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.     

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.     

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.     

In Vitro effects of zinc on markers of bone formation

Zinc deficiency is associated with a reduced rate of bone formation that can be corrected by supplementation of the deficient diet with adequate amounts of zinc. This study was conducted to examine the effects of zinc on bone cell parameters associated with bone formation. Tibiae were removed from 19-d-old chicken embryos and incubated for 48 h in Dulbecco’s modified Eagle’s medium supplemented with antibiotics, bovine serum albumin, and HEPES. The addition of zinc (25–200 Μg/dL) to tibial cultures resulted in a concentrationdependent increase in alkaline phosphatase activity, an increase in the incorporation of proline into bone protein and an increase in the posttranslational oxidation of proline to peptidyl hydroxyproline. These effects of zinc were all diminished by the addition of 2,6-pyridine dicarboxylic acid, a chelator of zinc. The addition of either cycloheximide (10^-5M), dactinomycin (10^-8M), or hydroxyurea (10^-3M) to tibial cultures also attenuated the effects of zinc. The effect of zinc on bone cell DNA synthesis was measured by following the incorporation of³H-thymidine into DNA and by fluorometric measurement of cellular DNA content. These methods revealed that the addition of zinc to cultured tibiae resulted in a concentration-dependent increase in tibial DNA content and synthesis rate. The magnitude of the zinc-induced DNA increase was similar to the magnitude of the zinc-induced increases in alkaline phosphatase activity, proline incorporation, and hydroxyproline synthesis. Normalization of these latter responses to tibial DNA content yield data indicating that the effect of zinc on bone formation results from a zinc-induced increase in bone cell proliferation.     

DNA lesions,chromosomal aberrations and G2 delay in CHO cells cultured in medium containing bromo- or chloro-deoxyuridine

Experiments have been performed to investigate whether BrdUrd- and CldUrd-substituted DNA contains lesions causing a delay in cell-cycle progression and induction of chromosomal aberrations. The presence of lesions has been determined directly by alkaline sucrose gradient and nucleoid sedimentation analysis and indirectly by screening for induced chromosomal aberrations. The influence of inhibitors of DNA repair (caffeine and 3-aminobenzamide) or DNA synthesis (hydroxyurea) on the frequencies of such aberrations has been estimated. It is found that BrdUrd and CldUrd are cytotoxic when present in DNA. No randomly located DNA breaks could be detected under neutral conditions, but BrdUrd-substituted DNA was found to contain numerous alkali-labile sites. CldUrd at high concentrations causes G₂ delay, similar to the action of known DNA-damaging agents. The extent of delay depends on the pattern of incorporation of the analogue, i.e., incorporation for two cell cycles causes the longest delay, growth for 12 h in CldUrd followed by 12 h in dThd-containing medium causes a lesser delay and the delay is not significant when the cells are incubated in the analogue for only 12 h prior to fixation. Numerous chromatid type aberrations are present in cells incubated at the highest CldUrd concentration, and their induction follows the pattern of induction of G₂ delay, indicating the sharing of a common lesion. Caffeine, 3-aminobenzamide and hydroxyurea increase the number of chromosomal aberrations when added 2 h before fixation. The significance of these results is discussed.     

Hydroxyurea inhibits ODC induction, but not the G1 to S phase transition.

Chinese hamster ovary cells, selected in mitosis and plated into medium containing hydroxyurea, can progress through G₁ and enter S phase although bulk DNA synthesis is prevented. As the cells progress through G₁ in the presence of hydroxyurea, ornithine decarboxylase activity remains low while general protein synthesis appears unaffected. After hydroxyurea is removed, ornithine decarboxylase activity increases, but only after approximately 20% of the DNA has been replicated. These results suggest that ornithine decarboxylase induction is not essential for cellular progression into S phase but is required for the completion of DNA synthesis.     

Modulation of glycoprotein hormone alpha-subunit levels, alkaline phosphatase activity, and DNA replication by antimetabolites in HeLa cultures

Synthesis of the glycoprotein hormone common alpha-subunit and alkaline phosphatase (placental isozyme) has been examined in HeLa S3 cells. A variety of compounds that inhibit DNA synthesis lead to the increased production of both proteins. Experiments presented in this communication were undertaken to determine whether protein induction and DNA synthesis inhibition are coordinated. In general, nucleoside analogs and compounds that alter deoxynucleotide metabolism were good inducers of these ectopic products, whereas agents that altered DNA by intercalation, crosslinking, and covalent modification were poor inducers. The former class of effectors includes 5-bromo-2'-deoxyuridine, 5-fluoro-2'-deoxyuridine, 2'-deoxythymidine, 1-beta-D-arabinofuranosylcytosine, methotrexate, hydroxyurea, N-phosphonoacetyl-L-aspartic acid, and sodium butyrate; and the latter class of compounds includes ethidium bromide, acridine, bleomycin, mitomycin C, cesalin, macromomycin, and cis-diamminedichloroplatinum(II). A direct correlation between protein induction and DNA synthesis inhibition is unlikely based on the following observations: (i) for some effectors, the concentrations required to induce alpha-subunit and PAP were significantly different from those necessary to inhibit DNA synthesis; (ii) several agents inhibit DNA replication but do not enhance hormone or enzyme production; (iii) the kinetics of ectopic protein induction were similar for a number of inducers whereas the kinetics of DNA synthesis inhibition elicited by the same compounds were quite different. It is difficult from the data obtained, however, to rule out the possibility that inhibition of DNA synthesis may be required but is not sufficient for protein induction.     

Induction of Rat Liver Alkaline Phosphatase by Bile Duct Ligation

Bile duct ligation causes a five- to sevenfold increase in the activity of rat liver alkaline phosphatase within 12 hours after ligation and a similar rise in the activity of alkaline phosphatase in serum. The increased serum activity is due entirely to the appearance of a new isoenzyme that has the properties of rat liver alkaline phosphatase. The increase in both serum and liver alkaline phosphatase is prevented by the prior administration of cycloheximide in a dose that inhibits protein synthesis by 70%. Rat liver alkaline phosphatase was then purified to homogeneity. Antibody was raised to purified rat liver alkaline phosphatase in rabbits. The antibody was coupled to sepharose 4B and affinity columns made. ³-H-leucine was then injected into the portal veins of sham operated rats and rats with bile duct ligation four hours after ligation. One hour after injection and five hours after ligation, animals were sacrificed. Liver alkaline phosphatase was purified by means of affinity chromatography and double immunoprecipitation with rabbit antibody to rat liver alkaline phosphatase and goat anti-rabbit gamma globulin. Bile duct ligation increased the incorporation of ³-H-leucine into liver alkaline phosphatase more than threefold compared with sham operated rats, 164 CPM/mg protein vs. 49 CPM/mg protein (p < .001). The data indicate that the increased activity of rat liver alkaline phosphatase after bile duct ligation is due to enzyme induction rather than to activation of a pre-existing, relatively inactive enzyme.     

Tyrosine aminotransferase sensitivity to bromodeoxyuridine during restricted intervals of S phase in hepatoma cells

Synchronized hepatoma tissue culture (HTC) cells, accumulated at the G1/S boundary with aminopterin, were released into S phase with either thymidine or 5-bromodeoxyuridine (BUdR). Tyrosine aminotransferase (TAT) activity was found to be unaffected by BUdR over the initial 3 h of S phase, but then to rapidly decline to a new basal level of 40% of control by 9 h. There was no corresponding response in the activities of alcohol dehydrogenase, malate dehydrogenase, acid phosphatase, and alkaline phosphatase, or in the rate of protein and RNA synthesis. If BUdR incorporation was restricted to limited periods of S phase, TAT was found to be maximally suppressed by incorporation into the initial 40% of the DNA. Incorporation of the analogue into the latter 60% of DNA synthesized during S phase had no effect on TAT. This is the first report that the effect of BUdR on TAT in HTC cells is associated with incorporation of the analog into DNA synthesized during a specific interval of S phase.     

DNA synthesis in growth and encystment of Acanthamoeba castellanii.

Differentiation of Acanthamoeba castellanii into dormant cysts occurs spontaneously in stationary phase cultures, or can be induced experimentally by starvation. Although no further increase in cell density occurred after induction in either case, incorporation of [H]thymidine into DNA continued at a reduced rate through the period when differentiated products (cyst wall components) were formed. No net accumulation of DNA occurred during differentiation, indicating that the DNA synthesis occurring at this time was balanced by breakdown. When either 5-fluorodeoxyuridine (FUdR) or hydroxyurea was added to exponentially growing cultures, growth was terminated and the subsequent spontaneous encystment was delayed in comparison with untreated stationary phase cultures. A similar delay was observed for experimentally induced encystment of FUdR-pretreated cells. In all cases, delay of encystment was correlated with inhibition of 32PO4 incorporation into DNA, and unexpectedly also into RNA. Addition of FUdR at zero-time of experimental induction of cells not previously exposed to FUdR, on the other hand, had no effect on encystment or on 32PO4 incorporation. The delay of encystment produced by FUdR and hydroxyurea, therefore, appeared to reflect a requirement for normal synthesis of DNA and/or RNA not only during encystment, but also during the period of exponential growth just before encystment induction.     

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.     

Hypomethylation of repair patches in HeLa cells

In HeLa cells, under conditions where normal semiconservative synthesis is suppressed by hydroxyurea, the excision repair process after irradiation by UV results in a small amount of incorporation of nucleotides into nonreplicated DNA. By labelling the cytosine moieties of these repair patches, and measuring the ratio between cytosine and 5-methylcytosine, we have found that the level of methylation of cytosine in repair patches five hours after UV-irradiation of the cells is about half of that observed in normal semiconservatively synthesized DNA.     

The effects of aging on phosphofructokinase induction during lymphocyte mitogenesis in relation to DNA and protein synthesis

The incorporation of radiolabeled leucine into phytohemagglutinin-stimulated human lymphocytes increases by 9 hours after mitogen addition in the young whereas this process is delayed by two-fold in the aged (18 hours). Once induced, the leucine incorporation is about 56% less in the aged as compared to the young. The induction of phosphofructokinase (PFK) catalytic activity mimics the induction of protein synthesis in both young (9 hours) and aged (18 hours) subjects also taking twice as long to induce in the aged and attaining much lower levels of induction with increasing subject age. The increase of thymidine incorporation in mitogen-stimulated cells does not occur until 12 hours after the increase in leucine incorporation in both the young (21 hours) and aged (30 hours) which also represents a 9 hour age-related delay in induction. The marked increase in protein synthesis rate occurs in a concerted manner with the induction of glycolysis and the delay and impairment in protein biosynthesis in the aged appears to relate to the similar age-related findings for glycolytic enzyme induction. The mitogen-induced increase in DNA synthesis is a later event and the age-related delay in DNA synthesis induction may be secondary to the delay in the induction of protein synthesis. Other enzyme-dependent processes besides DNA synthesis and glycolysis may also be secondary to a primary slowing of protein synthesis in the aged and related to the delayed cell cycle time frequently observed in aged subjects.     

Induction of glycoprotein biosynthesis in activated B lymphocytes

Resting murine splenic B lymphocytes (B cells) can be stimulated to proliferate by exposure to a variety of polyclonal activators. To investigate changes in glycoprotein synthesis that occur during the activation process, N-glycosylation activity was assessed by following the incorporation of [2-3H]mannose into dolichol-linked oligosaccharide intermediates and glycoprotein after B cells were exposed to anti-immunoglobulin M (anti-mu). Stimulation of B cells by anti-mu resulted in a dramatic induction of N-glycosylation activity. The incorporation of radiolabeled mannose into oligosaccharide-lipid increased 9-fold while the rate of labeling of glycoprotein increased 27-fold between 18 and 38 h after exposure to anti-mu. Maximal stimulation of N-glycosylation activity was observed at an anti-mu concentration of 20-50 micrograms/ml. Similar results were obtained when B cells were activated by bacterial lipopolysaccharide (LPS), another polyclonal activating agent. The major dolichol-bound oligosaccharide labeled during the induction period was determined to be Glc3Man9GlcNAc2 by HPLC analysis. Nearly full induction of oligosaccharide-lipid synthesis and protein N-glycosylation was also seen when DNA synthesis was suppressed by activating B cells with anti-mu in a serum-free medium, or by activating with anti-mu or LPS in the presence of hydroxyurea. The results suggest that the N-glycosylation pathway is induced during the G0 to G1 transition or during the G1 period, and that entry into S phase is not required. These studies describe a striking developmental increase in N-glycosylation activity and extend the information on biochemical changes occurring during the activation of B cells.     

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.     

Continuous induction of unscheduled DNA synthesis by gamma irradiation

The induction of DNA-synthesis in non-S-phase cells is a very sensitive measure of a preceding damage of DNA. Usually, in an in vivo-in vitro test (treatment of an animal, incorporation of H3-thymidine in a cell suspension) the damaging of DNA takes place hours to days before the evaluation. In this case, the time course of the UDS-induction after a single dose of 1 Gy gamma irradiation was observed over a long period of time (21 months). C57 black mice served as test animals. In an age of about 80 days they were irradiated and the induction of unscheduled DNA synthesis was measured at ten time intervals during the whole life-span of the animals. Although the repair in this gamma radiation damage in DNA is a very quick process--with centrifugation in alkaline sucrose a half-life of some minutes is found--an induction of unscheduled DNA synthesis could be seen at the irradiated animals until the end of their life (640 days). The reason for this could be permanent disorders in cellular regulation caused by the gamma irradiation.