`
`Epidermal Growth Factor Receptor Expression in Human Lung Cancer Cell Lines‘
`
`Maria l-laeder,’ Martin Rotsch, Gerold Bepler, Cordula Hennig, Klaus Havemann, Barbara Heimann, and
`Karin Moelling
`Philipps-University Marburx, Department ofInternal Medicine, Division ofHematology/Oncology/Immunology, Baldingerstrasse. 3550 Marbmg [M. II., M. R., G. B.,
`C. 11., K. H.I, and Max-PIanck~In.rtitutji.tr Molekulare Generik, llinestrarre 73, I000 Berlin 33 (B. h'., K. M./, Federal Republic ofGermany
`
`ABSTRACT
`
`lung cancer
`lung cancer (NSCLC) and small cell
`Non-small cell
`(SCLC) cell lines were studied for epidermal growth factor (EGF) recep-
`tor expression. All NSCLC cell lines tested (eight of eight) had specific
`EGF binding sites, whereas only five of ll SCLC cell lines bound EGF.
`NSCLC and SCLC cell lines expressed the same type of high alfinity
`EGF binding sites with a K. of 0.5 to 4.5 nM; however, NSCLC cells
`bound significantly more EGF than SCLC cell lines. The amount of
`binding sites in NSCLC cells ranged between 71 and 1,000 fmol/mg of
`protein and in SCLC cells, between 26 and 143 fmol/mg of protein. The
`two SCLC cell lines with EGF binding values within the range of NSCLC
`belonged to the variant subtype of S-C113. By means of an anti-erbB
`serum and indirect radioimmunoprecipitation, a strong M, ~l70,000
`protein band could be detected in the NSCLC cell lines. This protein
`corresponds to the EGF receptor molecule. its identity was proven by
`competition with excess erbll antigen for the antibody during the radioim-
`muuoprecipitation. Furthermore, this M, 170,000 protein exhibited pro-
`tein lcinase activity as evidenced by in rirro autophosphorylation. The
`radioactivity incorporated into the M, 170,000 band in radioimmunopre
`cipitation and protein kinase assays was [0 to 100 times lower in these
`SCLC cell lines which were positive in the EGF binding assay compared
`to the NSCDC cell lines. We conclude that NSCLC in contrast to SCLC
`expresses high levels of EGF receptors which may be used to facilitate
`the differential diagnosis in some cases of lung cancer. These data suggest
`that EGF may play a role in growth and differentiation of NSCLC.
`
`INTRODUCTION
`
`Growth factor research and oncogene research are closely
`linked based on the hypothesis of autocrine secretion;
`i.e..,
`cancer cells can produce and respond to their own growth
`factors (I). The sequence homology between the v-erbB onco-
`gene product and the cytoplasmic and membrane part of the
`EGF3 receptor hints toward a relation between oncogenic stim-
`ulation of a cell and normal growth-regulatory mechanisms (2).
`The EGF receptor is a glycoprotein with a molecular weight of
`170,000 to 380,000 with an intrinsic tyrosine-specific protein
`kinase, which is stimulated upon EGF binding. The extracel-
`lular EGF binding domain of 621 amino acids is separated
`from the intracellular protein kinase domain of 542 amino
`acids by a short hydrophobic transmembrane part of 26 amino
`acids (3).
`Since the EGF receptor may play a central role in cancer
`growth control, we examined a series of human SCLC and
`NSCLC cell lines for the presence of EGF receptors using two
`approaches: indirect by determining EGF binding sites and
`
`Received 8/l2/86; revised l/20/87; 10/23/87; accepted ll /23/87.
`The costs of publication of this article were defrayed in part by the payment
`of page charges. This article must therefore be hereby marked advertisement in
`accordance with I8 U.S.C. Section 1734 solely to indicate this fact.
`‘ This work was supported by funds from the SF}! 2l5 of the German Research
`Society, by the Deutsche Krehshilfe e.V. (K. M.). and the Stiftung Unterberg (B.
`H.).
`‘ To whom requests for reprints should be addressed, at Klinikum der l’hilipps-
`Universitaet Marhurg. Zentrum fuer lnnere Medizin. Abteilung Haematologiel
`Onltologie/immunologic, Baldingerstrasse, D-3550 Marburg/Lalm, Federal Re-
`public of Germany.
`’ The abbreviations used are: EGF, epidermal growth factor, SCLC. small cell
`lung cancer: NSCLC, non-small cell lung cancer; l.—DOPA, 3.4—dihydroxy~L~
`phenylalanine; BSS, balanced salt solution; TCA, triehloroacetic acid; D'lT,
`dithiothreitol; PK, protein ltinase: RIP, radioimmunoprecipitation.
`
`direct by identifying the protein in metabolically labeled cells
`and by using autophosphorylation reaction. Concerning
`NSCLC, squamous cell carcinoma, adenocarcinoma, large cell
`carcinoma, and mesothelioma were studied. Here we provide
`data on the number and kinetics of membrane EGF binding
`sites and evidence for their nature as membrane receptors.
`
`MATERIALS AND METHODS
`
`Cell Lines
`
`The cell lines used in this study were the SCLC cell lines SCLC-
`16}lV, SCLC-21H, SCLC-22!-l, SCLC-24!-l, NCLH69, NCI-H82,
`NCI-H146, NC]-N417, NCI-H526. NC!-N592, and DMS-79, and the
`NSCLC cell lines U~l8l(l and LCLC—l03H (large cell carcinoma),
`EPLC~32Ml, EPLC-32M5, EPLC-65H, EPLC-65M2, and U-1752
`(squamous cell carcinoma), A549 and NCl~ll23 (adenocarcinoma), and
`MSTO-21 ill (mesothelioma). Details concerning the characteristics of
`these cell lines have been described elsewhere (4-10). In brief, cell lines
`SCLC-22H, SCLC—24l-l, NCl-H69, NCLHI46, and NCLN592 had
`detectable activities of L—DOPA decarboxylase and were classified as
`classic SCLC cell lines (8). All other cell lines of SCLC origin had
`absent or very low activities, a characteristic feature of the variant
`SCLC subtype (8). Neuron—specific enolase and creatine kinase 83
`levels were high in the listed SCLC cell lines and low in NSCLC cell
`lines. Cell line A431, a human vulva carcinoma cell line, and cell line
`5637, a human bladder carcinoma cell line, were used as reference cell
`lines of nonlung origin (4, ll). Cell lines of small cell origin grew as
`floating cell aggregates, and all other cell lines were substrate adherent.
`They were kept in RPMI i640 medium (No. 0414876; Gibco, Paisly,
`United Kingdom) supplemented with l0% fetal bovine serum (No. 01 l-
`6290; Gibco) in a well-humidified atmosphere of 5% CO; at 37‘C and
`were free of Mycoplasma contamination. For receptor binding studies,
`adherent growing cells were seeded in 60-mm Falcon plastic Petri
`dishes (No. 3006 optical; Falcon, Oxnard, CA) and used during loga-
`rithmic growth phase just before they reached confluency. Floating cell
`lines were kept in 75-cm’ culture flasks (No. 6581 70; Greiner, Nottin-
`gen, West Germany) and used during logarithmic growth phase 48 h
`after medium change.
`
`Reagents
`
`EGF (from mouse subrnaxillary glands) was purchased from Sigma,
`Deisenhofen, West Germany (No. E7755) and "51-EGF from Amer-
`sham lnternational, Buckinghamshire, United Kingdom (Code IM.
`124). EGF was labeled by chloramine-T—mediated iodination of mouse
`EGF and purified by gel chromatography. The specific activity was
`approximately 100 uCi/ug of total EGF. The protein assay (Coomassie
`Brillant Blue G-250) was obtained from Bio-Rad Laboratories,
`Munchen, West Germany (No. 500-0006). Antisera used in the indirect
`immunoprecipitation assay and protein kinase assay were described
`elsewhere (12).
`
`Protein Assay
`
`Cells were washed twice with 4-ml portions of Hanks’ BSS (No. 041-
`4175; Gibco). To precipitate the proteins, 2 ml of 10% TCA in double-
`distilled water were added to the NSCLC monolayer or to the centri-
`fuged SCLC cell lines (150 x g, 5 min). After 30 min at 4'C, the TCA
`solution was decanted. The proteins were dissolved in 1 ml of 0.2 N
`Naoll at room temperature. The protein content was measured with
`the Bio—Rad standard protein assay. There was no interfering effect
`H32
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`APOTEX EX. 1025-001
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`
`EGF RECEPTOR EXPRESSION iN HUMAN LUNG CANCER CELL LINES
`
`with NaOH up to 25 ug of protein/sample (corresponding to 100 to
`500 pg of protein/ml).
`
`Indirect lmmunoprecipitntion Assay (RIP)
`
`GF/B filters (Whatman, Maidstone, United Kingdom). The filters were
`dried and transferred into counting vessels, and the radioactivity was
`measured. Specific and nonspecific binding was determined as described
`for NSCLC.
`
`On a 9—cm Petri dish, 2 to 4 x 10'’ cells of each cell line were grown
`in logarithmic phase to about 50% conlluency. Cells were washed to
`remove medium and serum and incubated for 90 min with RPM! 1640
`medium free of unlabeled methionine supplemented with dialyzed
`serum and [”S}rnethionine (500 “Ci/ml). Cells were lysed immediately
`in lysis buffer (50 mM Tris-HCI (pH 7.4):l50 mM NaCl:1% Triton X-
`l00:l0% glycerolzl mM D'l'T:l0 mM NaF:l00 units/ml of trasylol:l
`mM phenylmethylsulfonyl fluoride) and treated with antiserum and
`Protein A for indirect immunoprecipitation. The antiserum against
`bacterially expressed erbB was prepared in rabbits as described (12).
`The precipitates were analyzed on i096 sodium dodecyl sulfate-poly-
`acrylamide gels, dried, and expressed for autoradiography (exposure,
`48 h). The specificity of the reaction was tested by competition with
`the antigen, i.e., by absorbing the antibodies with excess of the bacte-
`rially expressed erbB which had served as the antigen. After preincu-
`bation (10 pl of serum:20 pg of protein, 30 min, 4'C), the complex was
`applied to the lysate, and the immunoprecipitation procedure was
`perfonned as described above. Details have been published elsewhere
`(1 2).
`
`PK Assay
`
`lmmunopreeipitates were prepared from H)’ cells as described for
`the indirect immunoprecipitation procedure except that cells were not
`labeled with the isotope. The immunoprecipitates were washed 4 times
`with lysis buffer as described above except that Triton X-100 was 0.1%.
`The final wash was performed in a kinase buffer containing 50 mM
`Tris-HCl (pH 7.5), 50 mM NaCl, 0.l% Triton X-100, 10% glycol, l
`mM NaF, and l mM DTT. The protein ltinase reactions were performed
`in l00-pl total volume with 10 mM MnCl¢ added and with 20 pCi of
`[7-”l’]A'l”P (3000 Ci/mmol; Amersham, United Kingdom) without
`unlabeled ATP. incubation was performed for 10 min at 0'C and
`terminated by the addition of sodium dodecyl sulfate-containing gel
`electrophoresis buffer. The reaction products were directly applied to
`sodium dodecyl sulfate-polyacrylamide gels and processed for amora-
`diography (exposure, 30 min).
`For determining the relative protein kinase activity, the M, 170,000
`band was cut off the gel, and the incorporated radioactivity was counted.
`The com values obtained were expressed as relative values using SCLC-
`22H or SCLC-24H as standard.
`
`Radioactivity Measurements
`
`Radioactivity was determined with a gamma counter (Hydrogamma
`l6; Oaltefield Instruments, Ltd., Eynsham, Oxford, United Kingdom).
`The efficiency was 70%.
`
`RESULTS
`
`Effect of Temperature on Time Course of "51-EGF Binding.
`At 37‘C maximum binding was achieved after a 60-min incu-
`bation period in NSCLC and SCLC cell lines. After longer
`incubations the cell—bound radioactivity decreased to 65% of
`the maximum value (5-h incubation). At O'C the maximum
`binding (65% of the maximum value at 37'C) occurred after an
`incubation period of 90 min. No loss in cell-bound radioactivity
`was detected up to 5 h of incubation. Based on these results the
`"51-EGF binding assays were performed with an incubation
`period of 60 min at 37°C.
`Competition of "51-EGF. Different NSCLC and SCLC cell
`lines were studied in displacement experiments with a constant
`amount of the labeled derivative (0.5 nM) and varying quantities
`of unlabeled EGF in the concentration range of 0.l to 2 X 10’
`nM. The effect of increased concentrations of unlabeled EGF
`
`on ”’l~EGF binding is shown in Fig. L»! for three NSCLC cell
`lines (A549, EPLC-32M5, NCI~l-I23) and in Fig. U} for two
`SCLC cell lines (NC!-H146 and SCLC—22H). As can be seen
`from Fig. 1, differences in the maximum cell—bound radioactiv-
`ity for NSCLC and SCLC cell
`lines and among different
`NSCLC cell lines existed. The competition of labeled EGF with
`unlabeled was completed when unlabeled EGF was added in
`concentrations >100 nM for all NSCLC cell lines tested (list of
`cell lines tested in Table 1). No displacement could be obtained
`in the SCLC cell line NC!-H146. The residue level of cell-
`
`ml-EGF Binding Assay
`
`bound radioactivity was low, especially for NSCLC cell lines,
`suggesting low unspecific binding.
`Scatchard Analysis of '1‘!-EGF Binding. In Fig. 2 the effect
`of increased concentrations of labeled EGF on binding in
`NSCLC and SCLC cell lines is shown for cell lines EPLC-
`65M2 and DMS-79, respectively. Nonspecific and specific bind-
`NSCLC. Monolayer cultures were washed twice with 4~ml portions
`of prewarrned Hanks’ BSS. One ml of the prewarmed binding medium
`ing was determined as described in “Materials and Methods.”
`consisting of RPM! 1640 and 0.1% bovine serum albumin was added
`As anticipated from competition experiments, nonspecific bind-
`to each dish. Labeled and unlabeled EGFs (both dissolved in phosphate-
`ing was very low for EPLC-65M2 and slightly higher for DMS~
`buffered saline; M. 6100) were added, and a final volume of 1.5 ml/
`79. To analyze binding characteristics, Scatchard plots were
`dish was obtained. The corresponding cell density was about l0‘ cells]
`evaluated (inserts in Fig. 2). The maximal amount of binding
`dish as calculated with a Neubauer chamber after harvesting cells by
`sites (B....,.) was achieved from the x—intercept of the Scatchard
`trypsin treatment. After incubation at 37'C, unbound ""l~EGF was
`plot. About 487 fmol of EGF/mg of protein were maximally
`removed by washing the cells 5 times with 4-ml portions of cold Hanks’
`bound by EPLC-65M2 and about 143 fmol of EGF/mg of
`solution containing 0.1% bovine serum albumin. To solubilize the cells,
`2 ml of 0.! N NaOH were added to each culture dish. After 30 min at
`protein by DMS-79. Identical data were obtained regardless of
`37"C, the content of each dish was transferred into counting vials, and
`whether the NSCLC cell line El’LC~6SM2 was investigated by
`the radioactivity was measured. Nonspecific binding was determined
`the usual method for NSCLC cell lines or by the filtration
`by measuring the cell-bound radioactivity in the presence of 10 pg/dish
`method after solubilizing the cells by scraping. EGF binding
`(1 pm) unlabeled EGF. Specific binding was calculated from the differ-
`characteristics were established for 8 NSCLC and for It SCLC
`ence between cell-bound radioactivity in the presence and absence of
`cell lines. Evaluations of all cell lines tested are summarized in
`unlabeled EGF and analyzed by the method of Scatchard (13).
`Table 1. EGF binding sites were found in 8 of 8 NSCLC cell
`SCLC. Cells were washed twice with Hanks‘ BSS by centrifugation
`lines and 5 of ll SCLC cell lines. The dissociation constants
`at l50 x g for 5 min and adjusted to 4 to 6 X lo‘ cells/ml in RPMI
`were calculated from Scatchard plots and are very similar to
`1640 with 0.l% bovine serum albumin. One hundred ul of labeled and
`those for NSCLC and SCLC cell lines. However, a marked
`unlabeled EGF were added to 300 pl ofthe concentrated cell suspension.
`difference in the maximally bound EGF amounts was found
`After incubation at 37’C, unbound radioactivity was removed by wash-
`ing the cells twice with cold Hanks‘ solution containing 0.1% bovine
`between NSCLC and SCLC cell
`lines. The range of bound
`serum albumin in a filtration apparatus (Millipore, Bedford, MA) with
`EGF/mg of protein was 71 to 606 fmol/mg of protein in
`M33
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`APOTEX EX. 1025-002
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`
`EGF RECEPTOR EXPRESSION IN HUMAN LUNG CANCER CELL LINES
`
`Bound EGF cpm -10"’
`
` o—--4 EPLC -32 M5
`
`
`Swill!)
`
`nM EGF
`
`o--——¢ SCLC ~ 22H
`o———o SCLC ~ H1I.6
`
`
`
`0.1
`
`0.5
`
`1
`
`5
`
`K)
`
`50
`
`Kill
`
`SW
`
`new old
`
`Table 1 E617 binding sites in human lung cancer cell lines
`Maximum
`binding
`(fmol/mg)
`
`Cell line
`Squamous cell carcinoma
`EPLC-32M I
`EPLC—65H
`EPLC-6SM2
`U- I 752
`
`Adcnocarcinoma
`A549
`NC!-H23
`
`Large cell carcinoma
`LCLC«l03H
`U-l 8 I 0
`
`Mesotbeliorna
`MST()~2llH
`
`Small cell carcinoma, classic subtype
`SCLC-22H
`SCLC-24}-I
`NC!-H69
`NCI-H146
`NCl—N592
`
`Small cell carcinoma, variant subtype
`SCLC-I GHV
`SCLC-2 l H
`NCl—H82
`NC!-N417
`NCI-H526
`DMS-79
`
`Nonlung carcinoma
`A431
`5637
`
`' —. defined as negative.
`
`K.
`(ma)
`
`0.50
`2.60
`1.44
`1.29
`
`4.54
`0.59
`
`2.63
`1.48
`
`3 l0
`
`0.58
`0.50
`-—-
`-—-
`—
`
`2.60
`-—-
`-
`0.84
`—
`0.95
`
`1.78
`3.25
`
`486
`570
`487
`606
`
`553
`71
`
`522
`252
`
`W00
`
`26
`33
`-—‘
`-—-
`—
`
`100
`-—
`-—-
`26
`—
`143
`
`2370
`1430
`
`Fig. l. Competition of labeled and unlabeled EGF for binding to NSCDC cell
`lines A549, EPLC~32M$. and NCI-H23 (A) and to SCLC cell lines NCl—22H
`and NCl~Hl46 (B). Indicated amounts of unlabeled EGF and 0.5 nM “’l—EGF
`(I-$0.000 cpnx/ng) were added simultaneously to the culture dish. Ordinate, bound
`radioactivity (cpm); abscissa. unlabeled EGF (nM).
`
`NSCLC cell lines and 26 to M3 fmol/mg of protein in SCLC
`cell lines. Both cell lines of nonlung origin, the urinary bladder
`carcinoma cell line 5637 (14) and the vulvar carcinoma cell line
`A431 (15) used as controls, had an even higher binding poten-
`tial.
`
`tein kinase. This property is a further independent proof for
`the identification of the receptor. Therefore a protein kinase
`assay was performed using immunoprecipitated proteins from
`various cell lines. Incorporation of radioactively labeled ATP
`into the EGF receptor itself occurs in an autocatalytic fashion.
`Radioactively labeled EGF receptor was detected in A431,
`EPLC—6SM2, EPLC-32Ml, and MSTO—2l Ill cells. The result
`is shown in Fig. 3B. The appearance of autophosphorylated
`EGF receptor is in accordance with the presence of EGF
`receptors as evidenced by [”S]methionine—laheled M. 170,000
`molecules shown in Fig. 3A. These results indicate that indeed
`EGF receptors are highly expressed in NSCLC cells.
`While the incorporated radioactivity at a molecular weight
`of 170,000 in RIP and in the even more sensitive PK was quite
`strong for NSCLC cell lines, it was markedly reduced in the
`SCLC cell lines positive in the binding assays. Table 2 gives a
`comparison between the cpm values obtained for SCLC and
`NSCLC cell lines after the M, 170,000 band was cut off the
`gel. When the cprn values obtained are expressed as relative
`values (relative protein kinase activity), it can be seen that they
`are 10 to 100 times smaller in the live SCLC cell lines positive
`in EGF binding experiments compared to the values obtained
`for NSCLC cell lines and 1,000 times smaller compared to the
`control A431.
`
`Analysis of the EGF Receptor and its Associated Protein
`Kinase Activity. EGF binding to tumor cells only indirectly
`suggests that binding involves EGF receptors.
`in order to
`demonstrate EGF receptor molecules directly in the appropriate
`cells, antibodies which recognize the EGF receptor specifically
`were used for its identification in an indirect immunoprecipi—
`tation procedure. The antibody was directed against the onco-
`gene protein erbB, which represents a truncated EGF receptor
`(3). Properties of this antibody have been described elsewhere
`(12). [”S}Methionine-labeled cells were treated for indirect
`immunoprecipitation with the anti—erbB serum. The result is
`shown in Fig. 3. A strong band with an approximate molecular
`weight of 170,000 was detected with anti-erbB serum in the
`control A431 and was also detected in the NSCLC cell lines
`EPLC-65M2, EPLC-32Ml, and MSTO-21 lll cells. To dem-
`onstrate more clearly that the prominent band represents the
`EGF receptor molecule,
`the specificity of the reaction was
`proven by competition of the excess antigen which was the
`We have studied EGF receptor expression in human lung
`cancer cell lines. Displacement experiments demonstrated com-
`bacterially expressed erbB protein for the antibody. Presence of
`petition of labeled EGF with unlabeled EGF in all NSCLC cell
`the competing antigen is indicated by +C in Fig. 3.4. Clearly
`its presence completely erased the precipitation band.
`lines tasted but not in all SCLC cell lines. Time course experi-
`ments for NSCLC and SCLC cell lines demonstrated differ»
`The EGF receptor is associated with a tyrosine-specific pro-
`H34
`
`DISCUSSION
`
`APOTEX EX. 1025-003
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`EGF RECEPTOR EXPRESSION IN HUMAN LUNG CANCER CELL LINES
`
`
`
`
`
`
`
`
`
`A RIP
`
`l5 l
`
`aHzz
`
`
`
`
`
`Fig. 3. R11’ (A), indirect immunoprecipitation of EGF receptor proteins in
`{”S]methionine (2 to 4 x 10‘ cell each)-labeled cell lines. The sets used were 10
`pl of normal rabbit serum (NRS). 10 ,ul of a negative rabbit hypetimtnune serum
`1317/5 (a-erbli’), 10 ul of an anti-«bl! serum (aver-bB). Presence of purified
`bacterial MS2-erbB protein (50 ug) as competing antigen is indicated (-l-C). PK
`(B). analysis of protein kinase activity of the EGF receptor in vitro. Freshly
`harvested cells U0‘) were lysed and tested for protein ltinase activity by the
`addition of radioactively labeled ATP to preformed immune complexes. The
`reaction products were analysed by gel electrophoresis and autoradiography. For
`nomenclature of sera. see RIP.
`
`Table 2 Cpm values ofincorporated radioactivity in PK and relative protein
`kinase activity in SCLC and NSCLC cell lines
`Relative protein
`kinase activity
`
`cpm
`
`SCLC cell lines
`SCLC~l6HV
`DMS-79
`NCI-N417
`SCLC~22H
`SCLC~24H
`
`NSCDC cell lines
`EPLC-32Ml
`EPl.C~65H
`l.C1.C‘~ 103}!
`
`Nonlung carcinoma
`A431
`
`2,192
`377
`2,602
`244
`166
`
`11,173
`21,350
`9,960
`
`lo
`1
`10
`1
`1
`
`I00
`300
`100
`
`127,553
`
`1.000
`
`
`nuns-w-gu-no
`
`an-taunt-nan;
`
`01
`
`0.5
`
`1
`
`2
`
`3 nu ‘*5:-Ear
`
`Fig. 2. Effect of EGF concentration on binding of NSCLC cell line EPLC~
`65M2 (A) and SCLC cell line DMS~79 (B). Labeled EGI-1150.000 cpm/ng) was
`added as indicated to the culture dish without (0) and with (A) an excess amount
`of unlabeled EGF (1 mu). 0, specific binding. Ordinate, bound radioactivity
`(cptn); abscissa, "51-!-ZGF(nM). Inserts, Scatchard plot of binding data.
`
`were very similar and characteristic for EGF binding sites (16,
`19, 20) and classify the EGF binding sites as high affinity sites.
`Nonspecific binding was low, especially for the NSCLC cell
`lines. For most of the cell lines tested, the low bound-free ratio
`at low growth factor concentrations found in Scatchard plots
`ences in EGF binding at 37'C and 0°C, respectively. These
`can be explained by a longer time required for maximum
`results were in agreement with investigations on fibroblasts (16)
`binding. This agrees with data published for EGF binding to
`and nonlung cancer cell lines (17). The decrease in cell-bound
`human fibroblasts (16) and to the urinary bladder carcinoma
`radioactivity after an incubation period of I h at 37‘C may be
`explained by receptor internalization and degradation (16-18).
`cell line 5637. Both control lines A431 (squamous cell carci-
`noma) and the urinary bladder carcinoma cell line 5637 (ade~
`At 4‘C, internalization and degradation of receptor-bound EGF
`were negligible, but EGF binding was reduced, which may be
`nocarcinotna) had even higher levels of EGF binding sites than
`NSCLC cells. This may be caused by an amplification of the
`caused by conformational changes of proteins and phospho~
`EGF receptor gene, which is known for A431 but has not yet
`lipids at low temperature (18).
`been investigated in 5637. The levels of EGF binding sites in
`Detailed analysis of EGF binding sites in NSCLC and SCLC
`cell lines based on Scatchard analysis revealed a characteristic
`NSCLC are not unusually high and do thus not suggest an EGF
`receptor gene amplification in this type of lung cancer. Within
`difference in the amount of binding sites between the two lung
`the group of SCLC cell lines, low levels of EGF binding sites
`cancer groups (Table 1). The amount of binding sites in NSCLC
`were found in both subtypes of SCLC, classic and variant. The
`cells ranged from 71 to 1000 fmol/mg of protein and was
`highest levels, however, were found in cell lines of the variant
`significantly lower in SCLC cells (26 to 143 fmol/mg of pro-
`tein). In addition, all NSCLC cell lines tested (squamous cell
`subtype. In light of the recently published inverse relationship
`between EGF receptor expression and differentiation in tumors
`carcinoma, adenocarcinoma,
`large cell carcinoma, meso~
`thelioma) had EGF binding sites, and only 5 of 11 SCLC cell
`of neuroectodermal origin (21), our data suggest that the variant
`lines bound EGF (Table 1).
`SCLC subtype is less differentiated than the classic subtype.
`The amount of EGF molecules bound/cell (82,500 molecules
`This is in agreement with the reported loss of neuroendocrine
`for EPLC-65M2 and l7,000 for DMS-79) agreed with values
`features (I.—DOPA decarboxylase) in variant SCLC cell lines
`found in other cancer cell lines (15). The dissociation constants
`(8, l0).
`ll35
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`EGF RECEPTOR EXPRESSION IN HUMAN LUNG CANCER CELL UNES
`
`Our data generally confirm earlier results published by Sher-
`win er al. (22). in contrast to these authors, however, we found
`small amounts of EGF binding sites in 5 of II SCLC cell lines.
`including the only EGF binding—positive cell line (NC!-N417),
`which was stated to be a converter line, in the report of Sherwin
`er al. (22). Besides the possibility of conversion during pro-
`longed passage in vitro. these differences may be attributed to
`the fact that all except one EGF binding-positive SCLC cell
`line were established in laboratories (Marburg, Federal Repub-
`lic of Germany, and Dartmouth, NH) different from the Na-
`tional Cancer Institute (Bethesda, MD); the latter provided all
`the SCLC cell lines for the study of Sherwin er al. (22).
`In order to classify the EGF binding sites in lung cancer cells
`as EGF receptors, various cell lines were tested in a protein
`kinase assay and in an indirect immunoprecipitation assay with
`an antibody directed against
`the internal part of the EGF
`receptor. In the indirect immunoprecipitation assay, a strong
`band with an approximate molecular weight of 170,000 could
`be detected in the NSCLC cell
`lines EPLC-65M2, EPLC-
`32M 1. and MSTO-2} 11!. The specificity of the reaction was
`proven by competition for the antibody by excess antigen. The
`appearance of autophosphorylated EGF receptors in the protein
`kinase assay confirmed the results obtained by the indirect
`immunoprccipitation assay and provided more evidence for the
`characterization of the EGF binding sites as EGF receptors. As
`to what to expect from results of the binding assay, the incor-
`porated radioactivity at the M, 170.000 band in RIP and PK
`was strongly reduced in the live SCLC cell lines positive for
`EGF binding sites. The relative protein kinase activity revealed
`that the cpm values obtained for the SCLC cell lines were 10
`to 100 times decreased compared to the cpm values obtained
`for NSCLC cell lines (Table 2). Again, the even higher levels
`of EGF receptors in the control line A431 are demonstrable.
`We are currently conducting experiments on the proliferative
`effects of exogenous EGF in serum-free growing cell
`lines.
`Preliminary data have shown that the growth of NCI-H69 and
`SCLC-l6HV is independent of EGF. In contrast, 11- l 8 I 0 seems
`to require exogenous EGF for optimal serum-free culture
`growth. Data taken from the literature show that EGF stimu-
`lates epithelial growth in cells with moderate receptor levels
`but inhibits growth in malignant cells with high level receptor
`.
`expression (23-25).
`In summary, we have shown that NSCLC, in contrast to
`”'
`SCLC, expresses in all cases high levels of EGF receptors. This
`suggests that EGF may serve as a biological marker for NSCLC 20,
`and may play a role in the growth and differentiation control
`of human lung cancer.
`
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`l8.
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`2,
`
`ACKNOWLEDGMENTS
`
`22.
`
`We thank K. Bcisenherz, P. Olschewski. A. lmmel. and S. Sukrow
`
`for technical assistance; C. Born for typing the manuscript; and A.
`Gregory-Bepler for correcting the manuscript.
`
`23
`
`'
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`l 136
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`APOTEX EX