Proc. Nati. Acad. Sci. USA Vol. 75, No. 10, pp. 4972-4976, October 1978
Cell Biology
Coordinate control of corticotropin, f3-lipotropin, and f3-endorphin release in mouse pituitary cell cultures (radioimmunoassay/gel electrophoresis)
RICHARD G. ALLEN*, EDWARD HERBERT*t, MICHAEL HINMAN*, HARUO SHIBUYAf, AND CANDACE B. PERTt * Department of Chemistry, University of Oregon, Eugene, Oregon 97403; and * Section of Biochemistry, Adult Psychiatry Branch, National Institute of Mental Health, Bethesda, Maryland 20014
Communicated by George Streisinger, July 10, 1978
ABSTRACT Hypothalamic extract stimulates the release of corticotropin (ACH) and endorphins 2.5- to 30-fold in mouse pituitary tumor cell cultures (AtT-20/D16, line) and primary cell cultures from mouse anterior pituitar. ACTH and endorhin activities were measured by radioimmunoassay and immu* noprecipitation. Pretreatment of tumor cell cultures with 1 MM dexamethasone reduced the stimulatory effect of the extract on release of ACTH and endorphins. Pretreatment of primary cell cultures with 10-6 M dexamethasone reduced the stimulatory effect of both vasopressin and the extract on the release of ACTH and endorphins. Release of ACTR and endorphin was coupled in both kinds of cultures in the basal, stimulated, and inhibited states. The molecular weight forms of ACTH and endorphin in tumor cell culture medium were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Radioimmunoassay and immunoprecipitation show that the 13,000-dalton and 4500-dalton forms of ACTH were present in about equal amounts in medium from cultures incubated with or without hypothalamic extract for 15 min, 30 min, or 2 hr. Smaller amounts of the high molecular weight forms of ACTH (20,000- to 23,000-dalton and 31,000-dalton ACTIH) were observed in the culture medium at these times. The predominant forms of endorphin released after 20 min or 3 hr of incubation had molecular weights of 31,000, 11,700 (0-lipotropic hormone-size material) and 3500 (P-endorphin-size material). No degradation of the forms of endorphin released into the culture medium was observed after incubating the culture medium for 1.5 hr in the absence of cells. The proportions of the different forms of endorphin and ACTH present in the culture medium resembles that seen in cell extracts.
dition, the forms of ACTH and endorphin released by the tumor cells have been investigated. MATERIALS AND METHODS Culture Methods and Pretreatment with Dexamethasone. AtT-20/D16, cells were grown in 2.0 ml of Dulbecco-Vogt minimal essential medium containing 10% horse serum in 3.5-cm culture dishes as described (8). When the cultures reached 50% confluency, they were incubated for 48-52 hr with medium of the same composition with or without ,uM dexamethasone. The cultures were washed for 15 min at 370 in serum-free medium (8). The cultures were then incubated in serum-free test medium with or without hypothalamic extract (HE) or vasopressin for the times indicated. Preparation of Primary Cell Cultures from Mouse Anterior Pituitary. Pituitaries were dissected into Vale 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer (9), pH 7.2/3 mM EDTA and the lobes were separated. Anterior pituitary lobes were sliced several times with a razor blade and soaked 40 min at 37° in 1 ml of Vale-Hepes (9) containing 800 international units of collagenase (Sigma Cat. C0130) and 750 national formulary units of hyaluronidase (Sigma H2126) with 5 tyg/ml DNase (Worthington) added to prevent clumping. The pieces of pituitary were rinsed with Vale-Hepes buffer containing 3 mM EDTA, neuraminidase (Sigma Type V) at 4 tlg/ml, and bovine serum albumin (Sigma A4378) at 10 ,ug/ml and incubated for 10 min at 370 in 1 ml of the same solution. The pieces were then rinsed with Vale-Hepes buffer containing bovine serum albumin at 10 ,g/ml and DNase at 5 ,g/ml and flushed gently through a fire-polished siliconized pipette with 0.6 ml of the same solution, and the supernatant was transferred to a conical centrifuge tube. The flushing procedure was repeated twice and the combined supernatants (1.8 ml) were centrifuged for 1.5 min at 200-400 X g. The pellet was resuspended gently with the same pipette and 0.6 ml of the ValeHepes solution containing bovine serum albumin and DNase and centrifuged 1 min at 150 X g. The cells were resuspended three more times, using culture medium containing only 1.85 g of NaHCO3 per liter (low CO2 medium) plus 10% horse serum the last two times. The final pellet was resuspended in low CO2 medium/10% horse serum (0.3 ml per dish) and plated in plastic tissue culture dishes containing 1.2 ml. of the same medium that had been preincubated in a 5% CO2 incubator. Dispersed cells were incubated for 4 days before the experiments were performed. Extracts of 4-day cultures showed the same distribution of Abbreviations: ACTH, corticotropin; LPH, lipotropic hormone; NaDodSO4, sodium dodecyl sulfate; HE, hypothalamic extract; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; RIA, radioim-
Corticotropin (ACTH), ,B-endorphin, and ,B-melanocyte stimulating hormone-have recently been shown to be synthesized as part of a. high-molecular-weight precursor molecule in mouse pituitary tumor cells (AtT-20/Dl6v) (1-3). The same kind of precursor molecule is also present in extracts of anterior and intermediate lobes of mouse pituitary (4). Several observations suggest that release of the three biologically active polypeptides contained in the common precursor might be coupled. First, the blood levels of ACTH and f3-melanocyte stimulating hormone rise and fall together under some circumstances (5, 6). Second, the blood levels of ACTH and f3-endorphin rise together within minutes after stressing a rat by breaking its leg (7). In the present study, pituitary cell culture systems have been used to determine whether the release of ACTH and endorphin is coupled. Release of ACTH in these cultures is stimulated by corticotropic releasing hormone(s) and inhibited by glucocorticoids (8) in much the same manner as in primary pituitary cell cultures. AtT-20/D16, cell cultures have been used as a model system for determining whether ACTH and endorphin release is coupled in the basal, stimulated, and inhibited states. In adThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
munoassay. t To whom requests for reprints should be addressed.
4972
Proc. Natl. Acad. Sci. USA 75 (1978)
Cell Biology: Allen et al. molecular weight forms of ACTH and endorphin as did extracts of anterior pituitary (unpublished results). Preparation of Samples for Radioimmunoassay and Gel Electrophoresis. Medium was harvested as previously described for radioimmunoassay (RIA) (8). Cells were scraped from the plates, extracted in 200-300 ,l of 5 M acetic acid, heated at 700 for 1 hr, and then boiled for 1 min or left at room temperature for 1 hr. The acid extracts from each plate were lyophilized, resuspended in 100 Ml of 1.0% NaDodSO4/6 M urea/2.5% 2-mercaptoethanol, heated for 15 min at 850, and applied to the gels. Culture medium was prepared for gel electrophoresis by adding 50 til of the latter solution directly to 200 ,l of serum-free medium. The samples were heated at 1000 for 1 min and applied to the gels. NaDodSO4/Polyacrylamide Gel Electrophoresis. Gel electrophoresis of cell extracts and tissue culture medium was performed as described by Mains and Eipper (10) by using tube gels composed of 10% polyacrylamide/0.54% N,N'-methylenebisacrylamide. Dansylated yeast alcohol dehydrogenase, dansylated myoglobin, and 125I-labeled porcine ACTH were used as internal molecular weight markers. The gels were sliced and eluted as described by Mains and Eipper (10). Gel electrophoresis of immunoprecipitated culture medium was performed on 12% Bio-Phore gels. Sample preparation followed the method of Roberts and Herbert (2). Antisera. ACTH was assayed with an antiserum that is specific for the a(4-20) region of ACTH (8). This antiserum crossreacts with high molecular weight forms of ACTH (2) and a(1-39) ACTH but not with fl-lipotropic hormone (LPH) or (3-endorphin. The forms of endorphin were assayed with an antiserum (RB-100) that is specific for the (3-endorphin region of 3-LPH [(61-91)LPH]. This antiserum, which was a generous gift from Roger Guillemin of the Salk Institute, reacts equally well, on a molar basis, with ,B-LPH and f3-endorphin, but it does not react with ACTH (11). RB-100 also reacts with the high molecular weight (31,000) precursor forms of ACTH and B3-LPH (11). An antiserum (RB-66) to a-endorphin [p3(70-84)LPH] was also used in some of these studies. RB-66 recognizes the ,B(70-76) sequence of O-LPH. It reacts with a-endorphin but not with fl-endorphin or ,B-LPH (11). Another endorphin antiserum (F-F) was raised in a rabbit by injecting a-endorphin coupled to bovine serum albumin (12). F-F reacts equally well with ,B-endorphin and fl-LPH on a molar basis. Radioimmunoassay. The RIAs were performed according to the method of Rees et al. (13) modified by Herbert et al. (8). Porcine ACTH (Sigma Chemical Co.) was used as a standard after purification by gel-filtration chromatography on Sephadex G-50 columns (8). Synthetic porcine a- and O-endorphins used as standards in the endorphin RIAs were a gift from R. Guillemin, T. Vargo, and N. Ling of the Salk Institute. Porcine ACTH and a- and 3-endorphins were iodinated with hypochlorite (14). Immunoprecipitations. Immunoprecipitations were performed in one of two ways: (i) ACTH immunoprecipitations were performed by the double antibody method (10) with antiserum Bertha or (ii) endorphin immunoprecipitations were performed as described by Roberts and Herbert (2) with antiserum F-F and Staphylococcus aureus Cowan I. Completeness of immunoprecipitation was tested by adding a second aliquot of antibody to the supernatant remaining after the first immunoprecipitation; no further ACTH or endorphin was precipitated by the second aliquot of antibody. Extraction and Gel Filtration of Hypothalami. Crude extracts of rabbit hypothalamus (Pel-Freez Co.) (8) were used in the tumor cell experiments described in Figs. 1, 3, and 4. For experiments with primary cell cultures, five rabbit hypothalami
4973
were extracted (8), treated with iodoacetamide and phenylmethylsulfonyl fluoride (2, 3), and fractionated on a column of Sephadex G-25 superfine (1 X 50 cm) at 40 using 1% acetic acid for elution. Column fractions (0.9 ml) were lyophilized, redissolved in serum-free tissue culture medium, and tested for releasing activity as described in Fig. 1. RESULTS Effect of Dexamethasone and Crude HE on ACTH and Endorphin Levels in Tumor Cell Culture Medium. HEs contain an elusive entity named corticotropin releasing liormone (15). The AtT-20/D16, cell line is a useful system to study the effect of HE on release because of the large amounts of endorphin and ACTH the cells contain. Previous work has shown that stimulation of ACTH release in cultures of AtT-20/Dl6v cells by HE is dependent upon the dose of HE (8). In addition, the response to HE is decreased when the cells havebeen treatedwithidexamethasone (11 M1)forq 48 hr prior to addition of the extract (8). Because the tumor cells have been shown to contain endorphin (1-3), we repeated the experiment and assayed the culture medium after 3 hr of exposure to different doses of crude HE. The RIA-ACTH and RIA-endorphin results are shown in Fig. 1. Release of both ACTH and endorphin was stimulated by HE and inhibited by dexamethasone. The same result was observed when endorphin activity was assayed by the radioreceptor assay of Pert and Bowie (16) instead of by the RIA (results not shown). Polyacrylamide Gel Electrophoresis of Tumor Cell Extracts and Tumor Cell Culture Medium. When tumor cell extracts were fractionated by NaDodSO4/gel electrophoresis and eluates of gel slices were assayed with ACTH and gl-endorphin antisera, four major size classes of ACTH and three major size classes of g-endorphin were detected (Fig. 2). The 4500-dalton form of ACTH is a(I-39) ACTH (17, 4). The 13,000-dalton form of ACTH is a glycosylated form of a(1-39) ACTH (17). The 11,700-dalton form of f3-endorphin is the size of #-LPH and the 3500-dalton form is the size of f3-endorphin. The 31,000-dalton class of molecules contains the determinants 3.0k V. a)
Cn
2.0p-
4) Q .>+
CE0. . C
Co
0
_
1.01-
T.
I
0 0 0 0 0 0 25 100 200 0 25 100 He(MIl)0 FIG. 1. Effects of dexamethasone and HE on release of ACTH and f3-endorphin activities in AtT-20/D16v cultures. Cells were grown to half confluency in Dulbecco-Vogt minimal essential medium with 10% horse serum (9) in 3.5-cm culture dishes containing 2.0 ml of medium. The medium was changed and cultures were incubated for 48 hr with or without 1 MM dexamethasone (dex) with one change of medium at 24 hr. The medium was replaced with 2.0 ml of serum-free test medium containing the amounts of rabbit HE shown below the histogram, and the incubation was continued for 3.0 hr. Medium was harvested and either frozen or assayed directly by RIA with ACTH and fl-endorphin antisera. The results are the mean + SD of triplicate plates. O, RIA-fl-endorphin; O, RIA-ACTH. dex
-
201 .00
Cell Biology: Allen et al.
4974
Proc. Natl. Acad. Sci. USA 75 (1978) c
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Cell Biology
Coordinate control of corticotropin, f3-lipotropin, and f3-endorphin release in mouse pituitary cell cultures (radioimmunoassay/gel electrophoresis)
RICHARD G. ALLEN*, EDWARD HERBERT*t, MICHAEL HINMAN*, HARUO SHIBUYAf, AND CANDACE B. PERTt * Department of Chemistry, University of Oregon, Eugene, Oregon 97403; and * Section of Biochemistry, Adult Psychiatry Branch, National Institute of Mental Health, Bethesda, Maryland 20014
Communicated by George Streisinger, July 10, 1978
ABSTRACT Hypothalamic extract stimulates the release of corticotropin (ACH) and endorphins 2.5- to 30-fold in mouse pituitary tumor cell cultures (AtT-20/D16, line) and primary cell cultures from mouse anterior pituitar. ACTH and endorhin activities were measured by radioimmunoassay and immu* noprecipitation. Pretreatment of tumor cell cultures with 1 MM dexamethasone reduced the stimulatory effect of the extract on release of ACTH and endorphins. Pretreatment of primary cell cultures with 10-6 M dexamethasone reduced the stimulatory effect of both vasopressin and the extract on the release of ACTH and endorphins. Release of ACTR and endorphin was coupled in both kinds of cultures in the basal, stimulated, and inhibited states. The molecular weight forms of ACTH and endorphin in tumor cell culture medium were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Radioimmunoassay and immunoprecipitation show that the 13,000-dalton and 4500-dalton forms of ACTH were present in about equal amounts in medium from cultures incubated with or without hypothalamic extract for 15 min, 30 min, or 2 hr. Smaller amounts of the high molecular weight forms of ACTH (20,000- to 23,000-dalton and 31,000-dalton ACTIH) were observed in the culture medium at these times. The predominant forms of endorphin released after 20 min or 3 hr of incubation had molecular weights of 31,000, 11,700 (0-lipotropic hormone-size material) and 3500 (P-endorphin-size material). No degradation of the forms of endorphin released into the culture medium was observed after incubating the culture medium for 1.5 hr in the absence of cells. The proportions of the different forms of endorphin and ACTH present in the culture medium resembles that seen in cell extracts.
dition, the forms of ACTH and endorphin released by the tumor cells have been investigated. MATERIALS AND METHODS Culture Methods and Pretreatment with Dexamethasone. AtT-20/D16, cells were grown in 2.0 ml of Dulbecco-Vogt minimal essential medium containing 10% horse serum in 3.5-cm culture dishes as described (8). When the cultures reached 50% confluency, they were incubated for 48-52 hr with medium of the same composition with or without ,uM dexamethasone. The cultures were washed for 15 min at 370 in serum-free medium (8). The cultures were then incubated in serum-free test medium with or without hypothalamic extract (HE) or vasopressin for the times indicated. Preparation of Primary Cell Cultures from Mouse Anterior Pituitary. Pituitaries were dissected into Vale 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer (9), pH 7.2/3 mM EDTA and the lobes were separated. Anterior pituitary lobes were sliced several times with a razor blade and soaked 40 min at 37° in 1 ml of Vale-Hepes (9) containing 800 international units of collagenase (Sigma Cat. C0130) and 750 national formulary units of hyaluronidase (Sigma H2126) with 5 tyg/ml DNase (Worthington) added to prevent clumping. The pieces of pituitary were rinsed with Vale-Hepes buffer containing 3 mM EDTA, neuraminidase (Sigma Type V) at 4 tlg/ml, and bovine serum albumin (Sigma A4378) at 10 ,ug/ml and incubated for 10 min at 370 in 1 ml of the same solution. The pieces were then rinsed with Vale-Hepes buffer containing bovine serum albumin at 10 ,g/ml and DNase at 5 ,g/ml and flushed gently through a fire-polished siliconized pipette with 0.6 ml of the same solution, and the supernatant was transferred to a conical centrifuge tube. The flushing procedure was repeated twice and the combined supernatants (1.8 ml) were centrifuged for 1.5 min at 200-400 X g. The pellet was resuspended gently with the same pipette and 0.6 ml of the ValeHepes solution containing bovine serum albumin and DNase and centrifuged 1 min at 150 X g. The cells were resuspended three more times, using culture medium containing only 1.85 g of NaHCO3 per liter (low CO2 medium) plus 10% horse serum the last two times. The final pellet was resuspended in low CO2 medium/10% horse serum (0.3 ml per dish) and plated in plastic tissue culture dishes containing 1.2 ml. of the same medium that had been preincubated in a 5% CO2 incubator. Dispersed cells were incubated for 4 days before the experiments were performed. Extracts of 4-day cultures showed the same distribution of Abbreviations: ACTH, corticotropin; LPH, lipotropic hormone; NaDodSO4, sodium dodecyl sulfate; HE, hypothalamic extract; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; RIA, radioim-
Corticotropin (ACTH), ,B-endorphin, and ,B-melanocyte stimulating hormone-have recently been shown to be synthesized as part of a. high-molecular-weight precursor molecule in mouse pituitary tumor cells (AtT-20/Dl6v) (1-3). The same kind of precursor molecule is also present in extracts of anterior and intermediate lobes of mouse pituitary (4). Several observations suggest that release of the three biologically active polypeptides contained in the common precursor might be coupled. First, the blood levels of ACTH and f3-melanocyte stimulating hormone rise and fall together under some circumstances (5, 6). Second, the blood levels of ACTH and f3-endorphin rise together within minutes after stressing a rat by breaking its leg (7). In the present study, pituitary cell culture systems have been used to determine whether the release of ACTH and endorphin is coupled. Release of ACTH in these cultures is stimulated by corticotropic releasing hormone(s) and inhibited by glucocorticoids (8) in much the same manner as in primary pituitary cell cultures. AtT-20/D16, cell cultures have been used as a model system for determining whether ACTH and endorphin release is coupled in the basal, stimulated, and inhibited states. In adThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
munoassay. t To whom requests for reprints should be addressed.
4972
Proc. Natl. Acad. Sci. USA 75 (1978)
Cell Biology: Allen et al. molecular weight forms of ACTH and endorphin as did extracts of anterior pituitary (unpublished results). Preparation of Samples for Radioimmunoassay and Gel Electrophoresis. Medium was harvested as previously described for radioimmunoassay (RIA) (8). Cells were scraped from the plates, extracted in 200-300 ,l of 5 M acetic acid, heated at 700 for 1 hr, and then boiled for 1 min or left at room temperature for 1 hr. The acid extracts from each plate were lyophilized, resuspended in 100 Ml of 1.0% NaDodSO4/6 M urea/2.5% 2-mercaptoethanol, heated for 15 min at 850, and applied to the gels. Culture medium was prepared for gel electrophoresis by adding 50 til of the latter solution directly to 200 ,l of serum-free medium. The samples were heated at 1000 for 1 min and applied to the gels. NaDodSO4/Polyacrylamide Gel Electrophoresis. Gel electrophoresis of cell extracts and tissue culture medium was performed as described by Mains and Eipper (10) by using tube gels composed of 10% polyacrylamide/0.54% N,N'-methylenebisacrylamide. Dansylated yeast alcohol dehydrogenase, dansylated myoglobin, and 125I-labeled porcine ACTH were used as internal molecular weight markers. The gels were sliced and eluted as described by Mains and Eipper (10). Gel electrophoresis of immunoprecipitated culture medium was performed on 12% Bio-Phore gels. Sample preparation followed the method of Roberts and Herbert (2). Antisera. ACTH was assayed with an antiserum that is specific for the a(4-20) region of ACTH (8). This antiserum crossreacts with high molecular weight forms of ACTH (2) and a(1-39) ACTH but not with fl-lipotropic hormone (LPH) or (3-endorphin. The forms of endorphin were assayed with an antiserum (RB-100) that is specific for the (3-endorphin region of 3-LPH [(61-91)LPH]. This antiserum, which was a generous gift from Roger Guillemin of the Salk Institute, reacts equally well, on a molar basis, with ,B-LPH and f3-endorphin, but it does not react with ACTH (11). RB-100 also reacts with the high molecular weight (31,000) precursor forms of ACTH and B3-LPH (11). An antiserum (RB-66) to a-endorphin [p3(70-84)LPH] was also used in some of these studies. RB-66 recognizes the ,B(70-76) sequence of O-LPH. It reacts with a-endorphin but not with fl-endorphin or ,B-LPH (11). Another endorphin antiserum (F-F) was raised in a rabbit by injecting a-endorphin coupled to bovine serum albumin (12). F-F reacts equally well with ,B-endorphin and fl-LPH on a molar basis. Radioimmunoassay. The RIAs were performed according to the method of Rees et al. (13) modified by Herbert et al. (8). Porcine ACTH (Sigma Chemical Co.) was used as a standard after purification by gel-filtration chromatography on Sephadex G-50 columns (8). Synthetic porcine a- and O-endorphins used as standards in the endorphin RIAs were a gift from R. Guillemin, T. Vargo, and N. Ling of the Salk Institute. Porcine ACTH and a- and 3-endorphins were iodinated with hypochlorite (14). Immunoprecipitations. Immunoprecipitations were performed in one of two ways: (i) ACTH immunoprecipitations were performed by the double antibody method (10) with antiserum Bertha or (ii) endorphin immunoprecipitations were performed as described by Roberts and Herbert (2) with antiserum F-F and Staphylococcus aureus Cowan I. Completeness of immunoprecipitation was tested by adding a second aliquot of antibody to the supernatant remaining after the first immunoprecipitation; no further ACTH or endorphin was precipitated by the second aliquot of antibody. Extraction and Gel Filtration of Hypothalami. Crude extracts of rabbit hypothalamus (Pel-Freez Co.) (8) were used in the tumor cell experiments described in Figs. 1, 3, and 4. For experiments with primary cell cultures, five rabbit hypothalami
4973
were extracted (8), treated with iodoacetamide and phenylmethylsulfonyl fluoride (2, 3), and fractionated on a column of Sephadex G-25 superfine (1 X 50 cm) at 40 using 1% acetic acid for elution. Column fractions (0.9 ml) were lyophilized, redissolved in serum-free tissue culture medium, and tested for releasing activity as described in Fig. 1. RESULTS Effect of Dexamethasone and Crude HE on ACTH and Endorphin Levels in Tumor Cell Culture Medium. HEs contain an elusive entity named corticotropin releasing liormone (15). The AtT-20/D16, cell line is a useful system to study the effect of HE on release because of the large amounts of endorphin and ACTH the cells contain. Previous work has shown that stimulation of ACTH release in cultures of AtT-20/Dl6v cells by HE is dependent upon the dose of HE (8). In addition, the response to HE is decreased when the cells havebeen treatedwithidexamethasone (11 M1)forq 48 hr prior to addition of the extract (8). Because the tumor cells have been shown to contain endorphin (1-3), we repeated the experiment and assayed the culture medium after 3 hr of exposure to different doses of crude HE. The RIA-ACTH and RIA-endorphin results are shown in Fig. 1. Release of both ACTH and endorphin was stimulated by HE and inhibited by dexamethasone. The same result was observed when endorphin activity was assayed by the radioreceptor assay of Pert and Bowie (16) instead of by the RIA (results not shown). Polyacrylamide Gel Electrophoresis of Tumor Cell Extracts and Tumor Cell Culture Medium. When tumor cell extracts were fractionated by NaDodSO4/gel electrophoresis and eluates of gel slices were assayed with ACTH and gl-endorphin antisera, four major size classes of ACTH and three major size classes of g-endorphin were detected (Fig. 2). The 4500-dalton form of ACTH is a(I-39) ACTH (17, 4). The 13,000-dalton form of ACTH is a glycosylated form of a(1-39) ACTH (17). The 11,700-dalton form of f3-endorphin is the size of #-LPH and the 3500-dalton form is the size of f3-endorphin. The 31,000-dalton class of molecules contains the determinants 3.0k V. a)
Cn
2.0p-
4) Q .>+
CE0. . C
Co
0
_
1.01-
T.
I
0 0 0 0 0 0 25 100 200 0 25 100 He(MIl)0 FIG. 1. Effects of dexamethasone and HE on release of ACTH and f3-endorphin activities in AtT-20/D16v cultures. Cells were grown to half confluency in Dulbecco-Vogt minimal essential medium with 10% horse serum (9) in 3.5-cm culture dishes containing 2.0 ml of medium. The medium was changed and cultures were incubated for 48 hr with or without 1 MM dexamethasone (dex) with one change of medium at 24 hr. The medium was replaced with 2.0 ml of serum-free test medium containing the amounts of rabbit HE shown below the histogram, and the incubation was continued for 3.0 hr. Medium was harvested and either frozen or assayed directly by RIA with ACTH and fl-endorphin antisera. The results are the mean + SD of triplicate plates. O, RIA-fl-endorphin; O, RIA-ACTH. dex
-
201 .00
Cell Biology: Allen et al.
4974
Proc. Natl. Acad. Sci. USA 75 (1978) c
A
.C
7.5
U
._
31
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12SI-AcTH
0 A
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YADH
117
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