The Prostate 19:173-180 (1991)
Autocrine Regulation of DU145 Human Prostate Cancer Cell Growth by Epidermal Growth Factor-Related Polypeptides Jeanne M. Connolly and David P. Rose
Division o f Nutrition and Endocrinology, Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, New York The DU145 human prostate cancer cell line possesses epidermal growth factor (EGF) receptors and synthesizes both EGF and the related polypeptide transforming growth factora (TGF-a). A monoclonal antibody to the EGF receptor was used to determine whether these characteristics were indicative of a functional autocrine regulatory system. This antibody competed effectively with [‘z51]EGFfor binding to DU145 cell binding sites over a 1 x lo-” to 1 X lo-’ M concentration range, and did so with a capability similar to that of the two natural ligands. It inhibited growth of these cells in both 3% fetal bovine serum-supplemented and serum-free medium; in experiments with incubation times of 3-5 days there was a 45-50% reduction in cell number. Growth suppression by the EGF receptor blockade of cells plated at a density of 1.5 X lo4 cells/ml/well was reversed competitively by the addition of EGF to the medium; 0.3 nM completely eliminated the M antibody concentration. It is concluded that DU145 cell inhibitory effect of a 1 X growth is regulated by an EGF-mediated autocrine loop.
Key words: receptor-blocking monoclonal antibody
INTRODUCTION
Human cancer cell lines of various origin possess receptors for EGF (EGF.R) and secrete TGF-a, an EGF-related polypeptide which exerts its biological effects by binding to the EGF.R, into the culture medium [l-81. It has been proposed that these features constitute an autocrine loop for the regulation of tumor cell growth, and that in the case of hormone-responsive breast cancers, response to estrogens may involve the stimulation of TGF-a production [2]. Most prostate cancers are androgen-dependent during the early stages of their natural history, but eventually a point is reached when steroid hormone suppression therapy no longer controls growth [9]. We have shown previously that the DU145 androgen-independent human prostate cancer cell line [ 101 specifically binds EGF and produces both EGF, and, in lesser amounts, TGF-a [6]. At high cell densities, the addition of EGF to serum-free culture medium had little effect on DU 145 cell
Received for publication May 13, 1991; accepted June 10, 1991. Address reprint requests to D.P. Rose, Division of Nutrition and Endocrinology, Naylor Dana Institute for Disease Prevention, Valhalla, NY 10595.
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growth, and we suggested that this was because the amounts of EGF-related polypeptides being secreted by the cells were sufficient for optimal stimulation by these growth factors [6,8]. However, in the case of the MDA-MB-231 human breast cancer cell line, combined expression of EGF.R and TGF-a secretion does not appear to affect growth, suggesting that under some circumstances there may be circumvention of such a potential autocrine loop [ 1 I ] . In the present study, we used a blocking monoclonal antibody to the EGF.R to demonstrate the presence of intact autocrine regulation, the involvement of endogenously produced EGF-related polypeptides in DU145 prostate cancer cell growth, and the competitive reversal of anti-EGF.R growth inhibition by exogenous EGF. MATERIALS AND METHODS Materials Insulin was purchased from Sigma Chemical Co. (St. Louis, MO), delipidized bovine serum albumin (BSA) from Collaborative Research (Lexington, MA), ['2sII]humanEGF, 125 pgCi/pg, from Biomedical Technologies, Inc. (Cambridge, MA), a non-specific mouse IgG, from Chemicon (El Segundo, CA), and human EGF, human TGF-a, and monoclonal anti-human EGF.R (catalogue No. 05-101; lot number 10508) from Upstate Biotechnology Inc. (Lake Placid, NY). RPMI-1640 culture medium was obtained from Gibco (Grand Island, NY) and fetal bovine serum (FBS) was from Hyclone (Logan, UT). Radioreceptor assays for EGF-related polypeptides performed in our laboratory [12] did not detect EGF.R-binding growth factors in 3-10% FBS-supplemented RPMI- 1640 medium. Cell Culture The DU145 prostate cancer cell line was obtained from the American Type Culture Collection (Rockville, MD) and was cultured routinely in RPMI-1640 medium with 5% FBS plus 100,000 units/L penicillin and 100 mg/L streptomycin in a 95% air/5% C 0 2 incubator at 37°C. Binding Experiments The DU145 cells were plated at 1.5 X lo4 cells/ml/well in 5% FBS-supplemented phenol red-free RPMI-1640. After 48 h of incubation at 37"C, and at approximately 70% confluence, the cells were refed. Two hours before beginning a binding experiment, the medium was changed to phenol red-free RPMI- 1640 containing 0.1% delipidized BSA. At time zero, the medium was again changed to incorporate one of the three competitors, EGF, TGF-a or anti-EGF.R, at concentrations ranging from lo-'' to lo-' M. The ['2'I]EGF in 100 p1 of RPMI-1640 plus 0.1% BSA was immediately added to each well to a final concentration of 8 x lo-'' M, mixed, and incubation was performed at 0°C (ice-bath) for 2 h. All concentrations of each competing ligand were set up in triplicate. A range of concentrations of non-specific mouse IgG, was set up as a negative control, together with triplicate wells to provide for the total cell counts. Growth Experiments
These were performed in 24-well plates (Costar, Cambridge, MA). The cells were first plated at a density of 1.5 x lo4 cells/ml/well, and incubated for 24 h in 5%
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Fig. 1 . Competition between [1Z51]EGFand unlabeled EGF (A),TGF-a ( O ) , and EGF.R monoclonal antibody ( 0 )for binding to DU145 cells. Binding is expressed as percent of maximum [1251]EGFbinding. Incubation was for 2 hr at 0°C.
FJ3S-supplemented phenol red-free RPMI-1640 to allow attachment. They were then washed with unsupplemented RPMI- 1640, and the experimental medium was added to the wells, which were set up in triplicate. Incubation was continued for 2 or 3 days, or for 5 days with refeeding after 3 days, and the cells were counted with an electronic particle counter (Model F; Coulter Electronics, Hialeah, FL). Statistical Comparisons These were made using Student's unpaired t test; values of P < .05 were regarded as significant. RESULTS competition for the EGF.R
Competitive binding experiments showed that EGF, TGF-a, and anti-EGF. R were all effective, and to similar degrees, in blocking the binding of [1251]EGFto DU145 prostate cancer cells (Fig. 1). The non-specific IgG, had no effect on the binding of labeled EGF to the receptor. Effects of Anti-EGF.R on DU145 Cell Growth
In the first growth experiment, 1.5 X lo4 DU145 cells were plated, allowed to attach in 5% FBS-RPMI-1640 medium for 24 h, and then cultured in RPMI-1640 supplemented with 3% FBS for 2, 3, and 5 days; corresponding triplicate sets of wells contained a 2 X lop9 M concentration of EGF.R antibody. Figure 2 shows that at each of the three time points blocking the EGF.R resulted in a 45-50% inhibition of cell growth compared with corresponding control. When only 0.5 X lo4 cells were plated the degree of growth inhibition was reduced to 25%. The relationship between anti-EGF.R concentration and DU 145 cell growth inhibition was examined in RPMI-1640 supplemented with 3% FBS with a 3 day incubation period. Anti-EGF.R at levels ranging from 1 X lo-'' to 1 X lo-' M was
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Fig. 2. Effect of EGF.R antibody ( 2 x IO-'M) on DU145 cell growth over 2 , 3 , or 5 days with a plating density of 1.5 x 10' cells/ml/well ("Materials and Methods"), and over 3 days with a plating density of 0.5 x lo4 cells/ml/well. The solid bars represent the cell number (mean ? SE for triplicate wells) in the absence, and the cross-hatched bars in the presence of the EGF.R antibody. The levels of growth inhibition as a percent of control cell numbers were "23% (not significant. P = .08);b50% ( P < .001); '44% ( P < ,001);d458 ( P < .oOl).
added to triplicate wells. Figure 3 shows that an anti-EGF.R concentration-related growth inhibition occurred, but which plateaued in the presence of 1 X lop9 M and above of the antibody. A 1 x lo-' M concentration of non-specific IgG, had no demonstrable effect on cell growth (data not shown). An experiment was performed to determine whether the growth inhibitory effect of anti-EGF.R was reversible by high concentrations of exogenous EGF. One set of DU145 cells was initially plated at a density of 1.5 X lo4 cells/ml/well and again grown for 3 days in RPMI-I640 plus 3% FBS. The results are shown in Figure 4.In the absence of added EGF, the anti-EGF.R at a concentration of I X M caused a 44% inhibition of cell growth compared with the control wells (9.98 ? 0.39 vs. 5.56 ? 0.28 x lo4 cells, P < .001). At 0.3 nM, EGF completely eliminated the inhibitory activity of anti-EGF.R (control wells, 9.98 0.39; anti-EGF.R plus 0.3 nM EGF, 10.20 2 1.40 x lo4 cells). In this experiment, EGF in the absence of the antibody caused stimulation of DU 145 growth in the FBS-supplemented medium 0.39 vs. 12.71 ? 0.33 x lo4 cells/ml/well; P < .001), and at an EGF (9.98 concentration of 10 nM the level of antibody used was insufficient to prevent a growth response. Another set of cells was plated at a density of only 0.5 x lo4 ml/well in order to reduce the levels of endogenously secreted EGF and TGF-a in the medium. Here, the inhibitory effect of anti-EGF.R in the absence of added EGF was only 23% and did not achieve statistical significance (P = .08);this modest growth inhibition by the antibody was eliminated completely by adding EGF at concentrations of 2 and 10 nM (Fig. 5). Although radioreceptor assays for EGF-like polypeptides using the A43 1 cell EGF.R had failed to detect EGF/TGF-a in medium supplemented with up to 10%
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Fig. 3. Effect of EGF.R antibody concentration (lo-'' to lo-' M) on DU145 cell growth over a 3 day incubation period. Cell number (mean & SE for triplicate wells) in the presence of the antibody was significantly reduced compared with control wells: ' P < .05; * P < .01; **P < ,001.
Fig. 4. Reversal of EGF.R antibody ( 1 X M) inhibition of DU145 cell growth by human EGF after plating at a density of 1.5 X lo4 cells/ml/well and 3 days incubation. The cross-hatched bars represent the cell number (mean SE for triplicate wells) after growth in the presence of EGF alone (0-20 nM), and the solid bars in the presence of both EGF and EGF.R antibody ( I x M). Significantly different from corresponding cell number with EGF alone: ' P < .05; **P < ,001,
*
FBS, the effect of anti-EGF.R on DU145 prostate cancer cell growth in serum-free medium was also assessed. The cells were plated at 1.5 X lo4 cells/ml/well, incubated for 24 h in 5% FBS-supplemented RPMI-1640, washed with the basal medium, and then cultured in phenol red-free RPMI-1640 plus BSA, 1.25 mg/ml, and linoleic M anti-EGF.R for 3 days. Triplicate acid, 750 ng/ml, with or without 5 X control wells contained 5% FBS-supplemented medium. Table 1 shows that the cells grew well in the serum-free medium, the cell number after 3 days of growth being 85% of that reached in 5% FBS-supplemented RPMI-1640; the presence of the anti-EGF.R caused a 46% inhibition of cell growth (P < .002).
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Fig. 5. Effects of EGF.R antibody ( I X lo-' M) and human EGF on DU145 cells plated at a density of 0.5 x lo4 cells/ml/well with a 3 day incubation. The cross-hatched bars represent the cell number (mean 2 SE for triplicate wells) after growth in the presence of EGF alone (0-20 nM) and the solid bars in the presence of both EGF and EGF.R antibody ( I x lo-' M). None of the statistical comparisons achieved statistical significance at the P < .05 level.
TABLE I. The Effect of Anti-EGF.R on DU145 Prostate Cancer Cell Growth in Serum-Free Medium Addition None anti-EGF.R. 5 x lo-' M 5% FBS
Cell number x 10' 5.13 2 0.37 2.78 2 0.37* 6.01 2 0.20
* P < .002 compared with the serum-free control medium.
DISCUSSION
We reported previously that the addition of EGF to serum-free medium has some stimulatory effect on [3H]thymidine incorporation into DU I45 cells growing at low density, but without a corresponding increase in cell number [ 6 ] .It was suggested that failure of the cells to undergo mitosis was due to a lack of essential nutrients or non-EGF-related growth factors contained in serum. This conclusion is supported by the observation that while growth of the androgen-responsive LNCaP prostate cancer cell line is stimulated by exogenous EGF there is only a weak reponse by these cells in the absence of charcoal-stripped serum [ 131, and by the present finding that DU 145 cell proliferation in the presence of 3% FBS-supplemented medium, which did not contain detectable levels of EGF-related polypeptides, is stimulated by exogenous EGF. Androgen-independent DU 145 prostate cancer cells produce EGF and secrete relatively large amounts into the culture medium; in a previous study we found that when normalized for cell number the levels detected were approximately 14-fold those found in medium conditioned by androgen-responsive LNCaP cells [ 81. Moreover, after plating at an initial low cell density, EGF secretion by DU145 cells is highest on the day before a phase of rapid cell growth [14]. In addition to EGF,
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DU145 and LNCaP cells produce TGF-a, albeit at considerably lower concentrations [6,81. These observations are all consistent with autocrine stimulation of DU145 cells by tumor-secreted EGF and TGF-a and suggest that this may provide, at least in part, the mechanism for escape from androgen dependence. The present demonstrationthat inhibition of binding of the natural ligands to the EGF.R suppresses DU145 cell growth, and that this effect is reversed by high concentrations of exogenous EGF, provides direct evidence for the existence of a functioning EGF/TGF-a-mediated autocrine loop. At low cell density, the anti-EGF.R had relatively little effect on DU145 cell growth, a result consistent with there being only a low level of autocrine activity when EGF/TGF-a concentrations produced by the cells are limited by their population density. Antibodies to the EGF.R have been used previously to evaluate the autocrine regulation of human cancer cells [ 11,151. Taetle et al. [ 151 observed growth inhibition of MDA-468 breast cancer and A431 vulvar epidermoid carcinoma cells, both of which exhibit amplified EGF.R expression, each having approximately 3 x lo6 sitedcell [12,15]. However, the same two anti-EGF.Rs had no effect on ovarian and epidermoid carcinoma cell lines with an estimated 120 and 400 X lo' receptorskell, respectively. Inhibition of MDA-468 cell growth by one of these monoclonal antibodies was confirmed by Ennis et al. [l 11, who also reported no growth suppression of the MDA-231 breast cancer cell line, despite the capacity of these cells to bind EGF-related polypeptides with approximately 257 X lo3 EGF.R siteskell [ 161, and to transcribe TGF-a mRNA. It was concluded that the potential autocrine loop was non-functional in MDA-23 1 cells, perhaps because they express an activated protooncogene which permits by-pass of the EGF/TGF-a-mediated system of growth regulation. These results, together with those of Taetle et al. [ 151 might be interpreted as indicating that the EGF.R antibody exerts an inhibitory effect only on cancer cell lines with an abnormally high number of receptor sites per cell. However, the present results with prostate cancer cells show that overexpression of the EGF.R is not a prerequisite for EGF/TGF-a autocrine stimulation of human cancer cell growth; DU145 cells possess only 180 x 10' binding sitedcell [17], or 106 x 10' highaffinity receptorskell [6], which is similar to the EGF.R number of the unregulated MDA-MB-231 breast cancer cell line. In the case of the estrogen-dependentMCF-7 breast cancer cell line, Bates et al. [ 181 have demonstrated that anti-EGF.R inhibits estradiol-stimulated cell proliferation, and similar experiments utilizing the hormone-responsive LNCaP human prostate cancer cell line [8] are indicated to investigate further the relationship between androgenic growth stimulation and the EGF/TGF-a autocrine loop.
REFERENCES 1. Salomon DS, Zwiebel JA, Ban0 M, Losonczy I, Fehnel P, Kidwell WR: Presence of transforming
growth factors in human breast cancer cells. Cancer Res 44:4069-4077, 1984. 2. Lippman ME, Dickson RB, Gelmdnn EP, Rosen N, Knabbe C, Bates S , Bronzert D, Huff K, Kdsid A: Growth regulation of human breast carcinoma occurs through regulated growth factor secretion. J Cell Biochem 35:l-16, 1987. 3. Coffey RJ Jr, Goustin AS, Mangelsdorf' Soderquist A, Shipley GD, Wolfshohl J , Carpenter G ,
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Moses HL: Transforming growth factor a and p expression in human colon cancer lines: implications for an autocrine model. Cancer Res 47:4590-4594, 1987. 4. Derynck R , Goeddel DV, Ullrich A, Gutterman JU, Williams RD, Bringman TS, Berger WH: Synthesis of messenger RNAs for transforming growth factors a and p and the epidermal growth factor receptor by human tumors. Cancer Res 47:707-712, 1987. 5 . Imanishi K, Yamaguchi K, Suzuki M, Honda S , Yanaihara N, Abe K: Production of transforming growth factor-a in human tumour cell lines. Br J Cancer 59:761-765, 1989. 6. Connolly JM, Rose DP: Secretion of epidermal growth factor and related polypeptides by the DU145 human prostate cancer cell line. The Prostate 15:177-186, 1989. 7. Wilding G, Valverius E, Knabbe C, Gelman EP: Role of transforming growth factor-a in human prostate cancer cell growth. The Prostate 15:l-12, 1989. 8. Connolly JM, Rose DP: Production of epidermal growth factor and trandorming growth factor a by the androgen-responsive LNCaP human prostate cancer cell line. The Prostate 16: IW-218, 1990. 9. Hodges CV: Hormone therapy of prostatic cancer. In: Rose DP (ed): “Endocrinology of Cancer,” Vol. 2. Boca Raton: CRC Press, 1979, pp 57-67. 10. Stone KR. Mickey DD, Wunderli H, Mickey GH, Paulson DF: Isolation of a human prostate carcinoma cell line (DU145). Int J Cancer 21:274-281, 1978. 1 I . Ennis BW, Valverius EM, Bates SE. Lippman ME, Bellot F, Kris R, Schlessinger J, Masui H, Goldenberg A, Mendelsohn J , Dickson RB: Anti-epidermal growth factor receptor antibodies inhibit the autocrine-stimulated growth of MDA-468 human breast cancer cells. Mol Endocrinol 3:18301838, 1989. 12. Connolly JM. Rose DP: Quantitative differences in the effects of mouse and human epidermal growth factors on A431 human tumor cells. Cancer Lett 37:241-249. 1987. 13. Schuurmans ALG, Bolt J, Mulder E: Androgens stimulate both growth rate and epidermal growth factor activity of the human prostate tumor cell LNCaP. The Prostate 1 2 5 - 6 3 , 1988. 14. Tillotson JK, Rose DP: Epidermal growth factor-mediated regulation of DU145 prostate cancer cell growth. Proc Am Assoc Cancer Res 32:5 I, 1991. 15. Taetle R, Honeysett JM, Houston LL: Effects of anti-epidermal growth factor (EGF) receptor antibodies and an anti-EGF receptor recombinant-ricin A chain immunoconjugate on growth of human cells. JNCI 80:1053-1059, 1988. 16. Davidson NE, Gelmann EP, Lippman ME. Dickson RB: Epidermal growth factor receptor gene expression in estrogen receptor-positive and negative human breast cancer cell lines. Mol Endocrinol 1:216-223, 1987. 17. Wilding G, Zugmeier G , Knabbe C, Valverius E, Flanders K, Gelmann EP: The role of transforming growth factors a and p in human prostate cancer cell growth. Proc Am Assoc Cancer Res 29:241, 1988. 18. Bates SE, Davidson NE, Valverius EM, Freter CE. Dickson RB, Tam JP, Kudlow JE, Lippman ME, Salomon DS: Expression of transforming growth factor a and its messenger ribonucleic acid in human breast cancer: its regulation by estrogen and its possible functional significance. Mol Endocrinol 2543-555, 1988.
Autocrine Regulation of DU145 Human Prostate Cancer Cell Growth by Epidermal Growth Factor-Related Polypeptides Jeanne M. Connolly and David P. Rose
Division o f Nutrition and Endocrinology, Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, New York The DU145 human prostate cancer cell line possesses epidermal growth factor (EGF) receptors and synthesizes both EGF and the related polypeptide transforming growth factora (TGF-a). A monoclonal antibody to the EGF receptor was used to determine whether these characteristics were indicative of a functional autocrine regulatory system. This antibody competed effectively with [‘z51]EGFfor binding to DU145 cell binding sites over a 1 x lo-” to 1 X lo-’ M concentration range, and did so with a capability similar to that of the two natural ligands. It inhibited growth of these cells in both 3% fetal bovine serum-supplemented and serum-free medium; in experiments with incubation times of 3-5 days there was a 45-50% reduction in cell number. Growth suppression by the EGF receptor blockade of cells plated at a density of 1.5 X lo4 cells/ml/well was reversed competitively by the addition of EGF to the medium; 0.3 nM completely eliminated the M antibody concentration. It is concluded that DU145 cell inhibitory effect of a 1 X growth is regulated by an EGF-mediated autocrine loop.
Key words: receptor-blocking monoclonal antibody
INTRODUCTION
Human cancer cell lines of various origin possess receptors for EGF (EGF.R) and secrete TGF-a, an EGF-related polypeptide which exerts its biological effects by binding to the EGF.R, into the culture medium [l-81. It has been proposed that these features constitute an autocrine loop for the regulation of tumor cell growth, and that in the case of hormone-responsive breast cancers, response to estrogens may involve the stimulation of TGF-a production [2]. Most prostate cancers are androgen-dependent during the early stages of their natural history, but eventually a point is reached when steroid hormone suppression therapy no longer controls growth [9]. We have shown previously that the DU145 androgen-independent human prostate cancer cell line [ 101 specifically binds EGF and produces both EGF, and, in lesser amounts, TGF-a [6]. At high cell densities, the addition of EGF to serum-free culture medium had little effect on DU 145 cell
Received for publication May 13, 1991; accepted June 10, 1991. Address reprint requests to D.P. Rose, Division of Nutrition and Endocrinology, Naylor Dana Institute for Disease Prevention, Valhalla, NY 10595.
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growth, and we suggested that this was because the amounts of EGF-related polypeptides being secreted by the cells were sufficient for optimal stimulation by these growth factors [6,8]. However, in the case of the MDA-MB-231 human breast cancer cell line, combined expression of EGF.R and TGF-a secretion does not appear to affect growth, suggesting that under some circumstances there may be circumvention of such a potential autocrine loop [ 1 I ] . In the present study, we used a blocking monoclonal antibody to the EGF.R to demonstrate the presence of intact autocrine regulation, the involvement of endogenously produced EGF-related polypeptides in DU145 prostate cancer cell growth, and the competitive reversal of anti-EGF.R growth inhibition by exogenous EGF. MATERIALS AND METHODS Materials Insulin was purchased from Sigma Chemical Co. (St. Louis, MO), delipidized bovine serum albumin (BSA) from Collaborative Research (Lexington, MA), ['2sII]humanEGF, 125 pgCi/pg, from Biomedical Technologies, Inc. (Cambridge, MA), a non-specific mouse IgG, from Chemicon (El Segundo, CA), and human EGF, human TGF-a, and monoclonal anti-human EGF.R (catalogue No. 05-101; lot number 10508) from Upstate Biotechnology Inc. (Lake Placid, NY). RPMI-1640 culture medium was obtained from Gibco (Grand Island, NY) and fetal bovine serum (FBS) was from Hyclone (Logan, UT). Radioreceptor assays for EGF-related polypeptides performed in our laboratory [12] did not detect EGF.R-binding growth factors in 3-10% FBS-supplemented RPMI- 1640 medium. Cell Culture The DU145 prostate cancer cell line was obtained from the American Type Culture Collection (Rockville, MD) and was cultured routinely in RPMI-1640 medium with 5% FBS plus 100,000 units/L penicillin and 100 mg/L streptomycin in a 95% air/5% C 0 2 incubator at 37°C. Binding Experiments The DU145 cells were plated at 1.5 X lo4 cells/ml/well in 5% FBS-supplemented phenol red-free RPMI-1640. After 48 h of incubation at 37"C, and at approximately 70% confluence, the cells were refed. Two hours before beginning a binding experiment, the medium was changed to phenol red-free RPMI- 1640 containing 0.1% delipidized BSA. At time zero, the medium was again changed to incorporate one of the three competitors, EGF, TGF-a or anti-EGF.R, at concentrations ranging from lo-'' to lo-' M. The ['2'I]EGF in 100 p1 of RPMI-1640 plus 0.1% BSA was immediately added to each well to a final concentration of 8 x lo-'' M, mixed, and incubation was performed at 0°C (ice-bath) for 2 h. All concentrations of each competing ligand were set up in triplicate. A range of concentrations of non-specific mouse IgG, was set up as a negative control, together with triplicate wells to provide for the total cell counts. Growth Experiments
These were performed in 24-well plates (Costar, Cambridge, MA). The cells were first plated at a density of 1.5 x lo4 cells/ml/well, and incubated for 24 h in 5%
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Fig. 1 . Competition between [1Z51]EGFand unlabeled EGF (A),TGF-a ( O ) , and EGF.R monoclonal antibody ( 0 )for binding to DU145 cells. Binding is expressed as percent of maximum [1251]EGFbinding. Incubation was for 2 hr at 0°C.
FJ3S-supplemented phenol red-free RPMI-1640 to allow attachment. They were then washed with unsupplemented RPMI- 1640, and the experimental medium was added to the wells, which were set up in triplicate. Incubation was continued for 2 or 3 days, or for 5 days with refeeding after 3 days, and the cells were counted with an electronic particle counter (Model F; Coulter Electronics, Hialeah, FL). Statistical Comparisons These were made using Student's unpaired t test; values of P < .05 were regarded as significant. RESULTS competition for the EGF.R
Competitive binding experiments showed that EGF, TGF-a, and anti-EGF. R were all effective, and to similar degrees, in blocking the binding of [1251]EGFto DU145 prostate cancer cells (Fig. 1). The non-specific IgG, had no effect on the binding of labeled EGF to the receptor. Effects of Anti-EGF.R on DU145 Cell Growth
In the first growth experiment, 1.5 X lo4 DU145 cells were plated, allowed to attach in 5% FBS-RPMI-1640 medium for 24 h, and then cultured in RPMI-1640 supplemented with 3% FBS for 2, 3, and 5 days; corresponding triplicate sets of wells contained a 2 X lop9 M concentration of EGF.R antibody. Figure 2 shows that at each of the three time points blocking the EGF.R resulted in a 45-50% inhibition of cell growth compared with corresponding control. When only 0.5 X lo4 cells were plated the degree of growth inhibition was reduced to 25%. The relationship between anti-EGF.R concentration and DU 145 cell growth inhibition was examined in RPMI-1640 supplemented with 3% FBS with a 3 day incubation period. Anti-EGF.R at levels ranging from 1 X lo-'' to 1 X lo-' M was
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Fig. 2. Effect of EGF.R antibody ( 2 x IO-'M) on DU145 cell growth over 2 , 3 , or 5 days with a plating density of 1.5 x 10' cells/ml/well ("Materials and Methods"), and over 3 days with a plating density of 0.5 x lo4 cells/ml/well. The solid bars represent the cell number (mean ? SE for triplicate wells) in the absence, and the cross-hatched bars in the presence of the EGF.R antibody. The levels of growth inhibition as a percent of control cell numbers were "23% (not significant. P = .08);b50% ( P < .001); '44% ( P < ,001);d458 ( P < .oOl).
added to triplicate wells. Figure 3 shows that an anti-EGF.R concentration-related growth inhibition occurred, but which plateaued in the presence of 1 X lop9 M and above of the antibody. A 1 x lo-' M concentration of non-specific IgG, had no demonstrable effect on cell growth (data not shown). An experiment was performed to determine whether the growth inhibitory effect of anti-EGF.R was reversible by high concentrations of exogenous EGF. One set of DU145 cells was initially plated at a density of 1.5 X lo4 cells/ml/well and again grown for 3 days in RPMI-I640 plus 3% FBS. The results are shown in Figure 4.In the absence of added EGF, the anti-EGF.R at a concentration of I X M caused a 44% inhibition of cell growth compared with the control wells (9.98 ? 0.39 vs. 5.56 ? 0.28 x lo4 cells, P < .001). At 0.3 nM, EGF completely eliminated the inhibitory activity of anti-EGF.R (control wells, 9.98 0.39; anti-EGF.R plus 0.3 nM EGF, 10.20 2 1.40 x lo4 cells). In this experiment, EGF in the absence of the antibody caused stimulation of DU 145 growth in the FBS-supplemented medium 0.39 vs. 12.71 ? 0.33 x lo4 cells/ml/well; P < .001), and at an EGF (9.98 concentration of 10 nM the level of antibody used was insufficient to prevent a growth response. Another set of cells was plated at a density of only 0.5 x lo4 ml/well in order to reduce the levels of endogenously secreted EGF and TGF-a in the medium. Here, the inhibitory effect of anti-EGF.R in the absence of added EGF was only 23% and did not achieve statistical significance (P = .08);this modest growth inhibition by the antibody was eliminated completely by adding EGF at concentrations of 2 and 10 nM (Fig. 5). Although radioreceptor assays for EGF-like polypeptides using the A43 1 cell EGF.R had failed to detect EGF/TGF-a in medium supplemented with up to 10%
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Fig. 3. Effect of EGF.R antibody concentration (lo-'' to lo-' M) on DU145 cell growth over a 3 day incubation period. Cell number (mean & SE for triplicate wells) in the presence of the antibody was significantly reduced compared with control wells: ' P < .05; * P < .01; **P < ,001.
Fig. 4. Reversal of EGF.R antibody ( 1 X M) inhibition of DU145 cell growth by human EGF after plating at a density of 1.5 X lo4 cells/ml/well and 3 days incubation. The cross-hatched bars represent the cell number (mean SE for triplicate wells) after growth in the presence of EGF alone (0-20 nM), and the solid bars in the presence of both EGF and EGF.R antibody ( I x M). Significantly different from corresponding cell number with EGF alone: ' P < .05; **P < ,001,
*
FBS, the effect of anti-EGF.R on DU145 prostate cancer cell growth in serum-free medium was also assessed. The cells were plated at 1.5 X lo4 cells/ml/well, incubated for 24 h in 5% FBS-supplemented RPMI-1640, washed with the basal medium, and then cultured in phenol red-free RPMI-1640 plus BSA, 1.25 mg/ml, and linoleic M anti-EGF.R for 3 days. Triplicate acid, 750 ng/ml, with or without 5 X control wells contained 5% FBS-supplemented medium. Table 1 shows that the cells grew well in the serum-free medium, the cell number after 3 days of growth being 85% of that reached in 5% FBS-supplemented RPMI-1640; the presence of the anti-EGF.R caused a 46% inhibition of cell growth (P < .002).
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EQF
(nM)
Fig. 5. Effects of EGF.R antibody ( I X lo-' M) and human EGF on DU145 cells plated at a density of 0.5 x lo4 cells/ml/well with a 3 day incubation. The cross-hatched bars represent the cell number (mean 2 SE for triplicate wells) after growth in the presence of EGF alone (0-20 nM) and the solid bars in the presence of both EGF and EGF.R antibody ( I x lo-' M). None of the statistical comparisons achieved statistical significance at the P < .05 level.
TABLE I. The Effect of Anti-EGF.R on DU145 Prostate Cancer Cell Growth in Serum-Free Medium Addition None anti-EGF.R. 5 x lo-' M 5% FBS
Cell number x 10' 5.13 2 0.37 2.78 2 0.37* 6.01 2 0.20
* P < .002 compared with the serum-free control medium.
DISCUSSION
We reported previously that the addition of EGF to serum-free medium has some stimulatory effect on [3H]thymidine incorporation into DU I45 cells growing at low density, but without a corresponding increase in cell number [ 6 ] .It was suggested that failure of the cells to undergo mitosis was due to a lack of essential nutrients or non-EGF-related growth factors contained in serum. This conclusion is supported by the observation that while growth of the androgen-responsive LNCaP prostate cancer cell line is stimulated by exogenous EGF there is only a weak reponse by these cells in the absence of charcoal-stripped serum [ 131, and by the present finding that DU 145 cell proliferation in the presence of 3% FBS-supplemented medium, which did not contain detectable levels of EGF-related polypeptides, is stimulated by exogenous EGF. Androgen-independent DU 145 prostate cancer cells produce EGF and secrete relatively large amounts into the culture medium; in a previous study we found that when normalized for cell number the levels detected were approximately 14-fold those found in medium conditioned by androgen-responsive LNCaP cells [ 81. Moreover, after plating at an initial low cell density, EGF secretion by DU145 cells is highest on the day before a phase of rapid cell growth [14]. In addition to EGF,
DU145 Cells and EGF
179
DU145 and LNCaP cells produce TGF-a, albeit at considerably lower concentrations [6,81. These observations are all consistent with autocrine stimulation of DU145 cells by tumor-secreted EGF and TGF-a and suggest that this may provide, at least in part, the mechanism for escape from androgen dependence. The present demonstrationthat inhibition of binding of the natural ligands to the EGF.R suppresses DU145 cell growth, and that this effect is reversed by high concentrations of exogenous EGF, provides direct evidence for the existence of a functioning EGF/TGF-a-mediated autocrine loop. At low cell density, the anti-EGF.R had relatively little effect on DU145 cell growth, a result consistent with there being only a low level of autocrine activity when EGF/TGF-a concentrations produced by the cells are limited by their population density. Antibodies to the EGF.R have been used previously to evaluate the autocrine regulation of human cancer cells [ 11,151. Taetle et al. [ 151 observed growth inhibition of MDA-468 breast cancer and A431 vulvar epidermoid carcinoma cells, both of which exhibit amplified EGF.R expression, each having approximately 3 x lo6 sitedcell [12,15]. However, the same two anti-EGF.Rs had no effect on ovarian and epidermoid carcinoma cell lines with an estimated 120 and 400 X lo' receptorskell, respectively. Inhibition of MDA-468 cell growth by one of these monoclonal antibodies was confirmed by Ennis et al. [l 11, who also reported no growth suppression of the MDA-231 breast cancer cell line, despite the capacity of these cells to bind EGF-related polypeptides with approximately 257 X lo3 EGF.R siteskell [ 161, and to transcribe TGF-a mRNA. It was concluded that the potential autocrine loop was non-functional in MDA-23 1 cells, perhaps because they express an activated protooncogene which permits by-pass of the EGF/TGF-a-mediated system of growth regulation. These results, together with those of Taetle et al. [ 151 might be interpreted as indicating that the EGF.R antibody exerts an inhibitory effect only on cancer cell lines with an abnormally high number of receptor sites per cell. However, the present results with prostate cancer cells show that overexpression of the EGF.R is not a prerequisite for EGF/TGF-a autocrine stimulation of human cancer cell growth; DU145 cells possess only 180 x 10' binding sitedcell [17], or 106 x 10' highaffinity receptorskell [6], which is similar to the EGF.R number of the unregulated MDA-MB-231 breast cancer cell line. In the case of the estrogen-dependentMCF-7 breast cancer cell line, Bates et al. [ 181 have demonstrated that anti-EGF.R inhibits estradiol-stimulated cell proliferation, and similar experiments utilizing the hormone-responsive LNCaP human prostate cancer cell line [8] are indicated to investigate further the relationship between androgenic growth stimulation and the EGF/TGF-a autocrine loop.
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