Human placental cells transformed by tsA mutants of simian virus 40 : A model system for the study of placental functions ( human chorionic gonadotropin / placental alkaline phosphatase / thymidine kinase )

Human placental cells were transformed with wild-type simian virus 40 (SV40) and temperature-sensitive SV40 mutants of the A and B classes. Four criteria for transformation were used: decreased generation time, increased saturation density, increased efficiency of growth on plastic, and ability to overgrow a nontransformed monolayer. Cell lines transformed by tsA mutants lost the transformed phenotype at the restrictive temperature (400); therefore, the A function of SV40 is required for the maintenance of the transformed phenotype in human placental cells. A decrease in alkaline phosphatase activity, an inhibition of human chorionic gonadotropin synthesis, and an increase in thymidine kinase activity were seen when human-placental cells transformed by wild-type or tsB mutants of SV40 were grown at 330 or 400 and when tsA transformants were grown at 33°. When tsA transformants were grown at 400, alkaline phosphatase activity and human chorionic gonadotropin synthesis were greatly stimulated and thymidine kinase activity was greatly reduced, approximating their levels in the placenta. Placental functions in vitro have usually been studied by using explants and primary cultures which are difficult to establish and maintain in tissue culture. Choriocarcinoma cells (malignant cells derived from the placenta) have been used extensively as an in vitro system to study placental functions (1-4); these cells do not, however, control the synthesis of certain proteins as does the placenta in vivo. Because neither placental cells in vitro nor choriocarcinoma cells have proven to be a suitable model for placental cells in vivo, I thought that placental cells transformed by temperature-sensitive A (tsA) mutants of simian virus 40 (SV40) might be a better system. The A function of SV40 is required for the initiation of viral DNA replication (5, 6), for the induction of host DNA synthesis (7), for the stimulation of cellular thymidine kinase activity (8), and for the maintenance of the transformed phenotype (9-12). Cell lines induced by tsA mutants should behave like transformed cells at the permissive temperature, allowing propagation and cloning. At the nonpermissive temperature, these cells should regain their nontransformed phenotype. Placental cell lines transformed by wild-type SV40 and its temperature-sensitive mutants have therefore been established. This report demonstrates that the A function of SV40 is required for the maintenance of the transformed phenotype in human placental cells and that normal placental functions are expressed when tsA-transformed cells are incubated at the restrictive temperature. MATERIALS AND METHODS Virus. The ts mutants and wild-type SV40 virus have been described (13). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertsement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1409 Cells and Culture Conditions. Primary placental cells were obtained by collagenase digestion (0.1%, Worthington Biochemical Co., Freehold, NJ) of human term placenta. The primary cultures were grown at 370 in a modified minimal essential medium supplemented with 10% fetal bovine serum (aMEM-10, Flow Laboratories, Rockville, MD), streptomycin (100 Mg/ml), and penicillin (100 units/ml). At confluence, the cells were trypsinized and subcultured. These secondary placental cells in medium supplemented with 8% dimethyl sulfoxide were stored in a liquid nitrogen freezer for subsequent use. These cells, designated "TP" cells (term placental cells), were used for transformation. Transformation. TP cells (passage 2) were grown to 10-20% of confluence (approximately 2 X 105 cells per 25-cm2 flask), the medium was removed, and wild-type SV40 or a ts mutant of SV40 was allowed to adsorb for 2-3 hr at 330 (multiplicity of infection = 5-10). Medium supplemented with 4% fetal bovine serum was then added to the infected flask, and the cells were incubated at 330. After 24 hr of incubation, the cells were suspended after incubation with trypsin and serially diluted. Medium (aMEM-4) was replaced twice weekly. Clones were identifiable after 4-5 weeks and were selected and purified by growing in low-serum medium (aMEM-4) at low cell density. The transformation efficiency was 0.5-1%. More than 50 clones were isolated. Recloning of wild-type transformed and mutant-transformed cells was generally successful; however, for unknown reasons, the life-span of the doubly cloned cells was considerably shortened. Therefore, only singly cloned cultures were used in this study. Cultures of TP cells from the same placenta were transformed by viral strains A30, A209, B201, and WT. Mutants tsA30 and tsA209 are independent mutants of the SV40 A gene and mutant tsB201 is a mutant of the B gene. The A function of SV40, but not the B function, is required for the maintenance of transformed phenotype; thus, cells induced by tsB mutants manifest the transformed phenotype at both 330 and 400. The transformed cell lines are identified by their TP origin, the virus by which they were transformed, and their isolation rumber. The transformed lines were thus designated, TPA30-1, TPA30-6, TPA209-9, TPWT-2, and TPB201-1. Efficiency of Growth on Plastic. Cells were serially diluted starting with approximately 105 cells per 25-cm2 flask. Duplicate dilutions were prepared, and the efficiencies of clone formation at 400 and 330 were determined. Medium was replaced twice weekly at 330 (aMEM-4) and every other day at 400 (aMEM-10). After 2 weeks, cells were fixed and stained. Overgrowth of Nontransformed Placental Cell Layer by Transformed Cells. TP cells were treated with trypsin and Abbreviations: ts, temperature-sensitive; SV40, simian virus 40; TP cells, term placental cells; aMEM, a minimal essential medium; hCG, human chorionic gonadotropin. Proc. Natl. Acad. Sci. USA 75 (1978) suspended in aMEM-4 at a density of 2 X 105 cells per ml. Each well of the 24-well plates (Cooke Laboratory Products, Dynatech Laboratories, Inc., Alexandria, VA) received enough of the suspension (1.5 ml) to produce a confluent monolayer immediately after attachment. Each well also received one of four serial (1:3) dilutions of a transformed cell culture. The first well of each series received 5-10 X 104 transformed cells. Medium was replaced twice weekly at 330 (aMEM-4) and every other day at 400 (aMEM-10). After 2 weeks, the cells were fixed and stained. Fixing and Staining. After removal of the medium, the flasks or plates were washed with phosphate-buffered saline, rinsed with absolute methanol, fixed in absolute methanol for 20 min at 250, and allowed to dry. The cells were stained with 0.1% Evans blue in phosphate-buffered saline for 20 min at room temperature, rinsed with water, and dried. Glucose-6-Phosphate Dehydrogenase. The electrophoretic mobility of these enzyme species in SV40-transformed placental cells was determined by using Cellogel electrophoresis as described by Rattazzi et al. (14). Normal A and B types, kindly provided by L. Corash, were used as standards. Preparation of Cell Extracts. Cells were harvested by scraping with a rubber policeman. The pellet was suspended in buffer (0.01 M Tris.HCl, pH 7.4/0.15 M KCl/3mM 2-mercaptoethanol/l mM EDTA) and the cells were ruptured by sonication (Raytheon Magnetostrictive Oscillator, model DF-101, 250-W, 10 kHz, Raytheon Company, Manchester, NH) for 2 min at maximal power. The sonicates were centrifuged at 10,000 X g for 15 min; the supernatant solutions were used immediately for measurement of alkaline phosphatase and thymidine kinase activities. For gel electrophoresis, heat denaturation, and immunoprecipitation studies, alkaline phosphatase was partially purified by butanol extraction and acetone precipitation of the tsA-transformed cells grown at 400 (15). Assay of Alkaline Phosphatase and Thymidine Kinase. Alkaline phosphatase activity was measured by the release of p-nitrophenol from p-nitrophenyl phosphate at pH 10.7 and 370 (16). Thymidine kinase was assayed by the method of Kit and Dubbs (17), which measures the rate of conversion of [2-14C]thymidine to a form that can be adsorbed by polyethyleneimine-cellulose. Protein was determined by the method of Lowry et al. (18). Radioimmunoassays. A double-antibody radioimmunoassay using human chorionic gonadotropin (hCG) and hCG-a as reference standard was used to measure hCG and hCG-a in unfractionated medium (19). Antisera used were: anti-hCG-#3 (Sb6) for hCG (20), and anti-hCG-a (CA3) for hCG-a. Purified preparations of hCG (CR119, 11,600 international units/mg, ventral prostate assay, Second International Standard hCG) and of hCG-a (CR117) were radioiodinated and used to determine a standard curve for the assay. Complete hCG (CR119) has slight (1-3%) crossreactivity in the hCG-a assay. hCG-a has no crossreactivity in the hCG assay. Complete medium not exposed to cells had no detectable hCG or hCG-a. The limit of detection for hCG or hCG-a in the radioimmunoassays was 0.1 ng. Antisera, standard hCG, and hCG-a were kindly provided by K. Catt, G. Hodgen, and H. Chen.