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ICANCER RESEARCH 49. 1130-1137, March 1. 1989] Epidermal Growth Factor Receptor Protein-Tyrosine Kinase Activity in Human Cell Lines Established from Squamous Carcinomas of the Head and Neck1 Steve A. Maxwell, Peter G. Sacks, Jordan U. Gutterman, and Gary E. Gallick2 Departments of Tumor Biology [S. A, M., P. G. S., G. E. G.], Clinical Immunology [S. A. M., J. U. G.], and Head and Neck Surgery [P. G. S.J, The University of Texas M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77030 ABSTRACT Two cell lines established from tumors of the head and neck area at different clinical stages were found to differ in the expression and in the tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Cell line 183A was derived from an early-stage tumor and cell line 1483 was derived from a tumor that had metastasized to lymph nodes. The 1483 cells displayed a higher plating efficiency and clonogenicity in soft agar, suggesting a more tumorigenic phenotype over the 183A cells. Analyses of EGF receptor levels by using Rl anti-EGF receptor serum indicated that the 1483 cells expressed 5-fold more receptor than did the 183A cells. EGF receptors isolated from each cell line were active for kinase activity in an immune complex kinase assay, using monoclonal Rl anti-EGF receptor antibody. The autophosphorylation activity of both receptors was stimulated by addition of EGF to isolated membrane preparations and intact cells, although the EGF receptor of the 1483 cells was much less responsive to EGF than the receptor from 183A cells. In addition, the 1483 receptor consistently incorporated about twice as much phosphate as did the 183A receptor in an immune complex kinase assay. These data suggest that the basal tyrosine kinase activity of the EGF receptor from 1483 cells may be more active than the EGF receptor kinase from 183 cells. INTRODUCTION Aberrant expression of the epidermal growth factor receptor may be involved in the genesis and progression of several malignant diseases. A role for elevated expression of the EGF3 receptor in tumorigenesis is exemplified by consistent obser vations of augmented levels of the EGF receptor in several types of malignancies. For example, amplification of the glycosylated M, 170,000 EGF receptor has been found in primary brain tumors of glial origin (1) as well as in the epidermoid carcinoma cell line designated A431 (2). High numbers of EGF receptors also occur in several types of malignancies including bladder tumors (3), breast carcinomas (4, 5), and squamous cell carcinoma cells derived from human head and neck cancers (6, 7). Many studies have demonstrated that the receptors for EGF (8-11), platelet growth factor (12), insulin (13), and insulinlike growth factor 1(14) have an intrinsic kinase activity specific for tyrosine residues. Upon binding their respective ligands, the tyrosine kinase activity becomes stimulated severalfold as in dicated by enhanced autophosphorylation of the receptor, in- creased phosphorylation of exogenous substrates in vitro, and elevated phosphorylation at the tyrosine residues of several proteins in vivo. Constitutive activation of the tyrosine kinase of the EGF receptor may play a role in AEV-mediated trans formation (15-18). The \-erbB oncogene product encoded by the AEV appears to represent a truncated EGF receptor con taining the cytoplasmic kinase domain but lacking any sequence corresponding to the extracellular ligand-binding domain (2, 16). Furthermore, the \-erbB protein has undergone a C-terminal modification in which 32 amino acids of the EGF recep tor protein are replaced by 4 amino acids encoded by the retroviral genome and as such lacks the major autophospho rylation site of the native receptor (Tyr1173)(2). Thus, the verbB protein represents a truncated EGF receptor which may be constitutively activated for tyrosine kinase activity (19, 20). These examples of aberrant EGF receptor expression and kinase activity in several malignant diseases and in AEV trans formation suggest that defective control of the EGF receptormediated mitogenic signal may be involved in common malig nancies of brain and head and neck areas. Although expression of the EGF receptor has been examined in many squamous carcinoma samples, few or no studies have been made on the tyrosine kinase activity of the EGF receptor. To investigate the biochemical activity of the EGF receptor in malignant lesions of squamous epidermoid origin, we used cell lines established from squamous carcinomas of the head and neck (21). One cell line, designated 183A, was isolated from a poorly differentiated squamous carcinoma of the tonsil. Another cell line was derived from a well-differentiated squamous carcinoma of the retromolar trigone and was designated 1483. The higher plating efficiency and clonogenicity in soft agar of the 1483 cell line indicated that it was more tumorigenic than the 183A cell line. Our characterization of EGF receptor expression in these squa mous carcinoma cells has yielded important differences between the two cell lines, not only in the expression but also in the tyrosine protein kinase activity of the EGF receptor protein. MATERIALS AND METHODS Cell Lines Cell lines were established from tumor specimens obtained immedi ately after surgery as previously described (21). Cell lines 183A and 1483 were recently established from squamous cell carcinomas of the The costs of publication of this article were defrayed in part by the payment head and neck. Line 183A was derived from a 54-year-old male patient of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. diagnosed as having a TjN0Mo tumor (greatest diameter of the primary ' This work was supported in part by the Clayton Foundation for Research to tumor was greater than 4 cm; no positive lymph nodes, no distant J. U. G., a Senior Clayton Foundation Investigator. This work was also aided by mÃ©tastases)that was histologically and pathologically described as a Grant RR5511-23 to S. A. M. and Grant CA39803 to G. E. G. from the NIH. 2 To whom reprints should be addressed, at Department of Tumor Biology, poorly differentiated squamous cell carcinoma of the tonsil. Line 1483 Box 79, M. D. Anderson Hospital and Tumor Institute, 1515 Holcombe Boule was derived from a 66-year-old male diagnosed as having a T2N|M0 vard, Houston, TX 77030. tumor (greatest diameter of primary tumor was 2-4 cm; single positive * The abbreviations used are: EGF, epidermal growth factor; SDS, sodium lymph node, no distant mÃ©tastases).The lesion was identified as a welldodecyl sulfate; AEV, avian erythroblastosis; RIPA A, cell lysis buffer containing 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 150 min NaCl, 5 mM differentiated squamous cell carcinoma of the retromolar trigone. A431 EDTA. 1% aprotinin, 5 mM phenylmethylsulfonyl fluoride, 10 fig/ml leupeptin, cells, an established cell line from a human epidermoid carcinoma 1 mM sodium vanadate, 5 mM sodium pyrophosphate, 20 mM sodium phosphate, which has been shown to overexpress the EGF receptor (2), were used pH 7.4; RIPA B buffer, same as RIPA A but lacking SDS. sodium deoxycholate, as control for identifying the EGF receptor in the 1483 and 183A cell sodium pyrophosphate, and sodium vanadate; HEPES, 4-(2-hydroxyethyl)-llines. piperazineethanesulfonic acid. 1130 Received5/16/88;revised11/1/88;accepted11/22/88. Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1989 American Association for Cancer Research. EGF RECEPTOR-TYROSINE KINASE ACTIVITY IN CARCINOMA CELL LINES Metabolic Labeling and Immunoprecipitation For metabolic labeling, cells at approximately 70-80% confluency were incubated overnight (16 h) in media lacking either methionine or phosphate and were then subsequently supplemented with 0.5-1 mCi [35S]methionine (1 nmol) (New England Nuclear and ICN) or 0.5 mCi [32P]orthophosphate (New England Nuclear) per ml, respectively, and 2% dialyzed fetal calf serum. After incubation with radioactive medium, cell monolayers were rinsed twice on ice with cold Ca2+-, Mg2*-free phosphate-buffered saline. Five to 7 ml of RIPA "A" lysis buffer were added to 32PÂ¡-labeled cells and 4 to 5 ml of RIPA "B" buffer were added to [35S]methionine-labeled cell monolayers. Cells were then scraped into the RIPA buffer and lysed in a loose-fitting Wheaton homogenizer with 20 strokes. The lysate was incubated on ice for 5-10 min and clarified by centrifuging at 10,000 x g for 10 min. Clarified lysates from each cell line were adjusted to equivalent protein (1-3 mg/ml) by the BCA protein assay method (Pierce Chemical Co., Rockford, IL), using bovine serum albumin as a standard. Immunoprecipitation of EGF receptor was accomplished with monoclonal antibody Rl (100 Mg/ml) purchased from Amersham Biologicals (Arlington Heights, IL). The supernatants were incubated for l h on ice with 5 M' of Rl antiserum/ml extract. The immune complexes were immunoprecipitated with a 15-min incubation on ice with 50 ii\ pansorbin (10% w/v solution) for each ml of 32P-labeled extract and with 50 p\ of protein A-Sepharose 4B (Sigma Chemical Co., St. Louis, MO; 4 mg/ml) for each ml of 35S lysate. The immune precipitates were washed two or three times with RIPA A, were drained, and were suspended in Laemmli sample buffer for electrophoresis on a SDS polyacrylamide gel (22). Autoradiography was used to visualize 32P-labeled proteins and fluorography was used to visualize 35S-labeled proteins. Preparation of Membranes Membranes were harvested by a modification of the procedure of Resh and Erikson (23). Cells were washed twice with cold Ca2*-, Mg2+free phosphate-buffered saline solution and disrupted in 2 ml of hypotonic HEPES buffer (0.2 HIMMgCh, 20 mM HEPES, pH 7.0) with 50 strokes in a tight-fitting Wheaton Dounce homogenizer. After the homogenates were incubated for 10 min on ice, EDTA was added to a concentration of 5 mM. After further Dounce homogenization, the lysates were centrifuged 10 min at 1,000 x g. Aprotinin and leupeptin were then immediately added to the supernatants to final concentrations of 2% and 20 Mg/ml, respectively. The pellet was reextracted with hypotonie HEPES buffer and the second clarified lysate was combined with the first extract. The extract was centrifuged at 100,000 x g for 60 min and the pelleted crude membranes resuspended in 20 mM HEPES, pH 7.0. The membranes were washed once in 20 mM HEPES, pH 7.0, by centrifugation at 100,000 x g for 60 min and the pellet was gently suspended in 20 mM HEPES, pH 7.O. The crude membrane preparation was used either immediately for the kinase assay or stored at -70-C. Kinase Assays Immune Complex Kinase Assay. Immune complexes for kinase assays were prepared as described for immunoprecipitation by lysing 35Slabeled cells in RIPA B. The protein A-Sepharose-precipitated immune complexes were washed twice with 0.1% Triton X-100 and 150 mM NaCl in 10 mM sodium phosphate, pH 7.4. The washed immune precipitates were then resuspended in 0.1% Triton X-100 in 20 mM HEPES, pH 7.0, and divided into S-M!aliquots. The aliquots were adjusted to 100 /IM sodium vanadate and the kinase reaction was initiated by addition of 50 n\ of 20 mM HEPES containing 5 MCi [32P]ATP and 6 mM MnCl2. The reaction was allowed to proceed at 30Â°Cfor 10 min and was terminated by addition of 1 ml of cold RIPA A. The complexes were then washed twice with RIPA A and the phosphorylated proteins were prepared for electrophoresis according to the procedure of Laemmli (22). Membrane Kinase Assay. Kinase assays of membrane preparations were performed by adjusting 30-50 Mgof membrane protein to 50 M' in 20 mM HEPES, pH 7.0, and adding 50 Â¡A of a solution containing 20 MCi[32P]ATP (0.07 MMfinal concentration) and 6 mM MnCh in 20 mM HEPES, pH 7.0. Kinase assays were performed for 10 min on ice and were terminated by addition of 1 ml of RIPA A. The mixture was then disrupted in a tight-fitting Wheaton Dounce homogenizer with 20 strokes and clarified at 100,000 x g for 60 min. The supernatant was incubated with 5 M'of Rl monoclonal anti-EGF receptor antibody for I h and the immune complexes were collected and prepared as described for the immunoprecipitation procedure. Analysis of Enzyme Activity For analysis of enzyme activity, cells were incubated for 16 h in methiomne-free modified Eagle's medium containing 0.5-1 mCi/ml [35S]methionine and 2% dialyzed fetal calf serum. Cell lysates were prepared according to immune complex kinase assay. Before antiserum was added to the lysates, 1 ml of the clarified cell extract was subjected to protein precipitation with 20% trichloroacetic acid. The trichloroacetic acid-precipitated protein pellet was resuspended in 100 /il of RIPA A and analyzed for specific activity by monitoring 35Scounts/min/Mg protein. Each cell lysate from 183A and 1483 cells was normalized for equivalent protein (2-3 mg/ml). Immune complexes were harvested from cell lysates as described for the immune complex kinase assay, and each precipitate was suspended in 0.1% Triton X-100 and 20 mM HEPES, pH 7.0. The suspension was separated into two equal sets of 200 fi\. One set was further divided into 50-Ml aliquots, washed three times in RIPA A, and suspended in sample buffer. The other set was subjected in SU-M!portions to the kinase assay in the presence of various concentrations of ATP (0.23 MMat 3000 MCi/nmol or 6 MMATP at 90-100 MCi/nmol) and angiotensin II (0.3-3 mM). After incubation at 30Â°C,the assays were immediately terminated by immersion in an ice bath followed by pelleting of the Sepharose beads in a microfuge at 4Â°C.The supernatants were aspirated and "quick-frozen" in an isopropyl alcohol/dry ice bath. The frozen supernatants were then lyophilized, resuspended in pH 3.5 pyridine acetate buffer, and spotted on thinlayer cellulose acetate plates. Phosphorylated angiotensin was separated from labeled ATP by electrophoresis at constant voltage (400 V) for 2 h. The protein A-Sepharose pellets from the kinase assays were washed with RIPA A and the 32P-labeled proteins were subjected to SDS polyacrylamide gel electrophoresis and autoradiography. The 35S-labeled proteins were visualized on a separate gel by fluorography. The [35S]methionine label was not detectable in the autoradiographic pro cedure in the time used to expose the 32P-labeIed proteins on Kodak XAR-5 film. Autophosphorylation and kinetic analyses of angiotensin II phosphorylation determinations are expressed as the average of values obtained from at least two independent experiments. Quantitation of |35S|Methionine- and 32P-labeled Proteins The 35S-labeled proteins were eluted from the gel slice by incubation overnight at 37Â°Cin 0.5 ml of NCS tissue solubilizer (Amersham) solution. Five ml of Atomlight fluor (New England Nuclear) were then added and the eluate was analyzed for cpm along with a parallel blank. Given the specific activity of cellular protein in the initial cell extract, an estimate of the amount of EGF receptor could be made in the immune complex kinase assay. For analyses of 32P-labeled proteins, gel slices were incubated for 2 h at 80Â°Cin 1 ml of 6 N HC1. Five ml of water were then added and the eluates were monitored for cpm along with a parallel blank. Under these conditions, less than 0.004% of the 35S radioactivity could be detected relative to cpm obtained under conditions using Atomlight fluor. Thus, the cpm from 35Srepresented an insignificant contamination under the conditions used to monitor 32P. A known amount of [32P]ATP was analyzed under conditions identical to those used for eluting 32P-labeled proteins, which allowed a molar determination of incorporated phosphate to be made. Quantitation of Phosphorylated Angiotensin II The phosphorylated angiotensin II peptide was resolved from labeled ATP by electrophoresis on a thin-layer cellulose plate in a pH 3.5 pyridine acetate buffer (pyridine:acetic acid:water; 5:50:945) at constant voltage (400 V) for 2 h. Area corresponding to phosphorylated angio tensin II was localized by autoradiography and eluted by incubating overnight in 5 ml Atomlight fluor at 37Â°C.Moles of 32P incorporation 1131 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1989 American Association for Cancer Research. EGF RECEPTOR-TYROSINE KINASE ACTIVITY IN CARCINOMA CELL LINES B 3 Fig. 1. Analysis of EGF receptor protein and kinase activity of 183A and 1483 cell lines. Protein levels were examined by metabolic labeling with [35S]methionine (I), and kinase activity was analyzed by immune complex ki nase assays (A). Lane I, control serum, 183A; Lane 2, RI anti-EGF receptor serum, 183A; Lane 3, control serum, 1483; Lane 4, antiEGF receptor, 1483. "S- and 32P-labeled pro teins were fractionated on individual 8% SDS polyacrylamide gels. The "S-labeled proteins were treated with En'hance (NEN) and local ized by fluorography with a 20-h exposure on Kodak XAR-5 film. The 33P-labeled proteins were visualized by autoradiography for 4 h by using Kodak XAR-S film and a DuPont en hancing screen. The 3!S was not detected in the 4 li autoradiograph procedure on Kodak XAR-S film and DuPont enhancing screens. Ordinate, molecular weight in thousands. 4 200- P170- P170- 92.5- 68- 43- 18.4- were determined by monitoring cpm relative to a known standard of the original [32P]ATP. Phosphoamino Acid Analyses of 32P-labeled Proteins Phosphoamino acid analysis was performed according to the method of Hunter and Sefton (24). 32P-labeled EGF receptor was electroeluted from the polyacrylamide gel fragments and precipitated with trichloroacetic acid. 32P-labeled angiotensin II was eluted in pH 3.5 pyridine acetate (pyridine:acetic acid:water; 5:50:945) and lyophilized to a pow der. The peptide or protein residue was suspended in 6 N HCl, sealed in glass ampules, and heated in mineral oil at 1IOC for 2 h. After the hydrolysis, the HCl was removed under vacuum and the residue was suspended in pH 3.5 pyridine acetate containing 1 mg/ml each of phosphoserine, phosphothreonine, and phosphotyrosine markers. Phosphoamino acids were resolved by thin-layer electrophoresis at constant voltage (400 V) for 2 h. Phosphoamino acid markers were visualized by staining with ninhydrin and 32P-labeled amino acids were visualized by autoradiography. RESULTS Properties of Head and Neck Squamous Carcinoma Cell Lines. The cell lines designated 183A and 1483 exhibit epithelial morphology and grow in a cobblestone-like pattern (21). When injected s.c. into nude mice, both lines were tumorigenic (IO7 cells/mouse) and generated tumors in all tested mice. Both cell lines have doubling times of approximately 36 h. The higher plating efficiency and clonogenicity in soft agar of the 1483 cell line indicates that it exhibits a more tumorigenic phenotype than does the 183A line. EGF at 10 ng/ml was found to stimulate the growth of the 183A cells whereas the growth of the 1483 cells was inhibited.4 Analysis of Steady-State Levels of EGF Receptor in Cell Lines. Expression of the EGF receptor was examined by metabolically labeling cells to steady state with [35S]methionine, followed by 1 Unpublished data. immunoprecipitation with the Rl anti-EGF receptor antibody. The R l antibodies react with the extracellular domain of the EGF receptor but do not interfere with EGF binding to the ligand-binding domain (25). The EGF receptor was identified based on a relative mobility of 170,000 (Fig. \A, Lanes 2 and 4), on comigration with the EGF receptor from A431 cells (Fig. 3, I and H). and on a specific reactivity with Rl antibodies (compare Fig. \A, Lanes 1 and 3 with Lanes 2 and 4). The results of a 16-h [35S]methionine incorporation revealed that 1483 has a 5-fold elevated level of the EGF receptor (Fig. \A, Lane 4) over that of 183A (Fig. IA, Lane 2) as analyzed by the quantitation technique described in "Materials and Methods." Quantitation on a molar basis yielded 0.144 pmol EGF receptor for the 183A immunoprecipitate and 0.719 pmol for that of 1483. Two additional experiments yielded results similar to those illustrated in Fig. \A. These results are in agreement with the levels of EGF receptor previously determined by Western immunoblotting for both 183A and 1483 cells (21). Kinase Activity of EGF Receptor in Cell Lines. The kinase activity of the EGF receptor from each cell line was examined, since the 183A and 1483 cells differed in their growth response to EGF. Rl immune complexes containing the EGF receptor from either the 183A or 1483 cells were tested for kinase activity. Fig. IB, Lanes 2 and 4, show that both cell lines produce an enzymatically active EGF receptor. Phosphoamino acid analyses of each phosphorylated EGF receptor yielded exclusively phosphotyrosine (Fig. 2A, Lanes 1 and 2). Response of the EGF receptor kinase activity of each cell line to EGF stimulation was investigated by using isolated crude membrane preparations. The EGF receptor kinase from A431 cells was stimulated approximately 4-fold in response to bind ing of EGF to isolated membrane preparations (Fig. 3A, Lane 2), which is similar to other reported data (8, 9). The ability of the EGF receptors from 183A and 1483 cells to be stimulated by EGF was also examined in vitro with isolated membrane 1132 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1989 American Association for Cancer Research. EGF RECEPTOR-TYROSINE K1NASE ACTIVITY IN CARCINOMA CELL LINES B pserpthrPtyr- Fig. 2. Phosphoamino acid analyses of phosphorylated EGF receptor (A) and the peptide substrate, angiotensin II (B). A, Lane 1, 183A; Lane 2, 1483. B, Lane 1, angiotensin II phosphorylated by 183A receptor; Lane 2, an giotensin II phosphorylated by 1483 receptor. pser, phosphoserine; pthr, phosphothreonine; ptyr, phosphotyrosine. ptyr- phosphopeptide - O- O- with [32P]PÂ¡ and then treating with 100 ng EGF for 10 min, preparations. Membrane preparations of 1483 cells were di luted approximately 5-fold to obtain a receptor level which was followed by immunoprecipitation. The 1483 cell lysate was equivalent to that of the 183A preparation. Immunoblotting of again diluted 5-fold to approximate an amount of receptor membrane preparations confirmed that equivalent amounts of equivalent to that of the 183A preparation. Similar results to receptor were present in both the 1483 and 183A membrane those obtained in the membrane kinase assays were observed kinase assays (data not shown). The basal level of autophosfor EGF stimulation of each cell line, indicating that differences phorylation activity of each receptor appeared to be similar in in EGF stimulation of kinase activity was not an in vitro artifact the isolated membrane preparations (Fig. 3B, Lanes 1 and 3). (Fig. 3C). Scanning of the autoradiograph of EGF receptors and analyses of peak areas However, a significant difference was consistently observed in metabolically labeled with [32P]PÂ¡ the ability of each kinase to be stimulated by EGF. The autoindicated that EGF produced a 4.5-fold stimulation of 183A receptor phosphorylation (Fig. 3C, Lane 2) and a 1.9-fold phosphorylation of the EGF receptor from 183A was enhanced 6.6-fold upon addition of EGF to the membranes, as determined increase in 1483 receptor phosphorylation (Fig. 3C, Lane 4). by scintillation counting of radioactivity (Fig. 3B, Lane 2), Kinetic Analyses of EGF Receptor Activity. Other possible whereas only a 2.2-fold increase in EGF receptor activity in variations in kinase activity between the 183A and 1483 EGF response to EGF addition was observed in the 1483 membrane receptors were investigated by using the neuropeptide angioten sin II. Angiotensin II has been used to study EGF receptorkinase assays (Fig. 3B, Lane 4). Stimulation of EGF kinase activity upon addition of EGF to isolated membrane prepara tyrosine protein kinase activity from A431 cells (26, 27) and tions from 183 and 1483A cells was also analyzed by using the has the advantage of containing a single tyrosine residue and peptide substrate angiotensin II. The EGF receptor in mem no serine or threonine residues. branes from 183A cells exhibited a 2.3-fold increase in EGFThe specificity of EGF receptor kinase activity toward angio stimulated phosphorylation (Table 1), whereas the 1483 mem tensin II was analyzed with the receptor isolated in the Rl brane preparation displayed a 1.7-fold increase upon EGF immune complex. The phosphorylation of the angiotensin II addition (Table 1). Response of EGF receptor kinase to EGF peptide was specific only for immune complexes containing stimulation in vivo was studied by metabolically labeling cells either the 183A or the 1483 EGF receptor, as indicated by the 1133 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1989 American Association for Cancer Research. EOF RECEPTOR-TYROSINE KINASE ACTIVITY IN CARCINOMA CELL LINES B 1234 P170- 92.5â€” 68â€” 43- 18.4Fig. 3. Analyses of response of EGF receptor kinase activity in 183A and 1483 cells and their derived membrane preparations to EGF stimulation. Activity was analyzed by membrane kinase assays (A and B) and by metabolic incorporation of ["?]?, (Q. A, Lane 1, A431, untreated control; Lane 2, A431, treatment with 100 ng EGF. B, Lane 1, 183A, no EGF; Lane 2, 183A, treated with EGF; Lane 3, 1483, no EGF; Lane 4, 1483, treated with EGF. C, Lane l, 183A, no EGF; Lane 2, 183A cells treated with 100 ng/ml EGF; Lane 3, 1483, no EGF; Lane 4, 1483 cells treated with EGF. Proteins were visualized by autoradiography and exposure on Kodak XAR-5 film with DuPont enhancing screens for 3 h (A), 16 h (B), and 12 h (C). Ordinate, molecular weight in thousands. Table 1 Rate ofangiotensin II phosphorylation by 183 and 1483 membrane preparations in presence and absence of EGF Rates are expressed as fmol 1:l' incorporated into angiotensin II min ni: membrane protein. Each value represents the mean of triplicate determinations. (-)EGF (+)EGF Cell line 183A 1483 14.8 21.2 34.1 36.9 absence of detectable peptide phosphorylation in immune com plexes obtained with control serum (data not shown). As ex pected, phosphoamino acid analysis of phosphorylated angio tensin II yielded exclusively phosphotyrosine in each case (Fig. IB, Lanes 1 and 2). These data indicate that the EGF receptor in the immune complex kinase assay covalenti) modified angio tensin II with a phosphate residue at tyrosine. Phosphorylation ofangiotensin II over a reaction time period at 0.23 UM[32P]ATP and 3 mivi angiotensin II was monitored. A sigmoidal curve was generated, suggesting the occurrence of a biphasic reaction mechanism (Fig. 4A). A pause in activity was observed from 1 to 10 min for the 183A receptor kinase assay, whereafter the rate accelerated up to 15 min (Fig. 4A). A less distiguishable pause in activity was also apparent for the 1483 receptor assay. The pause in reaction may be due to a requirement of enzyme activation for autophosphorylation (26, 27) and/or ATP binding (28). To investigate this possibility further, a reaction time course was performed at a higher concentration of ATP (6 MM).If autophosphorylation and/or activation by ATP is involved, the pause phase in activity would be expected to be shortened. As observed in Fig. 5A, a reaction time course at a higher ATP concentration (6 MM)still yielded a sigmoidal curve but with a much shorter pause lasting ap proximately 6 min. For the 183A receptor kinase, the rate of reaction (slope of the curve) after the reaction pause greatly increased 127-fold from 0.003 pmol/min at 0.23 MMATP to 0.38 pmol/min at 6 MMATP, whereas the reaction rate for the 1483 receptor kinase accelerated 34-fold from 0.019 pmol/min to 0.64 pmol/min. These data suggested that the EGF receptors from the 183A and 1483 cell lines require either autophos phorylation or ATP binding or both for maximal activity. Furthermore, a difference in the requirement of each receptor for activation by ATP was evident from these changes of reaction rate after the pause phase of the time course. The incorporation of phosphate into the EGF receptors from 183A and 1483 cells was monitored overtime in the presence of angiotensin II at 0.23 and 6 MMATP to further examine the dependence of enzyme activity for autophosphorylation. At 0.23 MMATP, the 1483 and 183A receptors autophosphorylated at different rates and to different degrees (Fig. 4B). While the 183 receptor autophosphorylation appears to slow after 5 min, the autophosphorylation of the 1483 receptor continues 1134 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1989 American Association for Cancer Research. EOF RECEPTOR-TYROSINE KINASE ACTIVITY IN CARCINOMA CELL LINES 10 15 (mir,) B I Fig. 4. Time course of EOF receptor kinase activity at 0.23 JIM ATP. A, phosphate incorporation into angiotensin II. Concentration of peptide was 3 HIM. Points, determined from an average of two individual assays. (â€¢)183 receptor assays; (A) 1483 receptor assays, li. course of autophosphorylation in the presence Fig. 5. Time course of EGF receptor activity at 6 ^M ATP. A, phosphate of angiotensin II. Concentration of peptide in each experiment was 0.3 HIM.(â€¢) incorporation into angiotensin II. Concentration of angiotensin was 3 HIM.A. 183 receptor assays; (A) 1483 receptor assays. Points, average of determinations points, determined from an average of two independent assays. (â€¢)183 receptor assays; (A) 1483 receptor assays. li. course of autophosphorylation in presence of from two individual assays from two independent experiments. angiotensin II. Concentration of peptide in each experiment was 0.3 HIM.Points, average of determinations from two individual assays from two independent to increase up to 15 min. The data support the observation in experiments. (â€¢)183 receptor assays; (A) 1483 receptor assays. Figs. 4A and 5A that a particular degree of autophosphorylation is required for optimal activity toward the peptide substrate, since the incorporation of phosphate into the receptors at a level of approximately 0.35 and 0.65 mol phosphate/mol of receptor occurred for the 183 and 1483 receptors (Fig. 4fi), respectively, before activity for the exogenous substrate in creases (Fig. 4A). At 6 /J.M ATP, the autophosphorylation reaction proceeded much faster and to a higher degree, as would be expected (Fig. SB). Under these conditions, a higher amount of phosphate was incorporated into the 1483 receptor (0.87 mol phosphate/mol receptor) than into the 183A receptor (0.44 mol phosphate/mol receptor) after 5 min of reaction time. Although both enzymes appear to be activated for angiotensin II after about 6-7 min, the 1483 receptor incorporated almost twice as much phosphate as the 183A receptor, which also suggested that the 1483 receptor may have more activity under these conditions. Reciprocal plots of enzyme activity versus substrate concen tration confirmed a higher basal activity (Vmax)for the 1483 EGF receptor over the 183 receptor (Table 2). A higher Km value of the 1483 receptor for angiotensin suggests a difference in affinity for angiotensin as a substrate (Table 2). Table 2 Apparent Kmand y^, determinations for 183 and 1483 EGF receptors obtained at 0.23 tiM A TP Apparent Kmand Vmâ€ž data are expressed as the average of two determinations obtained from two independent experiments. â€ž¿â€ž (fmol J2P/min/ fmol EGF-R) Cell line (mM) I83A 1483 0.6 1.3 0.16 2.3 from them (6, 7) as well as in glioma (1) and breast carcinoma cells (4, 5). These findings have led to speculation that overexpression of the EGF receptor might be involved in tumorigenicity. Evidence for this possibility has been obtained by Santon et al. (29), who isolated variant A431 cells with different levels of EGF receptor and showed that higher concentrations of the EGF receptor appeared to provide the cells with a growth advantage in vitro and in vivo. In addition, A431 cells selected for decreased EGF receptor expression exhibited a greater capacity to undergo terminal differentiation (30). While in creased density of the EGF receptor appears to correlate with tumorigenicity, as yet no biochemical differences in EGF recep tor activity have been correlated with different levels of expres sion. Since the tyrosine kinase activity of the EGF receptor is DISCUSSION important for transmitting a mitogenic signal upon EGF bindThe EGF receptor has been found to be frequently elevated ing, we examined the activity of the EGF receptor in the 183A din squamous carcinoma tumor samples and cell lines derived and 1483 squamous carcinoma cell lines. We have previously 1135 Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1989 American Association for Cancer Research. EGF RECEPTOR-TYROSINE KINASE ACTIVITY IN CARCINOMA CELL LINES demonstrated that 1483 cells expressed elevated amounts of the EGF receptor as compared to the 183A cells. In this report, we observed three important differences between the kinase activities of the 1483 and 183A receptors: (a) the 1483 receptor kinase was much less responsive to EGF than that observed for 183A; (b) the 1483 receptor autophosphorylated in vitro to a greater degree than the 183A receptor; and (c) the 1483 receptor exhibited a higher activity and a lower affinity for angiotensin in Rl immune complexes. These differences in enzymic activity between the two EGF receptors may explain the different biological responses of the 183A and 1483 cells to EGF. The lower stimulation of the EGF receptor kinase of the 1483 cells in the presence of EGF as compared to the receptor of the 183A cells in membrane preparations may be the result of either a difference in topology or conformation between the two receptors in the membrane. The latter possibility is more likely, based upon the difference in degree of in vitro autophosphorylation between these two receptors in the immune com plex kinase assay. While the 1483 receptor underwent a similar amount of autophosphorylation as the 183A receptor in mem branes and intact cells, the 1483 receptor was found to undergo greater (2-fold) phosphorylation over the 183A receptor when assayed isolated in the Rl immune complex under both 0.23 and 6 MMATP. The greater autophosphorylation of the 1483 receptor in vitro is probably not the result of lower endogenous phosphorylation, since both the metabolic labeling and mem brane kinase assays suggested that a similar amount of phos phorylation occurred for both receptors in the absence of EGF. One possible interpretation of these results is that additional phosphorylation sites may be exposed on the 1483 receptor in an isolated state in vitro, whereas these sites are sequestered in vivo. Alternatively, the higher degree of autophosphorylation may reflect a greater activity of the 1483 receptor kinase as compared to the 183 receptor. The EGF receptors from 183A and 1483 cells appeared to be dependent on autophosphorylation for optimal activity for angiotensin II. A similar effect of autophosphorylation on the A431 EGF receptor kinase activity has also been observed in that preincubation of the receptor with saturating ATP concen tration prior to the kinase assay resulted in an enhancement of enzyme activity (26, 27). It was proposed that autophospho rylation activated the receptor by removing an inhibitory con straint so that exogenous substrates can have greater access to the enzyme-active site. The higher degree and faster rate of autophosphorylation of the 1483 receptor may allow for a greater activity toward the peptide substrate by increasing ac cessibility for the active site and by increasing the affinity for ATP. Thus, this higher degree of autophosphorylation observed in vitro may reflect a partial activation of the 1483 receptor. Furthermore, a higher activity of the 1483 receptor over the 183 receptor also appears evident from the greater incorpora tion of 32P into angiotensin II, even though approximately with receptor oligomers. The monomeric receptors possess low ligand and reduced kinase activity, whereas the oligomeric receptors have high EGF-binding activity and activated kinase activity. Several recent studies have supported this model (32, 33). Subsequent EGF receptor clustering appears to be a nec essary and sufficient signal for mitogenesis induced by EGF (34). In light of these observations, the higher concentration of EGF receptor in the 1483 cell membranes may favor or drive the aggregation process resulting in enhanced kinase activation when compared to receptor activity in 183A cells. The changes in ligand binding after aggregation predicted by this model are very similar to those we have observed in 1483 cells relative to 183 cells. Thus, the clustering of EGF receptor in squamous carcinoma cells may result in partial activation of the tyrosine kinase activity. Analysis of EGF receptor kinase activity in membranes of variant 1483 cells expressing different levels of the EGF receptor should provide more insight into any involve ment of the density of the EGF receptor in increased tumorigenicity. Furthermore, analyses of additional cell lines derived from squamous carcinomas of the head and neck, as well as primary tumor samples, will be necessary before differences in EGF receptor kinase activity observed here can be conclusively correlated with the biological phenotype of the tumor. REFERENCES equivalent amounts of 183 and 1483 receptors were present in the assay. Any intrinsic mechanism which might account for the differ ences in activity between the two kinases must be subtle since no apparent difference in mobility on SDS gel electrophoresis was observed when the proteins were directly isolated from the cells. 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