Effects of dexamethasone on the growth and epidermal growth factor receptor expression of the OVCA 433 ovarian cancer cells

molecular and Cel~l~larEnd~rinolo~, 0 1992 Elsevier Scientific Publishers

MOLCEL

83 (1992) 183- 193 Ireland, Ltd. 0303-7207/92/$05.~

183

02692

Effects of dexamethasone on the growth and epidermal growth factor receptor expression of the OVCA 433 ovarian cancer cells G. Ferrandina ‘, G. Scambia ‘, P. Benedetti Panici ‘, G. Bonanno ‘, R. De Vincenzo ‘, C. Rumi h, S. Bussa ‘, M. Genuardi ‘, V. Spica Roman0 ’ and S. Mancuso a ‘IDepartment of Obstetrics and Gynecology, h Department of Hematoio~l. and ” ~epartrner~t ofHuman Genetics, Catholic Unitwsity, Rome, Itaiy (Received

Key words: Dexamethasone;

Epidermal

growth

10 August

factor

1991; accepted

receptor;

Ovarian

21 October

cancer

1991)

cell

Summary We studied the correlation between dexamethasone (Dex> induced growth effects and modulation of epidermal growth factor receptor (EGFR) expression in OVCA 433 ovarian cancer cells. These cells express specific high and low affinity ‘2sf-EGF binding sites and are growth stimulated by EGF. Dex exhibits mitoinhibito~ effects by recruiting OVCA 433 cells in the Go-G, phase of the cycle, but increases the number of both the high and the low affinity EGFR in a dose dependent manner. The maximal EGFR expression increase occurs after 24 h of Dex treatment consistently with Northern blot studies. The mitogenic activity of EGF in OVCA 433 cells is not affected by the presence of Dex. Moreover Dex growth inhibition occurs in JAl cells, an ovarian cancer cell line which expresses unfunctional EGFR and which is unresponsive to EGF. Our results indicate that the Dex induced growth effects occur independently of EGFR expression.

Introduction Epidermal growth factor (EGF), a peptide factor with growth promoting effects on several cancer cell lines, acts by binding with a specific plasma membrane receptor (EGFR) encoded by the EGFR gene (Carpenter, 1987; Cohen, 1987). Steroid hormones can affect the expression of EGFR and other growth factor receptors (Baker et al., 1978; Tantus et al., 1982; Horwitz et al., 1985; Murphy et al., 1985; Knutson, 19861, and

Correspondence to: Salvatore Mancuso, MD, Department of Gynecology and Obstetrics, Catholic University, Largo A. Gemelli 8, 00168 Rome, Italy.

this modulation has been considered as an indirect mechanism through which steroid hormones may influence cell proliferation. In particular the modulation of EGFR expression in the presence of glucocorticoids has been studied in fibroblasts (Hosokawa et al., 19861, hepatocytes (Glaudhaug et al., 19891, and HeLa cancer cells (Fanger et al., 1986). However, the correlation between changes in EGFR expression and celluIar growth is not yet clear. It has been suggested that the EGF-EGFR system may play a role in regulating the growth of ovarian cancer cells. In fact, the presence of EGFR has been observed in normal and neopiastic ovarian tissues (Gullick et al., 1986; Battaglia et al., 1989) and EGF/transforming growth fac-

184

tor-a activity can be detected in most ovarian adenocarcinomas and normal ovaries (Bauknecht et al., 1986). In the present study we investigated the interactions between steroidal and peptide growth factor regulating cell growth systems in the OVCA 433 human ovarian cancer cells. This cell line expressing high levels of EGFR and sensitive to the mitogenic action of EGF (Scambia et al., 1991) provides a good experimental model since it contains specific high affinity receptors for glucocorticoids (GR) and is growth inhibited by dexamethasone (Dex> (Amin et al., 1987).

Materials

and methods

Materials ‘“51-EGF (854,000 Ci/M) was purchased from Amersham International, UK, and unlabelled EGF was obtained from Boehringer-Mannheim (Germany). Unlabelled EGF was resuspended in 25 mM Tris, 20% glycerol, 5 mM NaN, and 0.1% bovine serum albumin (TGNB). 1,2,4(n)-3H-Dex (29.3 Ci/mM) was purchased from Amersham International, UK. Dex and the other steroids were obtained from Sigma, St. Louis, MO, USA. Quercetin (3,3’,4’,5,7-pentahydroxiflavone) was obtained from Aldrich, Steinhein (Germany).

Cell culture conditions OVCA 433 ovarian cancer cells (Bast et al., 1981) were provided by Dr. B.A. Littlefield of Yale University, USA. This cell line was grown in Eagle’s minimum essential medium with Earle’s salts (MEM), supplemented with 10% heat-inactivated fetal calf serum (HI-FCS) (Flow Laboratories, Irvine, UK), 1 mM sodium pyruvate, 2 mM r_-glutamine and antibiotics. The JAl ovarian cancer cell line (Hill et al., 1987) was obtained from Dr. B. Hill (Imperial Cancer Research Fund, UK) and grown in RPM1 1640 with glutamine (Gibco, Scotland, UK) supplemented with 10% HI-FCS 20 mM Hepes buffer and antibiotics. Cells were grown in monolayer culture in 75 cm2 tissue culture flasks, and were subcultured by harvesting with calcium-free and magnesium-free Hanks’ balanced salt solution supplemented with

0.05% trypsin and 0.2% traacetic acid (EDTA).

ethylenediaminote-

Measurement of 12j1-EGF binding OVCA 433 cells were plated at a density of 100,000 cells/ml in their own medium supplemented as described above. 24 h after plating, the medium was removed and replaced with MEM without red phenol, containing 10% HI-FCS stripped with charcoal (HI-DCC-FCS), and various concentrations of steroid hormones or equivalent amounts of ethanol in control cells to give a final ethanol concentration of 1%. After 24 h of treatment, cells were washed twice and incubated in serum-free MEM with ‘251-EGF (1 nM) in the presence or absence of an excess of unlabelled EGF (1 PM) for 100 min at 37°C. Binding was stopped by two MEM washes. Cells were dissolved with 1 M NaOH and radioactivity was measured in a Beckman gamma counter for 1 min. Specific binding was calculated as the difference between the level of radioactivity in the absence (total bound) and in the presence (nonspecific bound) of unlabelled EGF. A similar procedure was employed to test the effects of quercetin on 12”1-EGF binding in OVCA 433 cells. In order to verify the presence of a 12”1-EGF internalization process occurring during the assay at 37°C experiments were carried out in which, following the incubation with 12’1-EGF (1 nM), cells were incubated for 6 min on ice with 0.7 ml of acetic acid (0.2 M, pH 2.5) with 0.5 M NaCl (Haigler et al., 1979). Cells were then washed twice with MEM and lysed with 1 M NaOH. The radioactivity was taken as a measure of internalized EGF. In order to study the effect of Dex on EGFR occupancy we removed the receptor bound endogenous ligand by washing the cell monolayer, before performing the ‘*‘I-EGF binding assay, with the same acidic buffer used for the internalization studies. In down-regulation experiments, after a 24 h exposure to Dex (lo-’ M) OVCA 433 cells were washed with serum-free MEM and cultured with EGF (lo-” M), Dex (lo-’ M), EGF (lo-” M) + Dex (lo-’ M), and vehicle alone for 6-8 h at 37°C. Then, in order to remove EGF from its

185

receptor, we washed the cell monolayer with the acidic buffer for 6 min at 0°C before performing the ‘251-EGF binding assay in which the incubation was carried out at 37°C for 100 min. Since we demonstrated that some modifications of 12”1-EGF internalization occurred when the assay was performed at 37°C for 100 min, the analysis of saturation binding data was carried out incubating the cells at 0°C for 4 h with increasing ‘251-EGF concentrations (from 0.07 nM to 4.80 nM) to avoid the internalization and degradation of EGFR. Preliminary experiments showed that ‘251-EGF binding reached equilibrium after about 4 h according to Fitzpatrick et al. (1984). Results were analyzed by the methods of Scatchard (1949) and Rosenthal (1967). Measurement of specific 3H-Dex binding

JAl cells were plated at a density of 100,000 cells/ml in the specific medium supplemented as described above. After 24 h cells were washed twice and incubated in serum-free RPM1 with a saturating “H-Dex concentration (40 nM) in the presence or absence of an excess of unlabelled Dex (4 X 10ph M) for 2 h at 37°C. Binding was stopped by two RPM1 washes and cells were lysed with 1 M NaOH. The radioactivity was measured in a Beckman beta counter for 1 min. Growth experiments

OVCA 433 cells and JAl cells were plated at a density of 100,000 cells/ml on culture dishes (35 mm) containing the specific medium supplemented as described above. 24 h after plating, cells were washed twice and cultured for 72 h in phenol red-free MEM with 10% HI-DCC-FCS containing increasing amounts of EGF, Dex, or vehicle alone. Quadruplicate hemocytometer counts of triplicate culture dishes were performed and the results were expressed as a percentage of control. Cell cycle analysis

OVCA 433 cells were plated at a density of 100,000 cells/ml in 75 cm2 tissue culture flasks. After 24 h exposure to Dex (lop7 M), cells were detached by a cell scraper, centrifuged at 1500 X g for 5 min at 20°C and washed twice by resuspending in phosphate buffered saline (PBS). Cell

preparation was performed according to the procedure described by Hollander et al. (1988) and modified for single laser flow cytometry (488 nm>. Briefly, the cells were incubated for 30 min on ice with an anti-EGFR monoclonal antibody (Oncogene Science) and, after washing, for 30 min on ice with fluorescein isothiocyanate (FIT0conjugate goat anti-mouse Ig (BectonDickinson). After washing, cells were fixed by resuspending in 10% methanol, 40% acetone PBS cold solution for 15 min and incubated for 1 h at room temperature in the dark in a solution of propidium iodide (50 pug/ml) (Sigma) and ribonuclease A (1 pg/ml) (Sigma). Stained cells were analyzed by Facscan cytometer (BectonDickinson). In the DNA histograms obtained, we selected specific ‘gates’ corresponding to the different cell cycle phases, and for every ‘gate’ we studied the distribution of EGFR positive cells. Cell cycle phases were also studied with the sum of broadened rectangle model on DNA histograms. RNA blot analysis

OVCA 433 ovarian cancer cells were plated at a density of 100,000 cells/ml in 75 cm2 tissue culture flasks. After 24 h the medium was replaced with fresh medium containing Dex (lop7 M). After 2, 4, 8 and 12 h of Dex treatment total RNA was isolated using the guanidine isothiocyanate/CsCl method as described by Chirgwin et al. (1979). Total RNA from each sample was loaded on a formaldehyde-agarose gel by the method described by Maniatis et al. (1982). RNA was transferred by Northern blotting (Maniatis et al., 1982) onto a nylon membrane (Hybond Nf, Amersham, UK) and subsequently hybridized to the EGFR cDNA insert from plasmid pE7 (Xu et al., 1984) and to P-actin and glyceraldehyde3-phosphate-dehydrogenase (GAPDH) probes. “2P-Deoxycytidine 5’-triphosphate was purchased from NEN DuPont de Nemours (Deutschland). The probes were “2P-oligolabelled according to the random primer method (Feinberg et al., 1983) obtaining specific activities varying from 0.7 x lo9 to 1 X 10” cpm/pg for EGFR, /3-actin, and GAPDH probes. The quantification of mRNA was carried out by densitometric scanning of autoradiograms using an AUS JENA densitometer.

1X6

Statistical analysis was performed dent’s r-test.

using the Stu-

300 -

Statistical analysis Statistical analysis of growth curve and 12’1EGF binding data was performed by using the Student’s t-test. r= 0.95

Results ““I-EGF binding characteristics Fig. 1 shows a representative example of saturation 12’I-EGF binding data in OVCA 433 cells. Two classes of binding sites with apparent K, of 0.13 nM and 1.25 nM and a number of 16,000 and 47,000 sites/cell respectively are detected. In Table 1 the estimate of EGF binding parameters in control and Dex treated cells is shown. There is no difference in the apparent K, for the high and the low affinity binding sites in Dex treated cells with respect to control cells, but there is an increase in total number of sites. This set of experiments was performed under conditions (4 h, OOC)which prevent EGF processing. Fig. 2 shows the effect of different concentrations of Dex on ‘251-EGF binding to OVCA 433 cells after 24 h treatment. We observe a dose dependent increase of ‘251-EGF binding starting at a Dex concentration of lO_” M and reaching the maximal value at lo-’ M. The time course of the Dex induced increase in 12”I-EGF binding demonstrates that the stimulatory effect is already present after 2 h, with a maximal increase at 24 h (Fig. 3). We also investigated the specificity of the response to Dex. Significant increases in ““I-EGF binding are seen only in the presence of Dex (254.4 k 7.6) ( p < O.Ol), triamcinolone (187.2 St 7.5) (p < 0.05), and cortisol (165.1 + 10) (p < 0.05) while all other steroids tested are ineffective (Table 2). To investigate if the process of ‘251-EGF internalization and/or modifications of EGFR occupancy could be relevant in producing the Dex induced ““I-EGF binding increase, two additional sets of experiments were performed using an acidic buffer which is able to remove the endogenous ligand from the membrane receptor. When washing with the acidic buffer was per-

Total (PM)

-I 2

3

4

log Free (PM)

0

I (Ml

20’)

hound (pM) binding data in OVCA 433 cells. Fig. 1. Analysis of “‘I-EGF Cells were cultured for 24 h and incubated for 4 h at 0°C h with increasing ‘*“I-EGF concentrations (from 0.07 nM to 4.80 nM) in the presence or absence of an excess of unlabelled EGF (1 PM). At the end of the incubation period cells were lysed with 1 M NaOH. Binding data of one of three similar experiments were analyzed plotting bound ligand against free ligand (A), bound ligand against log free ligand (E), and bound/free against bound ligand (C). Each point represents the mean of triplicate determinations. Intra-experimental variance was about 5%.

formed at the end of the assay, the percentage of acid-resistant radioactivity was found to be slightly reduced after Dex treatment, suggesting that Dex

187 TABLE

1

‘=I-EGF BINDING CHARACTERISTICS AND DEX TREATED OVCA 433 CELLS

IN CONTROL

24 h after plating, cells were exposed to Dex (lo-’ M) and vehicle alone in phenol red-free MEM with 10% HI-DCC-FCS for 24 h. Then cells were washed twice and incubated in serum-free MEM with increasing ‘*‘I-EGF concentrations (from 0.07 nM to 4.80 nM1 in the presence or absence of unlabelled EGF (1 FM) for 4 h at 0°C. At the end of the incubation period cells were lysed with 1 M NaOH. Data are the mean f SD of three different experiments performed in triplicate. Intra-experimetnal variance was less than 10%.

K,, (nM) K,? (nM) Sites, /cell Sites, /cell

Control

Dex treated

0.12* 0.03 1.27i 0.20 17,000 k 800 45,000 + 2,800

O.lOk 0.01 1.29+_ 0.10 43,000 f 1,500 117,000 f 7,000

Fig. 3. Time course of Dex induced effects on “‘I-EGF binding in OVCA 433 cells. Cells were cultured in the presence of Dex (lo-’ M) for the time indicated. The incubation with lz51-EGF (1 nM) in the presence or absence of an excess of unlabelled EGF (1 PM) was carried out at 37°C for 100 min. Results represent the mean*SD of three different experiments performed in triplicate. Intra-experimental variance was less than 10%. ;5250_

induced lz51-EGF binding increase may be only relatively influenced by the EGFR internalization steps in the conditions (37°C for 100 min) we experienced. Results obtained when the acidic buffer was used just before the assay, demonstrate that the enhancement of EGFR was not a consequence of Dex induced modifications of EGFR occupancy (Table 3). Finally, Dex treatment does not alter the EGF induced down-regulation of EGFR (Table 4).

L ‘: 8

Growth experiments

100

-10 Dexamethasone

-9

-8 concentrations

Fig. 2. Effects of different Dex concentrations on “‘1-EGF binding in OVCA 433 cells. Specific binding was calculated after 24 h exposure to Dex. Incubation with lzI-EGF (1 nM) in the presence or absence of unlabelled EGF (1 PM) was carried out at 37°C for 100 min. Results are the mean + SD of three different experiments performed in triplicate. Intra-experimental variance was less than 10%.

In Fig. 4 the growth effects of different EGF concentrations on OVCA 433 cells are shown. The proliferative effect was present at 5 ng/ml (p < 0.05) and was maximal at 100 ng/ml (p < 0.01). In the same figure we demonstrate the dose dependent inhibitory activity of Dex at concentrations from lo-” M to lo-” M. To investigate the influence of Dex on EGF proliferative effects we exposed OVCA 433 cells to Dex (lo-’ Ml throughout the experiment and after 24 h of exposure we added EGF (50 ng/ml).

188 TABLE

TABLE

2

EFFECTS OF DIFFERENT STEROID HORMONES ON ‘2’I-EGF BINDING IN OVCA 433 OVARIAN CANCER CELLS This table summarizes the ability of different steroid hormones at IO-’ M concentration to affect “‘I-EGF binding in OVCA 433 cells after 24 h exposure in phenol red-free MEM with 10% HI-DCC-FCS. Cells were washed twice and incubated in serum-free MEM with “‘1-EGF (1 nM) in the presence or absence of an excess of unlabelled EGF (1 FM) for 100 min at 37°C. At the end of the incubation period cells were lysed with 1 M NaOH. Data are the mean k SD of three different experiments performed in triplicate. Intra-experimental variance was less than 10%. Steroids

Specific ‘zsI-EGF binding (% of control)

Aldosterone Progesterone Testosterone Methyltrienolone Estradiol Tamoxifen Triamcinolone Cortisol Dexamethasone

95.0* 3.7 92.Ok 4.6 85.6+ 4.5 93.5+ 7.0 100.8+ 4.5 94.2k 5.4 I87.2+ 7.5 165.1 f 10 254.4k 7.6

(R1881)

As shown in Fig. 5, even EGF is able to exhibit Moreover at days 3 and 4 not significantly different TABLE

4

EFFECTS EGFR

OF DEX

ON THE

DOWN-REGULATION

OF

This table shows the effects of a 24 h Dex treatment (lo-’ M) on the down-regulation of EGFR. After Dex treatment OVCA 433 cells were washed twice, incubated in serum-free MEM and exposed to EGF (lo-” MI, Dex (lo-’ MI, and Dex+ EGF for 7 h at 37°C. An acid washing at 0°C for 6 min was performed and then the 12’I-EGF binding assay was carried out at 37°C for 100 min. At the end of the incubation period cells were lysed with 1 M NaOH. Results are the mean + SD of three different experiments performed in triplicate. Intraexperimental variance was less than 10%. Treatment

Specific ““I-EGF binding (% of control)

EGF (lo-’ MI Dex (lo-’ MI Dex + EGF

9.5 * 1.5 242.9 k 5.6 9.0 + 3.4

in the presence of Dex its mitogenic effects. the number of cells was compared to the num80

3

EFFECTS OF ACID WASHING “‘1-EGF TO OVCA 433 CELLS

ON THE

BINDING

510

OF

This table shows the effects of 24 h exposure of Dex (lo-’ Ml on ““I-EGF binding in OVCA 433 cells. After treatment cells were washed twice and incubated in serum-free MEM with “‘I-EGF (1 nMI in the presence or absence of an excess of unlabelled EGF (1 PM) for 100 min at 37°C. Results are the mean k SD of three different experiments performed in triplicate. Intra-experimental variance was less than 10%.

I 20

50

100

EGF (ng/ml)

iJO_

60_

40_

20_

‘251-EGF bound

Treatment

Acid washing None Dex (lo-’

MI

31,062+ 2,480 76,162 f 6,800

a

(dpm/ml) Acid washing

b

6,958 f 765 10,05 1 f 947

Acid washing was carried out before (“I and after th) performing the “‘1-EGF binding assay in order to evaluate the effects of Dex treatment on EGFR occupancy and EGFR internalization, respectively.

I

-10

I

I

I

-7

-6

Fig. 4. OVCA 433 cell growth response to different concentrations of EGF and Dex. Cell counts were performed after 3 days of exposure to the substances tested. Each value represents the mean k SD of three different experiments performed in triplicate.

I89

increase in EGFR mRNA levels in Dex treated cells compared to control. The enhancement was present after 2 h of Dex treatment and was maximal after 12 h (p < 0.05). This effect was specific for the EGFR transcript since Dex had no effect on p-actin and GAPDH mRNA levels. JA 1 cells 0

1

Fig. 5. OVCA 433 cell growth response to EGF (50 ng/ml) addition after 24 h of Dex (IO-’ M) treatment. Data are the meant_ SD of three different experiments performed in triplicate. (0) Control cells; (A ) Dex treated cells; (01 EGF addition in control cells; ( A) EGF addition in Dex treated cells.

ber of cells exposed only to EGF. The latter results were also obtained when cells were plated at low density (data not shown) excluding that conditions near cell confluence may affect the results. Northern blotting analysis

Fig. 6 shows a representative example of Northern blot analysis in control and Dex treated OVCA 433 ovarian cancer cells. We observe an

A

In order to test the hypothesis that Dex induced growth effects are not mediated by a modulation of EGFR expression, we studied the Dex induced effects on ‘251-EGF binding in JAl ovarian cancer cells which express EGFR (Scambia et al., 1991) but which are unresponsive to EGF (Fig. 7). In this cell line containing specific glucocorticoid receptors (data not shown), Dex induces a dose dependent growth inhibition (Fig. 71, and an increase in EGFR expression (p < 0.01) (Table 5). Cell cycle experiments

Table 6 shows the percentage distribution of OVCA 433 cells in the different phases of the cycle and the percentage of EGFR+ cells according to the cell cycle phases in control and in Dex treated cells. It is evident that Dex induced a recruitment of OVCA 433 cells in the G,,-G,

r

B mRNA Levels

t-

actin

+GAPDH

:

C

2

4

8

12

hours

0

Fig. 6. Northern blot analysis of EGFR expression in control and Dex treated OVCA 433 cells. Cells were exposed for 2, 4, 8 and 12 h to Dex (lo-’ M) before assay. For details see Materials and methods. (A) Histogram showing variations in the amount of EGFR specific RNA at different times after Dex treatment. EGFR expression is measured as the intensity ratio (mean + SD of three different experiments) between EGFR and p-actin specific bands. Statistical analysis was performed using the Student’s r-test. (B) A representative example of Northern blot analysis of Dex treated and untreated (C) OVCA 433 cells with EGFR, p-actin, and GAPDH probes.

190 TABLE

5

‘IsI-EGF

BINDING

IN JAl

CELLS

After 24 h Dex treatment JAl cells were washed twice and incubated in serum-free RPM1 with a single “‘I-EGF saturation concentration (3.6 nM) in the presence or absence of an excess of unlabelled EGF (1 FM) for 100 min at 37°C. At the end of the incubation period cells were lysed with I M NaOH. Data are the mean+ SD of three different experiments performed in triplicate. Intra-experimental variance was less than 10%‘. Treatment

lob

i0

EGF (ng/ml)

100 153.7 * 2.8

Ml

phase with a reduction of cells in the S phase (p < 0.05). As expected we observed a global increase of EGFR’ cells after Dex treatment. The distribution of EGFR+ cells in the different phases of cell cycle was identical in both Dex treated and control cells. In order to confirm that the Dex induced EGFR increase is not due to cell cycle effects, we measured EGFR levels in OVCA 433 cells after 24 h exposure to different concentrations of quercetin, a bioflavonoid whose ability to recruit OVCA 433 cells in the G,,-G, phase has been documented (Scambia et al., 1990). As shown in Table 7, quercetin, at concentrations effective on cell growth (Scambia et al., 1990) is not able to modify EGFR expression. Discussion Although the effects growth (Braunschweiger TABLE

;o

5;o

“s1-EGF binding (% of control)

Control Dex (lO_’

CELL

I

of glucocorticoids on cell et al., 1981, 1983; Amin

8

20

-I 1

/

-4

-10

Dex(Log

I

-8

I

-7

-6

M)

Fig. 7. JAl cell growth response to different concentrations of EGF and Dex. Cell counts were performed after 3 days of exposure to the substances tested. Each value represents the mean + SD of three different experiments performed in triplicate

et al., 1987) have been widely demonstrated, the mechanisms through which they occur are not yet clear. It has been hypothesized that the modulation of peptide growth factor production (Syms et al., 1984), or of capacity/affinity of their receptors (Baker et al., 1978; Braunschweiger et al., 1981, 1983), may be involved. In this study we

6 CYCLE

ANALYSIS

IN OVCA

433 CELLS

This table summarizes the distribution of OVCA 433 cells in the different phases of the cell cycle in control and Dex treated cells. For detailed procedure see Materials and methods. The respective percentage of EGFR positive cells in each phase is also shown. Data are the mean f SD of three different experiments performed in triplicate. Cycle phases

Go-G, S G,-M All cells

Control

cells

Dex treated

% all cells

% EGFR+

32.1 f 2.0 56.3 of 2.1 11.6+0.6

72.7+5.5 79.1 f 6.3 83.6 + 7.2 78.4 k 6.7

cells

cells

% all cells

% EGFR+

45.2+2.5 40.0 f 0.9 14.8 f 0.4

93.4 f 8.9 90.5 + 7.5 94.1 * 7.2 92.6 k 7.8

cells

191 TABLE EFFECTS

7 OF QUERCETIN

ON ‘*‘I-EGF

BINDING

The ability of different concentrations of quercetin to affect ““I-EGF binding in OVCA 433 cells was tested. After 24 h treatment cells were washed twice and incubated in serum-free MEM with ‘251-EGF (1 nM) in the presence or absence of an excess of unlabelled EGF (1 PM) at 37°C for 100 min. At the end of the incubation period cells were lysed with 1 M NaOH. Data are the mean + SD of three different experiments performed in triplicate. Intra-experimental variance was less than 10%. Treatment

‘251-EGF binding t% of control)

Q (10-s M) Q (lo-’ M) Q (lOmh MI

98.2 k 1.8 90.5 * 3.0 101.6? 2.6

analyzed the relationship between Dex induced growth effects and EGFR expression in OVCA 433 ovarian cancer cells. This cell line, which is sensitive to the growth inhibitory activity of Dex (Amin et al., 1987), contains two affinity binding sites for 1251-EGF and is highly sensitive to the growth promoting effects of EGF. Growth inhibitory effects induced by Dex are not associated with the negative modulation of EGFR expression. In fact we demonstrated that Dex increases EGF binding in OVCA 433 ovarian cancer cells in a dose dependent manner. Our results showed that Dex induces an enhancement of both binding sites but does not modify their affinities according to Wu et al. (1981). On the other hand Dex induces a more pronounced increase in high affinity than in low affinity EGF binding sites in HeLa S3 cells (Fanger et al., 1986). These discrepancies may be due to the different experimental conditions, in particular to the use of incubation temperatures that influence the process of internalization. The Dex induced EGFR increase is probably mediated through an interaction with GR: (i) the effects of Dex on EGFR expression are present at physiological concentrations comparable to the K, of GR; (ii) EGFR expression increase is specifically induced only by glucocorticoids and not by the other steroids tested. Northern blot analysis demonstrated an increase in EGFR transcription at times and levels

compatible with the results obtained in time course experiments of ‘251-EGF binding assay. Further studies are in progress in our laboratory to establish which are the specific mechanisms responsible for the enhancement of RNA levels in Dex treated cells. There is evidence (Fanger et al., 1984) that Dex may be effective in inducing EGFR at levels of transcription and translation in HeLa S3 cells. OVCA 433 cells did not show any cell cycle dependent variations in basal EGFR levels. Moreover Dex induced EGFR expression enhancement did not appear to be related to a recruitment of OVCA 433 cells in a specific phase of the cycle according to other studies (Fanger et al., 1986). It has been observed that growth inhibition induced by different agents is accompanied by EGFR up-regulation (Jetten et al., 1980; Zuckier et al., 1983) and it has been hypothesized that the increased receptor expression could be only part of a homeostatic cellular response to heavy antiproliferative stimuli. However, the bioflavonoid quercetin, an antiproliferative agent able to recruit OVCA 433 cells in the G,-G, phase (Scambia et al., 19901, had no effect on EGFR expression. This set of data indicates that the growth effects induced by Dex occur independently of EGFR expression modulation. This is consistent with the paper by Wu et al. (1981) showing the lack of connection between the growth effects and the modification of EGF binding in the presence of hydrocortisone. The demonstration that in JAl cells which express high affinity 1251-EGF binding sites but which are unresponsive to EGF, the exposure to Dex is followed by a growth inhibition, represents a further confirmation of our results. It has been reported that the overexpression of EGFR is associated with a growth inhibition induced by EGF (Gill et al., 1981; Barnes et al., 1982; Filmus et al., 19851. However, our results showed that the Dex induced up-regulation of EGFR does not influence EGFR function or intracellular signal transduction steps. There are several observations of the ability of peptide growth factors to overcome glucocorticoid growth inhibition on different normal and

192

neoplastic cell types (Syms et al., 1984; Smith et al., 1985; Rao et al., 1987). In particular the EGF induced reversion of Dex growth inhibition in HBLlOO cells leads to hypothesize that growth factor pathways may be dominant with respect to those by which glucocorticoids inhibit cell growth (Rae et al., 1987). In conclusion, our data demonstrate that EGFR expression modulation is not associated with the effects on ceIi growth induced by Dex on OVCA 433 cell growth. It is possible that the EGFR modulation induced by Dex may only be part of a more complex biochemical cascade in which EGFR expression enhancement or reduction are only incidentally related to growth effects. As far as Dex inhibitory growth effects are concerned, it may be hypothesized that glucocorticoids could also facilitate the establishment of a growth inhibitory activity by activating specific receptors (such as type II estrogen binding sites or transforming growth factor-13 receptors) for growth inhibitor substances (~arkaverich et ai., 1981; Berchuck et al., 1990; Scambia et al., 1990). Recently it has been reported that a monoclonal antibody (MoAb) anti-EGFR (Masui et al., 1988) and the MoAb anti-EGFR-gelonin conjugate (Ozawa et al., 19891 may inhibit breast cancer cell growth and squamous cell carcinoma cell lines. It is worth noting that the addition of EGF to OVCA 433 cells at the time of maximal Dex induced EGFR enhancement did not modify the proliferative response to EGF. It has been suggested that the growth effects by EGF are mediated by occupation of only a negligible fraction of binding sites (Schechter et al., 1979). Therefore the Dex induced EGFR enhancement could represent a biochemical basis for increasing the antiproliferative activity of Dex by immunologic approaches. References Amin, W., Karlan, B.Y. and Littlefield, B.A. (1987) Cancer Res. 47, 6040-6045. Baker, J.B., Barsh, G.S., Carney, D.H. and Cunningham, D.D. (1978) Proc. Nat]. Acad. Sci. USA 75(4), 1882-1886. Barnes, D.W. (1982) J. Ceil Biol. 93, 1-4. Bast, R.C., Feeney, M., Lazarus, H., Nadler, L.M., Calvin, R.B. and Knapp, R.C. (1981) J. Clin. Invest. 68,1331-1337.

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