5-Bromo-2'-deoxyuridine induces placental alkaline phosphatase biosynthesis in cultured choriocarcinoma cells

5-Bromo-2'-deoxyuridine (BrdUrd) stimulated the biosynthesis and hence increased the activity of placental alkaline phosphatase in choriocarcinoma cells. While BrdUrd had no effect on the rate of degradation or processing of placental alkaline phosphatase, it increased the rate of phosphatase synthesis. The stimulation of enzyme activity could be completely accounted for by the increase in alkaline phosphatase protein. Both control and BrdUrd-induced cells contained polypeptides of 61,500 and 64,500 Da, identified as the precursor and fully processed forms of placental alkaline phosphatase monomer. The half-life of this enzyme monomer in both control and BrdUrd-treated cells was estimated to be 36 h. BrdUrd induced a specific increase in the placental alkaline phosphatase mRNA leading to the observed enhancement of biosynthesis. The continued rise in alkaline phosphatase biosynthesis in BrdUrd-induced cells following BrdUrd removal indicated that this analog acted by incorporation into DNA.     

Induction of placental alkaline phosphatase in choriocarcinoma cells by 5-bromo-2'-deoxyuridine.

Growth of choriocarcinoma cells in the presence of 5-bromo-2'-deoxyuridine (BrdUrd) results in a 30- to 40-fold increase in alkaline phosphatase activity. The effects of BrdUrd is specific for phosphatase with an alkaline pH optimum. The induction by BrdUrd is probably not due to the production of an altered enzyme, since the induced enzyme resembles the basal enzyme in thermal denaturation and kinetic properties. Enzyme induction can be prevented by thymidine but not by deoxycytidine or deoxyuridine. The induction of alkaline phosphatase appears to require incorporation of the BrdUrd into cellular DNA. The presence of BrdUrd in the growth medium is not necessary for alkaline phosphatase induction in proliferating cells containing BrdUrd-substituted genomes. However, enzyme induction and maintenance of the induced levels of alkaline phosphatase in nonproliferating cells containing BrdUrd-substituted DNA requires the presence of the analogues in the medium. The induction of alkaline phosphatase by BrdUrd in probably an indirect process.     

Induction of placental alkaline phosphatase in choriocarcinoma cells by 5-bromo-2′-deoxyuridine

Summary Growth of choriocarcinoma cells in the presence of 5-bromo-2′-deoxyuridine (BrdUrd) results in a 30- to 40-fold increase in alkaline phosphatase activity. The effect of BrdUrd is specific for phosphatase with an alkaline pH optimum. The induction by BrdUrd is probably not due to the production of an altered enzyme, since the induced enzyme resembles the basal enzyme in thermal denaturation and kinetic properties. Enzyme induction can be prevented by thymidine but not by deoxycytidine or deoxyuridine. The induction of alkaline phosphatase appears to require incorporation of the BrdUrd into cellular DNA. The presence of BrdUrd in the growth medium is not necessary for alkaline phosphatase induction in proliferating cells containing: BrdUrd-substituted genomes. However, enzyme induction and maintenance of the induced levels of alkaline phosphatase in nonproliferating cells containing BrdUrd-substituted DNA requires the presence of the analogues in the medium. The induction of alkaline phosphatase by BrdUrd in probably an indirect process.     

Multiple forms of alkaline phosphatase in untreated and 5-bromo-2'-deoxyuridine-treated choriocarcinoma cells

The alkaline phosphatases present in choriocarcinoma cells, either untreated or treated with 5-bromo-2′-deoxyuridine (BrdUrd), were purified and characterized. Three forms of phosphatase [I, IIa (or IIIa), and IIb (or IIIb)]were isolated from both the untreated and BrdUrd-treated cells. Although BrdUrd induced the synthesis of all three forms of alkaline phosphatase in these cells, the synthesis of forms IIa and IIb was, however, preferentially stimulated. The forms of phosphatase in choriocarcinoma cells resembled each other in their kinetic properties and thermal lability, but differed in their molecular weights and in their electrophoretic mobilities in nondenaturing polyacrylamide gels. All three phosphatases were inactivated by antiserum to term-placental alkaline phosphatase. The alkaline phosphatases from choriocarcinoma cells differed, however, from the enzyme from term placentas in several physicochemical properties. The phosphatases from choriocarcinoma cells had a lower K_m value for p-nitrophenyl phosphate, were more sensitive to inhibition by l-leucine, levamisole, l-p-bromotetramisole, and EDTA, and were more heat-labile. Phosphatase I comigrated with term-placental alkaline phosphatase on nondenaturing polyacrylamide electrophoretic gels, but phosphatases IIa and IIb migrated more slowly. The apparent molecular weights of phosphatase forms I, IIa, and IIb were estimated by gel filtration and polyacrylamide gel electrophoresis to be 115,000, 240,000, and 510,000, respectively. Although three molecular forms of alkaline phosphatase occurred in choriocarcinoma cells, the subunit molecular weight of these phosphatases appeared to be identical to each other and to the subunit of term-placental alkaline phosphatase (63,000 MW). The alkaline phosphatase in choriocarcinoma cells therefore exists in the dimeric, tetrameric, and octameric forms.     

Effect of pyrimidine deoxynucleosides and sodium butyrate on expression of the glycoprotein hormone alpha-subunit and placental alkaline phosphatase in HeLa cells

Production of the glycoprotein hormone common alpha-subunit and placental alkaline phosphatase activity can be modulated in HeLa cells by a variety of deoxynucleosides. Dose response curves for thymidine (Thd), fluorodeoxyuridine (FdUrd), bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) demonstrate that, in general, alkaline phosphatase was increased by lower concentrations of inducer than was alpha-subunit. The deoxynucleosides were not as effective as sodium butyrate as inducers of either protein. Whereas Thd and the halogenated dUrd derivatives enhanced protein expression, deoxycytidine (dCyd) had negative effects. Induction by deoxynucleosides of both alkaline phosphatase and alpha-subunit was inhibited by dCyd, but induction of alkaline phosphatase by butyrate was more sensitive to dCyd inhibition than was the butyrate-mediated induction of alpha-subunit. These results suggest that the two proteins are not regulated in a coordinate manner. Reversal of alkaline phosphatase induction by dCyd was not observed in cells preincubated with sodium butyrate for 6-24 h before the addition of dCyd, indicating that the deoxynucleoside interferes with an early event in the butyrate-mediated response. Combinations of butyrate with Thd, BrdUrd or IdUrd were synergistic with respect to the induction of HeLa-alpha. It is concluded that incorporation of the deoxynucleosides into DNA may not be required for the synergistic response since 2',5'-dideoxythymidine was an effective as Thd. Cytoplasmic dot hybridizations demonstrate that a primary effect of the various effectors is to increase the steady-state levels of alpha-subunit mRNA. There was a good correlation between alpha-subunit accumulation and corresponding levels of alpha-mRNA, suggesting that regulation occurs at a pretranslational site. Although the mechanism(s) is not understood, these data provide evidence that nucleosides or their derivatives can significantly affect gene expression.     

Effect of pyrimidine deoxynucleosides and sodium butyrate on expression of the glycoprotein hormone α-subunit and placental alkaline phosphatase in HeLa cells

Production of the glycoprotein hormone common α-subunit and placental alkaline phosphatase activity can be modulated in HeLa cells by a variety of deoxynucleosides. Dose response curves for thymidine (Thd), fluorodeoxyuridine (FdUrd), bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) demonstrate that, in general, alkaline phosphatase was increased by lower concentrations of inducer than was α-subunit. The deoxynucleosides were not as effective as sodium butyrate as inducers of either protein. Whereas Thd and the halogenated dUrd derivatives enhanced protein expression, deoxycytidine (dCyd) had negative effects. Induction by deoxynucleosides of both alkaline phosphatase and α-subunit was inhibited by dCyd, but induction of alkaline phosphatase by butyrate was more sensitive to dCyd inhibition than was the buryrate-mediated induction of α-subunit. These results suggest that the two proteins are not regulated in a coordinate manner. Reversal of alkaline phosphatase induction by dCyd was not observed in cells preincubated with sodium butyrate for 6–24 h before the addition of dCyd, indicating that the deoxynucleoside interferes with an early event in the butyrate-mediated response. Combinations of butyrate with Thd, BrdUrd or IdUrd were synergistic with respect to the induction of HeLa-α. It is concluded that incorporation of the deoxynucleosides into DNA may not be required for the synergistic response since 2′,5′-dideoxythymidine was an effective as Thd. Cytoplasmic dot hybridizations demonstrate that a primary effect of the various effectors is to increase the steady-state levels of α-subunit mRNA. There was a good correlation between α-subunit accumulation and corresponding levels of α-mRNA, suggesting that regulation occurs at a pretranslational site. Although the mechanism(s) is not understood, these data provide evidence that nucleosides or their derivatives can significantly affect gene expression.     

Cyclic adenosine 3',5'-monophosphate analogues modulate rat placental cell growth and differentiation

Cyclic adenosine 3',5'-monophosphate (cAMP) has been implicated in the control of placental function. The present investigation was designed to evaluate the actions of cAMP analogues on the control of rat placental development. Two model systems were used to assess the actions of cAMP in the placenta: 1) a rat placental cell line and 2) rat labyrinth placental explants. Elevation of intracellular cAMP via treatment with cAMP analogues, 3-isobutyl-1-methylxanthine, forskolin, or cholera toxin inhibited placental cell DNA synthesis whereas treatment with an analogue to cyclic guanosine 3',5'-monophosphate was without effect. The inhibitory actions of dibutyryl cAMP on DNA synthesis were at least partially reversible and were not the result of metabolic toxicity. Dibutyryl cAMP had dramatic effects on the organization and morphology of placental cells growing in vitro and diminished the ability of the placental cells to grow following transplantation into allogeneic hosts. Differentiation-associated characteristics of rat placental cells were also affected by cAMP. cAMP analogues stimulated placental cell progesterone release and inhibited placental cell alkaline phosphatase activity. Dibutyryl cAMP had effects on placental labyrinth explants similar to its effects on the placental cell line. Dibutyryl cAMP inhibited explant outgrowth while stimulating explant release of progesterone. In summary, cAMP effectively modulates the growth and differentiation of rat placental cells in vitro.     

A heat-stable alkaline phosphatase from Penaeus japonicus Bate (Crustacea: Decapoda): a phosphatidylinositol-glycan anchored membrane protein

1. A heat-stable alkaline phosphatase was purified from Penaeus japonicus, with a final specific activity of 21,280 U/mg of protein. 2. In polyacrylamide-gel electrophoresis under non-denaturing conditions, the purified shrimp alkaline phosphatase was found to have an identical molecular size and surface charge as the human placental enzyme. 3. By using SDS-PAGE, the monomers of shrimp alkaline phosphatase were discovered to have a Mr 55,000 but those of human placental enzyme with a Mr 70,000. Deglycosylation decreases the Mr values of the subunits to 33,000 for shrimp alkaline phosphatase. 4. The purified alkaline phosphatase from shrimp was recovered with both the attachment sites for sialic acids and phosphatidylinositol. 5. The shrimp alkaline phosphatase has an isoelectric point (pI) of 7.6 and the human placental enzyme has a pI of 4.8.     

RhoE is regulated by cyclic AMP and promotes fusion of human BeWo choriocarcinoma cells

Fusion of placental villous cytotrophoblasts with the overlying syncytiotrophoblast is essential for the maintenance of successful pregnancy, and disturbances in this process have been implicated in pathological conditions such as pre-eclampsia and intra-uterine growth retardation. In this study we examined the role of the Rho GTPase family member RhoE in trophoblast differentiation and fusion using the BeWo choriocarcinoma cell line, a model of villous cytotrophoblast fusion. Treatment of BeWo cells with the cell permeable cyclic AMP analogue dibutyryl cyclic AMP (dbcAMP) resulted in a strong upregulation of RhoE at 24 h, coinciding with the onset of fusion. Using the protein kinase A (PKA)-specific cAMP analogue N(6)-phenyl-cAMP, and a specific inhibitor of PKA (14-22 amide, PKI), we found that upregulation of RhoE by cAMP was mediated through activation of PKA signalling. Silencing of RhoE expression by RNA interference resulted in a significant decrease in dbcAMP-induced fusion. However, expression of differentiation markers human chorionic gonadotrophin and placental alkaline phosphatase was unaffected by RhoE silencing. Finally, we found that RhoE upregulation by dbcAMP was significantly reduced under hypoxic conditions in which cell fusion is impaired. These results show that induction of RhoE by cAMP is mediated through PKA and promotes BeWo cell fusion but has no effect on functional differentiation, supporting evidence that these two processes may be controlled by separate or diverging pathways.     

1,25-Dihydroxyvitamin D3 increases bone alkaline phosphatase isoenzyme levels in human osteogenic sarcoma cells

The specific activity of alkaline phosphatase was increased in two human osteogenic sarcoma cell lines, SAOS and TE85, after treatment with 1,25 dihydroxy-vitamin D3 (1,25(OH)2D3). Enzyme activity increased when the cells were incubated with concentrations of 1,25(OH)2D3 between 10(-9) and 10(-7) M and cell growth was not inhibited at these concentrations. The specific activity of alkaline phosphatase was 4- to 7-fold higher than that in the control cells after 5 to 7 days of continuous exposure to 1,25(OH)2D3. Immunochemical studies demonstrated that the enzyme from both control and 1,25(OH)2D3-treated cultures cross-reacted with antisera specific for the phosphatase isoenzyme produced by normal human bone, and did not cross-react with antisera specific for the placental alkaline phosphatase isoenzyme. The increased enzyme activity in cultures induced with 1,25(OH)2D3 correlated with an absolute increase in the number of bone-specific phosphatase molecules, as determined by radioimmunoassay. No effect on alkaline phosphatase activity was observed when the cells were treated with other vitamin D metabolites or with 5-bromo-2'-deoxyuridine. Comparative studies demonstrated that hydrocortisone, another steroid hormone, increased the phosphatase activity with a different time course than did 1,25(OH)2D3. High affinity cytoplasmic receptors for 1,25(OH)2D3 and hydrocortisone were found in the SAOS and TE85 cells.     

Expression and cyclic AMP-dependent regulation of a high affinity serotonin transporter in the human placental choriocarcinoma cell line (JAR).

The JAR human placental choriocarcinoma cell line transports serotonin, accumulating the monoamine inside the cell against a concentration gradient. The transport is energized by an NaCl gradient. Tricyclic (imipramine and desipramine) and non-tricyclic (paroxetine and fluoxetine) antidepressants inhibit the transporter markedly, but reserpine and 5-hydroxytryptophan do not. Ouabain, gramicidin, and nigericin, which reduce or abolish the transmembrane Na+ gradient, and phloridzin, which interferes with glucose transport into the cells, inhibit the transport. Preincubation of the cells with glucose-free medium also causes similar inhibition. The activity of the serotonin transporter in this cell line is stimulated in response to overnight (16-h) incubation with increasing concentrations of cholera toxin (0.1-1,000 ng/ml). Under these conditions the stimulation is maximal at 10 ng/ml cholera toxin (3.1 +/- 0.2-fold). Cholera toxin increases the cAMP content of these cells by several hundredfold within 2 h. Isobutylmethylxanthine (100 microM), dibutyryl cAMP (100 microM), and forskolin (100 microM) mimic the action of cholera toxin, eliciting a 1.6-2.5-fold stimulation of the serotonin transporter activity. The stimulatory effect of cholera toxin is antagonized significantly by simultaneous incubation of the cells with 50 microM N-(2-aminoethyl)-5-isoquinolinesulfonamide, a protein kinase inhibitor. The effect of cholera toxin on serotonin transport is specific because, under similar conditions, cholera toxin inhibits 3-O-methyl-D-glucose transport and does not influence taurine transport in this cell line. There is also no significant change in the protein content of the cells after cholera toxin treatment. Kinetic analysis reveals that cholera toxin causes an increase in the maximal velocity (7.89 +/- 0.67 to 17.55 +/- 1.06 pmol/mg of protein/5 min) and a decrease in the Michaelis-Menten constant (0.52 +/- 0.09 to 0.29 +/- 0.04 microM). These data show that the JAR human placental choriocarcinoma cell line expresses a high affinity serotonin transporter that is sensitive to inhibition by antidepressants and that the activity of the transporter is under cAMP-dependent regulation.     

Biosynthesis of placental alkaline phosphatase and its post-translational modification by glycophospholipid for membrane-anchoring

The biosynthesis and post-translational modification of placental alkaline phosphatase were studied in human choriocarcinoma cells, JEG-3. Pulse-chase experiments with [35S]methionine demonstrated that placental alkaline phosphatase was synthesized as a major precursor form with Mr 63,000, which was then converted to a mature form with Mr 66,000, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type. In addition, the two forms of the protein were found to be modified by a glycophospholipid, components of which were characterized by metabolic incorporation into placental alkaline phosphatase of 3H-labeled compounds such as myo-inositol, palmitic acid, stearic acid, mannose, glucosamine, and ethanolamine. When placental alkaline phosphatase labeled with these compounds was treated with phosphatidylinositol-specific phospholipase C or papain, the phospholipase C removed only the 3H-labeled fatty acids, whereas papain, that is known to cleave the C-terminal region, released all the radioactive glycolipid components including [3H]ethanolamine. More detailed analysis with shorter pulse-chase experiments demonstrated that placental alkaline phosphatase was primarily synthesized as a form with Mr 64,500 which was not yet labeled with [3H]palmitic acid. This form was converted by papain digestion to the above-mentioned major precursor with Mr 63,000. Taken together, these results suggest that placental alkaline phosphatase is initially synthesized as the precursor with Mr 64,500, which is immediately converted to the intermediate form with Mr 63,000 by simultaneously occurring proteolysis of the C terminus and replacement by the glycophospholipid, and finally to the mature form with Mr 66,000 by terminal glycosylation of its N-linked oligosaccharides. The glycophospholipid thus attached is considered to function as the membrane-anchoring domain of placental alkaline phosphatase.     

Estrogen synthetase (aromatase) in cultured human term placental cells and neoplastic human trophoblast

Estrogen synthetase (aromatase) is present in large amounts in human term placenta. However, the localization of aromatase within the cellular structure of the placental villus is obscure. By immunocytochemical techniques using antibodies that separately recognize each component of the aromatase cytochrome P-450 enzyme system, the fraction of term placental trophoblast cells in primary culture expressing each aromatase component antigen increased from 20% in fresh mononucleated cells to about 65% for multinucleated giant cells after 72 h. In contrast, about 80% of human choriocarcinoma cells in continuous culture (JAr line) expressed each aromatase component antigen. The fraction of trophoblast cells in primary culture containing human chorionic gonadotropin increased from about 14% in fresh mononucleated cells to about 45% after 72 h and was about 30% in the choriocarcinoma cells. Fibroblast cells in culture, derived from trypsin-treated placental villi, contained aromatase activity, albeit much lower than term placental trophoblast cells. Aromatase specific activity in these placental fibroblasts did not change following growth with dibutyryl cAMP plus theophylline for 72 h.     

Effects of sodium butyrate on human colonic adenocarcinoma cells. Induction of placental-like alkaline phosphatase

We have examined the effects of the "differentiating agent," sodium butyrate, on the induction of alkaline phosphatase in human colonic tumor cell line LS174T. Culture of these cells in the presence of 2 mM butyrate caused this activity to increase from less than 0.0001 unit/mg of protein to greater than 0.7 unit/mg of protein over an 8-day period. This induction proceeded in a nonlinear fashion with a lag time of 2-3 days occurring before enzymatic activity began to rise. These increases in activity were accompanied by elevations in the content of a placental-like isozyme of alkaline phosphatase as demonstrated by "Western" immunoblots. Dome formation, indicative of differentiation in cultured cells, also required 3 days treatment with butyrate before becoming evident. The rate of biosynthesis of the enzyme, examined using metabolic labeling with L-[35S]methionine and immunoprecipitation, was found to increase continuously between days 2 and 6 of butyrate treatment. "Northern" blot analysis indicated that treatment of these cells with butyrate caused greater than 20-fold induction of a 2700-base mRNA that hybridized to a cDNA probe for placental alkaline phosphatase. The mRNA for alkaline phosphatase produced by these cells upon butyrate treatment was approximately 300-400 bases smaller than the mRNA for alkaline phosphatase found in placenta. Human small intestine also contained two mRNAs that hybridized relatively weakly with the placental alkaline phosphatase probe. These results indicate that a placental alkaline phosphatase-like protein and mRNA are induced by butyrate in LS174T cells with a time course consistent with cellular differentiation preceding induction.     

Placental ceruloplasmin homolog is regulated by iron and copper and is implicated in iron metabolism

We previously reported an endogenous, membrane-bound Cu oxidase with homology to ceruloplasmin in BeWo cells, a placental choriocarcinoma cell line. In this previous study, ceruloplasmin immunoreactivity was localized to the perinuclear region and non-brush-border membranes. Here, we show that azide-sensitive oxidase activity is enriched in the same fractions, correlating subcellular localization of enzyme activity with ceruloplasmin immunoreactivity. Expression of the placental Cu oxidase is inversely proportional to Fe status and directly proportional to Cu status at enzyme and protein levels. To identify a role for the Cu oxidase, cells were exposed to (59)Fe-transferrin for 18 h in an environment of 20% O(2) or 5% O(2). At 5% O(2), Cu-deficient cells retain significantly more (59)Fe than control cells. This excess in (59)Fe accumulation is caused by a significant decrease in (59)Fe release. These results indicate that downregulation of the placental Cu oxidase in BeWo cells impairs Fe release. This effect is only apparent in an environment of limited O(2).     

Synthesis of first trimester placental alkaline phosphatase in cultured human term placental cells

Alkaline phosphatase activity in human placental cells transformed by a tsA mutant of simian virus 40 (SV40) can be greatly induced by growing these cells at 40 degrees C, the temperature at which the tsA transformants regain their nontransformed phenotype. The induction of alkaline phosphatase in these cells requires the synthesis of both RNA and protein. The induced alkaline phosphatase from a SV40 tsA30 mutant-transformed term placental cell line (TPA30-1) was purified, characterized, and compared with alkaline phosphatase from term placenta and first trimester placenta. The form of alkaline phosphatase found in TPA30-1 cells differs from the phosphatase of term placenta in physiochemical and immunological properties. The TPA30-1 phosphatase is, however, indistinguishable from the alkaline phosphatase of human first trimester placenta by several criteria, including electrophoretic mobility, apparent molecular weight (Mr = 165,000), size of monomeric subunit (Mr = 77,000), heat lability, and sensitivity to inhibition by amino acids and EDTA. In addition, alkaline phosphatase from both TPA30-1 cells and first trimester placenta can be inactivated by antiserum to liver alkaline phosphatase but not by antiserum to term placental alkaline phosphatase. The induction of first trimester phosphatase in cells derived from term placenta provides a system for the study of alkaline phosphatase gene regulation in human placenta.     

Inhibitory effect of dibutyryl cyclic adenosine monophosphate on the induction of alkaline phosphatase in human fetal liver cell line

When human fetal liver cells (HuL-1-317), cultured continuously in a serum-free medium, were incubated with a combination of prednisolone, butyrate and a hypertonic concentration of NaCl at 37 degrees C, alkaline phosphatase activity increased. However, the addition of dibutyryl adenosine cyclic monophosphate (Bt2cAMP) to these agents inhibited the increase in alkaline phosphatase activity in a dose-dependent manner: the inhibitory effect of Bt2cAMP was significant at 0.05 mM, but disappeared at 0.01 mM. Both cycloheximide and actinomycin D inhibited the increase in alkaline phosphatase activity with the combination described above. Western blotting showed that this enzyme activity increase was a consequence of greater biosynthesis of enzyme molecules in HuL-1-317 cells, and that Bt2cAMP regulated the synthesis of enzyme molecules. We conclude that the changes in alkaline phosphatase activity under various conditions are based on the changes in the number of enzyme molecules in HuL-1-317 cells.