Proc. Natl. Acad. Sci. USA Vol. 76, No. 1, pp. 1323-327, January 1979
Cell Biology
Regulation of alkaline phosphatase expression in human choriocarcinoma cell lines (isoenzymes/two-dimensional electrophoresis)
THOMAS A. HAMILTON, AGNES W. TIN, AND HOWARD H. SUSSMAN Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305
Communicated by Roy Hertz, October 13, 1978
The coincident expression of two structurally ABSTRACT distinct isoenzymes of human alkaline phosphatase was demonstrated in two independently derived gestational choriocarcinoma cell lines. These proteins were shown to have enzymatic, antigenic, and physical-chemical properties resembling those of isoenzymes from term placenta and adult liver. The regulation of these isoenzymes has been studied during the ex sure of both cell lines to 5-bromodeox ridine and dibutyryl cyclic AMP. The responses of the alkaline phosphatase isoenzymes to these agents have also been compared with the response of another protein phenotypic to lacenta, the a subunit of chorionic gonadotropin. The results show that (i) the separate structural genes coding for placental and liver alkaline phosphatases are regulated in a noncoordinate fashion; (ii) both alkaline phosphatase genes respond independently of the a subunit; and (iii) the induction of the placental type isoenzyme occurs via at least two independent pathways.
At least two different genetically coded human alkaline phosphatases have been identified: that expressed by the full-term placenta [placental alkaline phosphatase (PAP)] and that expressed in adult liver [liver alkaline phosphatase (LAP)] (1-3). While these isoenzymes are characteristic of placenta and liver, they are not necessarily unique to these organs (3-5). Of interest in this respect is the observation that an LAP-like enzyme is expressed in early placental tissue (4). The present report considers the regulation of synthesis of these enzymes in cell cultures, where their expression is phenotypic of the differentiated tissue from which the cell line was derived. For this purpose, we examined the alkaline phosphatase isoenzyme composition of two gestational choriocarcinoma cell lines, BeWo (6, 7) and JEG-3 (6, 8), a clonal derivative of the same tumor developed independently. Analysis of the antigenic, functional, and physical-chemical properties of the enzymes from these cell lines indicated that both PAP and LAP are expressed in each. Alkaline phosphatase is inducible in a variety of human tumor cell lines. Agents that cause a significant elevation in enzyme specific activity include corticosteroid hormones (9), sodium butyrate (10), 5-bromodeoxyuridine (BrdUrd) (11, 12), and N6,02'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) (10, 13, 14). We measured the effect of both BrdUrd and Bt2cAMP on the expression of both classes of alkaline phosphatase contained in these two cell lines as well as the expression of a third phenotype-specific gene, the a subunit of human chorionic gonadotropin (a). The results show that (i) PAP is induced while LAP is unaffected; (ii) both phosphatase genes are regulated independently of a and (iii) induction of PAP can occur via at least two distinct molecular pathways.
MATERIALS AND METHODS Cell Culture. BeWo cells were obtained from the American Type Culture Collection (CCL 98), and JEG-3 cells were kindly provided by Saul Rosen. Both cell lines were grown in polystyrene T-flasks as described (7, 8). Cells were routinely plated between 5 X 104 and 1 X 105 cells per cm2 as indicated in legends to the figures. Unless otherwise indicated, cells were harvested at confluence. Cells were plated in medium containing the indicated amount of BrdUrd or Bt2cAMP. Enzyme Preparation. Cells were harvested after two washes in cold 0.85% NaCl by scraping with a rubber policeman into TNM buffer (20 mM Tris-HCI, pH 7.4/150 mM NaCl/2 mM MgCl2) and then sonicated with 20 0.5-sec 50-W pulses. Alkaline phosphatase activity was measured in crude sonicates according to Bessey et al. (15); 1 unit equals 1 grmol of p-nitrophenylphosphate cleaved per min. Protein content was determined by the method of Schaffner and Weissmann (16). The sonicates were adjusted to a final concentration of 1% Triton X-100, extracted at room temperature for 10 min, and centrifuged at 110,000 X g for 60 min. The supernatant was used directly for immunoprecipitation. Enzyme extraction with 1-butanol (17) was occasionally substituted, but no variation in isoenzyme content or behavior was observed. Immunoassay. Isoenzymes were identified by an enzyme activity-based double antibody precipitation assay that has been described elsewhere (1). The proportion that precipitated was determined as the difference between soluble enzyme activity in a control tube (nonimmune serum) and in tubes containing specific antisera. 32P Labeling and Two-Dimensional Electrophoresis. Between 0.01 and 0.05 unit of alkaline phosphatase was immunoprecipitated as above, except that a high-titer rabbit antiserum to goat gamma globulin was used as the second antibody. The immunoprecipitates were washed three times with TNM buffer, then resuspended in 200 Al of this buffer. This resuspended precipitate was labeled with 32P04 (carrier free, New England Nuclear) according to Milstein (18) as modified by Carlson et al. (19). The covalently labeled enzyme was then subjected to two-dimensional polyacrylamide gel electrophoresis as described fully elsewhere (20). Radioimmunoassay of PAP and a. 125I tracer double antibody-based immunoassays of PAP and a were conducted according to standard radioimmunoassay methods. PAP was purified according to Sussman et al. (1) and a was purified from pregnancy urine (Organon) according to Bahl (21). Antisera were prepared in goats by Antibodies, Inc. (Davis, CA). Anti-
The 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.
Abbreviations: PAP, placental alkaline phosphatase; LAP, liver alkaline phosphatase; a subunit of human chorionic gonadotropin; Bt2cAMP, N6,02'-dibutyryladenosine 3',5'-cyclic monophosphate; TNM, Tris/ a,
NaCI/MgCl2.
323
324
Proc. Nati. Acad. Sci. USA 76 (1979)
Cell Biology: Hamilton et al.
gens were iodinated with chloramine-T (22), and unlabeled purified proteins were used as standards. The a assay showed
Cell Biology
Regulation of alkaline phosphatase expression in human choriocarcinoma cell lines (isoenzymes/two-dimensional electrophoresis)
THOMAS A. HAMILTON, AGNES W. TIN, AND HOWARD H. SUSSMAN Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305
Communicated by Roy Hertz, October 13, 1978
The coincident expression of two structurally ABSTRACT distinct isoenzymes of human alkaline phosphatase was demonstrated in two independently derived gestational choriocarcinoma cell lines. These proteins were shown to have enzymatic, antigenic, and physical-chemical properties resembling those of isoenzymes from term placenta and adult liver. The regulation of these isoenzymes has been studied during the ex sure of both cell lines to 5-bromodeox ridine and dibutyryl cyclic AMP. The responses of the alkaline phosphatase isoenzymes to these agents have also been compared with the response of another protein phenotypic to lacenta, the a subunit of chorionic gonadotropin. The results show that (i) the separate structural genes coding for placental and liver alkaline phosphatases are regulated in a noncoordinate fashion; (ii) both alkaline phosphatase genes respond independently of the a subunit; and (iii) the induction of the placental type isoenzyme occurs via at least two independent pathways.
At least two different genetically coded human alkaline phosphatases have been identified: that expressed by the full-term placenta [placental alkaline phosphatase (PAP)] and that expressed in adult liver [liver alkaline phosphatase (LAP)] (1-3). While these isoenzymes are characteristic of placenta and liver, they are not necessarily unique to these organs (3-5). Of interest in this respect is the observation that an LAP-like enzyme is expressed in early placental tissue (4). The present report considers the regulation of synthesis of these enzymes in cell cultures, where their expression is phenotypic of the differentiated tissue from which the cell line was derived. For this purpose, we examined the alkaline phosphatase isoenzyme composition of two gestational choriocarcinoma cell lines, BeWo (6, 7) and JEG-3 (6, 8), a clonal derivative of the same tumor developed independently. Analysis of the antigenic, functional, and physical-chemical properties of the enzymes from these cell lines indicated that both PAP and LAP are expressed in each. Alkaline phosphatase is inducible in a variety of human tumor cell lines. Agents that cause a significant elevation in enzyme specific activity include corticosteroid hormones (9), sodium butyrate (10), 5-bromodeoxyuridine (BrdUrd) (11, 12), and N6,02'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) (10, 13, 14). We measured the effect of both BrdUrd and Bt2cAMP on the expression of both classes of alkaline phosphatase contained in these two cell lines as well as the expression of a third phenotype-specific gene, the a subunit of human chorionic gonadotropin (a). The results show that (i) PAP is induced while LAP is unaffected; (ii) both phosphatase genes are regulated independently of a and (iii) induction of PAP can occur via at least two distinct molecular pathways.
MATERIALS AND METHODS Cell Culture. BeWo cells were obtained from the American Type Culture Collection (CCL 98), and JEG-3 cells were kindly provided by Saul Rosen. Both cell lines were grown in polystyrene T-flasks as described (7, 8). Cells were routinely plated between 5 X 104 and 1 X 105 cells per cm2 as indicated in legends to the figures. Unless otherwise indicated, cells were harvested at confluence. Cells were plated in medium containing the indicated amount of BrdUrd or Bt2cAMP. Enzyme Preparation. Cells were harvested after two washes in cold 0.85% NaCl by scraping with a rubber policeman into TNM buffer (20 mM Tris-HCI, pH 7.4/150 mM NaCl/2 mM MgCl2) and then sonicated with 20 0.5-sec 50-W pulses. Alkaline phosphatase activity was measured in crude sonicates according to Bessey et al. (15); 1 unit equals 1 grmol of p-nitrophenylphosphate cleaved per min. Protein content was determined by the method of Schaffner and Weissmann (16). The sonicates were adjusted to a final concentration of 1% Triton X-100, extracted at room temperature for 10 min, and centrifuged at 110,000 X g for 60 min. The supernatant was used directly for immunoprecipitation. Enzyme extraction with 1-butanol (17) was occasionally substituted, but no variation in isoenzyme content or behavior was observed. Immunoassay. Isoenzymes were identified by an enzyme activity-based double antibody precipitation assay that has been described elsewhere (1). The proportion that precipitated was determined as the difference between soluble enzyme activity in a control tube (nonimmune serum) and in tubes containing specific antisera. 32P Labeling and Two-Dimensional Electrophoresis. Between 0.01 and 0.05 unit of alkaline phosphatase was immunoprecipitated as above, except that a high-titer rabbit antiserum to goat gamma globulin was used as the second antibody. The immunoprecipitates were washed three times with TNM buffer, then resuspended in 200 Al of this buffer. This resuspended precipitate was labeled with 32P04 (carrier free, New England Nuclear) according to Milstein (18) as modified by Carlson et al. (19). The covalently labeled enzyme was then subjected to two-dimensional polyacrylamide gel electrophoresis as described fully elsewhere (20). Radioimmunoassay of PAP and a. 125I tracer double antibody-based immunoassays of PAP and a were conducted according to standard radioimmunoassay methods. PAP was purified according to Sussman et al. (1) and a was purified from pregnancy urine (Organon) according to Bahl (21). Antisera were prepared in goats by Antibodies, Inc. (Davis, CA). Anti-
The 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.
Abbreviations: PAP, placental alkaline phosphatase; LAP, liver alkaline phosphatase; a subunit of human chorionic gonadotropin; Bt2cAMP, N6,02'-dibutyryladenosine 3',5'-cyclic monophosphate; TNM, Tris/ a,
NaCI/MgCl2.
323
324
Proc. Nati. Acad. Sci. USA 76 (1979)
Cell Biology: Hamilton et al.
gens were iodinated with chloramine-T (22), and unlabeled purified proteins were used as standards. The a assay showed
