lnt. J . Cancer: 45, 131-135 (1990) 0 1990 Wiley-Liss, Inc.
Publication of the International Union Against Cancer Publication de I'Union Internationale Contre le Cancer
EGF and TGF-a, THE LIGANDS OF HYPERPRODUCED EGFR IN HUMAN ESOPHAGEAL CARCINOMA CELLS, ACT AS AUTOCRINE GROWTH FACTORS Kazuhiro YOSHIDA'~~, Ekai Kuol, Toshitaka TSUDA~, Tetsuhiro TSUJINO',Masanori [TO', Mhoru NIIMOTO~ and Eiichi TAHARA'.~ 'First Department of Pathology, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734; and zDepartment of Surgery, Research Institute for Nuclear Medicine and Biology, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan. In order to ascertain autocrine growth factors in esophageal carcinomas, we analysed expression of mRNAs and proteins for epidermal growth factor (EGF), transforming growth factor-a (TGF-a) and epidermal growth factor receptor (EGFR) in 6 esophageal carcinoma cell lines. Gene alterations were also examined. All of the esophageal carcinoma cell lines expressed mRNA for EGFR and TGF-or genes. Interestingly, EGF mRNA of about 5.0 kb was also detected in TE-I, TE-2, and TE-8 cells. Production of protein was also confirmed by binding assay and ELISA on 3 of the 6 cell lines. The cells had a relatively high number of EGFRs and produced TGF-ar and EGF protein at the same time. Furthermore, antiEGF (KEM-10) and anti-TGF-a (WA-3) monoclonal antibodies (MAbs) inhibited spontaneous uptake of tritiated thymidine CH-TdR) by TE-I cells which expressed EGF, TGF-a and EGFR mRNA and protein. These results strongly suggest that EGF and/or TGF-a produced by carcinoma cells function as autocrine growth factors for human esophageal carcinomas.
Esophageal carcinoma exhibits amplification and overexpression of the EGFR gene (Hollstein et al., 1988; Lu et al., 1988; Hunts et al., 1985). However, this is detected not only in esophageal carcinomas but also in a variety of tumors such as brain tumors (Wong et al., 1987; Maiden et al., 1988; Libermann et al., 1985), breast carcinomas (Ro et al., 1988; Filmus et al., 1985) and gastric carcinoma (Yoshida et al., 1989b). Moreover, it is not known whether the ligands of hyperproduced EGFRs in esophageal carcinomas (Ozawa et al., 1988, 1987a) are EGF and/or TGF-a. EGF and TGF-a act through EGFR and share many biological activities. EGF stimulates accumulation of mRNA for EGFR (Yoshida et al., 1989a; DePalo and Das, 1988; Earp et al., 1986) as in the case of TGF-a in breast carcinoma cells (Bjorge et al., 1989). Moreover, EGF and TGF-a induce TGF-a gene expression in human keratinocytes (Coffey et al., 1987), reminiscent of mechanisms of autocrine growth control (Sporn and Roberts, 1985). Stem et al. (1987) have shown in an autocrine model that fibroblasts transfected with synthetic sequences encoding EGF in the expression vector exhibited uncontrolled proliferation and transformation. Velu et al. (1987) noted that NIH 3T3 cells were transformed by transfection with EGFR gene in retrovirus vector and that they developed a fully transformed phenotype in the presence of EGF. In this study, we first detected expression of mRNA and protein for EGF, TGF-a and EGFR by 6 esophageal carcinoma cell lines. Second, we examined whether anti-EGF and anti-TGF-a MAbs inhibited spontaneous growth of TE- 1 esophageal carcinoma cells, in an attempt to elucidate the possible autocrine growth mechanisms acting via EGFRs.
noma; TE-8 and TE- 12, moderately differentiated squamous carcinoma) were kindly provided by Dr. T. Nishihira (Tohoku University School of Medicine). Cells were routinely grown in monolayer culture at 37°C in a 5% C02/95% humidified-air atmosphere. The medium used was RPMI-1640 (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (Whittaker M.A. Bioproducts, Walkersville, MA). RNA preparation and Northern blot analysis RNA was extracted by the guanidium isothiothianatelcesium chloride method (Maniatis et al., 1984). Ten micrograms of poly(A)+ selected RNA were electrophoresed in 1.0% agaroselformaldehyde gel and blotted onto zeta-probe nylon filter membrane (BioRad, Richmond, CA). Filters were baked for 2 hr at 80°C under vacuum. After pre-hybridization had been attained, hybridization was performed at 42°C for 12-15 hr, using 32P-multi-prime-labelledprobe. Hybridization solution 5 x Dencontained 0 . 1 PIPES-NaOH ~ (PH 6.8), 0 . 6 5 NaCl, ~ hardt's solution (1 X Denhardt's = 0.02% w/v each of bovine serum albumin, Ficoll, and polyvinyl pyrrolidine), 0.1% sodium dodecyl sulfate (SDS), 50% de-ionized formamide, 10% Dextran sulfate and 100 Fg/ml salmon sperm DNA. Filters were washed twice in 0.1 X SSC-0.1 X SDS (1 X SSC = 0 . 1 5 ~NaCl and 0 . 0 1 5 ~sodium citrate) for 30 min at room temperature, then washed twice in 0.1 X SSC-0.1 X SDS for 60 min at 65°C and rinsed in 0.1 x SSC. Filters were autoradiographed overnight at - 80°C with Kodak XAR-5 films with an intensifying screen. DNA preparation and Southern blot analysis High-molecular-weight DNAs were prepared by the phenolchloroform method (Maniatis et al., 1984). DNAs were digested with EcoRI and electrophoresed on 0.8% agarose gel. DNAs were denatured, neutralized and then transferred to nitro-cellulose filter membrane. Hybridization and washing procedures were performed as described above. DNA probes The 1.9-kb human cDNA insert from phEGF15 was kindly provided by Dr. G.I. Bell (Bell et al., 1986), 1.4-kb human TGF-a cDNA by Dr. R. Derynck (Derynck et al., 1987) and 2.4-kb human EGFR cDNA in pE7 (Xu et al., 1984) by the Japanese Cancer Research Resources Bank (JCRB). p-actin probe was purchased from Oncor, Gaithersburg, MD. EGF receptor binding assay Recombinant human EGF (hEGF) and TGF-a (hTGF-a) were kindly provided by Wakunaga Pharmaceuticals Hiroshima, Japan. EGFRs were evaluated by lz5I-EGF specific binding to human esophageal carcinoma cells (Ochiai et al., 1988), hEGF being iodinated by the chroramine-T method.
MATERIAL AND METHODS
Cell culture Six cell lines established from esophageal carcinomas (TE1, well differentiated squamous carcinoma; TE-2 and TE-5, poorly differentiated squamous carcinoma; TE-7, adenocarci-
3To whom requests for reprints should be addressed. Received July 14, 1989 and in revised form August 21, 1989.
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YOSHIDA ET AL.
Cells were washed extensively with 0. IM phosphate-buffered saline (PBS). One milliliter of serum-free medium, containing 0.1% bovine serum albumin (BSA) and 100 PM '*ThEGF with or without 20 n M unlabelled hEGF, was added to each dish. After incubation for a designated time, the medium was removed and the dishes were washed 4 times with 2 ml PBS. Cells were dissolved in 0.1 N NaOH containing 0.05% SDS and dishes were rinsed with more water. The radioactivity was measured with a liquid scintillation counter. The specific binding was calculated as the difference between radioactivity in the absence or presence of unlabelled hEGF. The binding of 1251-labelledanti-TGF-a (WA-3)or anti-EGF (KEM-10)specijk MAb to carcinoma cells Preparation and characterization of MAbs against hEGF (KEM-10) and hTGF-a (WA-3) have been previously described (Imanishi et al., 1989). All MAbs used were purified by DEAE negative-ion exchange chromatography. Purity of MAbs was over 95% by gel-permeation HPLC. Membrane-bound TGF-a (Teixido et al., 1987) and EGF were examined by 127-labelled anti-TGF-cw (WA-3) or antiEGF (KEM-10) MAb binding assay in TE-1, TE-5 and TE-8. Cells were trypsinized and plated out as 1 X lo6 cells per 6-well plate (NUNC, Roskilde, Denmark) 24 hr prior to initiation of the assay. Two hours before the assay, the medium was replaced by RPMI 1640 containing 20 m~ HEPES, PH 7.4. The cells were incubated for 1 hr at 37°C and then cooled on ice. The medium was replaced by ice-cold binding buffer (RPMI 1640 containing 0.5% of BSA and 20 m~ HEPES, PH 7.4). For the binding assay, wells were set up in triplicate, containing 0.1 p g of 1251-labelledanti-TGF-a (WA-3) or antiEGF (KEM-10) MAb, and incubated for 3 hr on ice. The samples were washed 3 times with binding buffer, lysed with 1 N NaOH and then counted in a gamma counter. Non-specific binding was determined in the presence of 100 pg of unlabelled MAbs. EGF assay EGF was assayed by a highly specific sandwich ELISA with anti-EGF MAbs (KEM-I, KEM-10) that do not react with human TGF-a (Tahara et al., 1986). Briefly, cell pellets (1 X lo7 cells) were homogenized in 1 ml of extraction buffer (10 m Tris-HC1 PH 7.2, 0.15 M NaCl, 0.02% NaN,, 0.5% NP40). Aliquots (100 PI) of the extract were added to antiEGF MAb (KEM-10)-coated plates and incubated at 4°C overnight. After washing, horse-radish peroxidase-labelled antiEGF MAb (KEM-1 HRPO) was added. After 3 hr incubation, the substrate was added. Standard and cell samples were assayed in triplicate. Inhibition of spontaneous 3H-TdR uptake of TE-1 cells To examine whether EGF and/or TGF-a act as an autocrine growth factor, we examined the inhibition of 3H-TdR uptake of TE-1 cells by specific MAbs for EGF and TGF-a. Cells were inoculated at 1 x lo5 cells per well in 24-well plates (Falcon, Oxnard, CA) in 1 ml of RPMI 1640 medium supplemented with 10% FCS, and after 24 hr the medium was replaced by fresh medium supplemented with 0.5% FCS. After 24 hr, the cells were cultured with various concentrations of MAbs specific or non-specific to EGF and TGF-cw for 24 hr. The cells were pulsed with 0.2 pCi per well of 3H-TdR (5 p C i / w , New England Nuclear, Boston, MA) for the last 4 hr of incubation. RESULTS
Expression of mRNA for EGFR, EGF and TGF-a genes The initial approach was performed to test for EGFR mRNA in the 6 esophageal carcinoma cell lines. As shown in Figure 1,
EGFR
EGF
TGF-
O(
FIGUREI - Expression of EGFR, EGF and TGF-a mRNA in esophageal carcinoma cell lines by Northern hybridization; 10 k g of poly(A)+ selected KNA were applied in each lane. p-actin was used as an internal control.
EGFR transcripts of 10.6 and 5.8 kb were clearly detected in all the cell lines tested. TE-8 cells revealed rather high levels of EGFR mRNA and exhibited additional bands at 3.7 and 2.9 kb. To examine whether EGFR-expressing tumors express its ligands for autocrine growth promotion, we investigated the level of expression of mRNA for EGF and TGF-a genes. Expression of mRNA for EGF of about 5.0 kb was evident in TE-1, TE-2, and TE-8 cells, but it was not detected in TE-5, TE-7 or TE-12 cells. As for TGF-cw mRNA, all the 6 cell lines expressed fairly high levels of 4.8 kb TGF-a mRNA. In TE-1, TE-2 and TE-12 cells which expressed high levels of 4.8-kb transcript, a 1.6-kb transcript was also detected (Bates et al., 1988). The same filter was re-hybridized with p-actin probe as an internal control (Fig. 1). These results strongly suggest that EGF and/or TGF-cw act as autocrine growth factors through EGFR in esophageal carcinoma cells. In order to ascertain that EGFR, EGF and TGF-a mRNA expression was due to gene alterations, Southern blot analysis was performed. EGFR gene amplification was detected in TE1 and TE-8 cells (Yamamaoto et al., 1986), but neither amplification nor rearrangement was observed in the analysis of EGF and TGF-a genes (data not shown). EGFK binding assay The number of surface EGF receptors per cell was obtained by conducting a saturation binding study at 4°C with increasing concentrations of 1251-EGF(Ochiai et al., 1988). The binding
EXPRESSION OF
EGF,
133
TGF-a AND EGFR IN ESOPHAGEAL CARCINOMAS
TABLE I - EXPRESSION OF EGFR, TGF-u AND EGF IN ESOPHAGEAL CARCINOMA CELL LINES
EGER
TE- 1 TE-2 TE-5 TE-7 TE-8 TE-12
mRNA
Binding capacity' (sitedcell)
+++ +++ +++++ ++++ +++
9.1 x 105 NE 4.0 X 105 6.0 x 105 NE NE
TGF-a
KD
(M)
2.1 x 10-9 2.0 x 10-9 1.3 x 10-9
EGF
mRNA
Membrane (pg/lOJ'cells) recurs09
++++ +++ ++ ++ ++ +++
42.4 NE 60.2 38.0 NE NE
Content3 (pg/106 cells)
mRNA
+++ +-
210 NE 15 33 NE
-
+-
m
'Specific binding was determined by 1251-hWjF.-2Bindmg of lz5I-WA-3 to carcinoma cells (cprn). 1,000cpm = lOpg TGPu prec~sor.-~EGF content was determined by ELISA. NE,not examined.
assay and its Scatchard analysis were carried out on TE- 1, TE-5 and TE-7 cells (Table I). EGFR numbers ranged from 4 M. to 9 X 105/cell with a K, value of the order of
estingly, anti-TGF-a MAb (WA-3) also reduced 3H-TdR uptake to 40% of control levels at the concentration of 1.O mg/ml, but anti-IgE MAb (negative control) did not inhibit 3H-TdR incorporation. Our results indicate that anti-EGF (KEM- 10) and anti-TGF-a (WA-3) MAbs inhibit the biological activity of The binding of J251-labelledanti-TGF-a (WA-3) and anti-EGF (KEM-10) MAbs to esophageal carcinoma cells and EGF assay endogenous EGF and/or TGF-a by blocking their binding to We next determined the specific binding of 1251-labelled receptors. anti-TGF-a (WA-3) and anti-EGF (KEM-10) MAbs to TE-1, DISCUSSION TE-5 and TE-7 cells to detect membrane-bound TGF-a and EGF, respectively. Various levels of specific binding of EGF is a polypeptide which plays an important role in mam1251-labelledTGF-a MAb (WA-3) were detected in carcinoma mary tumorigenesis (Kurachi et al., 1985) and some breast cells (Fig. 2). A431 was used as a positive control and binding carcinoma cells express EGF mRNA with its expression being ranged from 3797 to 6016 cpm. Specific binding of increased by progestins (Murphy et al., 1988). Moreover, EGF 1251-labelled anti-EGF MAb (KEM-10) was not detected in increases synthesis of its receptors (Yoshida et al., 1989a; these cells. The amount of 25 kDa TGF-a precursor level was DePalo and Das, 1988; Earp et al., 1986) and stimulates accalculated as pg/106 cells, on the assumption that a single cumulation of TGF-a mRNA in MDA 468 cells (Bjorge et al., antibody binds to a single antigen (Table I). 1989). Gastric carcinomas express mRNA for EGF, TGF-a On the other hand, EGF assay was performed using ELISA and EGFRs (data not shown) and co-expression of EGF and in these cells. EGF protein was present in the lysates of esoph- EGFRs correlates with tumor invasion and patient prognosis ageal carcinoma cells (Table I). TE-1 cells which revealed a (Yasui et al., 1988a,b; Tahara et al., 1986). fairly high level of EGF mRNA had EGF proteins present at a However, little is known as yet about EGF mRNA expresconcentration of 210 pg/106 cells. EGF protein was detected, sion by esophageal carcinoma cells. In this study, EGF mRNA though at a rather low level, in TE-5 and TE-7, in which of about 5.0 kb was clearly detected in TE-1, TE-2 and TE-8 mRNA was not detected. cells. Moreover, EGF protein was also detected at high concentrations in TE-1 but at low levels in TE-5 and TE-7 in which Inhibition of spontaneous 3H-T& uptake by TE-I cells EGF mRNA was not detected. Ozawa et al. (19876) reported In order to show directly that EGF and/or TGF-a function as that EGF stimulates growth of EGFR-hyperproducing tumor autocrine growth factors to control growth of esophageal car- cells in athymic mice. EGF also stimulates growth of gastric cinoma cells, we attempted to examine whether EGF- and carcinoma cells which have high-affinity receptors (Ochiai et TGF-a-specific MAbs inhibit growth of TE-1 cells which si- al., 1988). On the other hand, EGF inhibits growth of squamultaneously express EGFR, EGF and TGF-a mRNA with mow-cell carcinoma cells under certain circumstances (Kaprotein (Fig. 3). 3H-TdR uptake by TE-1 cells when treated with MAbs was compared to that of the non-treated group. Anti-EGF MAb (KEM-10) reduced 3H-TdR incorporation to 80% of control values at the concentration of 0.1 mg/ml and to 100 a final level of 40% at the concentration of 1.0 mg/ml. Inter-
de
80
m
Y
8000 I
I
k
2 6000 Ln 2
4000
n Y
40
20
1 1 .o
0
0.1
concentration of MAb (mglml)
2000
0
.-m
60
0
r
.-F
9 $
5
D
-P
2
TE- 1
TE-5
TE-7
A431
FIGURE 2 - Binding of 1251-labelledTGF-a specific MAb (WA-3) to esophageal carcinoma cells. A431 was used as a positive control. Binding was measured by gamma counter (cpm).
FIGURE3 - Inhibition of spontaneous 3H-TdR incorporation by MAbs specific to EGF and TGF-a. TE-1 cells were cultured for 24 hr in the presence of various concentrations of anti-EGF MAb (KEM-10, U), anti-TGF-a (WA-3, Ed) and anti-IgE MAb (25B4, 0).The data show 3H-TdR incorporation in the experimental groups as a percentage of spontaneous 3H-TdR incorporation and represent means of triplicate determinations.
134
YOSHIDA ET AL.
mata et al., 1986). One of the reasons for such differences in growth might be related to the affinity to receptors or to the concentration of EGF in the medium. The mechanisms of this inhibitory effect remain unknown. As for EGFR, all the 6 carcinoma cell lines exhibited relatively high levels of EGFR mRNA, and the EGFR gene was amplified in TE-1 and TE-8 cells (Yamamoto et al., 1986). The number of EGFRs is about 4-9 X los siteskell, which is more than 10 times higher than that of gastric carcinoma cells (Ochiai et al., 1988). Moreover, most esophageal carcinoma tissues obtained from surgical specimens express higher levels of EGFR, EGF and TGF-a mRNAs than normal esophageal tissue (data not shown). TGF-a is regulated by estrogens and EGF in breast carcinoma cells (Bates et al., 1988; Bjorge et al., 1989) and is expressed in a variety of tumor cells as an autocrine secretion (Derynck et al., 1987; Ellem et al., 1988; Smith et al., 1987). In the present study, TGF-a mRNA was clearly detected in all the 6 carcinoma cell lines and so was a membrane-bound precursor. Membrane-bound EGF, however, was not evident, in spite of positive detection by ELISA. This might be explained by low levels of cell-surface EGF, compared to those of TGF-a. Membrane-bound pro-TGF-a binds to EGFR on the surface of contiguous cells and induces receptor autophosphorylation, leading to signal transduction (Brachmann et al., 1989; Wong et al., 1989). Further work should elucidate
whether pro-TGF-a produced by tumor cells has biological activities in esophageal carcinoma cells. We have shown that 3H-TdR incorporation by TE-1 cells was inhibited by anti-EGF (KEM-10) and anti-TGF-ol (WA-3) MAbs, which might have blocked the biological activities of endogenous EGF and TGF-a by inhibiting their bindings to EGF receptors in TE-1 cells. In addition, concomitant treatment with anti-EGF (KEM-10) and anti-TGF-a (WA-3) MAbs might also have suppressive effects on 3H-TdR uptake by TE-1 cells. Overall, our results strongly suggest that EGF and/or TGF-a produced by tumor cells may play an important role in the control of growth of esophageal carcinoma cells, acting as autocrine growth regulators. Expression of multiple growth factors has been reported to occur in brain tumors (Nister et al., 1988), breast carcinomas (Peres et al., 1987) and gastric carcinomas (Yoshida et al., 1989c; Bennett et al., 1989). Further work should elucidate whether esophageal carcinomas produce other growth factors and receptors than EGF, TGF-a and EGFRs. ACKNOWLEDGEMENTS
This study was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare for the Comprehensive 10-Year Strategy for Cancer Control, Japan. The authors thank Miss M. Ito, Miss A. Ohta, and Miss S . Yasuda for technical assistance.
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