Induction of alkaline phosphatase activity in HeLa cells by sodium butyrate

Summary The induction of HeLa cell alkaline phosphatase activity by sodium butyrate could be inhibited by the coadministration of caffeine or theophylline. The inhibitions were dose dependent, and at any given concentration the potency was theophylline > caffeine. Although the induction by sodium butyrate was more sensitive to the inhibition by the xanthines than was that produced by 5-iodo-2′-deoxyuridine, the magnitudes of the increases in cyclic AMP concentrations after treatment with the xanthines were similar in the inhibition of both types of induction. The induction of alkaline phosphatase activity by sodium butyrate also produced a shift in the thermostability pattern of the enzyme, with a proportionately greater increase in the heat-labile, rather than heat-stable, from of the activity. Supported by National Cancer Institute Grant CA16460.     

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.     

The inhibition by xanthine phosphodiesterase inhibitors of the induction of alkaline phosphatase activity in HeLa cells: relationship of enzyme activity to cyclic AMP concentrations.

The three xanthine derivatives, caffeine, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) produced dose-dependent increases in cyclic AMP concentrations in HeLa cells after long term treatment. Only IBMX produced increases over the first 60 minutes, with a peak of approximately 5-fold control values five to 10 minutes after the addition of the drug. About four hours after the addition of either 0.67 or 1.0 mM IBMX there was a second peak in the concentration of cyclic AMP which was at least as large and usually larger than the peak observed at five to ten minutes. Neither caffeine nor theophylline increased cyclic AMP concentrations above control values until one hour after addition of the compounds, and there was no indication of a peak in the concentration at four hours. Between 24 and 72 hours, all three compounds produced elevations in cyclic AMP levels that were steadily maintained. At any given concentration, the order of potency was IBMX greater than theophylline greater than caffeine. If the xanthine derivatives were removed from the medium after 24 hours of treatment, the cyclic AMP concentrations fell to control levels within one hour. Treatment with 5-iodo-2'-deoxyuridine (IdUrd) or hydrocortisone alone did not change the levels of cyclic AMP, nor did the presence of these inducers of alkaline phosphatase activity alter the effects of the xanthine derivations on cyclic AMP concentrations. The data showed a significant correlation between the magnitude of the increase in cycli AMP concentrations over the period from 24 to 72 hours and the degree of inhibition by the xanthine derivatives of the induction of alkaline phosphatase activity.     

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%.     

Differential theophylline inhibition of alkaline phosphatase and 5'-nucleotidase of bovine milk fat globule membranes

1. The effects of theophylline (1,3-dimethylxanthine) on alkaline phosphatase and 5'-nucleotidase activities of bovine milk fat globule membranes (MFGM) were examined. 2. Theophylline inhibited MFGM alkaline phosphatase in a concentration-dependent manner with 50% inhibition produced by 99 +/- 28 microM theophylline. 3. The 5'-nucleotidase activity was resistant to theophylline inhibition with 50% inhibition produced by 33.9 +/- 3.1 mM theophylline. 4. Theophylline was an uncompetitive inhibitor of MFGM alkaline phosphatase with a Ki of 126 +/- 15 microM. 5. The extent of theophylline inhibition of alkaline phosphatase activity was independent of the substrate utilized in the assay. 6. The effect of theophylline on bovine MFGM alkaline phosphatase was similar to theophylline effects on other mammalian alkaline phosphatases of liver/bone isoenzyme origin.     

A study of adenosine 3':5'-cyclic monophosphate, sodium butyrate and cortisol as inducers of HeLa alkaline phosphatase

The role of adenosine 3′:5′-cyclic monophosphate in the cortisol-mediated induction of HeLa 65 alkaline phosphatase was investigated. Although growth of these cells with 0.5–1.0 mmN₆,O²′-dibutyryl adenosine 3′:5′-cyclic monophosphate induces a 5- to 8-fold increase in cellular phosphatase activity after 72 hr, neither cAMP nor theophylline induce at concentrations up to 1 mm. Sodium butyrate induces the enzyme as well as dibutyryl cAMP. Moreover, induction kinetics show sodium butyrate to be a more efficient inducer than dibutyryl cAMP, inducing activity as quickly as cortisol. This suggests that the butyric acid cleaved from dibutyryl cAMP by HeLa cells is the mediator of induction when the cyclic nucleotide derivative is used.     

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.     

Differential effects of sodium butyrate and hyperosmolality on the modulation of alkaline phosphatases of LoVo cells

LoVo cells produce term-placental and intestinal alkaline phosphatases. Hyperosmolality and sodium butyrate increase the levels of both, but the effect of sodium butyrate is more pronounced on the intestinal enzyme. When applied together, induction of term-placental alkaline phosphatase is additive and that of the intestinal enzyme is synergistic. Induction by either stimulus or by their mixture is independent of cell density. However, whereas the effect of hyperosmolality is readily reversible, induction by sodium butyrate is not. No synergistic increase in intestinal alkaline phosphatase activity occurs when cells are sequentially treated with hyperosmolality and sodium butyrate or vice versa. This indicates that only when applied concurrently does one inducer amplify the effect of the other. Since the normal colonic mucosa produces intestinal alkaline phosphatase, its predominant induction by sodium butyrate in LoVo cells may reflect a more differentiated state.     

Modulation of alkaline phosphatases in LoVo, a human colon carcinoma cell line

LoVo, a continuous cell line derived from a human colon carcinoma produces two alkaline phosphatases: the heat-labile, L-homoarginine-insensitive, intestinal form, characteristic of its tissue of origin and the heat-stable, term-placental form, ectopically produced by a variety of tumors. Under basal conditions the activity levels of both enzymes are similar. Hyperosmolality and sodium butyrate induce increased levels of activity of the two alkaline phosphatases in a disparate fashion; whereas hyperosmolality augments the activity of both to the same extent, the effect of butyrate is more pronounced on the activity of the intestinal enzyme. When the two inducers are combined, induction of term-placental alkaline phosphatase is additive and that of the intestinal enzyme is synergistic. The effect of hyperosmolality is blocked by cycloheximide, and induction by sodium butyrate is inhibited by thymidine, cordycepin and cycloheximide. The known alkaline phosphatase inducer, prednisolone, has no effect on the enzymes of LoVo cells. Our results suggest that in these tumor cells the activity levels of the closely homologous term-placental and intestinal alkaline phosphatases appear to be independently controlled.     

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.     

Enhancement by theophylline of the butyrate-mediated induction of choriogonadotropin alpha-subunit in HeLa cells. I. Lack of correlation with cAMP.

The glycoprotein hormone common alpha-subunit can be induced in HeLa and other nontrophoblastic tumor cell lines by sodium butyrate. This report demonstrates that production of alpha-subunit can be further modulated by theophylline, especially in conjunction with butyrate. This synergism was not observed with other phosphodiesterase inhibitors such as xanthine, caffeine, theobromine, or methylisobutylxanthine. Induction by a combination of the short chain fatty acid plus the methylxanthine results from a decrease in the lag time after effector addition as well as a change in the rate of subunit accumulation. The increase in alpha-subunit is correlated with an increase in the levels of alpha-subunit mRNA, suggesting that induction is manifest at a pretranslational stage. The production of alpha-subunit was only marginally affected in cultures treated with 8-Br-cAMP or forskolin. Intracellular levels of cAMP were increased approximately threefold by methylisobutylxanthine, twofold by theophylline, fourfold by forskolin, and about 50% by butyrate, yet significant induction was achieved only by butyrate and theophylline. Taken together, these data suggest that the synergism between butyrate and theophylline is not mediated by cAMP.     

Effects of dibutyryl adenosine 3':5'-cyclic monophosphate and other agents on induction of alkaline phosphatase activity in monkey kidney cells

The induction of alkaline phosphatase (ALP) by dibutyryl adenosine 3':5'-cyclic monophosphate (Bt2cAMP) was investigated in strain JTC-12 . P3 cells derived from monkey (Maccaca irus) kidney cortex. ALP activity was increased by Bt2cAMP in a dose-dependent manner, reaching a plateau at concentrations higher than 5 mM with the activity being about 4 times that of the controls. The concentration of Bt2cAMP required for half-maximal induction of ALP activity was about 0.8 mM. ALP activity was increased rapidly by Bt2cAMP for the first 5 days and then continued to increase gradually towards a plateau level. Removal of Bt2cAMP from the medium caused a rapid decrease in the activity, suggesting that the induction of ALP activity by Bt2cAMP is reversible. ALP activity was induced synergistically in the presence of 1 mM sodium butyrate together with Bt2cAMP at concentrations from 0.01 to 1 mM. It was also found that in the presence of 1 mM Bt2cAMP, sodium butyrate increased ALP activity in the same manner as Bt2cAMP did in the presence of 1 mM sodium butyrate. Although dexamethasone, a potent glucocorticoid, had no effect on ALP activity in control cells, the hormone suppressed the ALP activity induced by Bt2cAMP in a dose-dependent manner. At concentrations above 0.2 mM, two xanthine derivatives, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX), also inhibited the induction of ALP activity by 1 mM Bt2cAMP. Inhibitors of protein synthesis, cycloheximide (1.5 micrograms/ml) and pactamycin (10 micrograms/ml), as well as inhibitors of RNA synthesis, actinomycin D (2 micrograms/ml) and alpha-amanitin (50 micrograms/ml), suppressed the induction of ALP activity.     

In vitro stimulation of prostaglandin synthetase by methyl xanthines

The methyl xanthines theophylline, caffeine and 1-methyl-3-isobutyl xanthine (MIX) stimulated the formation of prostaglandins (PG's) by bovine seminal vesicle microsomes (BSVM) and rabbit renal papilla microsomes (RRPM). The endoperoxide synthetase is probably the primary target of methyl xanthine action, as all the different PG's produced by RRPM were increased. However, a compound tentatively identified as PGD₂ was more increased than other PG's. The experiments further indicate that the effect of methyl xanthines is dependent on a non-microsomal factor, or on removal of a rapidly formed toxic component, for maximal stimulation, and that the stimulation is probably not related to oxygen radical scavenging. MIX was the most potent drug in the BSVM-system (EC₅₀=0.9 mM), followed by caffeine (EC₅₀=1.3 mM) and theophylline (EC₅₀=2.7 mM). Methyl xanthines are well-known inhibitors of cyclic nucleotide phosphodiesterase (PDE) and BSVM was rich in PDE-activity. BSVM-PDE was inhibited by the methyl xanthines, but a non-xanthine, papaverine, which was a potent BSVM-PDE inhibitor, did not stimulate PG-synthesis. Furthermore, cyclic nucleotides had no effect on PG-synthesis. It is therefore concluded that the stimulation of PG's by methyl xanthines is not related to inhibition of PDE. The possibility of a mechanism dependent on impairment of PG-metabolism was also excluded. Thus, methyl xanthines have a direct stimulatory effect on PG-synthesis in vitro.     

Induction of alkaline phosphatase in choriocarcinoma cells by 1-beta-D-arabinofuranosyl-cytosine, mitomycin C, phleomycin, and cyclic nucleotides.

Alkaline phosphatase is induced in cultured human choriocarcinoma cells by three inhibitors of DNA synthesis which alter DNA structure: 1-β-D-arabinofuranosyl-cytosine, mitomycin C, and phleomycin. No induction is observed with the inhibitors, hydroxyurea and thymidine, which do not alter DNA structure. Cyclic AMP, analogs of cyclic nucleotides, and sodium butyrate also induce alkaline phosphatase in these cells. Among the cyclic nucleotides tested, dibutyryl cyclic AMP is the best inducer, whereas dibutyryl cyclic GMP is a poor inducer. Induction of alkaline phosphatase by inhibitors of DNA synthesis or by exposure to dibutyryl cyclic AMP appears to utilize different mechanisms. Maximum induction is observed after simultaneous addition of both types of inducers at the concentrations found to be optimal for each inducer alone. Under these conditions, the induced activity is equal to or greater than the sum of the activities induced by each inducer. RNA synthesis and protein synthesis are required for induction. Dibutyryl cyclic AMP added to cultures of choriocarcinoma cells is not degraded in the culture medium, but is extensively degraded in the cells. Nevertheless, significant amounts of dibutyryl and monobutyryl cyclic AMP are found intracellularly throughout the experiment. Since the cellular uptake of dibutyryl cyclic AMP is extremely slow, the amount of butyrate released by intracellular degradation cannot account for the observed induction. Neither the rate of uptake nor the stability of dibutyryl cyclic AMP are changed by the addition of 1-β-D-arabinofuranosyl-cytosine to the culture medium. Furthermore, 1-β-D-arabinofuranosyl-cytosine inhibits the induction by sodium butyrate. The results indicate that butyrate is not the major mediator of induction by dibutyryl cyclic AMP.     

Increased alkaline phophatase activity in HeLa cells mediated by aliphatic monocarboxylates and inhibitors of DNA synthesis

Alkaline phosphatese activity of HeLa cells is increased from 3- to 8-fold during growth in medium with certain aliphatic monocarboxylates. The four-carbon fatty acid salt, sodium butyrate, is the most effective “inducer” with propionate (C₃), pentanoate (C₅) and hexanoate (C₆) having lesser effects. Other straight-chain aliphatic monocarboxylates, branched-chain analogues of inducers, hydroxylated derivatives, and metabolytes structurally related to butyrate are ineffective in mediating an increase in enzyme activity, indicating stringent structural requirements for inducers. The kinetics of increase in alkaline phosphatase activity in HeLa cells shows a 20–30 h lag period after adding the aliphatic acid followed by a rapid linear increase of enzyme activity. Protein synthesis is required for “induction”. The isozyme of HeLa alkaline phosphatase induced by monocarboxylates is the carcinoplacental form of the enzyme as determined by stereospecific inhibition by the l-enantiomorphs of phenylalanine and tryptophan, heat stability, and immunoreactivity with antibody against the human placental enzyme.Monocarboxylates that mediate increased alkaline phosphatase activity inhibit HeLa cell multiplication. Inhibition of HeLa cell growth may be necessary for induction and this hypothesis is supported by the findings that three different inhibitors of DNA synthesis, i.e. hydroxyurea, 1-β-d-arabinfuranosyl cytosine and methotrexate, also increase alkaline phosphatase activity. These inhibitors are synergistic with butyrate in causing HeLa cells to assume a more spindle-like shape and in producing an up-to 25-fold increase of enzyme activity. Studies on the modulation of carcinoplacental alkaline phosphatase by monocarboxylates commonly used as antimicrobial food additives and by anti-neoplastic agents may provide methods to evoke “tumor markers” of human occult malignancies. These drug-induced elevations of fetal isozyme activity may further our understanding of gene expression in human cells.     

Potentiating effects of methylxanthines on teratogenicity of mitomycin C in mice

A previous study demonstrated that caffeine strongly potentiated the teratogenic action of mitomycin C in mice. In the present study the effect of methylxanthines including caffeine, theophylline, theobromine (theobromine sodium salicylate), paraxanthine, and 1-methylxanthine was compared in order to analyze the structure-activity relationship. Jcl:ICR mice were injected IP with 3 mg/kg of mitomycin C, immediately followed by SC injection of each methylxanthine on day 11 of gestation. The doses of methylxanthines were calculated so that the mice received 50 mg/kg of caffeine or the equimolecular amount of the other methylxanthines. Fetuses were examined for external malformations on day 18 of gestation. Mitomycin C at 3 mg/kg and the methylxanthines at the doses used were not teratogenic. Combined administration of caffeine or theophylline with mitomycin C produced more than 80% of malformed fetuses. Although less effective than caffeine or theophylline, paraxanthine also significantly increased the incidence of malformed fetuses. Theobromine and 1-methylxanthine were virtually ineffective. From these findings, it is suggested that the methyl group at N-1 position of the xanthines is important for the enhancement but the N-1 methylation alone is ineffective unless accompanied with the substitution of the methyl moiety at the other position(s).