Parathyroid hormone inhibition of induction of alkaline phosphatase activity in HeLa cells by 5-iodo-2′-deoxyuridine without increased adenylate cyclase activity or cellular cAMP concentration

Parathyroid extract (PTE) as well as purified parathyroid hormone (PTH) activators of adenylate cyclase in bone and kidney, produced dose-dependent decreases in the induction of alkaline phosphatase activity by 5-iodo-2′-deoxyuridine in HeLa cells. However, the combination of PTE and 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor which also inhibits the induction of alkaline phosphatase activity, in most cases produced less than additive inhibition of enzyme induction. PTE or PTH in concentrations of up to 10 times greater than that necessary to have maximal effects on the induction of alkaline phosphatase activity produced no increase in adenylate cyclase activity, nor did they increase intracellular cAMP concentrations. In addition, PTE did not potentiate the increase in cAMP concentration produced by IBMX. It thus appears that the inhibition of alkaline phosphatase activity by PTH is not mediated by cAMP.     

CELL CYCLE EVENTS IN THE HYDROCORTISONE REGULATION OF ALKALINE PHOSPHATASE IN HELA S3 CELLS

The increase in alkaline phosphatase in asynchronous cultures of HeLa S₃ cells grown in medium supplemented with hydrocortisone is characterized by a lag period of 10–12 hr. Present studies utilizing synchronous cell populations indicate: (a) a minimum of 8–10 hr of incubation with hydrocortisone is necessary for maximum induction of alkaline phosphatase; (b) the increase in enzyme activity produced by hydrocortisone is initiated exclusively in the synthetic phase of the cell cycle; (c) alkaline phosphatase activity does not vary appreciably over a normal control cell cycle. Radioactive hydrocortisone is rapidly distributed into HeLa cells irrespective of their position in the cell cycle, indicating that inductive effects are not governed by selective permeability during the cell cycle. Hydrocortisone-1,2-[³H] diffuses back from the cell into the medium when the cells are incubated in fresh medium containing no hydrocortisone, and the alkaline phosphatase induction, under these conditions, is completely reversible.     

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.     

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.     

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.     

Bromodeoxyuridine induced variations in the level of alkaline phosphatase in several human heteroploid cell lines.

The level of alkaline phosphatase in a number of established cell lines of human origin can be modified by exposure to non-lethal concentrations of bromodeoxyuridine (BRdU).In the several cell lines examined an inverse relationship between amount of induction and constitutive level of the enzyme was observed. Thus, the H.Ep 2 line, which had the highest basal level of enzyme, was reversibly repressed following exposure to the drug, whereas other cell lines with relatively low constitutive enzyme levels were induced to a maximum of 10-fold following exposure. Initiation of induction required from 24 to 48 hours, and as short an exposure ("pulse") as five hours was sufficient to produce induction. Exposure to visible light had no effect upon the repression of alkaline phosphatase in H.Ep 2 by BRdU. Induction did not occur in non-dividing, serum starved cells. The time course of induction by BRdU and hydrocortisone was similar, and simultaneous exposure of the cells to both agents resulted in no greater induction than that observed with either drug alone. Experiments utilizing mitomycin C yielded significant induction in the presence of this agent alone, and somewhat less induction when both mitomycin C and BRdU were added simultaneously. These results suggest that DNA synthesis is required for BRdU were added simultaneously. These results suggest that DNA synthesis is required for BRdU-mediated induction of alkaline phosphatase.     

Carcinoplacental Alkaline Phosphatase

Using HeLa TCRC-1, a cell line which is monophenotypic with respect to the Regan isoenzyme of alkaline phosphatase, we have examined the factors which influence its expression in relation to events of the cell cycle.
DNA synthesis is not required for hormone induction of the Regan isoenzyme as in the presence of hydroxyurea, a specific inhibitor of DNA synthesis, we found induction to occur. Additionally, when partially synchronised cells were allowed to leave the S period prior to hormone treatment, and hydroxyurea was added to prevent cells from entering the next S period, hormone induction of the Regan isoenzyme was still observed. This indicates that initiation of expression of hormone-induced carcinoplacental alkaline phosphatase occur prior to the DNA synthetic phase of the cell cycle.
We propose a hypothetical two-step mechanism of hormone induction to interpret the present findings in relation to previous results.     

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.     

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.     

Treatment of immature mice with gonadotrophins. Effects on some enzymic activities of unfractionated ovarian homogenates

Treatment of immature mice with both follicle-stimulating hormone and human chorionic gonadotrophin in vivo resulted in large increases in the specific activities of ovarian alkaline phosphatase and alkaline nucleotidase. The specific activities of other ovarian enzymes studied were not altered by gonadotrophin treatment. A simultaneous change in the Michaelis constant of ovarian alkaline phosphatase accompanied the increase in specific activity. These changes commenced 6-8h after injection of human chorionic gonadotrophin plus follicle-stimulating hormone. Injection of human chorionic gonadotrophin induced the change in Michaelis constant and increased ovarian alkaline phosphatase activity. Treatment with follicle-stimulating hormone had no effect on ovarian alkaline phosphatase. However, follicle-stimulating hormone synergistically augmented the response to human chorionic gonadotrophin. A latent period of about 24h elapsed before this augmentation was expressed. Augmentation of ovarian alkaline phosphatase was directly related to the dose of follicle-stimulating hormone at a fixed dose of chorionic gonadotrophin. No response of ovarian alkaline phosphatase was observed after treatment of immature mice in vivo with oestrogens, progesterone, growth hormone or prolactin. Unlike chorionic gonadotrophin, sheep luteinizing hormone over a wide dose range induced no response within 24h. However, a response in ovarian alkaline phosphatase was observed when sheep luteinizing hormone was administered in combination with follicle-stimulating hormone. The specific activity and K(m) of ovarian alkaline phosphatase increased during normal maturation. The Michaelis constant ceased to increase as sexual maturity was reached. The changes in alkaline phosphatase activity were of a similar magnitude to those induced by gonadotrophin treatment. It is concluded that the changes induced acutely by treatment in vivo with unphysiological doses of gonadotrophins occur in the maturing mouse under the influence of endogenous, homologous gonadotrophins at physiological concentrations.     

Induction of alkaline phosphatase and the glycoprotein hormone α subunit in HeLa cells by inhibitors of DNA polymerase

Levels of the glycoprotein hormone α subunit and alkaline phosphatase activity were increased in cultures of HeLa S₃ cells exposed to aphidicolin (0.2–10 μg/ml) or phosphonoformic acid (0.1–3 mm), inhibitors of DNA polymerase α. Induction was dependent on both the concentration and duration of exposure to the inhibitors and was prevented by cycloheximide and actinomycin D. Limited characterization of the induced α subunit and alkaline phosphatase activity suggest that they are similar to the uninduced proteins expressed by this cell line. Induction of both proteins by aphidicolin and phosphonoformic acid was enhanced by the simultaneous addition of 3 mm sodium butyrate but was depressed by 1 mm hydroxy urea. In contrast, both butyrate and hydroxy urea cause induction of these proteins when added alone to HeLa cultures. It is unlikely that a direct relationship exists between protein induction and the inhibition of DNA synthesis produced by aphidicolin and phosphonoformic acid since the concentrations required to produce half-maximal induction are 5 to 10 times greater than those needed to inhibit replication by 50%.     

Effect of hydrocortisone on the maturation of human foetal kidney explants in serum-free organ culture

The effect of hydrocortisone on the in vitro maturation of human foetal kidney was investigated. Following legal therapeutic abortions, explants of renal cortex from foetuses aged 13-18 weeks were cultured for 5 days in serum-free Leibovitz's L-15 medium at 37 degrees C in a mixture of 95% air - 5% CO2, without hormone (controls) or with hydrocortisone at concentrations of 12.5, 25, or 50 ng/mL, which are the levels representative of different gestational periods. During the studied period of culture, the overall architecture of the renal structures was preserved without any evident signs of nephrogenesis induced by hydrocortisone. DNA synthesis was measured by incorporation of [3H]thymidine and was stimulated on day 5 by 80% with the addition of hydrocortisone at 12.5 ng/mL, and by 131% with 50 ng/mL. In autoradiograms, the sites of [3H]thymidine incorporation were the same after hydrocortisone addition, but the number of labelled nuclei was higher in 5-day explants supplemented with hydrocortisone at 50 ng/mL. The activities of some brush border enzymes (leucylnaphthylamidase, maltase, and alkaline phosphatase) were not influenced by hydrocortisone when compared with controls. Trehalase activity was decreased on day 5 with 12.5 and 50 ng/mL. A concentration of 12.5 ng/mL diminished gamma-glutamyltransferase activity by 29% on day 5. The incorporation of [3H]leucine into proteins was not influenced by any concentration of the glucocorticoid hormone. This study indicates that hydrocortisone directly influences cell proliferation and certain brush border enzymic activities in human developing kidney maintained in organ culture.     

The effects of calcitonin, parathyroid hormone and prostaglandin E2 on cyclic AMP levels of isolated osteoclasts

Cyclic AMP (cAMP) levels were measured in both isolated and attached osteoclasts. The level of cAMP was 0.1 pmol/10(5) osteoclasts. No change in cAMP level of osteoclasts could be detected following calcitonin treatment. Parathyroid hormone (PTH) and prostaglandin E2 (PGE2) treatment stimulated cAMP production in proportion to alkaline phosphatase levels and divergent to acid phosphatase levels. This indicates that osteoblasts, not osteoclasts, were responsive to PTH and PGE2.     

Alkaline phosphatase activity and the regulation of growth in transformed mammalian cells

The relationship between alkaline phosphatase activity and cell growth has been studied in hamster cells transformed by different carcinogens. About 90% of normal hamster embryo cells were constitutively positive for alkaline phosphatase activity (AP⁺). However, there were no AP⁺ cells in cell lines transformed after treatment with the chemical carcinogens dimethylnitrosamine or 4-nitro-quinoline-N-oxide and 0.02% and 4% AP⁺ cells in cell lines transformed by polyoma virus or Simian virus 40. The glucocorticoid hormone, prednisolone, induced alkaline phosphatase activity in 12% and 44% of the enzyme-negative (AP^?) cells in cell lines transformed by polyoma or Simian virus 40, but this hormone did not induce alkaline phosphatase activity in AP^? cells from cell lines transformed after treatment with the chemical carcinogens. Treatment of polyoma transformed AP^? cells with the mutagen N-methyl-N′-nitro-N-nitro-soguanidine produced AP⁺ cells, whereas no AP⁺ cells were found after mutagen treatment of AP^? cells from the chemically transformed cell lines. Studies on spontaneous segregation in the polyoma transformed cell line has shown that AP⁺ cells segregated AP^? cells both in vitro and in vivo, although no spontaneous segregation was observed from AP^? to AP⁺ cells. AP⁺ cells, compared to AP^? cells, showed a decrease in DNA synthesis, cell multiplication, the ability to form colonies in soft agar and tumorogenicity in animals. AP^? cells induced for alkaline phosphatase activity by prednisolone, showed the same growth properties in vitro as uninduced AP^? cells. The decreased cell growth found in AP⁺ cells which were constitutive for alkaline phosphatase activity was therefore not found in the hormone induced AP^? cells. The results indicate that constitutive alkaline phosphatase activity appears to be related to the regulation of cell growth and that AP^? cells have a selective advantage over AP⁺ cells.     

Induction of alkaline phosphatase by retinoic acid

Treatment of mammalian cells in culture with retinoic acid causes a time- and concentration-dependent increase of the specific activity of alkaline phosphatase. The increase reaches a factor of 15 and more and begins at a concentration of 10(-8)M retinoic acid. The induction is inhibited by cycloheximide or actinomycin D. The same isoenzyme of alkaline phosphatase is expressed in control and in retinoic acid-treated cells as demonstrated by the inhibitions by amino acids and peptides. The enzyme induction occurs in rat heart, skeletal muscle, brain, lung cells and HeLa cells. No induction was found in two lines of human melanoma cells. After treatment of cells with tunicamycin, the induction of alkaline phosphatase is detectable only in the homogenate and no longer detectable by histochemical methods. This shows that the glycosylation of the protein is an important step in the insertion of this enzyme into the plasma membrane.     

Effect of hydrocortisone on the properties of rat liver polyribosomes, metabolism, and template activity of polysomal poly-A-containing RNA

The effect of hydrocortisone on the amount of newly synthesized polyribosomal poly-A+-RNA and its translation activity and the distribution of polyribosomes in the induction dynamics according to their size were studied. It was shown that 3-5 hours after intraperitoneal injection of hydrocortisone the incorporation of labelled precursors into polyribosomal poly-A+-mRNA is increased, which is accompanied by rapid accumulation of mRNA in the polyribosomes. Under prolonged induction those parameters come down to the initial level. 4-7 hours after the injection of the hormone the relative amount of heavy polyribosomes (350-412S) in liver cells is increased. It was found that hydrocortisone significantly changes the specific translation activity of polysomal poly-A+-mRNA: it shows an increase 2-4 hours after the hormone injection and returns to the initial level 12 hours after the injection.     

Recombinant human interleukin-1 inhibits the induction by dexamethasone of alkaline phosphatase activity in murine capillary endothelial cells

A mutual antagonism exists between interleukin-1s (IL-1s) as pro-inflammatory and glucocorticoids as anti-inflammatory mediators. This report examines the effects of IL-1 on the induction by dexamethasone of alkaline phosphatase in LEII murine endothelial cells. Dexamethasone increases the specific activity of alkaline phosphatase in a time- and dose-dependent fashion (maximum 14-fold induction at 10(-6) M, IC50 = 10(-8) M), and this induction can be completely inhibited by simultaneous incubation with picomolar concentrations of recombinant human IL-1 alpha or IL-1 beta. This IL-1-mediated antagonism of dexamethasone activity is not due to a down-regulation of glucocorticoid receptors in the cell line used, because the number of receptors and their affinity for dexamethasone is unchanged in IL-1-treated cells. However, induction of alkaline phosphatase by dexamethasone in LEII cells is receptor-mediated, since it can also be inhibited by glucocorticoid-receptor antagonists.