The Prostate 1953-61 (1991)
Density-Dependent Regulation of Epidermal Growth Factor Receptor Expression in DU 145 Human Prostate Cancer Cells Joanne K. Tillotson and David P. Rose Division of Nutrition and Endocrinology, American Health Foundation, Valhalla, New York Androgen-independent prostate cancer cells may rely on an autocrine loop for growth stimulation, and have been shown to express both the epidermal growth factor receptor (EGFR) and its stimulatory ligands. We have shown here that DU 145 prostate cancer cells have a decreased amount of EGFR in confluent cultures when compared to levels seen in subconfluent cultures. This down-regulation of EGFR numbers is not due to cell proliferation or nutrient depletion, but can be correlated only with whether cell-cell contact exists throughout the culture. This is reminiscent of the situation existing in some tumors whereby EGFR expression is higher in cells at the invading margins of the tumor.
Key words: cell density, autocrine stimulation, cell-cell contact
INTRODUCTION Most human prostate cancers are initially dependent on androgens for their growth, and the patients respond well to surgical or medical treatment directed at the suppression of androgen action [ 13. However, a hormone-unresponsive stage of tumor growth eventually emerges, and when the disease progresses, the therapeutic options are limited. Prostate cancer cells are of epithelial origin, and several human prostate cancer cell lines have been shown to express the epidermal growth factor receptor [3-61 and secrete one or both of its natural ligands, epidermal growth factor (EGF) and transforming growth factor alpha (TGF-a) [2-61. However, the importance of this potential autocrine loop in either transformation or growth stimulation has not been adequately studied to date. One of the cell lines, designated DU 145, was derived from a brain metastasis of prostate adenocarcinoma [7]. These human prostate cancer cells are unresponsive to dihydrotestosterone added in culture [7], express relatively high levels of EGF receptor (EGFR) with apparently normal binding affinities [5], and secrete considerable quantities of EGF together with lower levels of TGF-a [3-61. They form tumors readily after injection into nude mice [8,9]. We selected the DU 145 cell line for initial studies of EGFR expression because
Received for publication November 26, 1990; accepted February 22, 1991. Address reprint requests to Dr. J.K. Tillotson, Division of Nutrition and Endocrinology, Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, NY 10595. 0 1991 Wiley-Liss, Inc.
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they represent a form of human tumors which is androgen-independent, and, therefore, may have become dependent on EGF-related autocrine growth factors. Further understanding of the role of these growth factors and their receptors may serve to elucidate mechanisms which could be exploited in future treatment regimens for hormone-unresponsive prostate cancer patients. Initial characterization of EGFR levels in DU 145 cells included the observation that receptor down-regulation occurs in culture after growth to high cell density levels. We report here an analysis of this phenomenon and its possible causes. MATERIALS AND METHODS Reagents
The RPMI- I640 culture medium and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY), insulin and aprotinin from Sigma Chemical Co. (St. Louis, MO), and delipidized bovine serum albumin (BSA) from Collaborative Research (Lexington, MA). The 35S-methionine and 35S-cysteine were obtained from Amersham Corp. (Arlington Heights, IL), and EGFR (Ab-1), TGF-a, and phosphotyrosine monoclonal antibodies for immunoprecipitations were from Oncogene Science (Manhasset, NY). Each of these antibodies has been shown to be highly specific [ 10-1 21. Cell Culture
DU 145 cells were obtained from the American Type Culture Collection (Rockville, MD) and routinely maintained at 37°C and 5% C0,/95% air in RPMI-1640 medium with 5% FBS plus 100,OOO units/liter penicillin and 100 mg/liter streptomycin. Cell counts were performed using a Coulter counter (Hialeah, FL). Metabolic Labeling
In experiments to quantitate EGFR concentrations, metabolic labeling of the receptor protein was undertaken. After incubation for 1 h in methionine-free RPMI1640, supplemented with 10 pg/ml insulin and 1.25 mg/ml BSA, the medium was replaced with fresh medium containing 100 p W m l each of 35S-methionine and 35S-cysteine. The cells were then incubated for 4 h at 37"C, the medium removed, and the monolayers washed with cold PBS before lysis and immunoprecipitation. lmmunoprecipitation
Proteins were immunoprecipitated from cell lysates or conditioned medium using specific monoclonal antibodies. Briefly, cell monolayers in T25 flasks were lysed in 3 ml/sample of lysis buffer (50 mM HEPES, pH 7.4, 50 mM NaC1, 25 mM KCI, 4 mM EDTA, 2 mM NaF, 10 mM Na4P,0,, 2 mM Na3V04, 1% Triton X-100, 1,OOO U/ml aprotinin). After clarifying the solution by centrifugation at 100,OOOg for 20 min, a constant fraction of either cell lysate or conditioned medium was incubated with 1 pg purified IgG and protein A-agarose at 4°C overnight with mixing. The protein A-agarose conjugates were gently pelleted by centrifugation and washed 4 times with fresh cold lysis buffer. The final pellet was either quantitated or resuspended in 20 pl SDS-electrophoresis sample buffer, boiled for 5 min, and the proteins from the entire pellet resolved by SDS-PAGE in 7.5% acrylamide gels. Efficiency of recovery - of the immune complexes was monitored by the stained bands
Epidermal Growth Factor Receptors in DU 145 Cells
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for IgG heavy and light chains. For metabolic labeling experiments, the gels were dried and the radioactivity was identified and quantitated on an Ambis Radioanalytic Imaging System (Ambis Systems, San Diego, CA). For non-radioactive experiments, gels were fixed and silver-stained, and an estimate of staining intensity was obtained using an LKB Ultro Scan XL scanning densitometer. In all cases, cell number per flask was determined on parallel cultures, and the EGFR cpm or densitometry data were normalized to a consistent cell number. RESULTS On 4 consecutive days after plating, duplicate flasks of DU 145 cells were either harvested for cell counts or metabolically labeled as described in “Materials and Methods. ” The 35S-labeled EGFR was immunoprecipitated and counted directly. Figure 1 shows the results for cell number and EGFR cpm/104 cells in two experiments (A and B) with plating densities which were approximately 2-fold greater for the experiment shown in panel B. In both sets, there was a lag period of several days slow growth before a large increase in cell numbers on day 4. Concurrent with the increase in cell number, the amount of labeled EGFR protein dropped to 10-15% of the earlier levels. Since this alteration in EGFR labeling could have been due to the effects of a variety of biologic or methodologic factors, additional experiments were designed to evaluate EGFR levels as a function of cell density alone. Aliquots containing 2 X lo6 DU 145 cells were plated at 6 different densities (in 1, 2, 3, 5 , 10, or 20 T25 flasks), and 35S-labeled,immunoprecipitated proteins were separated on SDS-PAGE gels. Figure 2 shows autoradiograms of proteins from these lysates immunoprecipitated by monoclonal antibodies to EGFR (A), phosphotyrosine (B), and TGF-a (C), respectively. The same antibodies were also used to precipitate labeled proteins from the medium; none of these proteins was detectable in the medium under these conditions. It is evident in Figure 2A that EGFR expression occurred at a lower level in lanes 1-3 than in the less densely seeded cultures shown in lanes 4-6. In contrast to the EGFR, Figure 2B shows that in all cultures (with the possible exception of the most dense culture shown in lane 1) the P-chain of the insulin receptor (IRP) was synthesized and autophosphorylated with similar efficiency, regardless of the plating density. Tyrosine phosphorylation of the EGFR was not seen in any lane, consistent with the observation that EGF secretion into the media did not occur in any of the samples. Table I shows the amount of EGFR and IRP, as determined from densitometric scans of the autoradiograms shown in Figure 2A,B, adjusted for cell numbers and compared with the cell density. This table demonstrates that at low densities (lanes 4-6, below about 30,000/cm2), the DU 145 cells produced a fairly constant amount of EGFR protein. However, at higher cell densities (lanes 1-3, above 30,000/cm2), a lower amount of EGFR is seen. The boundary between high and low EGFR production is roughly at the cell density where it was observed that cell-to-cell contact occurs throughout the culture. Similar densitometric data on the IRP bands shown in Figure 2B demonstrate that this protein (and/or its phosphorylation state) is not down-regulated in lanes 2, 3, although at the highest density, shown in lane 1, its expression appears to be depressed. Figure 2C shows the immunoprecipitation patterns obtained from cell lysates using the TGF-a specific monoclonal antibody. Specific TGF-a bands were not
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Fig. 1. %-EGFR labeling and cell densities on 4 consecutive days in DU 145 cultures seeded at different densities. Proteins were labeled and immunoprecipitated as described in “Materials and Methods” before counting in scintillation fluid to determine activity incorporated into the EGFR-fraction.
Fig. 2. Equal numbers of cells were distributed between a variable number of flasks such that after 24 h incubation the cultures ranged from low density to confluent. In order to minimize the accumulation of autocrine growth factors, media was changed at 24 h; labeling was initiated at 48 h after plating. The increase in cells recovered over plating number ranged from 5% at the highest density to 25% at the lowest density. DU 145 proteins were metabolically labeled with 35S-methionineand cysteine, immunoprecipitated from the cell lysate, and separated on SDS-PAGE.Monoclonal antibodies used in each panel were specific for (A) EGFR, (B)phosphotyrosine residues, and (C) TGF-a. Lanes 1-6 in each autoradiograph show the proteins precipitated from cultures in which 2 x lo6 cells were plated in 1, 2, 3, 5, 10, or 20 T-25flasks, respectively.
Epidermal Growth Factor Receptors in DU 145 Cells
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TABLE I. Relative Expression of EGFR and Phosphotyrosine-ContainingIRP in DUL45 Cell Cultures at Different Densities Cell density ceIIs/cm* 84,000 48,000 29,600 19,200 10,Ooo 4,800
Total cm2 25 50 15 125 250 500
Total cell number at harvest 2.1 x 106 2.4 X lo6 2.2 x 106 2.4 x lo6 2.5 x lo6 2.5 x lo6
Relative EGFR/106 cellsa .66 .68 .57 .99 .93 1 .00
Relative phos-tyrl lo6 cellsb .I5 .91 1
.oo .89 .95 .99
"The autoradiogram shown in Figure 2A was scanned densitometrically, the background in each lane was subtracted from the area, the value was normalized to total cell number, and the result adjusted relative to a value of 1 .O for the highest expression. bValues were derived for the IRP band shown in Figure 2B in the same manner as for EGFR values.
detected in any of the lysates, demonstrating the lack of TGFa synthesis, as well as serving as a non-specific control for background bands. No secretion of either EGF or TGFa into the medium was observed in any of these cultures, although we had previously shown that EGF can be detected in the medium from similar cultures at a later time (data not shown). Since the experiment shown in Figure 2 only evaluated newly synthesized protein, a similar experiment was conducted to examine total EGFR protein. Using larger flasks, equal aliquots of cells were again plated and allowed to equilibrate as before for 48 h. The cells were harvested and total immunoprecipitated EGFR was visualized on SDS-PAGE gels by silver staining and quantitated by densitometry. As shown in Figure 3, the highest density cultures again resulted in relatively low EGFR levels while the lower density cultures showed a higher level of expression. In both experiments shown in Figures 2 and 3, there was no evidence of a linear relationship between cell density and EGFR synthesis. Rather, there was an abrupt transition between high and low receptor expression at about 30,000 cells/cm2. Our accumulated experience is that cultures which appear to be 95-100% confluent by visual inspection would correlate with approximately 30,000 cells/cm2 for DU145 cells.
DISCUSSION
The development of prostate cancer therapeutic strategies which are based on the interruption of an EGF/TGFa-mediated autocrine loop is an approach which does not require the preservation of androgen dependence. In support of this concept, growth of the PC3 androgen-independent and LNCaP androgen-dependent prostate cancer cell lines has already been shown to be suppressed by suramin, a polyanionic naphthylurea which inhibits binding of EGF to its receptor [13,14]. Moreover, preliminary experiments in our laboratory have shown that DU 145 cell growth is inhibited in the presence of a blocking antibody to the EGFR (Connolly, J.M. and Rose, D.P., unpublished data). However, the interpretation and future application of such experiments which attempt to disrupt these receptor-ligand interactions require a better understanding of the biological regulation of both EGFR and ligand expres-
Epidermal Growth Factor Receptors in DU 145 Cells
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CELLS PER SO. CY. Fig. 3. EGFR were immunoprecipitated from DU 145 cultures in which 7.5 X lo6 cells were plated in 1 , 2, 3, 5, or 10 T-150 flasks. The resulting preparations were separated on SDS-PAGE and silver stained. The 170 kD bands were quantitated by densitometry and the values adjusted by the number of cells in each flask at harvest.
sion in prostate cancer cells. We have begun to pursue this issue by examining EGFR regulation in DU 145 prostate cancer cells. The experiments reported here have demonstrated how culture conditions influence EGFR levels. They showed that an inverse non-linear relationship exists between cell density and EGFR expression in cultured DU 145 prostate cancer cells. The methodologies employed suggest that this phenomenon is independent of several variables which might otherwise be postulated to affect cellular synthesis of the receptors, including the influence of cell proliferation or depletion of nutrients from the culture medium. Although cell numbers increased concurrently with receptor down-regulation in the first experiment, very little proliferation was observed under the conditions of the later experiments, and cannot be responsible for the downregulation of EGFR. Second, the later experiments effectively eliminate the possibility that down-regulation of EGFR was a consequence of nutrient depletion in rapidly proliferating cultures kept for longer periods of time. The demonstration that insulin receptors were produced and autophosphorylated equally at all densities suggests the lack of a general down-regulation of growth-related proteins and their functions. If higher densities of cells were rapidly using up nutrients which affect EGFR synthesis, a linear relationship would be expected instead of the sudden down-regulation which we observed. In addition, there have been several published reports which document EGFR down-regulation with increasing density in a number of non-prostatic cell lines [ 15-17]. However, EGFR modulation in these experiments appeared to occur over a wide range of cell densities rather than the abrupt pattern demonstrated in the present study (Figs. 2, 3; Table I). Although this abrupt downregulation may be a biological phenomenon unique to the DU 145 prostate cancer cells, it seems more likely that methodological differences would have prevented the observation of a similar effect in earlier studies. Thus, reported high affinity '251-EGF
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binding data [ 15-1 71 would be affected by variables which modify both synthesis and phosphorylation of EGFR,while variations in cell culture conditions could also affect many parameters of growth and protein synthesis regulation. It is intriguing to postulate that the extensive cell-cell contact achieved at confluence in our DU 145 cultures might have triggered a “switch” which downregulates the level of EGFR protein expressed. This possibility is especially significant in light of observations in other EGFR-expressing tumors that these receptors are present at higher levels on the invading margins of tumors [ 181. If the upregulation of EGFR reported here in sub-confluent cultured cells corresponds to the increased expression seen at the periphery of invading tumor margins, then this culture system may provide a convenient model for studying invasive properties of tumors. Further characterization of agents which would be capable of modulating such a “confluence switch” will assist in elucidating the mechanism and the significance for this mode of EGFR modulation in prostate cancer cells. ACKNOWLEDGMENTS We are most grateful to Jeanne Connolly for performing the cell cultures. This work was supported by a SIG from the American Cancer Society. REFERENCES I . Hodges CV: Hormone therapy of prostatic cancer. In Rose DP (ed): “Endocrinology of Cancer,” Vol. 2. Boca Raton: CRC Press, 1979, pp 57-67. 2. Schuurmans ALG, Bolt J, Mulder E: Androgens stimulate both growth rate and epidermal growth factor receptor activity of the human prostate tumor cell LNCaP. The Prostate 12:55-63, 1988. 3. Wilding G, Zugmeier G, Knabbe C. Valverius E, Flanders. K, Gelmann E P The role of transforming growth factors a and p in human prostate cancer cell growth. Proc Am Assoc Cancer Res 29:241, 1988. 4. Wilding G, Valverius E, Knabbe C, Gelman E P Role of transforming growth factor-a in human prostate cancer cell growth. The Prostate l5:l-l2, 1989. 5. Connolly JM, Rose DP: Secretion of epidermal growth factor and related polypeptides by the DU 145 human prostate cancer cell line. The Prostate 15:177-186, 1989. 6. Connolly JM, Rose DP: Production of epidermal growth factor and transforming growth factor a by the androgen-responsive LNCaP human prostate cancer cell line. The Prostate 16:209-218, 1990. 7. Stone KR, Mickey DD. Wunderli H. Mickey GH, Paulson DF: Isolation of a human prostate carcinoma cell line (DU 145). Int J Cancer 21:274-281, 1978. 8. Mickey DD, Stone KR, Wunderli H, Mickey GH, Vollmer RT, Paulson D F Heterotransplantation of a human prostatic adenocarcinoma cell line in nude mice. Cancer Res 37:4049-4058, 1977. 9. Rose DP, Cohen LA: Effects of dietary menhaden oil and retinyl acetate on the growth of DU 145 human prostatic adenocarcinoma cells transplanted into athymic nude mice. Carcinogenesis 9603605, 1988. 10. Kawamoto T, Sato JD, Le A, Polikoff J, Sato GH, Mendelsohn J: Growth stimulation of A431 cells by epidermal growth factor: Identification of high-affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody. Proc Natl Acad Sci USA 80:1337-1341, 1983. 11. Sorvillo JM, McCormack ES. Yanez L,Valenzuela D, Reynolds FH: Preparation and characterization of monoclonal antibodies specific for human transforming growth factor a. Oncogene 5:377386, 1990. 12. Huhn RD, Posner MR, Rayter SI, Foulkes JG, Frackelton AR: Cell lines and peripheral blood leukocytes derived from individuals with chronic myelogenous leukemia display virtually identical proteins phosphorylated on tyrosine residues. Proc Natl Acad Sci USA 84:4408-4412, 1987. 13. Fruehauf JP, Myers CE, Sinha BK: Synergistic activity of suramin with tumor necrosis factor a and doxorubicin on human prostate cancer cell lines. JNCl 82:1206-1209, 1990.
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14. Berns E, Schuurmans A, Bolt J, Lamb DJ, Foekens JA, Mulder E: Antiproliferative effects of suramin on androgen responsive tumor cells. Eur J Cancer 26:470-474, 1990. 15. Rizzino A, Kazakoff P, Ruff E, Kuszynski C, Nebelsick J: Regulatory effects of cell density on the binding of transforming growth factor p, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor. Cancer Res 39:4266-4271, 1988. 16. Rizzino A, Kazakoff P, Nebelsick J: Density-induced down regulation of epidermal growth factor receptors. In Vitro Cell Dev Biol 26:537-542, 1990. 17. Hollenberg MD, Barrett JC, Ts’o POP, Berhanu P: Selective reduction in receptors for epidermal growth factor-urogastrone in chemically transformed tumorigenic Syrian hamster embryo fibroblasts. Cancer Res 39:4166-4169, 1979. 18. Kearsley JH, Furlong KL, Cooke RA, Waters MJ: An immunohistochemicalassessment of cellular proliferation markers in head and neck squamous cell cancers. Br J Cancer 61:821-827, 1990.
Density-Dependent Regulation of Epidermal Growth Factor Receptor Expression in DU 145 Human Prostate Cancer Cells Joanne K. Tillotson and David P. Rose Division of Nutrition and Endocrinology, American Health Foundation, Valhalla, New York Androgen-independent prostate cancer cells may rely on an autocrine loop for growth stimulation, and have been shown to express both the epidermal growth factor receptor (EGFR) and its stimulatory ligands. We have shown here that DU 145 prostate cancer cells have a decreased amount of EGFR in confluent cultures when compared to levels seen in subconfluent cultures. This down-regulation of EGFR numbers is not due to cell proliferation or nutrient depletion, but can be correlated only with whether cell-cell contact exists throughout the culture. This is reminiscent of the situation existing in some tumors whereby EGFR expression is higher in cells at the invading margins of the tumor.
Key words: cell density, autocrine stimulation, cell-cell contact
INTRODUCTION Most human prostate cancers are initially dependent on androgens for their growth, and the patients respond well to surgical or medical treatment directed at the suppression of androgen action [ 13. However, a hormone-unresponsive stage of tumor growth eventually emerges, and when the disease progresses, the therapeutic options are limited. Prostate cancer cells are of epithelial origin, and several human prostate cancer cell lines have been shown to express the epidermal growth factor receptor [3-61 and secrete one or both of its natural ligands, epidermal growth factor (EGF) and transforming growth factor alpha (TGF-a) [2-61. However, the importance of this potential autocrine loop in either transformation or growth stimulation has not been adequately studied to date. One of the cell lines, designated DU 145, was derived from a brain metastasis of prostate adenocarcinoma [7]. These human prostate cancer cells are unresponsive to dihydrotestosterone added in culture [7], express relatively high levels of EGF receptor (EGFR) with apparently normal binding affinities [5], and secrete considerable quantities of EGF together with lower levels of TGF-a [3-61. They form tumors readily after injection into nude mice [8,9]. We selected the DU 145 cell line for initial studies of EGFR expression because
Received for publication November 26, 1990; accepted February 22, 1991. Address reprint requests to Dr. J.K. Tillotson, Division of Nutrition and Endocrinology, Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, NY 10595. 0 1991 Wiley-Liss, Inc.
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they represent a form of human tumors which is androgen-independent, and, therefore, may have become dependent on EGF-related autocrine growth factors. Further understanding of the role of these growth factors and their receptors may serve to elucidate mechanisms which could be exploited in future treatment regimens for hormone-unresponsive prostate cancer patients. Initial characterization of EGFR levels in DU 145 cells included the observation that receptor down-regulation occurs in culture after growth to high cell density levels. We report here an analysis of this phenomenon and its possible causes. MATERIALS AND METHODS Reagents
The RPMI- I640 culture medium and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY), insulin and aprotinin from Sigma Chemical Co. (St. Louis, MO), and delipidized bovine serum albumin (BSA) from Collaborative Research (Lexington, MA). The 35S-methionine and 35S-cysteine were obtained from Amersham Corp. (Arlington Heights, IL), and EGFR (Ab-1), TGF-a, and phosphotyrosine monoclonal antibodies for immunoprecipitations were from Oncogene Science (Manhasset, NY). Each of these antibodies has been shown to be highly specific [ 10-1 21. Cell Culture
DU 145 cells were obtained from the American Type Culture Collection (Rockville, MD) and routinely maintained at 37°C and 5% C0,/95% air in RPMI-1640 medium with 5% FBS plus 100,OOO units/liter penicillin and 100 mg/liter streptomycin. Cell counts were performed using a Coulter counter (Hialeah, FL). Metabolic Labeling
In experiments to quantitate EGFR concentrations, metabolic labeling of the receptor protein was undertaken. After incubation for 1 h in methionine-free RPMI1640, supplemented with 10 pg/ml insulin and 1.25 mg/ml BSA, the medium was replaced with fresh medium containing 100 p W m l each of 35S-methionine and 35S-cysteine. The cells were then incubated for 4 h at 37"C, the medium removed, and the monolayers washed with cold PBS before lysis and immunoprecipitation. lmmunoprecipitation
Proteins were immunoprecipitated from cell lysates or conditioned medium using specific monoclonal antibodies. Briefly, cell monolayers in T25 flasks were lysed in 3 ml/sample of lysis buffer (50 mM HEPES, pH 7.4, 50 mM NaC1, 25 mM KCI, 4 mM EDTA, 2 mM NaF, 10 mM Na4P,0,, 2 mM Na3V04, 1% Triton X-100, 1,OOO U/ml aprotinin). After clarifying the solution by centrifugation at 100,OOOg for 20 min, a constant fraction of either cell lysate or conditioned medium was incubated with 1 pg purified IgG and protein A-agarose at 4°C overnight with mixing. The protein A-agarose conjugates were gently pelleted by centrifugation and washed 4 times with fresh cold lysis buffer. The final pellet was either quantitated or resuspended in 20 pl SDS-electrophoresis sample buffer, boiled for 5 min, and the proteins from the entire pellet resolved by SDS-PAGE in 7.5% acrylamide gels. Efficiency of recovery - of the immune complexes was monitored by the stained bands
Epidermal Growth Factor Receptors in DU 145 Cells
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for IgG heavy and light chains. For metabolic labeling experiments, the gels were dried and the radioactivity was identified and quantitated on an Ambis Radioanalytic Imaging System (Ambis Systems, San Diego, CA). For non-radioactive experiments, gels were fixed and silver-stained, and an estimate of staining intensity was obtained using an LKB Ultro Scan XL scanning densitometer. In all cases, cell number per flask was determined on parallel cultures, and the EGFR cpm or densitometry data were normalized to a consistent cell number. RESULTS On 4 consecutive days after plating, duplicate flasks of DU 145 cells were either harvested for cell counts or metabolically labeled as described in “Materials and Methods. ” The 35S-labeled EGFR was immunoprecipitated and counted directly. Figure 1 shows the results for cell number and EGFR cpm/104 cells in two experiments (A and B) with plating densities which were approximately 2-fold greater for the experiment shown in panel B. In both sets, there was a lag period of several days slow growth before a large increase in cell numbers on day 4. Concurrent with the increase in cell number, the amount of labeled EGFR protein dropped to 10-15% of the earlier levels. Since this alteration in EGFR labeling could have been due to the effects of a variety of biologic or methodologic factors, additional experiments were designed to evaluate EGFR levels as a function of cell density alone. Aliquots containing 2 X lo6 DU 145 cells were plated at 6 different densities (in 1, 2, 3, 5 , 10, or 20 T25 flasks), and 35S-labeled,immunoprecipitated proteins were separated on SDS-PAGE gels. Figure 2 shows autoradiograms of proteins from these lysates immunoprecipitated by monoclonal antibodies to EGFR (A), phosphotyrosine (B), and TGF-a (C), respectively. The same antibodies were also used to precipitate labeled proteins from the medium; none of these proteins was detectable in the medium under these conditions. It is evident in Figure 2A that EGFR expression occurred at a lower level in lanes 1-3 than in the less densely seeded cultures shown in lanes 4-6. In contrast to the EGFR, Figure 2B shows that in all cultures (with the possible exception of the most dense culture shown in lane 1) the P-chain of the insulin receptor (IRP) was synthesized and autophosphorylated with similar efficiency, regardless of the plating density. Tyrosine phosphorylation of the EGFR was not seen in any lane, consistent with the observation that EGF secretion into the media did not occur in any of the samples. Table I shows the amount of EGFR and IRP, as determined from densitometric scans of the autoradiograms shown in Figure 2A,B, adjusted for cell numbers and compared with the cell density. This table demonstrates that at low densities (lanes 4-6, below about 30,000/cm2), the DU 145 cells produced a fairly constant amount of EGFR protein. However, at higher cell densities (lanes 1-3, above 30,000/cm2), a lower amount of EGFR is seen. The boundary between high and low EGFR production is roughly at the cell density where it was observed that cell-to-cell contact occurs throughout the culture. Similar densitometric data on the IRP bands shown in Figure 2B demonstrate that this protein (and/or its phosphorylation state) is not down-regulated in lanes 2, 3, although at the highest density, shown in lane 1, its expression appears to be depressed. Figure 2C shows the immunoprecipitation patterns obtained from cell lysates using the TGF-a specific monoclonal antibody. Specific TGF-a bands were not
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Fig. 1. %-EGFR labeling and cell densities on 4 consecutive days in DU 145 cultures seeded at different densities. Proteins were labeled and immunoprecipitated as described in “Materials and Methods” before counting in scintillation fluid to determine activity incorporated into the EGFR-fraction.
Fig. 2. Equal numbers of cells were distributed between a variable number of flasks such that after 24 h incubation the cultures ranged from low density to confluent. In order to minimize the accumulation of autocrine growth factors, media was changed at 24 h; labeling was initiated at 48 h after plating. The increase in cells recovered over plating number ranged from 5% at the highest density to 25% at the lowest density. DU 145 proteins were metabolically labeled with 35S-methionineand cysteine, immunoprecipitated from the cell lysate, and separated on SDS-PAGE.Monoclonal antibodies used in each panel were specific for (A) EGFR, (B)phosphotyrosine residues, and (C) TGF-a. Lanes 1-6 in each autoradiograph show the proteins precipitated from cultures in which 2 x lo6 cells were plated in 1, 2, 3, 5, 10, or 20 T-25flasks, respectively.
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TABLE I. Relative Expression of EGFR and Phosphotyrosine-ContainingIRP in DUL45 Cell Cultures at Different Densities Cell density ceIIs/cm* 84,000 48,000 29,600 19,200 10,Ooo 4,800
Total cm2 25 50 15 125 250 500
Total cell number at harvest 2.1 x 106 2.4 X lo6 2.2 x 106 2.4 x lo6 2.5 x lo6 2.5 x lo6
Relative EGFR/106 cellsa .66 .68 .57 .99 .93 1 .00
Relative phos-tyrl lo6 cellsb .I5 .91 1
.oo .89 .95 .99
"The autoradiogram shown in Figure 2A was scanned densitometrically, the background in each lane was subtracted from the area, the value was normalized to total cell number, and the result adjusted relative to a value of 1 .O for the highest expression. bValues were derived for the IRP band shown in Figure 2B in the same manner as for EGFR values.
detected in any of the lysates, demonstrating the lack of TGFa synthesis, as well as serving as a non-specific control for background bands. No secretion of either EGF or TGFa into the medium was observed in any of these cultures, although we had previously shown that EGF can be detected in the medium from similar cultures at a later time (data not shown). Since the experiment shown in Figure 2 only evaluated newly synthesized protein, a similar experiment was conducted to examine total EGFR protein. Using larger flasks, equal aliquots of cells were again plated and allowed to equilibrate as before for 48 h. The cells were harvested and total immunoprecipitated EGFR was visualized on SDS-PAGE gels by silver staining and quantitated by densitometry. As shown in Figure 3, the highest density cultures again resulted in relatively low EGFR levels while the lower density cultures showed a higher level of expression. In both experiments shown in Figures 2 and 3, there was no evidence of a linear relationship between cell density and EGFR synthesis. Rather, there was an abrupt transition between high and low receptor expression at about 30,000 cells/cm2. Our accumulated experience is that cultures which appear to be 95-100% confluent by visual inspection would correlate with approximately 30,000 cells/cm2 for DU145 cells.
DISCUSSION
The development of prostate cancer therapeutic strategies which are based on the interruption of an EGF/TGFa-mediated autocrine loop is an approach which does not require the preservation of androgen dependence. In support of this concept, growth of the PC3 androgen-independent and LNCaP androgen-dependent prostate cancer cell lines has already been shown to be suppressed by suramin, a polyanionic naphthylurea which inhibits binding of EGF to its receptor [13,14]. Moreover, preliminary experiments in our laboratory have shown that DU 145 cell growth is inhibited in the presence of a blocking antibody to the EGFR (Connolly, J.M. and Rose, D.P., unpublished data). However, the interpretation and future application of such experiments which attempt to disrupt these receptor-ligand interactions require a better understanding of the biological regulation of both EGFR and ligand expres-
Epidermal Growth Factor Receptors in DU 145 Cells
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CELLS PER SO. CY. Fig. 3. EGFR were immunoprecipitated from DU 145 cultures in which 7.5 X lo6 cells were plated in 1 , 2, 3, 5, or 10 T-150 flasks. The resulting preparations were separated on SDS-PAGE and silver stained. The 170 kD bands were quantitated by densitometry and the values adjusted by the number of cells in each flask at harvest.
sion in prostate cancer cells. We have begun to pursue this issue by examining EGFR regulation in DU 145 prostate cancer cells. The experiments reported here have demonstrated how culture conditions influence EGFR levels. They showed that an inverse non-linear relationship exists between cell density and EGFR expression in cultured DU 145 prostate cancer cells. The methodologies employed suggest that this phenomenon is independent of several variables which might otherwise be postulated to affect cellular synthesis of the receptors, including the influence of cell proliferation or depletion of nutrients from the culture medium. Although cell numbers increased concurrently with receptor down-regulation in the first experiment, very little proliferation was observed under the conditions of the later experiments, and cannot be responsible for the downregulation of EGFR. Second, the later experiments effectively eliminate the possibility that down-regulation of EGFR was a consequence of nutrient depletion in rapidly proliferating cultures kept for longer periods of time. The demonstration that insulin receptors were produced and autophosphorylated equally at all densities suggests the lack of a general down-regulation of growth-related proteins and their functions. If higher densities of cells were rapidly using up nutrients which affect EGFR synthesis, a linear relationship would be expected instead of the sudden down-regulation which we observed. In addition, there have been several published reports which document EGFR down-regulation with increasing density in a number of non-prostatic cell lines [ 15-17]. However, EGFR modulation in these experiments appeared to occur over a wide range of cell densities rather than the abrupt pattern demonstrated in the present study (Figs. 2, 3; Table I). Although this abrupt downregulation may be a biological phenomenon unique to the DU 145 prostate cancer cells, it seems more likely that methodological differences would have prevented the observation of a similar effect in earlier studies. Thus, reported high affinity '251-EGF
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Tillotson and Rose
binding data [ 15-1 71 would be affected by variables which modify both synthesis and phosphorylation of EGFR,while variations in cell culture conditions could also affect many parameters of growth and protein synthesis regulation. It is intriguing to postulate that the extensive cell-cell contact achieved at confluence in our DU 145 cultures might have triggered a “switch” which downregulates the level of EGFR protein expressed. This possibility is especially significant in light of observations in other EGFR-expressing tumors that these receptors are present at higher levels on the invading margins of tumors [ 181. If the upregulation of EGFR reported here in sub-confluent cultured cells corresponds to the increased expression seen at the periphery of invading tumor margins, then this culture system may provide a convenient model for studying invasive properties of tumors. Further characterization of agents which would be capable of modulating such a “confluence switch” will assist in elucidating the mechanism and the significance for this mode of EGFR modulation in prostate cancer cells. ACKNOWLEDGMENTS We are most grateful to Jeanne Connolly for performing the cell cultures. This work was supported by a SIG from the American Cancer Society. REFERENCES I . Hodges CV: Hormone therapy of prostatic cancer. In Rose DP (ed): “Endocrinology of Cancer,” Vol. 2. Boca Raton: CRC Press, 1979, pp 57-67. 2. Schuurmans ALG, Bolt J, Mulder E: Androgens stimulate both growth rate and epidermal growth factor receptor activity of the human prostate tumor cell LNCaP. The Prostate 12:55-63, 1988. 3. Wilding G, Zugmeier G, Knabbe C. Valverius E, Flanders. K, Gelmann E P The role of transforming growth factors a and p in human prostate cancer cell growth. Proc Am Assoc Cancer Res 29:241, 1988. 4. Wilding G, Valverius E, Knabbe C, Gelman E P Role of transforming growth factor-a in human prostate cancer cell growth. The Prostate l5:l-l2, 1989. 5. Connolly JM, Rose DP: Secretion of epidermal growth factor and related polypeptides by the DU 145 human prostate cancer cell line. The Prostate 15:177-186, 1989. 6. Connolly JM, Rose DP: Production of epidermal growth factor and transforming growth factor a by the androgen-responsive LNCaP human prostate cancer cell line. The Prostate 16:209-218, 1990. 7. Stone KR, Mickey DD. Wunderli H. Mickey GH, Paulson DF: Isolation of a human prostate carcinoma cell line (DU 145). Int J Cancer 21:274-281, 1978. 8. Mickey DD, Stone KR, Wunderli H, Mickey GH, Vollmer RT, Paulson D F Heterotransplantation of a human prostatic adenocarcinoma cell line in nude mice. Cancer Res 37:4049-4058, 1977. 9. Rose DP, Cohen LA: Effects of dietary menhaden oil and retinyl acetate on the growth of DU 145 human prostatic adenocarcinoma cells transplanted into athymic nude mice. Carcinogenesis 9603605, 1988. 10. Kawamoto T, Sato JD, Le A, Polikoff J, Sato GH, Mendelsohn J: Growth stimulation of A431 cells by epidermal growth factor: Identification of high-affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody. Proc Natl Acad Sci USA 80:1337-1341, 1983. 11. Sorvillo JM, McCormack ES. Yanez L,Valenzuela D, Reynolds FH: Preparation and characterization of monoclonal antibodies specific for human transforming growth factor a. Oncogene 5:377386, 1990. 12. Huhn RD, Posner MR, Rayter SI, Foulkes JG, Frackelton AR: Cell lines and peripheral blood leukocytes derived from individuals with chronic myelogenous leukemia display virtually identical proteins phosphorylated on tyrosine residues. Proc Natl Acad Sci USA 84:4408-4412, 1987. 13. Fruehauf JP, Myers CE, Sinha BK: Synergistic activity of suramin with tumor necrosis factor a and doxorubicin on human prostate cancer cell lines. JNCl 82:1206-1209, 1990.
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14. Berns E, Schuurmans A, Bolt J, Lamb DJ, Foekens JA, Mulder E: Antiproliferative effects of suramin on androgen responsive tumor cells. Eur J Cancer 26:470-474, 1990. 15. Rizzino A, Kazakoff P, Ruff E, Kuszynski C, Nebelsick J: Regulatory effects of cell density on the binding of transforming growth factor p, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor. Cancer Res 39:4266-4271, 1988. 16. Rizzino A, Kazakoff P, Nebelsick J: Density-induced down regulation of epidermal growth factor receptors. In Vitro Cell Dev Biol 26:537-542, 1990. 17. Hollenberg MD, Barrett JC, Ts’o POP, Berhanu P: Selective reduction in receptors for epidermal growth factor-urogastrone in chemically transformed tumorigenic Syrian hamster embryo fibroblasts. Cancer Res 39:4166-4169, 1979. 18. Kearsley JH, Furlong KL, Cooke RA, Waters MJ: An immunohistochemicalassessment of cellular proliferation markers in head and neck squamous cell cancers. Br J Cancer 61:821-827, 1990.
