Regulation of alkaline phosphatase gene expression in cultured placental cells.
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Alkaline Phosphatase↗
Biomedical subjects
J Y Chou
Publications and source records attributed to J Y Chou.
Transformed liver cells have modified transplasma membrane redox activity which is sensitive to adriamycin.
Electron transport across the plasma membrane is found in all cells which have been tested. This activity has been implicated in control of cellular growth, transport and hormone response. In virus transformed cells and tumor cells we find the activity is decreased and becomes sensitive to the antitumor drug adriamycin. Inhibition of transmembrane redox by adriamycin parallels cytoxicity to transformed cells.
Animals↗
Biosynthesis and processing of placental alkaline phosphatase.
Polypeptides of 61,500 and 64,500 apparent molecular weights were the precursor and fully processed forms of placental alkaline phosphatase monomer synthesized by choriocarcinoma cells in vivo. [3H] Mannose was incorporated into both polypeptides whereas [3H] glucosamine was incorporated mainly into the 64,500-dalton polypeptide, suggesting processing by the addition of glucosamine moieties. In the absence of microsomal membranes, choriocarcinoma mRNA directed the cell-free synthesis of the preprotein form of alkaline phosphatase monomer of apparent Mr = 60,000. The unglycosylated monomer had an apparent Mr = 58,000. In the presence of microsomal membranes, the 60,000-dalton polypeptide was processed to a polypeptide of apparent Mr = 61,500, comigrating with the precursor form of alkaline phosphatase monomer.
Alkaline Phosphatase↗
Isolation of cells secreting specific polypeptides.
Antibody coupled erythrocytes in an agar overlay were used as a simple, nontoxic technique for the isolation of cell clones secreting proteins and hormones in vitro. The adaptation of the reverse hemolytic plaque assay was developed to provide a rapid, sensitive, and specific method for the screening of any antigen secreting cell without the need of prior isolation. This technique promises to be of use in the characterization and cloning of cells growing in mixed culture.
Animals↗
Control of placental protein production by retinoic acid in cultured placental cells.
The synthesis of human chorionic gonadotropin (HCG), the subunit of HCG (HCG alpha), and pregnancy-specific beta 1-glycoprotein (PS beta G) was studied in temperature sensitive (ts), simian virus 40 (SV40) tsA mutant-transformed human first trimester placental (SPA255-26) cells. Retinoic acid increased the production of HCG and PS beta G but inhibited the production of HCG alpha in these cells. Passage of SPA255-26 placental cells in medium containing retinoic acid induced a stable altered phenotype characterized by elevated levels of HCG and PS beta G and a reduced level of HCG alpha. The retinoic acid induced phenotypic changes in these placental cells were reversible; removal of retinoic acid immediately decreased the production of HCG and PS beta G while increasing the production of HCG alpha. The ratio of HCG to HCG alpha in control SPA255-26 cells was approximately 0.1; this ratio increased to 4.8 in cells maintained in medium containing retinoic acid. Similarly, the HCG-to-HCG alpha ratio increased in choriocarcinoma cells maintained in retinoic acid containing medium. Our data suggest that retinoic acid may be needed to maintain a balanced production of HCG, HCG alpha, and PS beta G in placental cells in vitro. Retinoic acid may also play a role in modulating placental protein production during pregnancy.
Cell Differentiation↗
Temperature-sensitive fetal liver cells: a model for hepatocellular differentiation.
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Albumins↗
Temperature-sensitive adult liver cell line dependent on glucocorticoid for differentiation.
A clonal rat adult hepatocyte cell line (RALA255-10G) was shown to be temperature sensitive (ts) for growth and differentiation. Glucocorticoid was necessary to maintain the maximal levels of differentiated functions in these cells. The RALA255-10G cell line was established by transforming primary adult hepatocytes with simian virus 40 tsA255 virus that is temperature sensitive for maintenance of transformation. At the permissive temperature (33 degrees C), RALA255-10G cells showed characteristics of malignant transformation, synthesized low levels of albumin and transferrin, and contained low levels of functional receptors for glucagon. At the nonpermissive temperature (40 degrees C), these cells regain the normal differentiated phenotype, and the levels of these three hepatic functions were increased. Induction of albumin and transferrin production by RALA255-10G cells at 40 degrees C was shown to be the result of the increase in the biosynthesis of these proteins. Furthermore, the albumin and transferrin produced by these cells were immunologically and electrophoretically indistinguishable from authentic rat albumin and transferrin. Glucocorticoid, which reduced the growth rate and saturation density of RALA255-10G cells at 33 degrees C, was absolutely required by these cells to synthesize albumin at both temperatures. This hormone also enhanced transferrin production and glucagon response. Our data indicate that glucocorticoid hormone is one of the factors that maintain adult hepatocytes in a differentiated state.
Albumins↗
Role of cyclic adenosine 3',5'-monophosphate in differentiation of fetal liver cells in vitro.
Regulation of the synthesis of alpha-fetoprotein (AFP), albumin, and transferrin was studied in temperature-sensitive (ts), simian virus 40 (SV40) tsA mutant-transformed rat fetal liver RLA209-15 cells. The intracellular cyclic AMP (cAMP) levels in RLA209-15 cells grown at the nonpermissive temperature (40 degrees C, the temperature at which the cells exhibit the differentiated phenotype) were higher than the levels in these cells grown at the permissive temperature (33 degrees C, the temperature at which these cells exhibit the transformed phenotype). We have shown previously that differentiation of RLA209-15 cells at 40 degrees C was accompanied by increases in AFP, albumin and transferrin. In the present study, we found that 8 bromo-cAMP (8 BrcAMP), cholera toxin and methylisobutylxanthine (MIX), agents that increase intracellular cAMP levels, greatly stimulated the production of AFP, albumin and transferrin. The phosphodiesterase inhibitor MIX was the most effective inducer of AFP and albumin. The kinetics of AFP induction in the presence of 8BrcAMP, cholera toxin and MIX were similar to the kinetics of albumin induction. The positive correlation of cAMP levels and the differentiated state of these cells indicate that cAMP may be one of the factors that maintains differentiation of fetal liver cells in vitro.
1-Methyl-3-isobutylxanthine↗
Production of pregnancy-specific beta 1-glycoprotein by human placental cells and human fibroblasts.
The synthesis and regulation of pregnancy-specific beta 1-glycoprotein (PS beta G) in secondary placental cells and human fibroblasts was studied in vitro. Both types of cells produced PS beta G in culture. The secondary placental cells, however, produced considerably higher levels of PS beta G than did the human fibroblasts in both the absence and presence of inducers. The PS beta G produced by the secondary placental cells resembled the purified placental PS beta G in chromatographic behavior, immunological reactivity, and affinity to concanavalin A. The synthesis of PS beta G in the secondary placental cells and human fibroblasts was induced by sodium butyrate and 5-bromo-2'-deoxyuridine (BrdUrd) but was not affected by agents that increase intracellular cAMP concentrations and retinoic acid. The synthesis of two other placental markers, human chorionic gonadotropin (hCG) and the alpha subunit of hCG (hCG alpha), was also studied in both types of cells. Neither the secondary placental cells nor the human fibroblasts secreted hCG in vitro. The human fibroblasts also did not secrete hCG alpha both in the absence and presence of inducers. The secondary placental cells, however, secreted low levels of hCG alpha in the absence of inducer and high levels of hCG alpha in the presence of 8-bromo-cAMP (8BrcAMP), cholera toxin, and methylisobutylxanthine (MIX). The presence of PS beta G in secondary placental cells and human fibroblasts suggests that PS beta G may be associated with growth and maintenance of nontransformed cells in vitro.
1-Methyl-3-isobutylxanthine↗
alpha-Fetoprotein biosynthesis and hepatocellular differentiation.
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Animals↗
Biosynthesis of alpha-fetoprotein in cultured hepatoma cells.
Pulse and pulse-chase experiments demonstrated that a heterogeneous polypeptide with an apparent Mr = 68,000 was the first intracellular anti-alpha-fetoprotein (AFP)-precipitable polypeptide synthesized by rat Mc-A-RH-7777 hepatoma cells. The 68,000-dalton polypeptide may consist of polypeptides with apparent molecular weights ranging from 68,000 to 70,000. It was the precursor of two intracellular anti-AFP-precipitable polypeptides of 69,000 and 73,000 apparent molecular weight. The latter were secreted into the medium without further processing. The anti-AFP-precipitable polypeptides in both cells and medium incorporated [3H]glucosamine, indicating that these polypeptides are at least partially glycosylated. The 68,000-dalton polypeptide in cells was bound mostly to concanavalin A-Sepharose, whereas the 69,000-dalton polypeptide was entirely unbound. The 73,000-dalton polypeptide consisted of concanavalin A-bound and -unbound variants. Tunicamycin completely abolished the uptake of [3H]glucosamine into anti-AFT-precipitable polypeptides in both cells and medium, and the resulting polypeptide of apparent Mr = 66,000 did not bind to concanavalin A-Sepharose. Tunicamycin did not affect the synthesis or secretion of AFP by hepatoma cells.
Animals↗
Inhibition of synthesis of alpha-fetoprotein by glucocorticoids in cultured hepatoma cells.
alpha-Fetoprotein (AFP) synthesis was studied in the presence and absence of glucocorticoids in rat hepatoma Mc-A-RH-7777 cells. Radioimmunoassay of media from cell cultures grown in the presence of glucocorticoid (dexamethasone or cortisol) showed a reduction in AFP, an increase in albumin, and no significant change in transferrin accumulation, as compared to controls. Labeling experiments with L-[35S]methionine indicated that in both cells and media of dexamethasone-treated cultures there was a 50--80% reduction in polypeptide precipitated by anti-AFP serum, as compared with controls; no change was seen in polypeptide precipitated by anti-transferrin serum. Pulse and pulse-chase experiments demonstrated that dexamethasone inhibited the synthesis of AFP but not its secretion. The half-time for secretion of AFP in the presence and absence of dexamethasone was 43 min.
Albumins↗
Effects of retinoic acid on differentiation of choriocarcinoma cells in vitro.
Choriocarcinoma cells maintain multiple hormonal functions in culture. We have found that these cells secreted no immunoreactive pregnancy-specific beta 1-glycoprotein (PS beta G), a placental protein. Choriocarcinoma cells can be induced to synthesize low levels of PS beta G by retinoic acid, 8-bromo-cAMP (8BrcAMP), cholera toxin, methyl-isobutylxanthine (MIX), and 5-bromo-2'-deoxyuridine (BrdUrd). The simultaneous addition of retinoic acid along with 8BrcAMP, cholera toxin, or MIX gave synergistic induction of PS beta G. The simultaneous addition of retinoic acid and BrdUrd failed to give even additive induction. In addition to stimulating PS beta G production, retinoic acid increased the production of hCG and its alpha-subunit (hCG alpha) by choriocarcinoma cells. The simultaneous addition of retinoic acid along with 8BrcAMP, cholera toxin, or MIX gave additive induction for hCG and hCG alpha. Passage of choriocarcinoma cells in medium containing retinoic acid induced a stable altered phenotype characterized by elevated levels of PS beta G, hCG, and hCG alpha. These retinoid-treated choriocarcinoma cells remained responsive to 8BrcAMP or compounds that increase intracellular cAMP concentrations and to BrdUrd; the production to PS beta G and hCG was greatly stimulated by 8BrcAMP, cholera toxin, or MIX, and the production of hCG and hCG alpha was greatly inhibited by BrdUrd. However, the production of hCG alpha was only slightly induced by these cAMP modulators, and the production of PS beta G was not increased by BrdUrd.
1-Methyl-3-isobutylxanthine↗
Human trophoblasts: cellular source of colony-stimulating activity in placental tissue.
Colony-stimulating activity (CSA) is a class of protein factors that stimulates the in vitro growth and differentiation of hematopoietic committed stem cells into mature granulocytes and macrophages. In man, production of CSA is mediated by monocytes-macrophages, lymphocyte, and endothelial cells. One of the best studied and most potent sources of CSA is conditioned media derived from cultured human placenta. The cellular source of this placental CSA was studied using cloned term placental cell lines induced by a simian virus 40 (SV-40) Wild-Type (wt) or temperature sensitive (tsA) mutants. Conditioned media from SV-40 wt-transformed placental cells grown at 37 degrees C and tsA-transformed placental cells grown at 33 degrees C (the temperature at which the cells exhibit the transformed phenotype) and at 40 degrees C (temperature at which the cells express a normal differentiated phenotype) contained high levels of CSA. At the restricted temperature (40 degrees C), these cell lines expressed normal trophoblastic characteristics in that they produced human chorionic gonadotropin, pregnancy-specific-beta1-glyco protein, and placental alkaline phosphatase, indicating that the SV-40 transformation has not interfered with normal cellular functions of these trophoblasts. The CSA released by these cell lines resemble those of CM from fresh placental cells, in that the level of activity is high and that formation of both granulocyte/macrophage and eosinophil colonies is stimulated.
Cell Transformation, Viral↗
Study of liver differentiation in vitro.
A clonal rat fetal liver cell line that expresses the functions of differentiated liver cells under controllable conditions has been established. Normal fetal liver cells were transformed by a temperature-sensitive A (tsA) mutant (tsA209) of simian virus 40. At the permissive temperature (33 degrees C), the tsA209-transformed liver cell line (RLA209-15) can be cultured indefinitely and cloned readily. The RLA209-15 cells were temperature sensitive for maintenance of the transformed phenotype. These transformed liver cells selectively lost four characteristics of the transformed phenotype at the restrictive temperature (40 degrees C): generation time of the cells increased, the saturation density decreased, the efficiency of growth on nontransformed cell layers decreased, and the ability to clone in soft agar was lost. The transformation can be reversed simply by a shift in temperature. RLA209-15 fetal liver cells synthesized alpha-fetoprotein albumin, and transferrin. At 33 degrees C, the levels of these liver proteins were relatively low. At 40 degrees C the transformed phenotype was lost and the levels of alpha-fetoprotein, albumin, and transferrin were greatly increased. At the restrictive temperature, maximal induction of the synthesis of alpha-fetoprotein, albumin, and transferrin was achieved 3-4 d after the upward shift in temperature. The synthesis of alpha-fetoprotein then decreased; the synthesis of albumin and transferrin, however, was maintained. A second phase of albumin and transferrin synthesis was observed in all cultures after 6 d or more at 40 degrees C. Alpha-Fetoprotein, albumin, and transferrin secreted by RLA209-15 cells were immunologically indistinguishable from authentic alpha-fetoprotein, albumin, and transferrin, respectively. RLA209-15 cells, like primary cultures of hepatocytes and a simian virus 40 tsA255-transformed fetal liver cell line (RLA255-4) reported earlier from this laboratory, responded to glucagon with markedly elevated levels of cyclic AMP. Thus, it appears that glucagon receptors characteristic of hepatocytes are retained in the simian virus 40 tsA-transformed fetal liver cells.
Albumins↗
Cytomegalovirus replication in primary and passaged human placental cells.
Human cytomegalovirus (HCMV) was found to replicate in passaged fibroblastic human first trimester and term placental cells. The time-course of viral DNA replication as well as virus production in these human placental fibroblasts was similar to that in human embryo fibroblast cultures. In contrast, HCMV did not replicate in primary placental epithelioid cells. Continued passage (5 or more) of primary placental epithelioid cells was necessary to convert these cells to a state of permissiveness. The permissive cells were, however, fibroblasts. HCMV DNA replication in passaged placental fibroblastic cells was not affected by treatment with insulin or human chorionic gonadotropin. Furthermore, no replication of HCMV DNA occurred in choriocarcinoma cells, the epithelioid cells derived from cancer of the placenta. These results suggest that epithelial placental trophoblasts, either normal or transformed, were nonpermissive for HCMV. The permissiveness of HCMV infection to secondary placental cells which was observed might be due to the strong selection of fibroblastic cells in vitro.
Cell Line↗
Regulation of human chorionic gonadotropin synthesis in cultured human placental cells by glucocorticoids.
The synthesis of hCG and its subunits in temperature-sensitive, simian virus 40 tsA mutant-transformed human first trimester and term placental cells and in choriocarcinoma cells was studied in the presence and absence of glucocorticoid hormones. The tsA-transformed placental cells were studied at 33 C (the temperature at which the cells exhibit the transformed phenotype) and at 40 C (the temperature at which the cells regain their differentiated nontransformed phenotype). The glucocorticoids cortisol and dexamethasone did not affect the synthesis of hCG or hCG alpha in either transformed term placental cells or choriocarcinoma cells. In the tsA-transformed first trimester placental cells, however, glucocorticoids greatly inhibited hCG synthesis but induced hCG alpha synthesis. The inhibition on hCG synthesis was immediate, stronger at 40 C than at 33 C, and independent of the growth stage of the cells. The glucocorticoid effects were specific; neither estradiol nor progesterone affected the synthesis of hCG or hCG alpha. Furthermore, progesterone did not block the action of the glucocorticoid. These selective effects in cultured first trimester placental cells suggest that glucocorticoids may play an important role in the regulation of hCG production during early pregnancy.
Cell Line↗
Production of pregnancy-specific beta 1-glycoprotein by cultured placental cells.
The synthesis of pregnancy-specific beta 1-glycoprotein (PS beta G) was studied in simian virus 40 temperature-sensitive A mutant-transformed human first trimester and term placental cells. At the permissive temperature (33 C, transformed phenotype), they produced low levels of PS beta G. At the restrictive temperature (40 C), the transformed phenotype was lost, and the production of PS beta G was greatly enhanced. The PS beta G produced by these transformed placental cells resembled the purified placental PS beta G by several criteria. Both cell and placental PS beta G bound to Concanavalin A-Sepharose and were, therefore, glycoproteins. The cell PS beta G cochromatographed with placental PS beta G on a Bio-Gel A-0.5m column. Furthermore, the slopes of the dose-response curves for the cell PS beta G were indistinguishable from that for placental PS beta G. The synthesis of PS beta G at both 33 and 40 C in these placental cells was greatly induced by sodium butyrate and 5-bromo-2'-deoxyuridine. Sodium butyrate was a more effective inducer at 33 C, whereas BrdUrd appeared to be a better inducer at 40 C.
Bromodeoxyuridine↗