L TR.
`ligand (5, 6).
`by an as yet unidentified
`is likely to be activated
`in a 1:1 mixture
`NIH 3T3 cells were cultured
`Cell Culture.
`neu oncogene sequence with
`Comparison of the transforming
`and Ham's nutrient
`modified Eagle's medium
`ofDulbe.cco's
`counterpart suggested
`that a
`its normal rat protooncogene
`(2 mM), penicillin
`with glutamine
`mixture F-12 supplemented
`in
`domain resulting
`point mutation in the transmembrane
`and 10% HyClone
`(100 µ.g/ml),
`streptomycin
`(100 units/ml),
`of a valine residue by glutamate unmasked the
`substitution
`under 5%
`incubator
`(Logan, Utah) calf serum in a humidified
`growth factor receptor
`of this putative
`potential
`transforming
`C02 in air atmosphere.
`(7). Analogously,
`have converted nor­
`alterations
`structural
`and Amplification. Plasmid DNA was intro­
`Transfections
`for macrophage colony­
`mal genes coding for the receptors
`duced into mammalian cells by the calcium phosphate
`factor type 1 and EGF into v·f ms (8) and v-erbB
`stimulating
`plates of cells (60
`method (21). Half-confluent
`coprecipitation
`respectively.
`(9) oncogenes,
`mm) were exposed to 5 µ.g of plasmid DNA in 1 ml of
`Southern analysis of primary human tumors and estab­
`shock
`glycerol
`for 6-8 hr. After a 20% (vol/vol)
`precipitate
`cell lines revealed amplification
`and in
`lished tumor-derived
`medium. Two days
`(22), the cells were fed with nonselective
`of the EGF receptor gene. Am­
`some cases rearrangement
`containing
`later, they were passaged into selective medium
`in squamous carcinomas
`was particularly apparent
`plification
`(0418) at 400 µ.g/ml.
`Geneticin
`(12). The HER2 gene was also
`(10, 11) and glioblastomas
`Clones were picked using glass cloning cylinders
`with
`found to be amplified in a human salivary gland adenocarci­
`petroleum jelly for the bottom seal. Colonies arising from
`(2), and a gastric
`noma (3), a mammary gland carcinoma
`for growth in G418 were picked,
`cells selected
`transfected
`et al. (14) demon­
`cancer cell line (13). Recently, Slamon
`into medium containing
`7%
`expanded, and subcultured
`strated that about 30% of primary human breast carcinoma
`dialyzed fetal bovine serum in place ofl0% calf serum and the
`HER2 gene. Although a few
`tumors contained an amplified
`for plasmid am­
`of methotrexate
`concentration
`appropriate
`in most tumors
`were detected,
`sequence rearrangements
`step removes trace amounts of
`(23). The dialysis
`plification
`between amplified
`and
`there were no obvious differences
`purines and pyrimidines present in serum that decrease the
`of the
`amplification
`normal HER2 genes. Furthermore,
`of the
`with the prognosis
`significantly
`HER2 gene correlated
`To apply selective
`selection.
`of the methotrexate
`efficiency
`the probability
`of relapse.
`disease and
`pressure,
`of methotrex­
`concentrations
`stepwise increasing
`between
`of the correlation
`the significance
`To investigate
`of 400 nM. To avoid
`ate were used with a final concentration
`as it has been
`transformation
`and cellular
`overexpression
`cells, cells were
`transformed
`for spontaneously
`enriching
`c-mos (15) and c-Ha-rasl
`(16),
`observed for protooncogenes
`was to amplify the
`An additional control
`kept subconfluent.
`vector and a selection
`we employed a HER2 expression
`the HER2 cDNA insert in
`CVN neo-DHFR vector without
`after transfec­
`sequence amplification
`scheme that permitted
`the NIH 3T3 recipient
`cell line.
`tion of mouse NIH 3T3 cells. We report here that amplifi­
`and Labeling. The G-H2CT17 anti­
`Immunoprecipitations
`HER2 gene in NIH 3T3 cells leads to
`cation of the unaltered
`17 amino acids of H ER2 was
`the C-terminal
`body recognizing
`of pl85HERl as well as cellular
`transformation
`overexpression
`peptide coitjugated
`with
`prepared in rabbits using a synthetic
`and tumor formation in athymic mice. These findings,
`in
`soybean trypsin inhibitor.
`
`in part by page charge
`costs of this article were defrayed
`The publication
`payment. This article must therefore be hereby marked "advertisement"
`with 18 U.S.C. §1734 solely to indicate
`this fact.
`in accordance
`
`Abbreviations: DHFR, dihydrofolate reductase; R, resistance; EGF.
`epidermal growth factor; RSV, Rous sarcoma virus; LTR, long
`terminal repeat.
`
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`Cell Biology: Hudziak et al.
`
`Proc. Natl. Acad. Sci. USA 84 (1987)
`
`Cells were harvested by trypsinization and counted by
`Coulter Counter, and 1.5 x 106 cells were plated per 60-mm
`culture dish. After 36 hr, the cells were lysed at 4°C with 0.4
`ml ofHNEG buffer per plate (50 mM Hepes, pH 7.5/150 mM
`NaCl/I mM EGT A/10% glycerol) containing 1.0% Triton
`X-100 detergent and 1 mM phenylmethylsulfonyl fluoride.
`After 10 min, 0.8 ml of lysis dilution buffer (HNEG buffer
`with 1% bovine serum albumin and 0.1% Triton X-100) was
`added to each plate and the extracts were pelleted at 12,000
`x g for 5 min.
`HER2 antibody was added to the cell extracts, which were
`then incubated at 4°C for 2 hr; this was followed by incubation
`with protein A-Sepharose beads for 20 min and three washes
`with 1 ml of HNEG buffer with 0.1% Triton X-100.
`Autophosphorylation reactions were carried out at 4°C in 50
`µ.I of HNEG wash buffer with 5 mM MnCl2 and 3 µ.Ci of
`[y-32P]ATP (Amersham, 5000 Ci/mmol; 1 Ci = 37 GBq) for
`20 min. Proteins were separated on 7.5% NaDodS04/poly­
`acrylamide gels and analyzed by autoradiography.
`Transformation Assays. The efficiency of colony formation
`in soft agar (24) was determined by plating 25,000 cells in 3
`ml of 0.2% agar (Difeo, ·'purified") over 4 ml of 0.4% agar in
`a 60-mm dish. After 2-4 weeks, colonies of about 100 cells or
`more were counted.
`The plating efficiency of cell lines (25) in 1 % calf serum was
`determined by plating equal numbers of cells into 100-mm
`plates with either 10% or 1 % calf serum. After 2-3 weeks, the
`plates were stained with crystal violet and colonies were
`counted.
`Mouse Tumorigenicity Assays. Athymic (nu/nu) mice were
`obtained from Charles River Breeding Laboratories. Control
`NLH 3T3 and NIH 3T3/CVN cells and experimental HER2-
`3400 cells were harvested by trypsinization and counted with
`a Coulter Counter. They were then collected by low-speed
`centrifugation and resuspended in ice-cold phosphate-buff­
`ered saline to either 2.5 x 106, 5.0 x 106, or 1.0 x 107 cells
`per ml. Animals were injected subcutaneously with 0.1-ml
`volume of the cell suspensions. Tumor occurrence and size
`were monitored twice weekly.
`
`RESULTS
`
`For expression of HER2 sequences in NIH 3T3 cells, a cDNA
`coding for the entire 1255-amino acid polypeptide (1) was
`placed under transcriptional control of the RSV L TR. Tran­
`scriptional termination signals and a poly(A) site were pro­
`vided by 3' sequences of the hepatitis virus surface antigen
`gene (20). In addition, the expression vector contained the
`neo resistance (neoR) gene, which confers cellular resistance
`to the aminoglycoside antibiotic 0418 (18) and therefore
`allows selection of primary transfectants, as well as the
`DHFR gene for methotrexate resistance, which was used to
`amplify transfected DNA sequences under selective pres­
`sure. Both drug resistance genes were under simian virus 40
`early promoter transcriptional control. Bacterial plasmid
`sequences, including an origin of replication and the gene for
`ampicillin resistance, allowed replication of the entire expres­
`sion plasmid in Escherichia coli.
`The transforming activity of HER2 sequences was initially
`tested using a conventional NIH 3T3 cell focus-formation
`assay. Under conditions that resulted in about 1G4 foci per µ.g
`of a v-f ms viral construct, we were unable to detect any
`HER2-transforming activity. Because of the recently report­
`ed finding that about 30% of mammary carcinomas contain
`amplified HER2 gene sequences without apparent sequence
`rearrangements (14), we investigated whether amplification
`of an unaltered HER2 gene could transform mouse fibro­
`blasts. NIH 3T3 cells were transfected with the pCVN/HER2
`construct. An identical plasmid missing the HER2 expression
`module was used as a control. Four independent primary
`
`Table 1. Assay for growth in soft agar and 1% calf serum of
`HER2 primary and amplified cell lines
`
`HER2 gene copies
`per haploid
`genome, no.
`
`Soft agar
`colonies,
`no.
`
`Plating
`efficiency
`in 1% calf
`serum, %
`
`0
`0
`3
`60
`2
`55
`4
`90
`1
`131
`
`0
`0
`1
`424
`0
`836
`0
`376
`0
`373
`
`0.27
`0
`3
`38
`1.4
`11.7
`0.2
`49
`0.6
`50.2
`
`Cell line
`
`NIH 3T3/CVN
`NIH 3T3/CVN<OO
`HER2·1
`HER2-1400
`HER2-3
`HER2·3400
`HER2-4
`HER2·4400
`HER2-83
`HER2-83400
`
`Two control lines were used. The first one was a NIH 3T3 line
`transfected with a plasmid containing only the neo and DHFR genes.
`The second control line contained the neo-DHFR plasmid and was
`amplified to resistance to 400 mM methotrexate. HER2 gene copy
`numbers were determined using a human DNA standard and
`densitometer scanning of Southern hybridization autoradiograms.
`Equal cell numbers (25,000) were plated in soft agar and colonies
`were counted after 2-4 weeks. The plating efficiency in 1% calf
`serum is relative to the number of colonies arising when an equal
`aliquot was simultaneously plated in medium containing 10% calf
`serum.
`
`G418-resistant clones (HER2-l, HER2-3, HER2-4, HER2-
`B3) were isolated. Cell lines containing amplified HER2
`coding sequences were generated from these parental clones
`by culturing the cells in gradually increasing concentrations
`of methotrexate up to 400 nM (HER2-1400, HER2-3400,
`HER2-4400, HER2-3B400). Southern hybridization analysis of
`parental and amplified cell lines demonstrated that the HER2
`cDNA copy number increased from 1-4 to 55-131 per haploid
`genome (Table 1).
`To test whether gene amplification resulted in overexpres­
`sion of the HER2 gene product, cell lysates were im­
`munoprecipitated with an antibody against the C-terminal 17
`amino acids of the HER2 sequence. As shown in Fig. 1,
`substantially increased levels of the pl85HERi gene product
`were found in amplified cell lines relative to their parental
`G418R transfectants. The parental cells had a normal mor­
`phology that was indistinguishable from NIH 3T3 cells.
`However, amplified cells had the typical refractile, spindle-
`
`123456789
`
`200-
`
`116
`
`•
`
`F1G. 1. Quantitation of p185HERz in four primary, unamplified cell
`lines and lines derived from them by amplification to resistance to 400
`nM methotrexate. Lane 1, neo-DHFR control; lanes 2 and 3, HER2-l
`parent and amplified lines; lanes 4 and 5, HER2-3 parent and
`amplified lines; lanes 6 and 7, HER2-4 parent and amplified lines;
`lanes 8 and 9, HER2-83 parent and amplified lines. Positions of the
`size markers myosin and f3-galactosidase are indicated in kDa.
`
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`Cell Biology: Hudziak et al.
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`Proc. Natl. Acad. Sci. USA 84 (1987)
`
`7161
`
`FIG. 3. Anchorage-independent growth of HERZ-transformed
`FIG. 2. Morphology of NIH 3T3 cells transfected with HER2
`expression construct. (/) NIH 3TI cells transfected with the control
`cells in soft agar. Cells were plated in 0.2% soft agar over a 0.4% agar
`neo-DHFR plasmid. (2) NIH 3T3 cells with the neo-DHFR plasmid
`lower layer. After 3 weeks the plates were photographed at 4-0x
`magnification using a Nikon microscope with phase-contrast optics.
`amplified to resistance to 400 mM methotrexate. (3) Primary G418R
`cell line HER2-l, an unamplified clone expressing low levels ofpl85.
`(Upper) Control untransformed NIH 3T3 cells. (Lower) Anchorage­
`(4) The same clone amplified to 400 mM methotrexate resistance.
`independent growth of the cell line HER2-3400 containing the HER2
`(X50.)
`expression plasmid and amplified to resistance to 400 mM methotrex­
`ate. (x 25 .)
`
`
`cells and grew in irregular, shaped appearance of transformed
`
`piled-up clumps (Fig. 2).
`was a 26-fold increase in the number of cells plating in 100 nM
`
`
`
`
`
`NIH 3T3 cell lines with an amplified HER2 gene and high
`
`
`methotrexate in the selected cells compared with the parental
`
`
`other char­levels of HER2 gene expression also displayed
`
`cells, implying that the unselected but linked DHFR gene had
`
`
`
`acteristics associated with a transformed phenotype. As
`also been coamplified.
`
`
`shown in Table 1 and Fig. 3, these cells all formed colonies
`The tumorigenicity
`of cell lines with a high HER2 cDNA
`in soft agar and were able to grow at low density at low serum
`copy number was tested in nude mice by subcutaneous
`
`
`
`concentration. In contrast, primary transfectants did not
`
`
`grow under these conditions. The primary transfectants did,
`
`however, grow to a higher saturation density than the
`
`parental NIH 3T3 cells.
`
`The correspondence between a transformed phenotype
`and HER2 gene amplification
`
`and overexpression was inde­
`
`
`pendently confirmed by directly selecting for transformed
`
`
`cells and then analyzing the resulting clones. For this purpose
`the parental cell line HER2-3, whfoh contains about two
`
`was cultured in copies of the HER2 expression construct,
`
`
`
`medium containing a low concentration of fetal calf serum
`
`
`(0.5%); control cells containing the expression vector without
`HER2 coding sequences (pCVN) were cultured in parallel.
`FIG. 4. Quantitation of pl85 in the primary line HER2-3 and cell
`
`After 5 weeks, a few colonies appeared in the control culture
`and roughly 10-fold more colonies appeared in dishes con­
`lines selected for growth in 0.5% calf serum. HERZ-encoded protein
`taining the HER2-3 cell line. These colonies appeared to be
`was immunoprecipitated and labeled. Lanes 1-3, three colonies
`picked and expanded from plates after selection for growth in 0.5%
`
`
`
`morphologically transformed and were subsequently ana­
`serum; lane 4, the starting cell line, HER2-3; lane 5, a pool of0.5%
`
`
`4, three lyzed for HER2 overexpression. As shown in Fig.
`individual clones as well as a �I of the remaining
`
`serum-selected colonies of HER2-3; lane 6, a clone derived from a
`colonies
`G418R control line selected for growth in 0.5% calf serum. The
`had elevated levels of p185HE 2 compared with the original
`expected for a protein of apparent molecular mass of 185
`position
`
`
`
`parental 0418-resistant HER2-3 cell line. In addition, there
`kDa is indicated.
`
`... .. -185
`
`I 2 3 4 5 6
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`Cell Biology: Hudziak et al.
`
`Proc. Natl. Acad. Sci. USA 84 (1987)
`
`Table 2. Tumorigenicity testing i;>f cell lines
`
`Cell line
`
`Cells injected, no.
`
`Mice with tumors/
`mice injected
`
`NIH 3T3
`
`NIH 3T3/CVN
`
`HER2-3400
`
`0.25 x 106
`0.5 x 106
`1.0 x 106
`0.25 x 106
`0.5 x 106
`1.0 x 106
`0.25 x 106
`0.5 x 106
`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 HER2-3 and two control lines, NIH 3T3 cells and a
`NIJi 3T3 line contai11ing the contrql plasmid, were irtjected subcu­
`taneously at three different dosages into nude mice. The time after
`injection before tumors became visible was dose related: 22 days
`(average) for 1 x 106 cells, 28 days (average) for 0.5 x 106 cells, and
`34 days (average) for 0.25 x 106 cells.
`
`injection of three different cell numbers per animal. As shown
`in Table 2, the overexpressing cell line HER2-3<400 was
`strongly tumorigenic at all dosages tested, whereas the
`control cell lines, NIH 3T3 cells and NIH 3T3 cells
`transfected with CVN vector without the HER2 insert, were
`both negative under the same conditions. Necropsy of three
`mice with well-established tumors failed to identify any
`metastasis. Cell lines reestablished from three excised tu­
`mors still expressed the G418R phenotype, were resistant to
`methotrexate, and expressed high levels of p185HER2 (not
`shown).
`
`DISCUSSION
`
`Amplificatiop of the HE�2 gene has been reported in a few
`primary human tumors (2, 3) and tumor-derived cell lines (13,
`26). Recently, Slamon et al. (14) found that the HER2 gene
`is amplified in 30% of primary brefist tumors, a common
`human malignancy that affects about 7% of all American
`females. Notably, only 3/189 tumors surveyed showed any
`gross rearrangement of the HER2 gene. To assess the role of
`the HER2 gene in the neoplastic process, we characterized
`the transforming potential of the HER2 gene in an in vitro cell
`culture system.
`Expression of the full-length cDNA in NIH 3T3 cells did
`not lead to transformation as determined by a standard
`focus-forming transfection assay. However, HER2 overex­
`pression caused by gene amplification transformed these
`cells. Colonies that survived methotrexate selection were
`morphologically transformed and exhibited loss of contact
`inhibition. Such cells also grew in soft agar and would grow
`in 1% calf serum. Furthermore, cells transformed by HER2
`were tumorigenic in athymic mice.
`Selection of cells transfected with the HER2 cDNA for
`growth in low serum provided independent evidence that
`high-level expression of an unaltered HER2 gene product
`caused cellular transformation. Since DNA introduced into
`mammalian cells by transfection is more labile than genomic
`DNA (23, 27), we reason.ed that selection for a property
`demonstrated by pharmacologically amplified HER2 lines­
`namely, growth in low serum-might lead to amplification or
`other changes resulting in overexpression of pl85HER2•
`Clones derived from an unamplified HER2 line by this selection
`procedure appeared morphologically transformed and exhibit­
`ed elevated levels of pl85HER2.
`Taken together, the characteristic morphological changes,
`res11lts of in· vitro and in vivo transformation and tumorigenic­
`ity assays, and the elevated levels of p185HERl in cells
`selecte<;I for a transformed phenotype imply that high-level
`expression of HER2 results in transformation of NIH 3T3
`
`cells. Another member of the tyrosine kinase gene family was
`recently found to be amplified 4- to 8-fold in spontaneously
`arising foci of NIH 3T3 cells (28). Amplification of the met
`gene appears to be a frequent event and, similar to HER2
`amplification in mammary carcinpmas, is rarely accompa­
`nied by grpss rearrangements. These findings and the dis­
`covery of amplified EGF receptor genes in primafY human
`tumors (10-12) suggest that overexpression pf other growth
`factor receptor
`genes will also lead to transformation and
`tumorigenesis. Whether susceptibility to spontaneous ampli­
`fication is caused by characteristics of the genetic loci for
`HER2, met and EGF receptor, or cell-specific selective
`advantages caused by receptor overexpression remains to be
`investigated.
`It is not yet clear whether transformation of cells
`overexpressing a growth factor receptor gene is dependerit on
`paracrine or autocrine stimulation by the appropriate ligand.
`For HER2, this question cannot yet be addressed since its
`ligand has not been identified. In the case of the EGF
`receptor, however, we were able to demonstrate that primary
`human tumors and tumor-derived cell lines fre�uently ex­
`press mRNAs for both the receptor and transforming growth
`factor type a ligand (29). The finding that mere overexpres­
`sii:>n of an intact growth factor receptor may subvert normal
`cellular growth control mechanisms and lead to tumorigenic
`growth provides new potential for diagnostic approaches and
`therapeutic strategies for treatment of human malignancies.
`We express appreciation for the superb technical assistance of
`Thomas Dull in constructing the HER2 expression plasmids and Bill
`Lagrimas for help with the mouse tumorigenicity assays. We are
`grateful to Jeanne Arch for her patience and skill in typing this
`manuscript and to Dr. Suzanne Pfeffer for her valuable editorial
`comments.
`
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`Cell Biology: Hudziak et al.
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`5 of 5
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