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Characterization of Epidermal Growth Factor Receptor Gene Expression In Proc. Nati. Acad. Sci. USA Vol. 81, pp. 7308-7312, December 1984 Biochemistry Characterization of epidermal growth factor receptor gene expression in malignant and normal human cell lines (c-erbB/transforming protein/DNA amplification/RNA blot analysis/immunoprecipitation) YOUNG-HUA Xu, NANCY RICHERT, SEWI ITO, GLENN T. MERLINO, AND IRA PASTAN Laboratory of Molecular Biology, Division of Cancer Biology and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205 Contributed by Ira Pastan, July 27, 1984 ABSTRACT To investigate the possibility that the epider- kilobases (kb), as well as minor RNAs of 6.3, 4.6, and 3.3 kb mal growth factor (EGF) receptor functions as an oncogene (18). Other groups have identified similar-sized EGF recep- product, we have determined the levels of EGF receptor pro- tor RNAs in A431 cells (21, 22). In A498 kidney carcinoma tein and RNA in a variety of malignant and normal human cells the only major RNAs that were found to hybridize to cells, using a specific polyclonal antibody to the EGF receptor this probe were the 10- and 5.6-kb species (18). and a cDNA clone (plasmid pE7) that encodes the EGF recep- To gain further information about the EGF receptor gene tor, respectively. Besides A431 epidermoid carcinoma cells, and its expression, a variety of cell lines were examined to which are known to make large amounts of EGF receptor, cell determine the quality and quantity of pE7-hybridizable lines from two ovarian cancers, two cervical cancers, and one RNAs. EGF receptor levels were measured by using a spe- kidney cancer were found to contain substantial amounts of cific rabbit polyclonal antibody to the EGF receptor (anti- receptor protein (11-22% of A431). Normal human fibro- EGFR) and RNA levels were quantified, utilizing the pE7 blasts (Detroit 551), a human lymphocyte line (IM-9), and a cloned EGF receptor cDNA (18) as a probe. The copy num- leukemic lymphocyte line (CEM) contained low or undetect- ber of the EGF receptor gene in these cell lines was deter- able levels of EGF receptor. RNA blot analysis showed that mined by using the same pE7 probe. among the human cell lines examined the levels of a 10- and a 5.6-kilobase species of pE7-specific RNA generally correlated with the amount of the EGF receptor protein. Genomic DNA MATERIALS AND METHODS blot analysis revealed that except for A431 none of these cell Cell Cultures. Cell lines were maintained in medium sup- lines expressing high levels of EGF receptor protein possessed plemented with 10% fetal bovine serum (GIBCO) unless stat- amplified receptor gene sequences. A431 cells are known to ed otherwise. The human epidermoid carcinoma cell lines secrete a truncated form of the EGF receptor. An abundant A431 (George Todaro, National Institutes of Health) and KB 2.9-kilobase RNA is found only in A431 cells; it could encode (American Type Culture Collection), and the human kidney the truncated form of the EGF receptor. carcinoma cell line A498 (G. Todaro) were maintained in Dulbecco's modified Eagle's medium (DME medium). Epidermal growth factor (EGF) receptor is a 170-kDa glyco- HTB32 (a cervical carcinoma cell line from the ATCC) and protein found in many cell types (1-6). The receptor has an IM-9 lymphocytes (J. Roth, National Institutes of Health) intrinsic tyrosine-specific protein kinase activity that is stim- were grown in RPMI 1640 medium. The ovarian cell lines ulated by EGF (6-8). EGF binding is known to cause "down were 1847 from S. Aaronson (National Institutes of Health), regulation" of the receptor (9-13). Downward et al. (14) and OVCAR2 and OVCAR3 (referred to in this paper as have recently isolated six separate peptides from the EGF OVCA2 and OVCA3, respectively) from T. Hamilton and R. receptor. The peptides were found to be very similar to the Ozols (National Institutes of Health). The ovarian lines were deduced amino acid sequence of the oncogene erbB protein maintained in RPMI 1640 medium with insulin at 10 ,ug/ml. product [v-erbB (15)] of the avian erythroblastosis virus). MCF-7 breast cancer cells (M. Lippmann, National Insti- The erbB gene product has been shown to cause erythroblas- tutes of Health) were grown in improved minimal Eagle's tosis and sarcomas in infected chickens (16, 17). The strong medium (National Institutes of Health Media Unit). Detroit similarity between the v-erbB protein and the EGF receptor 551 normal human fibroblasts (ATCC no. CCL10) were suggests that the human cellular homolog to v-erbB (c-erbB) grown in DME medium, 1 mM sodium pyruvate, and the and the EGF receptor gene are closely related or are identi- nonessential amino acids of minimal Eagle's medium at 0.1 cal. mM each. Human leukemic lymphocytes (CEM) were main- We have used a fragment of the v-erbB gene to isolate tained in Eagle's minimal spinner medium (HEM Research, cDNA clones structurally related to the EGF receptor gene Rockville, MD). (18). One 2.4-kilobase pair (kbp) cDNA clone (pE7) encodes Toxicity Assay. A conjugate of EGF and Pseudomonas three of the peptides sequenced by Downward et al. (14) and exotoxin (PE) was constructed by the disulfide exchange re- is highly homologous to a large portion of the v-erbB onco- action previously described (23). The conjugate has 2 mol of gene. It seems likely that clone pE7 encodes a portion of the EGF per mol of PE (Mr 66,000). Therefore the EGF concen- EGF receptor because such clones were easily isolated from tration in EGF-PE at 1 ,g/ml is 0.2 ug/ml. a cDNA library made from A431 epidermoid carcinoma For toxicity assays, various cell lines were plated in 24- cells, which have an unusually large number of EGF recep- well Costar dishes at 5 x 104 cells per well. The following tors (19, 20), and could not be detected in a cDNA library day 1:10 serial dilutions of EGF-PE were added to the wells from WI38 human fibroblasts, which have a relatively low at concentrations ranging from 100 to 0.01 ng/ml. The cells number of EGF receptors (unpublished data). Clone pE7 hy- were incubated with the toxin conjugate at 37°C for 48 hr. bridizes to three major A431 RNA species, of 10, 5.6, and 2.9 The dishes were stained with 0.5% methylene blue in etha- The publication costs of this article were defrayed in part by page charge Abbreviations: EGF, epidermal growth factor; anti-EGFR, antibody payment. This article must therefore be hereby marked "advertisement" to EGF receptor; kbp, kilobase pair; kb, kilobase(s); PE, Pseudo- in accordance with 18 U.S.C. §1734 solely to indicate this fact. monas exotoxin. 7308 Downloaded by guest on September 27, 2021 Biochemistry: Xu et al. Proc. NatL Acad. Sci. USA 81 (1984) 7309 nol/phosphate-buffered saline (Pi/NaCl) (1:1, vol/vol) to as- 30 min and adsorbed with 0.2 ml of Formalin-fixed Staphylo- sess cell killing. coccus aureus (10%, wt/vol) at 40C for 15 min prior to immu- Isolation of RNA and RNA Blotting. For RNA isolation noprecipitation. The protein concentration of the cell lysates cells were plated so that they were just confluent 24 hr later. was 0.5-1.0 mg/ml. Total RNA was isolated from various cell lines by guanidine To ensure that the precipitation was linear with extract isothiocyanate solubilization and centrifugation over a CsCl concentration, three aliquots of each cell lysate (50, 100, and cushion (24). Poly(A)+ RNA, purified by passage over oli- 250 ,1) were immunoprecipitated with 20 ,.g of rabbit antise- go(dT)-cellulose, was fractionated on 1% agarose/formalde- rum and 50 A.l of S. aureus, using the procedure previously hyde gels (5 ,ug per well) and transferred to nitrocellulose described (33). The immunoprecipitates were analyzed by (25). The resulting filter-bound RNA was prehybridized and NaDodSO4/PAGE, using 5-15% linear gradient gels and the hybridized (18, 25) for 36 hr with nick-translated (26) 32p_ Laemmli buffer system (34). After fluorography (35) and labeled cDNA inserts. Washing included 1 hr in 30 mM autoradiography, the gels were quantitated by excising the NaCl/3 mM sodium citrate/0.1% NaDodSO4 at 430C. Filters EGF receptor protein band immunoprecipitated from each were subjected to autoradiographic analysis, which was cell extract. The gel slices were extracted with 2 ml of 30% quantitated by microdensitometry. (wt/vol) H202/1% NH40H in scintillation vials at 390C for Isolation of DNA and Southern Blotting. High molecular 24 hr, then 20 ml of Aquasol (New England Nuclear) was weight genomic DNA was isolated by using NaDodSO4/pro- added for counting. Background radioactivity was deter- teinase K lysis, organic extraction, and extensive dialysis mined for each lane by excising another region of the gel of (27, 28). Routinely, 15 pug of genomic DNA was digested comparable size. with EcoRI, electrophoresed on 1% agarose, transferred to [355]Methionine incorporation into total cell protein was nitrocellulose (27, 29), and probed with 32P-labeled pE7 determined by precipitating duplicated aliquots of each cell cDNA insert. Prehybridization and hybridization were as de- extract with 1 ml of 20% (wt/vol) trichloroacetic acid, using scribed (27-29). To reduce probe-related nonspecific back- 200 pg ovalbumin as a carrier. Protein concentrations were ground, the pE7 probe was first exposed to a blank nitrocel- determined by the Bradford method (36), using bovine gam- lulose filter in complete hybridization buffer containing 10% ma globulin as a standard. dextran sulfate for 2-3 hr before hybridization to the DNA filter.
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