0013-7227/91/1292-0950$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 2 Printed in U.S.A.
Isolation and Characterization of a Human Amnion Epithelial Cell Line That Expresses the PregnancySpecific /?i-Glycoprotein Gene* CATHIE A. PLOUZEK AND JANICE YANG CHOU Human Genetics Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892
polypeptide. At the nonpermissive temperature (39.5 C), HAA58OD-8C cells exhibited a differentiated phenotype, expressed increased levels of PSG, hCG«, and hCG/3 mRNAs, and produced high levels of PSG polypeptides of 72K and 48K. Sodium butyrate induced PSG mRNA expression, and in the presence of butyrate, HAA58OD-8C cells produced high amounts of PSG polypeptides of 72K, 62K, and 48K. Ribonuclease protection analysis indicated that similar PSG transcripts were expressed by HAA58OD-8C cells and human term placenta. However, these amnion cells expressed selectively a certain population of PSG transcripts. Our results show that this amnion cell line provides a suitable model for studies of PSG gene expression and regulation. (Endocrinology 129: 950-958,1991)
ABSTRACT. Human pregnancy-specific /?i-glycoprotein (PSG) is a family of closely related glycoproteins of 72K, 64K, 62K, and 54K. Together with the carcinoembryonic antigen, they form new members of the immunoglobulin superfamily. To study the molecular mechanisms that regulate expression of the PSG gene, we established a human amnion cell line, HAA58OD8C, immortalized with an origin-defective simian virus-40 (SV40) temperature-sensitive A58 mutant virus. HAA58OD-8C cells were temperature sensitive for maintenance of transformation and expressed genes encoding PSG and the a- and /?subunits of hCG. At the permissive temperature (33 C; transformed phenotype), they expressed low levels of PSG, hCGa, and hCG/3 mRNAs and synthesized low levels of a 48K PSG
P
REGNANCY- specific /3rglycoprotein (PSG) was the first of a group of pregnancy-associated proteins to be identified (for reviews, see Refs. 1 and 2). It is primarily produced by the human placenta, but low levels are also synthesized by amnion tissues. The concentrations of PSG in maternal serum and amniotic fluid increase as pregnancy progresses, reaching 200-400 and 2 mg/liter, respectively, at term (3, 4). PSG has been used clinically to diagnose pregnancy and predict some pregnancy-related complications. For example, low PSG values are associated with poor pregnancy outcome in threatened abortions (5), intrauterine growth retardation (6), and fetal hypoxia (7). The clinical uses of PSG are not limited to pregnancy. It is found in the serum of most patients with hydatidiform mole, invasive mole, and choriocarcinoma (1), and it has been employed as a marker for monitoring the treatment of choriocarcinoma (1). In addition, PSG analysis of amniotic fluid is used to diagnose Meckel's syndrome (8). To uncover the functions of human PSG in pregnancy,
our laboratory has undertaken a number of studies to characterize this protein. We found that placental PSG represents a family of closely related glycoproteins of 72K, 64K, 62K, and 54K (9, 10). In addition, cDNA and genomic clones encoding members of the PSG family were recently isolated and characterized by our laboratory (9, 11) and others (12-20). Sequence analysis of PSG cDNA clones indicated that this protein family is related to the carcinoembryonic antigen and is a member of the immunoglobulin superfamily (12-14, 21). Although PSG is a major placental protein, cultured choriocarcinoma cells (malignant trophoblasts) do not produce detectable levels of this protein (22). We have previously immortalized human placental cells with the simian virus-40 (SV40) viruses and obtained cell lines capable of synthesizing PSG (23). However, the SV40immortalized human cells shed infectious virus, thus limiting their use as a model. To immortalize human cells free of infectious virions, Small and co-workers (24) engineered an origin-defective SV40 virus and showed that these SV40 mutants immortalized virus-free human cells with high efficiency. In the present study we immortalized human amnion cells with an origin-defective SV40 temperature-sensitive (ts) A58 mutant virus (Gluzman, Y., unpublished results) carrying an additional
Received March 15, 1991. Address all correspondence and requests for reprints to: Dr. Janice Yang Chou, Building 10, Room 9S-242, National Institutes of Health, Bethesda, Maryland 20892. * This work was performed while C.A.P. held a National Research Council (Human Genetics Branch, NICHHD) research associateship.
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PSG SYNTHESIS IN A HUMAN AMNION CELL LINE mutation in the SV40 A gene which is required for maintenance of transformation (25). Like other SV40 tsA mutant-transformed mammalian cells (23, 26), the origin-defective SV40 tsA58-immortalized human amnion cell line (HAA58OD-8C) is conditionally transformed and expressed the transformed phenotype only at the permissive temperature. At the nonpermissive temperature, these cells reverted to a differentiated phenotype and expressed high levels of the PSG gene. Moreover, expression of the PSG gene could be augmented by sodium butyrate. HAA580D-8C cells also express the hCG gene, a major placental glycoprotein hormone (27), and a product of the human amnion (28, 29). It has been demonstrated that classes I and II of the major histocompatibility antigens are not expressed by placenta or placenta-related tissues, with the exception of the «-chain of the human histocompatibility antigen HLA-G (30, 31). HLA-G is a class I antigen that is specifically expressed in placenta-related tissues, including the JEG-3 human choriocarcinoma cell line (30-32). As expected, the HAA580D-8C amnion cell line reported in this study also expresses the HLA-G gene. In contrast to choriocarcinoma cells and placenta, which do not express the major histocompatibility class II antigen HLA-DR0i (32), the transformed amnion cells expressed this class II antigen. Materials and Methods Transformation of human amnion epithelial cells Primary human amnion epithelial cells were obtained by collagenase digestion of human amnion membrane from a term pregnancy. The primary cultures were grown in a-Modified Minimum Essential Medium supplemented with 10% fetal bovine serum, streptomycin (100 Mg/nil), and penicillin (100 U/ml) and were transfected with the DNA (10 Mg/25 cm2) of an origin-defective SV40 tsA58 mutant virus by a calcium phosphate precipitation method (33). The transformed cultures were incubated at 33 C in medium supplemented with 4% fetal bovine serum, which was replaced twice weekly. Clones of transformed cells, identifiable after 3 months, were selected and recloned. The HAA58OD-8C cell line, which produced high levels of immunoreactive PSG, was analyzed in detail. Overgrowth of nontransformed fibroblasts by transformed cells Human fibroblasts were trypsinized and suspended in aMinimum Essential Medium with 4% fetal bovine serum at a density of 2 x 106 cells/ml. Each well of a 24-well plate (Cooke Laboratory Products, Dynatech Laboratories, Inc., Alexandria, VA) received 1.5 ml of the suspension, which produced a confluent monolayer after attachment. Each well also received transformed HAA58OD-8C cells at various concentrations. One set of plates was incubated at the permissive temperature (33 C), and a duplicate set of plates was incubated at the nonpermissive temperature (39.5 C). Medium was replaced twice weekly. After 2 weeks of incubation at either temperature,
951
plates were fixed in absolute methanol and stained with 0.1% Evans blue in PBS. Biosynthesis of PSG HAA58OD-8C cells were grown initially at 33 C (permissive temperature) for 3-5 days, after which time (day 0) the cultures were treated with control medium or medium containing sodium butyrate, and some of the cultures were shifted to 39.5 C (nonpermissive temperature). Medium was changed every 2 days. Cultures were labeled with L-[35S]methionine (100 /iCi/ ml) for 4 h, and PSGs in the medium were isolated by immunoprecipitation with anti-PSG serum and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and fluorography. Hybridization probes Sequence analysis of PSG cDNA and genomic clones indicated that PSG16 (9), PSG93 (9), PSG95 (11), PSGC (12), and PSGld (17) encode five PSG transcripts generated by alternative RNA splicing of the same gene at one of the four sites (Cl, C2, C3, and C4) in the 3' exon (11) (Lei, K.-J., and J. Y. Chou, unpublished results). These five transcripts share sequence homology in the 5'-untranslated and coding regions, but differ in sequence in the 3'-end coding and untranslated regions. The PSG probes (into pGEM vectors) used in this study are: PSG5' (nucleotides 1-142, the 5' EcoRI-BamHl fragment of PSG93), PSG3'-C1 (nucleotides 1411-1508, the 3' BamHlNcol fragment of PSG95), PSG3'-C2 (nucleotides 1849-3' end, the 3' Pstl-EcoRl fragment of PSG95), PSG3'-Cl+C2 (nucleotides 1411-3' end, the 3'-BamHl-EcoRl fragment of PSG95), PSG3'-C3 (nucleotides 15-8,5 of the PSG93-specific sequence), and PSG3'-C4 (nucleotides 1748-3' end of PSG16). Antisense riboprobes were produced following the procedures supplied by Promega Biotec (Madison, WI). Additional antisense riboprobes used were the 517-base Pstl fragment of HLA-DRfr (34), hCGa (35), and the 224-base, 5' EcoRl-Pstl fragment of hCG/3 (36). The 3'-untranslated region of HLA-G (37) was labeled by random priming. Nucleic acid hybridization Total RNA was isolated by the guanidinium thiocyanateCsCl method (38), and poly(A)+ RNA was obtained by oligo(dT)-cellulose chromatography. RNA was separated by electrophoresis in 1.2% agarose gels containing 2.2 M formaldehyde (39) and transferred to Zetabind (AMF, Meriden, CT) or Nytran (Schleicher and Schuell, Keene, NH) membranes by electroblotting. The filters were hybridized at 42 C (HLA-G), 55 C (PSG3'-C3), or 65 C (PSG-5', PSG3'-Cl+C2, PSG3'-C3, PSG3'-C4, hCG«, and HLA-DR/3,) in a buffer containing 50% formamide, 10% dextran sulfate, and a probe (106 cpm/ml), as previously described (10). Northern blots were washed twice in 300 mM NaCl-30 mM Na-citrate containing 1% SDS for 10 min at room temperature, then twice in 15 mM NaCl-1.5 mM Na citrate containing 0.5% SDS for 10 min at room temperature, followed by four washes in 15 mM NaCl-1.5 mM Na citrate containing 0.1% SDS for 60 min at 65 C.
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PSG SYNTHESIS IN A HUMAN AMNION CELL LINE
952 Ribonuclease protection assay
Ribonuclease protection assays were performed essentially as previously described (33). Briefly, poly(A+) RNA of HAA58OD-8C cells, total RNA of human placenta, or yeast tRNA was hybridized with an antisense riboprobe (5 X 105 cpm) in buffer containing 80% formamide, 400 mM NaCl, 40 HIM PIPES (pH 6.4), and 1 mM EDTA at 45 C for 18 h. The hybrids were digested with RNase-A (40 Mg/ml) and RNase-Tl (2 Mg/ml) for 60 min at 30 C and then electrophoresed on polyacrylamide-urea sequencing gels. Single base substitutions were normally not digested by these ribonucleases under the assay conditions used.
Results Isolation of temperature-sensitive human amnion cells It has been demonstrated that PSG is synthesized by both human placenta and amnion (2-4). Although PSGproducing human placental cell lines immortalized with the SV40 virus have been established, these cells are also capable of producing infectious virions in culture, a characteristic of SV40-transformed human cells. To establish PSG-producing cell lines devoid of infectious virions, we attempted to immortalize both human placental and amnion cells with an origin-defective SV40 tsA58 mutant virus. Unfortunately, we were unable to immortalize human placental cells, but several human amnion cell lines were successfully obtained. Three months after transfecting primary human amnion epithelial cells with the DNA of a SV40 origindefective tsA58 virus at 33 C, clones of transformed cells could be identified. Transformed colonies were selected and recloned. These cell lines were screened for their ability to synthesize PSG; PSG-producing lines were then selected for further characterization. HAA58OD8C, which secreted PSG and maintained the ultrastructure characteristics of amnion epithelial cells, as demonstrated by electron microscopy analysis (Fig. 1), was extensively characterized. The microvilli of HAA58OD8C cells were present on the cell surface, and contiguous cells were joined by the junctional complex of the desmosome type. Additionally, the nuclei of HAA58OD-8C cells had an irregular configuration, and microfilaments were located in the cytoplasm of the cells, characteristic of amnion epithelial cells (40). To determine if HAA58OD-8C cells were temperature sensitive for maintenance of the transformed phenotype, we examined the ability of these cells to overgrow nontransformed cell layers at the permissive (33 C) or nonpermissive (39.5 C) temperature (Fig. 2). At 33 C, HAA58OD-8C behaved as transformed cells and overgrew nontransformed cell layers. At 39.5 C, the ability of this cell line to overgrow nontransformed cell layers was greatly diminished, demonstrating that HAA58OD-8C
Kndo «1991 Vol 129 • No 2
cells were temperature sensitive for maintenance of transformation. Biosynthesis of PSG To determine whether HAA58OD-8C cells produced PSG, synthesis was examined in cells grown at the permissive (33 C) and nonpermissive (39.5 C) temperatures. At 33 C, HAA58OD-8C cells synthesized low levels of a 48K PSG (Fig. 3a). At 39.5 C, synthesis was increased (Fig. 3b). Moreover, at 39.5 C, two PSG polypeptides of 72K and 48K were seen in these cells. We have previously shown that human placenta and primary trophoblasts contain PSG species of 72K (major), 64K, 62K, and 54K (9, 10), and placental fibroblasts produce PSG of 72K (major), 62K, and 48K (41). Both the 54K and 48K PSGs have polypeptide backbones of 36K, suggesting that these two polypeptides differ primarily in protein glycosylation. Our results indicate that differentiated HAA58OD-8C cells (at 39.5 C) synthesized the major placental PSG of 72K. It has been demonstrated that sodium butyrate increased PSG synthesis in placental fibroblasts (41) and several human cell lines (23, 42). In the present study we showed that PSG synthesis in HAA58OD-8C cells was also induced by butyrate (Fig. 3). Butyrate increased PSG biosynthesis in a dose-dependent manner (Fig. 3a). Moreover, in the presence of butyrate, HAA58OD-8C cells synthesized three PSG species of 72K, 62K, and 48K at either temperature (Fig. 3b). Thus, in transformed amnion cells, induction of the 72K PSG synthesis was achieved either by shifting these cells to the nonpermissive temperature (differentiated phenotype) or by butyrate treatment. However, synthesis of the 62K PSG was demonstrated only in the presence of butyrate. Northern analysis To deduce possible mechanisms regulating PSG gene expression in HAA58OD-8C cells, we examined PSG mRNA expression by Northern analysis (Fig. 4). Sequence analysis of PSG cDNA and genomic clones indicates that PSGs are a family of closely related glycoproteins which are encoded by multiple genes (9, 11-20). These PSG transcripts share strong sequence similarity at the 5'-untranslated and coding region, but differ in sequence at the 3'-end coding and untranslated regions. The 3' end of PSG mRNAs are evolved by alternative RNA splicing of a given gene at one of the four sites (Cl, C2, C3, and C4) in the 3' exon. The probes used in this study, which were derived from the PSGGl (Lei, K.-J., and J. Y. Chou, unpublished results), encoded species that hybridized with the majority of PSG mRNAs. We previously showed (10) that placenta expresses three PSG mRNAs of 2.3, 2.2, and 1.7 kilobases (kb), identified by four different PSG probes. HAA58OD-8C
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PSG SYNTHESIS IN A HUMAN AMNION CELL LINE
953
a.
Fid. 1. Electron micrographs of HAA58OD-8C amnion epithelial cells. Microvilli (MV), desmosomes (D), filaments (F), and nucleus (N) are denoted, a, 10,500-fold magnification; b, 35,000fold magnification.
Transformants 10
10
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FlO. 2. The ability of HAA58OD-8C amnion epithelial cells to overgrow a monolayer of fibroblasts at 33 and 39.5 C. Each well contains 2 x 10R human fibroblasts and 0, 10, 102, 103, 3 x 103, 10\ 3 x 10\ and 10fi HAA58OD-8C cells. Duplicate plates were prepared; one was incubated at 33 C, and the other at 39.5 C. After 2 weeks of incubation, plates were fixed and stained, as described in Materials and Methods.
cells were found to express all three PSG mRNAs, as identified by PSG-5' (2.3, 2.2, and 1.7 kb), PSG3'-C3 (2.2 kb), PSG3'-C4 (2.2 kb), and PSG3 / -Cl+C2 (2.3, 2.2, and 1.7 kb) probes (Fig. 4). In the absence of butyrate, cells grown at 39.5 C expressed higher levels of PSG mRNAs than cells grown at 33 C. Moreover, at 39.5 C, a 2.3-kb transcript which hybridized with the PSG-5' and PSG3'-C1+C2 probes was preferentially induced (Fig. 4). Sodium butyrate increased the steady state levels of all identified PSG mRNAs at both 33 and 39.5 C (Fig. 4). However, in the presence of butyrate at 39.5 C, HAA58OD-8C expressed more of the 2.3- and 1.7-kb mRNAs that hybridized with the PSG-5' or the PSG3'C1+C2 probe. Ribonuclease protection analysis of PSG expression
Because of the polymorphic nature of the PSG gene and its transcripts, each of the discrete bands (2.3, 2.2, or 1.7 kb) observed on Northern gels actually represents
a group of PSG transcripts of similar size. To determine the dominant PSG transcripts expressed in HAA58OD8C cells, we employed a RNase protection assay (Fig. 5). Placental mRNA yielded two clusters of protected fragments of 87-89 and 46-62 bases with the PSG-5' probe (Fig. 5a). RNA from butyrate-treated HAA58OD-8C cells yielded protected fragments with sizes similar to those observed in placenta, but at reduced levels. RNA from control HAA58OD-8C cells yielded fewer protected bands and at reduced levels. The 87-89-base bands were low or absent, and the 46- and 62-base bands were the major protected species. Moreover, a 120-base protected fragment was observed in mRNA from control HAA58OD-8C cells grown at both 33 and 39.5 C and from butyrate-treated cells grown at 33 C (Fig. 5a). This 120-base band was absent from RNAs isolated from placenta or butyrate-treated HAA58OD-8C cells grown at 39.5 C. This suggests that HAA58OD-8C cells produce a PSG transcript that is not found in human placenta and was inhibited by butyrate treatment. Both HAA58OD-8C and placental mRNAs yielded two major protected fragments of 71 and 41 bases, using PSG3'-C3 probe (Fig. 5b). Butyrate induced the expression of both transcripts, but the 71-base protected species was preferentially induced at 39.5 C. Placental mRNA yielded two major protected fragments of 160 and 53-56 bases with the PSG-3'-C4 probe (Fig. 5c). However, HAA58OD-8C mRNA yielded only the 160-base protected fragment, and butyrate induced the expression of this transcript. These results show that HAA58OD-8C cells and placental PSG mRNAs share sequence similarity in the 3' regions. However, HAA58OD-8C cells did not express all PSG mRNA species found in human placenta.
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PSG SYNTHESIS IN A HUMAN AMNION CELL LINE
Endo'1991 Voll29«No2
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FIG. 6. Expression of hCG, HLA-G, and HLA-DR/3i by HAA58OD-8C cells, a, HCG/3 expression analyzed by ribonuclease protection. Poly(A)+ mRNA (10 jig) from HAA58OD-8C cells grown at 33 or 39.5 C, total RNA (5 Mg) from placenta, and yeast tRNA (10 ^g) were annealed to a uniformly labeled antisense riboprobe corresponding to nucleotides 1-224 of hCG/3, as described in Materials and Methods, b-d, HCGa, HLA-G, and HLA-DRft expression analyzed by Northern hybridization. Poly(A)+ mRNA (10 Mg) from HAA58OD-8C cells grown at 33 or 39.5 C, or JEG3 choriocarcinoma cells was separated by electrophoresis on formaldehyde-agarose gels, transferred to Nytran membrane, and hybridized with a uniformly labeled antisense hCGa or HLA-DRfr riboprobe or the random primed HLA-G probe, as described in Materials and Methods. Stern for HLA-G, and Dr. J. Fiddes for hCGa and hCG/? cDNAs. References 1. Tatarinov YS 1978 Trophoblast-specific beta!-glycoprotein as a marker for pregnancy and malignancies. Gynecol Obstet Invest 9:65-97 2. Sorensen S 1984 Pregnancy-"specific" /3i-glycoprotein (SPi): purification, characterization, quantification and clinical application in malignancies (a review). Tumor Biol 5:275-302 3. Heikinheimo M 1980 Pregnancy-specific ft-glycoprotein in amniotic fluid in high risk late pregnancy. Oncodev Biol Med 1:287290 4. Grudzinskas JG, Evans DG, Gordon YB, Jeffrey D, Chard T 1978 Pregnancy specific ft glycoprotein in fetal and maternal compartments. Obstet Gynecol 52:43-45 5. Masson GM, Anthony F, Wilson MS 1983 Value of schwangeshattsprotein (SPi) and pregnancy associated protein A (PAPPA) in the clinical management of threatened abortion. Br J Obstet Gynaecol 90:146-149 6. Tamsen L, Johansson SGO, Axelsson O 1983 Pregnancy-specific ft-glycoprotein (SPI) in serum from woman with pregnancies complicated by intrauterine growth retardation. J Perinat Med 11:19-25 7. MacDonald DJ, Scott JM, Gemmel RS, Mack DS 1983 A prospec-
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9. 10. 11.
12.
13.
tive study of three biochemical fetoplacental tests: serum placental lactogen, pregnancy-specific ft-glycoprotein, and urinary estrogen and their relationship to placental insufficiency. Am J Obstet Gynecol 147:430-436 Heikinheimo M, Aula P, Rapola J, Wahlstrom T, Jalanko H, Seppala M 1982 Amniotic fluid pregnancy-specific ft-glycoprotein (SPi) in Meckels' syndrome: a new test for prenatal diagnosis? Prenat Diagn 2:103-108 Watanabe S, Chou JY 1988 Isolation and characterization of complementary DNAs encoding human pregnancy-specific ft-glycoprotein. J Biol Chem 263:2049-2054 Chou JY, Zilberstein M 1990 Expression of the pregnancy-specific ft-glycoprotein gene in cultured human trophoblasts. Endocrinology 127:2127-2135 Leslie KK, Watanabe S, Lei K-J, Chou DY, Plouzek CA, Deng HC, Torres J, Chou JY 1990 Linkage of two human pregnancyspecific ft-glycoprotein genes: one is associated with hydatidiform mole. Proc Natl Acad Sci USA 87:5822-5826 Streydio C, Lacka K, Swillens S, Vassart G 1988 The human pregnancy-specific ft-glycoprotein (PS/3G) and the carcinoembryonic antigen (CEA)-related proteins are members of the same multigene family. Biochem Biophys Res Commun 154:130-137 Rooney BC, Home CHW, Hardman N 1988 Molecular cloning of a cDNA for human pregnancy-specific /3rglycoprotein: homology with human carcinoembryonic antigen and related proteins. Gene 71:439-449
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PSG SYNTHESIS IN A HUMAN AMNION CELL LINE
14. Chan W-Y, Borjigin J, Zheng Q-X, Shupert WL 1988 Characterization of cDNA encoding human pregnancy-specific /Jrglycoprotein from placenta and extraplacental tissues and their comparison with carcinoembryonic antigen. DNA 7:545-555 15. McLenachan T, Mansfield B 1989 Expression of CEA-related genes in the first trimester human placenta. Biochem Biophys Res Commun 162:1486-1493 16. Khan WN, Hammarstrom S 1989 Carcinoembryonic antigen gene family: molecular cloning of cDNA for a PS/3G/FL-NCA glycoprotein with a novel domain arrangement. Biochem Biophys Res Commun 161:525-535 17. Zimmermann W, Weiss M, Thompson JA 1989 cDNA cloning demonstrates the expression of pregnancy-specific glycoprotein genes, a subgroup of the carcinoembryonic antigen gene family, in fetal liver. Biochem Biophys Res Commun 163:1197-1209 18. Oikawa S, Inuzuka C, Kosaki G, Nakazato H 1988 Exon-intron organization of a gene for pregnancy-specific /3i-glycoprotein, a subfamily member of CEA family: implications for its characteristic repetitive domains and C-terminal sequences. Biochem Biophys Res Commun 156:68-77 19. Oikawa S, Inuzuka C, Kuroki M, Matsuoka Y, Kosaki G, Nakazato H 1989 A pregnancy-specific /3i-glycoprotein, a CEA gene family member, expressed in a human promyelocytic leukemia cell line, HL-60: structures of protein, mRNA and gene. Biochem Biophys Res Commun 163:1021-1031 20. Thompson J, Koumari R, Wagner K, Barnert S, Schleussner C, Schrewe H, Zimmermann W, Muller G, Schempp W, Zaninetta D, Ammaturo D, Hardman N 1990 The human pregnancy-specific glycoprotein genes are tightly linked on the long arm of chromosome 19 and are coordinately expressed. Biochem Biophys Res Commun 167:848-859 21. Watanabe S, Chou JY 1988 Human pregnancy-specific /?i-glycoprotein: a new member of the carcinoembryonic antigen gene family. Biochem Biophys Res Commun 152:762-768 22. Chou JY 1982 Effects of retinoic acid on differentiation of choriocarcinoma cells in vitro. J Clin Endocrinol Metab 54:1174-1180 23. Chou JY, Rosen SW, Mano T 1981 Production of pregnancyspecific /8i-glycoprotein by cultured placental cells. J Clin Endocrinol Metab 53:239-245 24. Small MB, Gluzman Y, Ozer HL 1982 Enhanced transformation of human fibroblasts by origin-defective simian virus 40. Nature 296:671-672 25. Martin RG, Chou JY 1975 SV40 function required for the establishment and maintenance of malignant transformation. J Virol 15:599-612 26. Chou JY 1989 Differentiated mammalian cell lines immortalized by temperature-sensitive tumor viruses. Mol Endocrinol 10:15111514 27. Goldstein DP, Aono T, Taymor MT, Jochelson K, Todd R, Hines E 1968 Radioimmunoassay of serum chorionic gonadotropin activity in normal pregnancy. Am J Obstet Gynecol 102:110-114 28. Koh SH, Cauchi MN 1981 The production of /^-specific pregnancy glycoprotein (SPi) by the amnion. Eur J Obstet Gynecol Reprod Biol 11:215-219
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29. Ho PC, Haynes WDG, Ing RMY, Jones WR 1982 Histological, ultrastructural and immunofluorescence studies on the amniochorionic membrane. Placenta 3:109-126 30. Kovats S, Main EK, Librach C, Stubblebine M, Fisher SJ, DeMars R1990 A class I antigen, HLA-G, expressed in human trophoblasts. Science 248:220-223 31. Hunt JS, Hsi B-L 1990 Evasive strategies of trophoblast cells: selective expression of membrane antigens. Am J Reprod Immunol 23:57-63 32. Rinke de Wit TF, Vloemans S, van den Elsen PJ, Haworth A, Stern PL 1990 Differential expression of the HLA class I multigene family by human embryonal carcinoma and choriocarcinoma cell lines. J Immunol 144:1080-1087 33. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) 1990 Current Protocols in Molecular Biology. Greene and Wiley-Interscience, New York 34. Long EO, Wake CT, Gorski J, Mach B 1983 Complete sequence of an HLA-DRjS chain deduced from a cDNA clone and identification of multiple non-allelic DRj3 chain genes. EMBO J 2:389-394 35. Fiddes JC, Goodman HM 1979 Isolation, cloning and sequence analysis of the cDNA for the a-subunit of human chorionic gonadotropin. Nature 281:351-356 36. Fiddes JC, Goodman HM 1980 The cDNA for the 0-subunit of human chorionic gonadotropin suggests evolution of a gene by readthrough into the 3'-untranslated region. Nature 386:684-687 37. Geraghty DE, Koller BH, Orr HT 1987 A human major histocompatibility complex class I gene that encodes a protein with a shortened cytoplasmic segment. Proc Natl Acad Sci USA 84:91459149 38. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299 39. Lehrach H, Diamond D, Wozney JM, Boedtker H 1977 RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16:4743-4751 40. Van Herendael BJ, Oberti C, Brosens I 1978 Microanatomy of the human amniotic membranes. A light microscopic, transmission, and scanning electron microscopic study. Am J Obstet Gynecol 131:872-880 41. Chou JY, Sartwell AD, Lei K-J, Plouzek CA 1990 Effects of sodium butyrate on the synthesis of human pregnancy-specific f3x-glycoprotein. J Biol Chem 265:8788-8794 42. Azer PC, Braunstein GD, Van de Velde RL, Kogan R, Engvall E 1980 Ectopic production of pregnancy-specific ft -glycoprotein by a nontrophoblastic tumor in vitro. J Clin Endocrinol Metab 50:234239 43. Jenkins DM, O'Neill M, Johnson PM 1983 HLA-DR-positive cells in the human amniochorion. Immunol Lett 6:65-67 44. Candido EPM, Reeves R, Davie JR 1978 Sodium butyrate inhibits histone in deacetylation cultured cells. Cell 14:105-113 45. Bohan CA, Robinson RA, Luciw PA, Srinivasan A 1989 Mutational analysis of sodium butyrate inducible elements in the human immunodeficiency virus type I long terminal repeat. Virology 172:573-583
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Vol. 129, No. 2 Printed in U.S.A.
Isolation and Characterization of a Human Amnion Epithelial Cell Line That Expresses the PregnancySpecific /?i-Glycoprotein Gene* CATHIE A. PLOUZEK AND JANICE YANG CHOU Human Genetics Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892
polypeptide. At the nonpermissive temperature (39.5 C), HAA58OD-8C cells exhibited a differentiated phenotype, expressed increased levels of PSG, hCG«, and hCG/3 mRNAs, and produced high levels of PSG polypeptides of 72K and 48K. Sodium butyrate induced PSG mRNA expression, and in the presence of butyrate, HAA58OD-8C cells produced high amounts of PSG polypeptides of 72K, 62K, and 48K. Ribonuclease protection analysis indicated that similar PSG transcripts were expressed by HAA58OD-8C cells and human term placenta. However, these amnion cells expressed selectively a certain population of PSG transcripts. Our results show that this amnion cell line provides a suitable model for studies of PSG gene expression and regulation. (Endocrinology 129: 950-958,1991)
ABSTRACT. Human pregnancy-specific /?i-glycoprotein (PSG) is a family of closely related glycoproteins of 72K, 64K, 62K, and 54K. Together with the carcinoembryonic antigen, they form new members of the immunoglobulin superfamily. To study the molecular mechanisms that regulate expression of the PSG gene, we established a human amnion cell line, HAA58OD8C, immortalized with an origin-defective simian virus-40 (SV40) temperature-sensitive A58 mutant virus. HAA58OD-8C cells were temperature sensitive for maintenance of transformation and expressed genes encoding PSG and the a- and /?subunits of hCG. At the permissive temperature (33 C; transformed phenotype), they expressed low levels of PSG, hCGa, and hCG/3 mRNAs and synthesized low levels of a 48K PSG
P
REGNANCY- specific /3rglycoprotein (PSG) was the first of a group of pregnancy-associated proteins to be identified (for reviews, see Refs. 1 and 2). It is primarily produced by the human placenta, but low levels are also synthesized by amnion tissues. The concentrations of PSG in maternal serum and amniotic fluid increase as pregnancy progresses, reaching 200-400 and 2 mg/liter, respectively, at term (3, 4). PSG has been used clinically to diagnose pregnancy and predict some pregnancy-related complications. For example, low PSG values are associated with poor pregnancy outcome in threatened abortions (5), intrauterine growth retardation (6), and fetal hypoxia (7). The clinical uses of PSG are not limited to pregnancy. It is found in the serum of most patients with hydatidiform mole, invasive mole, and choriocarcinoma (1), and it has been employed as a marker for monitoring the treatment of choriocarcinoma (1). In addition, PSG analysis of amniotic fluid is used to diagnose Meckel's syndrome (8). To uncover the functions of human PSG in pregnancy,
our laboratory has undertaken a number of studies to characterize this protein. We found that placental PSG represents a family of closely related glycoproteins of 72K, 64K, 62K, and 54K (9, 10). In addition, cDNA and genomic clones encoding members of the PSG family were recently isolated and characterized by our laboratory (9, 11) and others (12-20). Sequence analysis of PSG cDNA clones indicated that this protein family is related to the carcinoembryonic antigen and is a member of the immunoglobulin superfamily (12-14, 21). Although PSG is a major placental protein, cultured choriocarcinoma cells (malignant trophoblasts) do not produce detectable levels of this protein (22). We have previously immortalized human placental cells with the simian virus-40 (SV40) viruses and obtained cell lines capable of synthesizing PSG (23). However, the SV40immortalized human cells shed infectious virus, thus limiting their use as a model. To immortalize human cells free of infectious virions, Small and co-workers (24) engineered an origin-defective SV40 virus and showed that these SV40 mutants immortalized virus-free human cells with high efficiency. In the present study we immortalized human amnion cells with an origin-defective SV40 temperature-sensitive (ts) A58 mutant virus (Gluzman, Y., unpublished results) carrying an additional
Received March 15, 1991. Address all correspondence and requests for reprints to: Dr. Janice Yang Chou, Building 10, Room 9S-242, National Institutes of Health, Bethesda, Maryland 20892. * This work was performed while C.A.P. held a National Research Council (Human Genetics Branch, NICHHD) research associateship.
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PSG SYNTHESIS IN A HUMAN AMNION CELL LINE mutation in the SV40 A gene which is required for maintenance of transformation (25). Like other SV40 tsA mutant-transformed mammalian cells (23, 26), the origin-defective SV40 tsA58-immortalized human amnion cell line (HAA58OD-8C) is conditionally transformed and expressed the transformed phenotype only at the permissive temperature. At the nonpermissive temperature, these cells reverted to a differentiated phenotype and expressed high levels of the PSG gene. Moreover, expression of the PSG gene could be augmented by sodium butyrate. HAA580D-8C cells also express the hCG gene, a major placental glycoprotein hormone (27), and a product of the human amnion (28, 29). It has been demonstrated that classes I and II of the major histocompatibility antigens are not expressed by placenta or placenta-related tissues, with the exception of the «-chain of the human histocompatibility antigen HLA-G (30, 31). HLA-G is a class I antigen that is specifically expressed in placenta-related tissues, including the JEG-3 human choriocarcinoma cell line (30-32). As expected, the HAA580D-8C amnion cell line reported in this study also expresses the HLA-G gene. In contrast to choriocarcinoma cells and placenta, which do not express the major histocompatibility class II antigen HLA-DR0i (32), the transformed amnion cells expressed this class II antigen. Materials and Methods Transformation of human amnion epithelial cells Primary human amnion epithelial cells were obtained by collagenase digestion of human amnion membrane from a term pregnancy. The primary cultures were grown in a-Modified Minimum Essential Medium supplemented with 10% fetal bovine serum, streptomycin (100 Mg/nil), and penicillin (100 U/ml) and were transfected with the DNA (10 Mg/25 cm2) of an origin-defective SV40 tsA58 mutant virus by a calcium phosphate precipitation method (33). The transformed cultures were incubated at 33 C in medium supplemented with 4% fetal bovine serum, which was replaced twice weekly. Clones of transformed cells, identifiable after 3 months, were selected and recloned. The HAA58OD-8C cell line, which produced high levels of immunoreactive PSG, was analyzed in detail. Overgrowth of nontransformed fibroblasts by transformed cells Human fibroblasts were trypsinized and suspended in aMinimum Essential Medium with 4% fetal bovine serum at a density of 2 x 106 cells/ml. Each well of a 24-well plate (Cooke Laboratory Products, Dynatech Laboratories, Inc., Alexandria, VA) received 1.5 ml of the suspension, which produced a confluent monolayer after attachment. Each well also received transformed HAA58OD-8C cells at various concentrations. One set of plates was incubated at the permissive temperature (33 C), and a duplicate set of plates was incubated at the nonpermissive temperature (39.5 C). Medium was replaced twice weekly. After 2 weeks of incubation at either temperature,
951
plates were fixed in absolute methanol and stained with 0.1% Evans blue in PBS. Biosynthesis of PSG HAA58OD-8C cells were grown initially at 33 C (permissive temperature) for 3-5 days, after which time (day 0) the cultures were treated with control medium or medium containing sodium butyrate, and some of the cultures were shifted to 39.5 C (nonpermissive temperature). Medium was changed every 2 days. Cultures were labeled with L-[35S]methionine (100 /iCi/ ml) for 4 h, and PSGs in the medium were isolated by immunoprecipitation with anti-PSG serum and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and fluorography. Hybridization probes Sequence analysis of PSG cDNA and genomic clones indicated that PSG16 (9), PSG93 (9), PSG95 (11), PSGC (12), and PSGld (17) encode five PSG transcripts generated by alternative RNA splicing of the same gene at one of the four sites (Cl, C2, C3, and C4) in the 3' exon (11) (Lei, K.-J., and J. Y. Chou, unpublished results). These five transcripts share sequence homology in the 5'-untranslated and coding regions, but differ in sequence in the 3'-end coding and untranslated regions. The PSG probes (into pGEM vectors) used in this study are: PSG5' (nucleotides 1-142, the 5' EcoRI-BamHl fragment of PSG93), PSG3'-C1 (nucleotides 1411-1508, the 3' BamHlNcol fragment of PSG95), PSG3'-C2 (nucleotides 1849-3' end, the 3' Pstl-EcoRl fragment of PSG95), PSG3'-Cl+C2 (nucleotides 1411-3' end, the 3'-BamHl-EcoRl fragment of PSG95), PSG3'-C3 (nucleotides 15-8,5 of the PSG93-specific sequence), and PSG3'-C4 (nucleotides 1748-3' end of PSG16). Antisense riboprobes were produced following the procedures supplied by Promega Biotec (Madison, WI). Additional antisense riboprobes used were the 517-base Pstl fragment of HLA-DRfr (34), hCGa (35), and the 224-base, 5' EcoRl-Pstl fragment of hCG/3 (36). The 3'-untranslated region of HLA-G (37) was labeled by random priming. Nucleic acid hybridization Total RNA was isolated by the guanidinium thiocyanateCsCl method (38), and poly(A)+ RNA was obtained by oligo(dT)-cellulose chromatography. RNA was separated by electrophoresis in 1.2% agarose gels containing 2.2 M formaldehyde (39) and transferred to Zetabind (AMF, Meriden, CT) or Nytran (Schleicher and Schuell, Keene, NH) membranes by electroblotting. The filters were hybridized at 42 C (HLA-G), 55 C (PSG3'-C3), or 65 C (PSG-5', PSG3'-Cl+C2, PSG3'-C3, PSG3'-C4, hCG«, and HLA-DR/3,) in a buffer containing 50% formamide, 10% dextran sulfate, and a probe (106 cpm/ml), as previously described (10). Northern blots were washed twice in 300 mM NaCl-30 mM Na-citrate containing 1% SDS for 10 min at room temperature, then twice in 15 mM NaCl-1.5 mM Na citrate containing 0.5% SDS for 10 min at room temperature, followed by four washes in 15 mM NaCl-1.5 mM Na citrate containing 0.1% SDS for 60 min at 65 C.
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PSG SYNTHESIS IN A HUMAN AMNION CELL LINE
952 Ribonuclease protection assay
Ribonuclease protection assays were performed essentially as previously described (33). Briefly, poly(A+) RNA of HAA58OD-8C cells, total RNA of human placenta, or yeast tRNA was hybridized with an antisense riboprobe (5 X 105 cpm) in buffer containing 80% formamide, 400 mM NaCl, 40 HIM PIPES (pH 6.4), and 1 mM EDTA at 45 C for 18 h. The hybrids were digested with RNase-A (40 Mg/ml) and RNase-Tl (2 Mg/ml) for 60 min at 30 C and then electrophoresed on polyacrylamide-urea sequencing gels. Single base substitutions were normally not digested by these ribonucleases under the assay conditions used.
Results Isolation of temperature-sensitive human amnion cells It has been demonstrated that PSG is synthesized by both human placenta and amnion (2-4). Although PSGproducing human placental cell lines immortalized with the SV40 virus have been established, these cells are also capable of producing infectious virions in culture, a characteristic of SV40-transformed human cells. To establish PSG-producing cell lines devoid of infectious virions, we attempted to immortalize both human placental and amnion cells with an origin-defective SV40 tsA58 mutant virus. Unfortunately, we were unable to immortalize human placental cells, but several human amnion cell lines were successfully obtained. Three months after transfecting primary human amnion epithelial cells with the DNA of a SV40 origindefective tsA58 virus at 33 C, clones of transformed cells could be identified. Transformed colonies were selected and recloned. These cell lines were screened for their ability to synthesize PSG; PSG-producing lines were then selected for further characterization. HAA58OD8C, which secreted PSG and maintained the ultrastructure characteristics of amnion epithelial cells, as demonstrated by electron microscopy analysis (Fig. 1), was extensively characterized. The microvilli of HAA58OD8C cells were present on the cell surface, and contiguous cells were joined by the junctional complex of the desmosome type. Additionally, the nuclei of HAA58OD-8C cells had an irregular configuration, and microfilaments were located in the cytoplasm of the cells, characteristic of amnion epithelial cells (40). To determine if HAA58OD-8C cells were temperature sensitive for maintenance of the transformed phenotype, we examined the ability of these cells to overgrow nontransformed cell layers at the permissive (33 C) or nonpermissive (39.5 C) temperature (Fig. 2). At 33 C, HAA58OD-8C behaved as transformed cells and overgrew nontransformed cell layers. At 39.5 C, the ability of this cell line to overgrow nontransformed cell layers was greatly diminished, demonstrating that HAA58OD-8C
Kndo «1991 Vol 129 • No 2
cells were temperature sensitive for maintenance of transformation. Biosynthesis of PSG To determine whether HAA58OD-8C cells produced PSG, synthesis was examined in cells grown at the permissive (33 C) and nonpermissive (39.5 C) temperatures. At 33 C, HAA58OD-8C cells synthesized low levels of a 48K PSG (Fig. 3a). At 39.5 C, synthesis was increased (Fig. 3b). Moreover, at 39.5 C, two PSG polypeptides of 72K and 48K were seen in these cells. We have previously shown that human placenta and primary trophoblasts contain PSG species of 72K (major), 64K, 62K, and 54K (9, 10), and placental fibroblasts produce PSG of 72K (major), 62K, and 48K (41). Both the 54K and 48K PSGs have polypeptide backbones of 36K, suggesting that these two polypeptides differ primarily in protein glycosylation. Our results indicate that differentiated HAA58OD-8C cells (at 39.5 C) synthesized the major placental PSG of 72K. It has been demonstrated that sodium butyrate increased PSG synthesis in placental fibroblasts (41) and several human cell lines (23, 42). In the present study we showed that PSG synthesis in HAA58OD-8C cells was also induced by butyrate (Fig. 3). Butyrate increased PSG biosynthesis in a dose-dependent manner (Fig. 3a). Moreover, in the presence of butyrate, HAA58OD-8C cells synthesized three PSG species of 72K, 62K, and 48K at either temperature (Fig. 3b). Thus, in transformed amnion cells, induction of the 72K PSG synthesis was achieved either by shifting these cells to the nonpermissive temperature (differentiated phenotype) or by butyrate treatment. However, synthesis of the 62K PSG was demonstrated only in the presence of butyrate. Northern analysis To deduce possible mechanisms regulating PSG gene expression in HAA58OD-8C cells, we examined PSG mRNA expression by Northern analysis (Fig. 4). Sequence analysis of PSG cDNA and genomic clones indicates that PSGs are a family of closely related glycoproteins which are encoded by multiple genes (9, 11-20). These PSG transcripts share strong sequence similarity at the 5'-untranslated and coding region, but differ in sequence at the 3'-end coding and untranslated regions. The 3' end of PSG mRNAs are evolved by alternative RNA splicing of a given gene at one of the four sites (Cl, C2, C3, and C4) in the 3' exon. The probes used in this study, which were derived from the PSGGl (Lei, K.-J., and J. Y. Chou, unpublished results), encoded species that hybridized with the majority of PSG mRNAs. We previously showed (10) that placenta expresses three PSG mRNAs of 2.3, 2.2, and 1.7 kilobases (kb), identified by four different PSG probes. HAA58OD-8C
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PSG SYNTHESIS IN A HUMAN AMNION CELL LINE
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a.
Fid. 1. Electron micrographs of HAA58OD-8C amnion epithelial cells. Microvilli (MV), desmosomes (D), filaments (F), and nucleus (N) are denoted, a, 10,500-fold magnification; b, 35,000fold magnification.
Transformants 10
10
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FlO. 2. The ability of HAA58OD-8C amnion epithelial cells to overgrow a monolayer of fibroblasts at 33 and 39.5 C. Each well contains 2 x 10R human fibroblasts and 0, 10, 102, 103, 3 x 103, 10\ 3 x 10\ and 10fi HAA58OD-8C cells. Duplicate plates were prepared; one was incubated at 33 C, and the other at 39.5 C. After 2 weeks of incubation, plates were fixed and stained, as described in Materials and Methods.
cells were found to express all three PSG mRNAs, as identified by PSG-5' (2.3, 2.2, and 1.7 kb), PSG3'-C3 (2.2 kb), PSG3'-C4 (2.2 kb), and PSG3 / -Cl+C2 (2.3, 2.2, and 1.7 kb) probes (Fig. 4). In the absence of butyrate, cells grown at 39.5 C expressed higher levels of PSG mRNAs than cells grown at 33 C. Moreover, at 39.5 C, a 2.3-kb transcript which hybridized with the PSG-5' and PSG3'-C1+C2 probes was preferentially induced (Fig. 4). Sodium butyrate increased the steady state levels of all identified PSG mRNAs at both 33 and 39.5 C (Fig. 4). However, in the presence of butyrate at 39.5 C, HAA58OD-8C expressed more of the 2.3- and 1.7-kb mRNAs that hybridized with the PSG-5' or the PSG3'C1+C2 probe. Ribonuclease protection analysis of PSG expression
Because of the polymorphic nature of the PSG gene and its transcripts, each of the discrete bands (2.3, 2.2, or 1.7 kb) observed on Northern gels actually represents
a group of PSG transcripts of similar size. To determine the dominant PSG transcripts expressed in HAA58OD8C cells, we employed a RNase protection assay (Fig. 5). Placental mRNA yielded two clusters of protected fragments of 87-89 and 46-62 bases with the PSG-5' probe (Fig. 5a). RNA from butyrate-treated HAA58OD-8C cells yielded protected fragments with sizes similar to those observed in placenta, but at reduced levels. RNA from control HAA58OD-8C cells yielded fewer protected bands and at reduced levels. The 87-89-base bands were low or absent, and the 46- and 62-base bands were the major protected species. Moreover, a 120-base protected fragment was observed in mRNA from control HAA58OD-8C cells grown at both 33 and 39.5 C and from butyrate-treated cells grown at 33 C (Fig. 5a). This 120-base band was absent from RNAs isolated from placenta or butyrate-treated HAA58OD-8C cells grown at 39.5 C. This suggests that HAA58OD-8C cells produce a PSG transcript that is not found in human placenta and was inhibited by butyrate treatment. Both HAA58OD-8C and placental mRNAs yielded two major protected fragments of 71 and 41 bases, using PSG3'-C3 probe (Fig. 5b). Butyrate induced the expression of both transcripts, but the 71-base protected species was preferentially induced at 39.5 C. Placental mRNA yielded two major protected fragments of 160 and 53-56 bases with the PSG-3'-C4 probe (Fig. 5c). However, HAA58OD-8C mRNA yielded only the 160-base protected fragment, and butyrate induced the expression of this transcript. These results show that HAA58OD-8C cells and placental PSG mRNAs share sequence similarity in the 3' regions. However, HAA58OD-8C cells did not express all PSG mRNA species found in human placenta.
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PSG SYNTHESIS IN A HUMAN AMNION CELL LINE
Endo'1991 Voll29«No2
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FIG. 6. Expression of hCG, HLA-G, and HLA-DR/3i by HAA58OD-8C cells, a, HCG/3 expression analyzed by ribonuclease protection. Poly(A)+ mRNA (10 jig) from HAA58OD-8C cells grown at 33 or 39.5 C, total RNA (5 Mg) from placenta, and yeast tRNA (10 ^g) were annealed to a uniformly labeled antisense riboprobe corresponding to nucleotides 1-224 of hCG/3, as described in Materials and Methods, b-d, HCGa, HLA-G, and HLA-DRft expression analyzed by Northern hybridization. Poly(A)+ mRNA (10 Mg) from HAA58OD-8C cells grown at 33 or 39.5 C, or JEG3 choriocarcinoma cells was separated by electrophoresis on formaldehyde-agarose gels, transferred to Nytran membrane, and hybridized with a uniformly labeled antisense hCGa or HLA-DRfr riboprobe or the random primed HLA-G probe, as described in Materials and Methods. Stern for HLA-G, and Dr. J. Fiddes for hCGa and hCG/? cDNAs. References 1. Tatarinov YS 1978 Trophoblast-specific beta!-glycoprotein as a marker for pregnancy and malignancies. Gynecol Obstet Invest 9:65-97 2. Sorensen S 1984 Pregnancy-"specific" /3i-glycoprotein (SPi): purification, characterization, quantification and clinical application in malignancies (a review). Tumor Biol 5:275-302 3. Heikinheimo M 1980 Pregnancy-specific ft-glycoprotein in amniotic fluid in high risk late pregnancy. Oncodev Biol Med 1:287290 4. Grudzinskas JG, Evans DG, Gordon YB, Jeffrey D, Chard T 1978 Pregnancy specific ft glycoprotein in fetal and maternal compartments. Obstet Gynecol 52:43-45 5. Masson GM, Anthony F, Wilson MS 1983 Value of schwangeshattsprotein (SPi) and pregnancy associated protein A (PAPPA) in the clinical management of threatened abortion. Br J Obstet Gynaecol 90:146-149 6. Tamsen L, Johansson SGO, Axelsson O 1983 Pregnancy-specific ft-glycoprotein (SPI) in serum from woman with pregnancies complicated by intrauterine growth retardation. J Perinat Med 11:19-25 7. MacDonald DJ, Scott JM, Gemmel RS, Mack DS 1983 A prospec-
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12.
13.
tive study of three biochemical fetoplacental tests: serum placental lactogen, pregnancy-specific ft-glycoprotein, and urinary estrogen and their relationship to placental insufficiency. Am J Obstet Gynecol 147:430-436 Heikinheimo M, Aula P, Rapola J, Wahlstrom T, Jalanko H, Seppala M 1982 Amniotic fluid pregnancy-specific ft-glycoprotein (SPi) in Meckels' syndrome: a new test for prenatal diagnosis? Prenat Diagn 2:103-108 Watanabe S, Chou JY 1988 Isolation and characterization of complementary DNAs encoding human pregnancy-specific ft-glycoprotein. J Biol Chem 263:2049-2054 Chou JY, Zilberstein M 1990 Expression of the pregnancy-specific ft-glycoprotein gene in cultured human trophoblasts. Endocrinology 127:2127-2135 Leslie KK, Watanabe S, Lei K-J, Chou DY, Plouzek CA, Deng HC, Torres J, Chou JY 1990 Linkage of two human pregnancyspecific ft-glycoprotein genes: one is associated with hydatidiform mole. Proc Natl Acad Sci USA 87:5822-5826 Streydio C, Lacka K, Swillens S, Vassart G 1988 The human pregnancy-specific ft-glycoprotein (PS/3G) and the carcinoembryonic antigen (CEA)-related proteins are members of the same multigene family. Biochem Biophys Res Commun 154:130-137 Rooney BC, Home CHW, Hardman N 1988 Molecular cloning of a cDNA for human pregnancy-specific /3rglycoprotein: homology with human carcinoembryonic antigen and related proteins. Gene 71:439-449
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PSG SYNTHESIS IN A HUMAN AMNION CELL LINE
14. Chan W-Y, Borjigin J, Zheng Q-X, Shupert WL 1988 Characterization of cDNA encoding human pregnancy-specific /Jrglycoprotein from placenta and extraplacental tissues and their comparison with carcinoembryonic antigen. DNA 7:545-555 15. McLenachan T, Mansfield B 1989 Expression of CEA-related genes in the first trimester human placenta. Biochem Biophys Res Commun 162:1486-1493 16. Khan WN, Hammarstrom S 1989 Carcinoembryonic antigen gene family: molecular cloning of cDNA for a PS/3G/FL-NCA glycoprotein with a novel domain arrangement. Biochem Biophys Res Commun 161:525-535 17. Zimmermann W, Weiss M, Thompson JA 1989 cDNA cloning demonstrates the expression of pregnancy-specific glycoprotein genes, a subgroup of the carcinoembryonic antigen gene family, in fetal liver. Biochem Biophys Res Commun 163:1197-1209 18. Oikawa S, Inuzuka C, Kosaki G, Nakazato H 1988 Exon-intron organization of a gene for pregnancy-specific /3i-glycoprotein, a subfamily member of CEA family: implications for its characteristic repetitive domains and C-terminal sequences. Biochem Biophys Res Commun 156:68-77 19. Oikawa S, Inuzuka C, Kuroki M, Matsuoka Y, Kosaki G, Nakazato H 1989 A pregnancy-specific /3i-glycoprotein, a CEA gene family member, expressed in a human promyelocytic leukemia cell line, HL-60: structures of protein, mRNA and gene. Biochem Biophys Res Commun 163:1021-1031 20. Thompson J, Koumari R, Wagner K, Barnert S, Schleussner C, Schrewe H, Zimmermann W, Muller G, Schempp W, Zaninetta D, Ammaturo D, Hardman N 1990 The human pregnancy-specific glycoprotein genes are tightly linked on the long arm of chromosome 19 and are coordinately expressed. Biochem Biophys Res Commun 167:848-859 21. Watanabe S, Chou JY 1988 Human pregnancy-specific /?i-glycoprotein: a new member of the carcinoembryonic antigen gene family. Biochem Biophys Res Commun 152:762-768 22. Chou JY 1982 Effects of retinoic acid on differentiation of choriocarcinoma cells in vitro. J Clin Endocrinol Metab 54:1174-1180 23. Chou JY, Rosen SW, Mano T 1981 Production of pregnancyspecific /8i-glycoprotein by cultured placental cells. J Clin Endocrinol Metab 53:239-245 24. Small MB, Gluzman Y, Ozer HL 1982 Enhanced transformation of human fibroblasts by origin-defective simian virus 40. Nature 296:671-672 25. Martin RG, Chou JY 1975 SV40 function required for the establishment and maintenance of malignant transformation. J Virol 15:599-612 26. Chou JY 1989 Differentiated mammalian cell lines immortalized by temperature-sensitive tumor viruses. Mol Endocrinol 10:15111514 27. Goldstein DP, Aono T, Taymor MT, Jochelson K, Todd R, Hines E 1968 Radioimmunoassay of serum chorionic gonadotropin activity in normal pregnancy. Am J Obstet Gynecol 102:110-114 28. Koh SH, Cauchi MN 1981 The production of /^-specific pregnancy glycoprotein (SPi) by the amnion. Eur J Obstet Gynecol Reprod Biol 11:215-219
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29. Ho PC, Haynes WDG, Ing RMY, Jones WR 1982 Histological, ultrastructural and immunofluorescence studies on the amniochorionic membrane. Placenta 3:109-126 30. Kovats S, Main EK, Librach C, Stubblebine M, Fisher SJ, DeMars R1990 A class I antigen, HLA-G, expressed in human trophoblasts. Science 248:220-223 31. Hunt JS, Hsi B-L 1990 Evasive strategies of trophoblast cells: selective expression of membrane antigens. Am J Reprod Immunol 23:57-63 32. Rinke de Wit TF, Vloemans S, van den Elsen PJ, Haworth A, Stern PL 1990 Differential expression of the HLA class I multigene family by human embryonal carcinoma and choriocarcinoma cell lines. J Immunol 144:1080-1087 33. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) 1990 Current Protocols in Molecular Biology. Greene and Wiley-Interscience, New York 34. Long EO, Wake CT, Gorski J, Mach B 1983 Complete sequence of an HLA-DRjS chain deduced from a cDNA clone and identification of multiple non-allelic DRj3 chain genes. EMBO J 2:389-394 35. Fiddes JC, Goodman HM 1979 Isolation, cloning and sequence analysis of the cDNA for the a-subunit of human chorionic gonadotropin. Nature 281:351-356 36. Fiddes JC, Goodman HM 1980 The cDNA for the 0-subunit of human chorionic gonadotropin suggests evolution of a gene by readthrough into the 3'-untranslated region. Nature 386:684-687 37. Geraghty DE, Koller BH, Orr HT 1987 A human major histocompatibility complex class I gene that encodes a protein with a shortened cytoplasmic segment. Proc Natl Acad Sci USA 84:91459149 38. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299 39. Lehrach H, Diamond D, Wozney JM, Boedtker H 1977 RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16:4743-4751 40. Van Herendael BJ, Oberti C, Brosens I 1978 Microanatomy of the human amniotic membranes. A light microscopic, transmission, and scanning electron microscopic study. Am J Obstet Gynecol 131:872-880 41. Chou JY, Sartwell AD, Lei K-J, Plouzek CA 1990 Effects of sodium butyrate on the synthesis of human pregnancy-specific f3x-glycoprotein. J Biol Chem 265:8788-8794 42. Azer PC, Braunstein GD, Van de Velde RL, Kogan R, Engvall E 1980 Ectopic production of pregnancy-specific ft -glycoprotein by a nontrophoblastic tumor in vitro. J Clin Endocrinol Metab 50:234239 43. Jenkins DM, O'Neill M, Johnson PM 1983 HLA-DR-positive cells in the human amniochorion. Immunol Lett 6:65-67 44. Candido EPM, Reeves R, Davie JR 1978 Sodium butyrate inhibits histone in deacetylation cultured cells. Cell 14:105-113 45. Bohan CA, Robinson RA, Luciw PA, Srinivasan A 1989 Mutational analysis of sodium butyrate inducible elements in the human immunodeficiency virus type I long terminal repeat. Virology 172:573-583
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