In vivo regulation of human leukocyte 3-hydroxy-3-methylglutaryl coenzyme A reductase: increased enzyme protein concentration and catalytic efficiency in human leukemia and lymphoma.

The activity of microsomal HMG-CoA reductase in freshly isolated leukocytes from patients with a variety of hematologic malignancies was significantly increased (up to 20-fold) when compared to enzyme activity in leukocytes from normal subjects (average 10.3 +/- 0.8 pmol/min per mg). Increased enzyme activity was not due to nonspecific leukocyte stimulation or to the presence of a malignancy, since normal enzyme activity was observed in subjects with either viral illnesses or solid tumors. Increased HMG-CoA reductase activity accompanying hematologic malignancy could also not be attributed to alterations in enzyme-substrate kinetic parameters (Km), or to alterations in the phosphorylation state or thiol-disulfide status of the enzyme, nor was it correlated with differences in serum lipid or lipoprotein concentrations. The increase (3.6-fold) in HMG-CoA reductase activity in leukocytes from patients with preleukemia was due entirely to a rise in enzyme catalytic efficiency (specific activity), whereas the increase (4.3-fold) observed in leukocytes from patients with overt leukemia or non-Hodgkin's lymphoma was due to a concomitant increase in both enzyme catalytic efficiency (2.5-fold) and enzyme protein concentration (1.6-fold). Similar increases in HMG-CoA reductase activity and catalytic efficiency were also noted for both transformed, nonmalignant, and malignant cultured leukocytes, suggesting that increased enzyme catalytic efficiency is not a nonspecific consequence of physiological changes occurring in response to the malignancy but may be an integral aspect of the malignant phenotype. HMG-CoA reductase protein concentrations, however, were not elevated in either transformed, nonmalignant, or malignant cultured leukocytes, suggesting that increases in enzyme protein levels may be secondary to other physiological changes that occur during the development of overt leukemia. Taken together, these observations suggest that an increase in the activity of HMG-CoA reductase, the rate-controlling enzyme in cholesterol synthesis, is a common occurrence in human hematologic malignancies and that a biphasic elevation of enzyme activity may exist in malignant leukocytes, such that changes in catalytic activity may occur early in tumorigenesis and may be followed by secondary changes in enzyme levels.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in avian myeloblasts. Mode of action of 25-hydroxycholesterol

25-Hydroxycholesterol inhibits cholesterol biosynthesis by inhibiting the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Addition of 25-hydroxycholesterol to chicken myeloblasts caused a rapid inhibition of HMG-CoA reductase activity, producing approximately an 80% decrease in enzyme activity after 60 min. The mode of action of 25-hydroxycholesterol was determined by immunoprecipitating radiolabeled enzyme from 25-hydroxycholesterol-treated myeloblasts. The decline in enzyme activity due to addition of 25-hydroxycholesterol was not associated with increased levels of [32P]PO4 incorporation into the immunoprecipitated reductase polypeptide (Mr = 94,000). Hence, 25-hydroxycholesterol did not appear to regulate reductase activity by enzyme phosphorylation, as observed for other modulators of HMG-CoA reductase. However, 25-hydroxycholesterol was shown to inhibit reductase activity by causing a 350% increase in the relative rate of reductase degradation and a 72% decrease in the relative rate of reductase synthesis. These alterations in the rates of degradation and synthesis occurred rapidly (within 10-30 min after addition of 25-hydroxycholesterol) and can account completely for the 25-hydroxycholesterol-induced inhibition of enzyme activity. The rapid decline in the rate of synthesis of HMG-CoA reductase in 25-hydroxycholesterol-treated cells was not associated with concomitant changes in the levels of reductase mRNA; therefore, suggesting that 25-hydroxycholesterol must inhibit the rate of reductase synthesis by translational regulation. We also present evidence that mRNA purified from chicken myeloblasts codes for two reductase polypeptides of Mr = 94,000 and 102,000.     

Expression of thymidine kinase and dihydrofolate reductase genes in mammalian ts mutants of the cell cycle

Thymidine kinase and dihydrofolate reductase mRNA levels and enzyme activities were determined in two temperature-sensitive cell lines, tsAF8 and ts13, that growth arrest in the G1 phase of the cell cycle at the restrictive temperature. The levels of thymidine kinase mRNA and enzyme activity increased markedly in both cell lines serum stimulated from quiescence at the permissive temperature. At the nonpermissive temperature, the levels of thymidine kinase mRNA and enzyme activity remain at the low levels of quiescent G0 cells. The levels of dihydrofolate reductase mRNA as well as the enzyme activity also increase when both cell lines are serum stimulated at the permissive temperature. When ts13 cells are serum stimulated at the nonpermissive temperature dihydrofolate reductase enzyme activity declines rapidly and dihydrofolate reductase mRNA is below detectable levels. On the contrary, when tsAF8 cells are serum stimulated at the nonpermissive temperature dihydrofolate reductase enzyme activity increases and mRNA levels are detectable slightly above G0 levels, even though the cells are blocked in the G1 phase. Studies with 2 other cDNA clones (one with an insert whose expression is cell cycle dependent and the other with an insert whose expression is not cell cycle dependent) indicate that the results are not due to aspecific toxicity or the effect of temperature. We conclude that the expression of different genes is affected differently by the ts block in G1, even when these genes are all growth-related.     

Mitochondrial deoxyguanosine kinase mutations and mitochondrial DNA depletion syndrome

Mitochondrial deoxyguanosine kinase (dGK) catalyzes the initial phosphorylation of purine deoxynucleosides. Mutations in the dGK gene leading to deficiency in dGK activity is one of the causes of severe mitochondrial DNA depletion diseases. We used site-directed mutagenesis to introduce the clinically observed genetic alterations in the dGK gene and characterized the recombinant enzymes. The R142K enzyme had very low activity with deoxyguanosine and no activity with deoxyadenosine. The E227K mutant enzyme had unchanged K(m) values for all its substrates but very low V(max) values. C-terminal truncated dGK proteins were inactive. These results may help to define the role of dGK in mitochondrial DNA (mtDNA) precursor synthesis.     

Evidence for the involvement of substrate cycles in the regulation of deoxyribonucleoside triphosphate pools in 3T6 cells

Pool sizes of deoxyribonucleoside triphosphates (dNTPs) in cultured cells are tightly regulated by i.al., the allosteric control of ribonucleotide reductase. We now determine the in situ activity of this enzyme from the turnover of the deoxycytidine triphosphate (dCTP) pool in rapidly growing 3T6 mouse fibroblasts, as well as in cells whose DNA replication was inhibited by aphidicolin or amethopterin, by following under steady state conditions the path of isotope from [5-3H]cytidine into nucleotides, DNA, and deoxynucleosides excreted into the medium. In normal cells as much as 28% of the dCDP synthesized was excreted as deoxynucleoside (mostly deoxyuridine), leading to an accumulation of deoxyuridine in the medium. Inhibition with amethopterin slightly increased ribonucleotide reductase activity, while aphidicolin halved the activity of this enzyme (and thymidylate synthase). In both instances all dCDP synthesized was degraded and excreted as nucleosides. This continued synthesis and turnover in the absence of DNA synthesis is in contrast to the earlier found inhibition of dCTP (and dTTP) turnover when hydroxyurea, an inhibitor of ribonucleotide reductase, was used to block DNA synthesis. To explain our results, we propose that substrate cycles between deoxyribonucleosides and their monophosphates, involving the activities of kinases and phosphatases, participate in the regulation of pool sizes. Within the cycles, a block of the reductase activates net phosphorylation, while inhibition of DNA polymerase stimulates degradation.     

Alterations in the components of ribonucleotide reductase in hydroxyurea-resistant hamster cells

Two components of mammalian ribonucleotide reductase have been separated by blue dextran-Sepharose chromatography from a hydroxyurea-resistant cell line, NCR-30A2, and its parental wild type. Analysis of reductase activity in these cells and the enzyme components reveals that there are three alterations involving ribonucleotide reductase activity in NCR-30A2 cells. There is an elevation in the effector-binding (EB) component, an elevation in the non-heme-iron-containing (NHI) component, and an alteration in the NHI component that renders the enzyme less sensitive to inhibition by hydroxyurea. These findings easily account for the resistance of NCR-30A2 cells to the antitumor agent hydroxyurea, and to other drugs with a similar mode of action.     

Relationships between reversion of hydroxyurea resistance in hamster cells and the co-amplification of ribonucleotide reductase M2 component, ornithine decarboxylase and P5-8 genes

Hydroxyurea is a specific inhibitor of ribonucleotide reductase, which is a rate-limiting enzyme activity in DNA synthesis. Cells selected for resistance to hydroxyurea contain alterations in ribonucleotide reductase activity. An unstable hydroxyurea resistant population of hamster cells has been used to isolate a stable drug resistant cell line, and two stable revertant lines with different sensitivities to hydroxyurea cytotoxicity and different ribonucleotide reductase activity levels. We show for the first time that a decrease in hydroxyurea resistance is accompanied by a parallel decline in gene copies for the M2 component of ribonucleotide reductase, ornithine decarboxylase and a gene of unknown function called p5-8, indicating that the co-amplification of the three genes is associated with drug resistance, and supporting the concept that M2, ornithine decarboxylase and p5-8 are closely linked, and form part of a single amplicon in hamster cells.     

A flow microcalorimetric method for enzyme activity measurements: Application to dihydrofolate reductase

A flow microcalorimetric method was developed for the analysis of enzymatic activities in crude tissue homogenates. It can be applied whenever a heat exchange is involved in an enzymatic reaction. The consequent sensitivity obviously depends on the enthalpy variation observed. Dihydrofolate reductase was chosen as an example; this enzyme is the molecular target of methotrexate, a widely used anticancer agent. This calorimetric method, whose sensitivity limit is 1.48 × 10⁻⁴ units of dihydrofolate reductase per milliliter of reactant medium, allows enzyme activity measurements in tissues with low dihydrofolate reductase levels. A few examples of measurements in animal tissues are given. These measurements are of some interest; indeed, increased activity and increased levels of this enzyme are two of the mechanisms which may explain resistance to methotrexate.     

Mutational analysis of parasite trypanothione reductase: acquisition of glutathione reductase activity in a triple mutant

African trypanosomes contain a cyclic derivative of oxidized glutathione, N1,N8-bis(glutathionyl)spermidine, termed trypanothione. This is the substrate for the parasite enzyme trypanothione reductase, a key enzyme in disulfide/dithiol redox balance and a target enzyme for trypanocidal therapy. Trypanothione reductase from these and related trypanosomatid parasites is structurally homologous to host glutathione reductase but the two enzymes show mutually exclusive substrate specificities. To assess the basis of host vs parasite enzyme recognition for their disulfide substrates, the interaction of bound glutathione with active-site residues in human red cell glutathione reductase as defined by prior X-ray analysis was used as the starting point for mutagenesis of three residues in trypanothione reductase from Trypanosoma congolense, a cattle parasite. Mutation of three residues radically alters enzyme specificity and permits acquisition of glutathione reductase activity at levels 10(4) higher than in wild-type trypanothione reductase.     

Cholesterol Biosynthesis and Its Regulation in Dissociated Cell Cultures of Fetal Rat Brain: Developmental Changes and the Role of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase

Primary cultures of cells dissociated from fetal rat brain were utilized to define the developmental changes in cholesterol biosynthesis and the role of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the regulation of these changes. Cerebral hemispheres of fetal rats of 15-16 days of gestation were dissociated mechanically into single cells and grown in the surface-adhering system. Cholesterol biosynthesis, studied as the rate of incorporation of [14C]acetate into digitonin-precipitable sterols, was shown to exhibit two distinct increases in synthetic rates, a prominent increase after 6 days in culture and a smaller one after 14 days in culture. Parallel measurements of HMG-CoA reductase activity also demonstrated two discrete increases in enzymatic activity, and the quantitative and temporal aspects of these increases were virtually identical to those for cholesterol synthesis. These data indicate that cholesterol biosynthesis undergoes prominent alterations with maturation and suggest that these alterations are mediated by changes in HMG-CoA reductase activity. The timing of the initial prominent peak in both cholesterol biosynthesis and HMG-CoA reductase activity at 6 days was found to be the same as the timing of the peak in DNA synthesis, determined as the rate of incorporation of [3H]thymidine into DNA. The second, smaller peak in reductase activity and sterol biosynthesis at 14 days occurred at the time of the most rapid rise in activity of the oligodendroglial enzyme, 2':3'-cyclic nucleotide 3'-phosphohydrolase (CNP). These latter observations suggest an intimate relationship of the sterol biosynthetic pathway with cellular proliferation and with oligodendroglial differentiation in developing mammalian brain.     

Subcellular distribution and properties of acyl/alkyl dihydroxyacetone phosphate reductase in rodent livers

On subcellular fractionation, the enzyme acyl/alkyl dihydroxyacetone phosphate (DHAP) reductase (EC 1.1.1.101) in guinea pig and rat liver was found to be present in both the light mitochondrial (L) and microsomal fractions. By using metrizamide density gradient centrifugation, it was shown that the alkyl DHAP reductase activity in the "L" fraction is localized mainly in peroxisomes. From the distribution of the marker enzymes it was calculated that about two-thirds of the liver reductase activity is in the peroxisomes and the rest in the microsomes. The properties of this enzyme in peroxisomes and microsomes are similar with respect to heat inactivation, pH optima, sensitivity to trypsin, and inhibition by NADP+ and acyl CoA. The enzyme activity in the peroxisomes and microsomes from mouse liver is increased to the same extent by chronically feeding the animals clofibrate, a hypolipidemic drug. The kinetic properties of this enzyme in these two different organelles are also similar. From these results it is concluded that the same enzyme is present in two different subcellular compartments of liver.     

The role of deoxynucleoside triphosphate pools in the inhibition of DNA-excision repair and replication in human cells by hydroxyurea

The effects of hydroxyurea (HU) on the DNA-excision repair process in human cells has been systematically examined. It is demonstrated that HU induces DNA single-strand break accumulation in a dose-dependent fashion in ultraviolet-irradiated and MMS-treated confluent but not log-phase fibroblasts and that these breaks are clearly the consequence of the inhibition by HU of the enzyme, ribonucleotide reductase. The breaks form rapidly, are stable for at least 10 h and largely disappear by 20 h. The production of these DNA-strand breaks is antagonized by a combined treatment of 10 microM deoxyadenosine, deoxycytidine and deoxyguanosine whereas thymidine potentiates strand-break formation at low HU concentrations. It is also confirmed that HU, while inhibiting replicative synthesis has no apparent inhibitory effect on unscheduled DNA synthesis (UDS) although the increased uptake of labeled DNA precursors into HU-treated cells makes it difficult to assess the actual effects on the repair-synthetic process. Analysis of the effects of HU on deoxynucleoside triphosphate pool levels and the demonstration of the failure of the HU block to replicative synthesis to be reversed by high (1 mM) concentrations of added deoxynucleosides lend support to the notion of compartmentalized dNTP pools for repair and replication.     

Anaerobic respiratory growth of Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi with trimethylamine N-oxide, nitrate and fumarate: ecological implications

Two symbiotic species, Photobacterium leiognathi and Vibrio fischeri, and one non-symbiotic species, Vibrio harveyi, of the Vibrionaceae were tested for their ability to grow by anaerobic respiration on various electron acceptors, including trimethylamine N-oxide (TMAO) and dimethylsulphoxide (DMSO), compounds common in the marine environment. Each species was able to grow anaerobically with TMAO, nitrate or fumarate, but not with DMSO, as an electron acceptor. Cell growth under microaerophilic growth conditions resulted in elevated levels of TMAO reductase, nitrate reductase and fumarate reductase activity in each strain, whereas growth in the presence of the respective substrate for each enzyme further elevated enzyme activity. TMAO reductase specific activity was the highest of all the reductases. Interestingly, the bacteria-colonized light organs from the two squids, Euprymna scolopes and Euprymna morsei, and the light organ of the ponyfish, Leiognathus equus, also had high levels of TMAO reductase enzyme activity, in contrast to non-symbiotic tissues. The ability of these bacterial symbionts to support cell growth by respiration with TMAO may conceivably eliminate the competition for oxygen needed for both bioluminescence and metabolism.     

Enzymes in pancreatic islets that use NADP(H) as a cofactor including evidence for a plasma membrane aldehyde reductase.

Recent evidence of a pyruvate malate shuttle capable of transporting a large amount of NADPH equivalents out of mitochondria in pancreatic islets suggests that cytosolic NADP(H) plays a role in beta cell metabolism. To obtain clues about these processes the activities of several NADPH-utilizing enzymes were estimated in pancreatic islets. Low levels of pyrroquinolone quinone (PQQ) and low levels of enzyme activity that reduce PQQ were found in islets. Low activities of palmitoyl-CoA and stearoyl-CoA desaturases were also detected. Significant activities of glutathione reductase, aldose reductase (EC.1.1.1.21) and aldehyde reductase (EC.1.1.1.2) were present in islets. Potent inhibitors of aldehyde and aldose reductases inhibited neither glucose-induced insulin release nor glucose metabolism in islets indicating that these reductases are not directly involved in glucose-induced insulin reaction. Over 90% of aldose reductase plus aldehyde reductase enzyme activity was present in the cytosol. Kinetic and chromatographic studies indicated that 60-70% of this activity in cytosol was due to aldehyde reductase and the remainder due to aldose reductase. Aldehyde reductase-like enzyme activity, as well as aldose reductase immunoreactivity, was detected in rat islet plasma membrane fractions purified by a polyethylene glycol-Dextran gradient or by a sucrose gradient. This is interesting in view of the fact that voltage-gated potassium channel beta subunits that contain aldehyde and aldose reductase-like NADPH-binding motifs have been detected in plasma membrane fractions of islets [Receptors and Channels 7: 237-243, 2000] and suggests that NADPH might have a yet unknown function in regulating activity of these potassium channels. Reductases may be present in cytosol to protect the insulin cell from molecules that cause oxidative injury.     

Engineering of Sulfolobus solfataricus HMG-CoA reductase to a form whose activity is regulated by phosphorylation and dephosphorylation

There are two classes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase: the class I enzymes of eukaryotes and some archaea, and the class II enzymes of certain eubacteria. The activity of the class I Syrian hamster HMG-CoA reductase is regulated by phosphorylation-dephosphorylation of Ser871. Phosphorylation apparently prevents the active site histidine, His865, from protonating the inhibitory coenzyme A thioanion prior to its release from the enzyme. Structural evidence for this hypothesis is, however, lacking. The HMG-CoA reductase of the thermophilic archaeon Sulfolobus solfataricus, whose stability recommends it for physical studies, lacks both a phosphoacceptor serine and a protein kinase recognition motif. Consequently, its activity is not regulated by phosphorylation. We therefore employed site-directed mutagenesis to engineer an appropriately located phosphoacceptor serine and cAMP-dependent protein kinase recognition motif. Substitution of serine for Ala406, the apparent cognate of hamster Ser871, and replacement of Leu403 and Gly404 by arginine created S. solfataricus mutant enzyme L403R/G404R/A406S. The general properties of enzyme L403R/G404R/A406S (K(m) values, V(max), optimal pH and temperature) were essentially those of the wild-type enzyme. Exposure of enzyme L403R/G404R/A406S to [gamma-(32)P]ATP and cAMP-dependent protein kinase was accompanied by incorporation of (32)P(i) and by a parallel decrease in catalytic activity. Subsequent treatment with a protein phosphatase released enzyme-bound (32)P(i) and restored activity to pretreatment levels. The regulatory properties of enzyme L403R/G404R/A406S thus match those of the hamster enzyme. Solution of the three-dimensional structures of the phospho and dephospho forms of this mutant enzyme thus should reveal structural features critical for regulation of the activity of a class I HMG-CoA reductase.     

Correlation between levels of ferritin and the iron-containing component of ribonucleotide reductase in hydroxyurea-sensitive, -resistant, and -revertant cell lines.

The reduction of ribonucleotides to deoxyribonucleotides, a rate-limiting step in DNA synthesis, is catalyzed by ribonucleotide reductase. This enzyme is composed of two components, M1 and M2. Recent work has shown that inhibition of ribonucleotide reductase by the antitumor drug hydroxyurea leads to a destabilized iron centre in protein M2. We have examined the relationship between the levels of ferritin, the iron storage protein, and the iron-containing M2 component of ribonucleotide reductase. These studies were carried out with hydroxyurea-sensitive, -resistant, and -revertant cell lines. Hydroxyurea-resistant mouse L cells contained M2 gene amplification and elevated levels of enzyme activity, M2 message, and total cellular M2 protein concentration. Hydroxyurea-revertant cells exhibited a wild-type M2 gene copy number, and approximately wild-type levels of enzyme activity, M2 message, and M2 protein concentration. In addition, we observed that the hydroxyurea-resistant cells possessed elevated levels of L-chain ferritin message and total cellular H-chain ferritin protein when compared to wild-type cells. In contrast, the revertant cell population contained approximately wild-type levels of ferritin mRNA and protein. In keeping with these observations, obtained with mouse L cells, was the finding that hydroxyurea-resistant Chinese hamster ovary cells with increased ribonucleotide reductase activity exhibited elevated expression of both ferritin and M2 genes, which declined in drug-sensitive revertant hamster cell lines with decreased levels of ribonucleotide reductase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assay of ribonucleotide reductase activity in intact permeabilized hamster cells: an evaluation

An intact cell assay system, based on Tween-80 permeabilization can be used to investigate ribonucleotide reductase activity in a variety of mammalian cell lines. An important consideration in the use of intact cells is the presence of other nucleotide metabolizing activities. The influence of these activities on estimates of pyrimidine (CDP) and purine (ADP) reductase in permeabilized hamster cells has been examined. Studies on the incorporation of label from CDP and ADP into RNA indicated that a very small proportion of the reductase substrates was eventually incorporated into RNA during routine enzyme assays, and would have no detectable effect on activity estimates. The possibility that the products of the reaction (dCDP and dADP) were eventually phosphorylated and incorporated into DNA was also examined, and it was found that proper permeabilization of the cells eliminated or greatly reduced loss of deoxyribonucleotides to DNA. An analysis by HPLC of nucleotides present during CDP and ADP reductase reactions showed that various kinases and phosphatases were active in permeabilized cells, as all levels of phosphorylation of nucleotide substrates and allosteric effectors were detected. The base composition of the nucleotides added to the assay systems were not altered. Although movement of phosphates occurred during the assay, the concentrations of substrates quickly reached equilibrium (within 1 min) with their respective nucleosides and nucleotides, resulting in a relatively constant although reduced concentration of CDP or ADP substrates during the 20-min assay. Similarly the levels of allosteric effectors, ATP for pyrimidine and dGTP for purine reductase activities, declined within the first minute of the assays and quickly reached an equilibrium with their respective adenine or guanine containing nucleotides during most of the reaction time. Although useful approximations of intracellular reductase activity can be obtained without correcting for modified nucleotide concentrations, precise determinations can be calculated when these alterations are taken into consideration. For example, estimates of intracellular K_m values for CDP closely resembled those reported with highly purified mammalian enzyme preparations in other studies. Clearly, the intact cell assay system provides worthwhile information about mammalian ribonucleotide reductase in its physiologically relevant environment.     

Defective regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in a somatic cell mutant.

A somatic cell mutant of the CHO-K1 cell selected to be resistant to the killing effects of 25-hydroxycholesterol in the absence of cholesterol is shown to be defective in the inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity by 25-hydroxycholesterol, cholesterol, and lipoproteins, thus maintaining the enzyme activity found in cells in the absence of exogenous sterol constitutively. The mutants phenotype is shown to be dominant with respect to the wild type. Actinomycin D and cycloheximide prevent the increase of HMG-CoA reductase activity that occurs in the CHO-K1 cell when cholesterol is removed from medium. Degradation of the enzyme, measured during inhibition of protein synthesis by cycloheximide, occurs at the same rate in the mutant as in the wild type. Kinetic studies indicate that the Km for two substrates, the activation energy, and a break in the Arrhenius plot are the same for HMG-CoA reductase determined in wild type and mutant cells. From these studies it is concluded that the mutant is defective in the regulation of synthesis of HMG-CoA reductase. Of the four processes which determine cellular cholesterol levels: biosynthesis, esterification, efflux, and uptake, only biosynthesis is altered, demonstrating that these processes are not co-ordinately controlled as has been suggested previously.     

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.