Dose-dependent pharmacokinetics of methotrexate and 7-hydroxymethotrexate in the rat in vivo.

The pharmacokinetics of methotrexate (MTX) and 7-hydroxymethotrexate (7-OH-MTX) in bile, urine, and serum was studied in rats in vivo after short-time infusions of 10, 50, 250, and 1000 mg/kg MTX. All animals were anesthetized and drained of bile during experiments. The biliary secretion rate of MTX approached saturation when serum MTX levels surpassed 700-800 microM, causing a significant reduction in biliary recovery as the parent compound (49 to 32%) at MTX doses exceeding 50 mg/kg. The hepatic metabolism of MTX to the 7-hydroxy metabolite was not saturated at the doses used. Serum MTX pharmacokinetics demonstrated dose dependency, inasmuch as doses exceeding 10 mg/kg were accompanied by a reduced total body clearance (Clr) and biliary clearance (ClB). A significant finding in relation to acute hepatotoxicity reported after high-dose MTX in humans was the occurrence of cholestasis 30-90 min after drug infusion and the observation of macroscopic precipitations in the bile duct in five of six animals treated with 1000 mg/kg MTX. In these five animals, cessation of bile secretion occurred at similar bile 7-OH-MTX levels [9800 +/- 1100 (SD) microM], while the single rat that secreted bile throughout the experiment had a 5-fold lower peak 7-OH-MTX concentration in bile. Analysis of biliary precipitates formed in vivo and in vitro found 7-OH-MTX to constitute 97% and MTX 3% of the drug content of the precipitated material.

Formation and elimination of 7-hydroxymethotrexate in the rat in vivo after methotrexate administration.

Bile, urine, and serum concentrations of methotrexate (MTX) and 7-hydroxy-methotrexate (7-OH-MTX) were monitored in rats in vivo following a short-time infusion of 10 mg/kg [3H]MTX. The experiments were performed in one group of anesthetized, bile-drained rats and in two control groups, one anesthetized and one unanesthetized, that were not bile-drained. Peak biliary levels of MTX (3.8 x 10(-3) M) and 7-OH-MTX (1.8 x 10(-4) M) appeared within 15 min after cessation of infusions. For two log ranges of serum MTX concentrations, biliary levels remained 180-fold higher. High bile 7-OH-MTX levels appeared few min after start of MTX administration, and were 720 times higher than the peak serum concentrations, indicating that the liver is a major site of 7-OH-MTX formation in the rat. 7-OH-MTX concentrations in bile declined monophasically with a half-life of 29.4 min, while MTX showed a biphasic elimination with initial and second phase half-lives of 23.1 and 86.4 min, respectively. Bile was the major excretory route for MTX and 7-OH-MTX, with 50% of the dose recovered as the parent compound and 3.6% as the metabolite. There was no difference in urinary recovery of MTX in bile-drained and control animals, indicative of insignificant enterohepatic circulation of MTX. This was further corroborated by the finding of just 2.1% urinary recovery of MTX in rats who received previously collected MTX-containing bile through a duodenal catheter. Serum concentration curves were analyzed according to a three-compartment open model with an initial elimination half-life of 1.7-3.3 min, a second phase half-life of 15.4-21.0 min, and a terminal phase half-life of 119-240 min. Our finding of 7-OH-MTX formation and high biliary levels of the metabolite in the rat, can be used as basis for studies of interactions with in vivo MTX conversion to the 7-hydroxy metabolite.

Induction of HL-60 cell differentiation by 3-deaza-(+/-)-aristeromycin, an inhibitor of S-adenosylhomocysteine hydrolase.

The metabolically stable inhibitor of S-adenosylhomocysteine hydrolase (AdoHcyase), 3-deaza-(+/-)-aristeromycin (dzAri) has recently been shown to induce differentiation in HL-60 cells. The present study was undertaken to characterize the cytostatic, cytotoxic, and differentiation inducing properties of dzAri in HL-60 cells and to investigate biochemical consequences of AdoHcyase inhibition. A dye exclusion test and a clonogenic assay were used to test cytotoxic and cytostatic properties. dzAri had reversible cytostatic effects on HL-60 cells at concentrations lower than 10 microM and partially reversible cytotoxic effects above 10 microM. The induction of differentiation was dependent upon concentration and time of exposure, with maximal effect after 6 days incubation with 5-10 microM dzAri. Washout experiments demonstrated that the cells were not committed to differentiation after 48 h of incubation with dzAri. The AdoHcyase of HL-60 cells was inhibited with a Ki of 20 nM. The concentration of S-adenosylhomocysteine increased dose dependently 48 h after incubation with 0.1-100 microM dzAri. The incorporation of [3H]methyl from [methyl-3H]methionine into 5-methylcytosine of DNA was reduced by 26% at 5 microM dzAri. The findings indicate that continuous presence of dzAri is necessary to induce differentiation and inhibit proliferation in HL-60 cells. The inhibition of AdoHcyase perturbs levels of transmethylation metabolites and the incorporation of [3H]methyl into 5-methyl-cytosine of DNA.

The c-fos cAMP-response element: regulation of gene expression by a beta 2-adrenergic agonist, serum and DNA methylation.

The transcription control region of the proto-oncogene c-fos contains multiple cis-acting elements to which specific trans-acting factors bind. One such well-studied binding motif in the c-fos promoter is the major cyclic AMP response element (CRE) TGACGT located at -62/-57. In this study we investigated the role of this element in gene regulation by beta 2-adrenergic/adenylate cyclase signalling and DNA methylation. By transient gene expression assays we were able to show that the c-fos regulatory sequences spanning nucleotides -361 to +13 could mediate gene expression by the beta 2-adrenergic agonist isoproterenol and the phosphodiesterase inhibitor theophylline. For isoproterenol however, a stimulating effect was observed in serum-starved cells, while an inhibitory effect was measured in cells supplemented with serum. The gene regulation by the cAMP elevating agents could be due, at least partially, to the major CRE since this isolated motif mediated gene expression by these drugs. Distinct protein-DNA complexes were obtained with nuclear extracts prepared from cells exposed to isoproterenol or/and theophylline under different serum conditions. We further show that DNA methylation of this CRE may also be involved in gene regulation as methylation of the CRE motif strongly reduced the binding of nuclear proteins.

Synergistic increase in c-fos expression by simultaneous activation of the ras/raf/map kinase- and protein kinase A signaling pathways is mediated by the c-fos AP-1 and SRE sites.

Expression of the c-fos proto-oncogene is induced by numerous stimuli some of which are transmitted through the Ras/Raf/MAP kinase or the cAMP-dependent protein kinase (PKA) pathways. The effect of cell-specific interactions between these pathways on c-fos expression was investigated by exposing quiescent NIH3T3 cells to serum, forskolin, or a combination. Co-stimulation with serum and forskolin resulted in a more than additive increase in c-fos transcription. Synergistic increase in c-fos promoter activity was also observed in transient transfection studies after co-stimulation with serum plus forskolin or co-transfection with c-Raf and PKA expression plasmids. Analysis of the cAMP signaling pathway revealed that the synergy was neither due to an increase in PKA activity nor to Ser-133 phosphorylation/activation of CREB. The activation status of the MAP kinases ERK1 and ERK2 in co-treated cells was comparable to that in serum-treated cells. Co-stimulation with forskolin did not alter the phosphorylation state of Elk-1 compared to serum-induced phosphorylation of Elk-1. Deletion of c-fos promoter elements previously shown to be important for regulation of c-fos expression in response to mitogens indicates a role for SRE and FAP-1 elements.

The human promyelocytic leukemia cell (HL-60 cell) beta-adrenergic receptor.

We have characterized the beta-adrenergic receptor binding site and the beta-adrenergic cAMP response of the HL-60 cell. The hydrophilic ligand [3H]-(-)-CGP-12177 was specifically and reversibly bound to one single class of binding sites (Kd 220 pM and Bmax 1,970 sites/cell). The adrenergic agonists inhibited the specific radioligand binding. The order of potency was isoproterenol greater than epinephrine greater than norepinephrine. The beta-2 selective antagonist ICI 118551 had a binding affinity 3 orders of potency higher than the beta-1 selective antagonist, atenolol. The adrenergic agonists elevated the cAMP accumulation in a concentration-dependent mode. The order of potency was isoproterenol greater than epinephrine greater than norepinephrine. Both the binding and the functional studies revealed stereospecificity and reversibility. The present data show that HL-60 cells possess beta-2 adrenergic receptors functionally coupled to adenylate cyclase.

Amiodarone inhibits the conversion of thyroxine to triiodothyronine in isolated rat hepatocytes.

Previous studies have shown decreased T3 and increased T4 and rT3 serum levels in subjects treated with amiodarone. We studied the acute effect of amiodarone on the in vitro conversion of T4 to T3 in suspensions of isolated rat hepatocytes. Iodine and melperone, another class III antiarrhythmic drug, were included in the study as controls. Amiodarone (60 microM) totally blocked the formation of T3 from T4, whereas concentrations of 6 and 0.6 microM reduced T3 formation to 13.7 +/- 10.6% and 63.9 +/- 15.9% (+/- SD), respectively, compared with untreated controls. The drug solvent did not affect the conversion rate. Iodide (120 microM) had an inhibitory effect of approximately 10% compared with the controls, whereas melperone did not affect this in vitro conversion. Amiodarone (60 microM) caused a slight but significant reduction of the cellular uptake of [125I]T4 after 10 min of incubation, whereas the 60 min values were unaltered. Our study indicates that amiodarone inhibits the 5'-monodeiodination of T4 to T3 in a dose-related manner. It is suggested that the antiarrhythmic activity of the drug is related to its inhibitory effect on the conversion of T4 to T3.

Interethnic difference in thiopurine methyltransferase activity.

A number of metabolic pathways are subject to both genetic polymorphism and interethnic differences. A catabolic pathway of 6-mercaptopurine, red blood cell (RBC) thiopurine methyltransferase (TPMT) activity showed genetic polymorphism in Caucasians, but variation according to ethnicity has not been studied. We investigated if red blood cell thiopurine methyltransferase was subject to interethnic variation in a Saami (Lappish; n = 36) and a Caucasian population (n = 50). The Saami population sample had 29% higher thiopurine methyltransferase activity, 17.0 +/- 3.3 U/ml red blood cell compared with the Caucasian population sample, 13.1 +/- 2.9 U/ml red blood cell (p much less than 0.001). Probit plots and frequency distribution histograms supported bimodality consistent with genetic polymorphism in both study populations. Differences in chronic diseases, drug consumption, age, or gender could not explain the interethnic difference in red blood cell thiopurine methyltransferase activity. The higher red blood cell thiopurine methyltransferase activity in the Saami population group indicates that these subjects may require higher dosages of thiopurine drugs than Caucasians.

Thiopurine methyltransferase activity in a Korean population sample of children.

Thiopurine methyltransferase (TPMT) is a cytoplasmic enzyme that catalyzes the S-methylation of the cytotoxic drugs 6-mercaptopurine and azathioprine. Red blood cell (RBC) TPMT activity is subject to genetic polymorphism, and we have previously demonstrated an interethnic difference in TPMT activity. To investigate whether there was a race-related difference in RBC TPMT activity, TPMT was measured in a Korean population sample of 309 healthy children. Mean TPMT activity in healthy Korean children was 12.4 +/- 2.4 units/ml RBC, which is similar to the earlier reported TPMT activities in white populations. In contrast to the bimodal or trimodal frequency distributions of RBC TPMT activity in most other population samples, the frequency distribution histogram, the probit plot, and the Shapiro-Wilk test supported a normal distribution of TPMT activity in this Korean population sample of healthy children. Mean RBC TPMT activity showed a tendency to decrease with age, but it was not statistically significant. No gender-related difference in RBC TPMT activity was found.

Detection of one single mutation predicts thiopurine S-methyltransferase activity in a population of Saami in northern Norway.

Thiopurine S-methyltransferase (TPMT) activity exhibits genetic polymorphism. The purpose of this investigation was to identify TPMT mutant alleles in the Saami population as a basis of developing genotyping tests for prediction of TPMT activity. The most predominant allele in Saamis (n = 194) was the TPMT*3C allele (A719G mutation) representing 92% of the mutant alleles, with an estimated allelic frequency of 3.3%. The most frequent allele in Caucasians (n = 66) living in the same geographic area was the TPMT*3A (A719G and G460A mutations) representing 91% of the mutant alleles, with an estimated allelic frequency of 3.4%. A test for one mutation, A719G, may prospectively identify more than 90% of the Saami individuals who require reduction in thiopurine dose to avoid hematopoietic toxicity. In a Norwegian population, comprising both the major Caucasian population and a minor Saami population, the same genotyping tests (eg, tests for the A719G and G460A mutations) may be used.

Human thiopurine methyltransferase pharmacogenetics: gene sequence polymorphisms.

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs. TPMT activity is regulated by a common genetic polymorphism that is associated with large individual variations in thiopurine toxicity and efficacy. We previously cloned the functional gene for human TPMT and reported a common variant allele for low enzyme activity, TPMT*3A, that contains point mutations at cDNA nucleotides 460 and 719. In the present study, we set out to determine the number, types, and frequencies of TPMT variant alleles associated with low enzyme activity in clinical laboratory samples in the United States and to compare those results with data obtained from two different ethnic groups. We identified a total of six different variant alleles for low TPMT activity in the 283 clinical laboratory samples studied. The most common variant was *3A; the second most frequent variant allele, *3C, contained only the nucleotide 719 polymorphism; and four other variant alleles were detected. TPMT*3A also appeared to be the most common variant allele in a Norwegian white population sample, but it was not found in a population sample of Korean children. However, *3C was present in samples from the Korean children, as was novel allele, *6. Characterization of variant alleles for low TPMT enzyme activity will help make it possible to assess the potential clinical utility of deoxyribonucleic acid-based diagnostic tests for determining TPMT genotype.

Clinical pharmacokinetics of phenylbutazone.

More than 25 years after phenylbutazone was introduced as a non-steroidal anti-inflammatory agent, basic knowledge is still accumulating on its pharmacokinetics in man. Phenylbutazone is almost completely absorbed after oral administration. A large fraction of the drug in plasma is bound to proteins, and the drug has a small volume of distribution. Phenylbutazone is eliminated by metabolism, only 1% being excreted unchanged in the urine. Approximately 10% of a single dose of phenylbutazone is excreted in bile as metabolites. About 60% of the urinary metabolites have been identified. A novel type of drug metabolite in man, the C-glucuronide, is formed by direct coupling of the pyrazolidine ring of phenylbutazone to glucuronic acid via a C-C bond. Phenylbutazone is oxidised in a phenyl ring or in the side chain to hydroxylated metabolites, which may undergo subsequent O-glucuronidation. After a single dose, C-glucuronidation seems to be the dominant reaction, while oxidation becomes increasingly important after repeated administration. Due to different pharmacokinetic properties of the metabolites, the C-glucuronides are detected in highest concentrations in the urine, while the pharmacologically active compounds oxyphenbutazone and gamma-hydroxyphenbutazone predominate in plasma. The biological (elimination) half-life of phenylbutazone in man is long, with a mean of about 70 hours, and exhibits large interindividual and intraindividual variation. The interindividual variation is largely due to genetic factors. The intraindividual variation is dose and time dependent. In an individual there may be several critical dose levels where a change in the elimination kinetics takes place. Since there is no correlation between the plasma level and the clinical or toxic effects of phenylbutazone, there is at present no need for routine monitoring of plasma concentrations of the drug.

Sex-dependent induction of alcohol dehydrogenase activity in rats.

The glycolethers 2-methoxyethanol (2-ME), 2-ethoxyethanol (2-EE), and 2-butoxyethanol are widely used organic solvents with teratogenic, spermatotoxic, and hematotoxic effects due to the respective alkoxyacetic acid metabolites formed via alcohol dehydrogenase (ADH). ADH displays sexually dimorphic activities in adult rats, and is probably at least in part under the control of testosterone. The aim of this study was to investigate whether induction of ADH is also sex-dependent. Ethanol, 2-ME, and 2-EE were tested as inducers of hepatic and gastric ADH in female, male, and castrated male rats. The activity of hepatic ADH was higher in female than in male rats, while the activity of gastric ADH was higher in male than in female rats. The activities of ADH increased with increasing chain length of the glycolethers and alcohols. Castration of male rats led to a female pattern of ADH activity, i.e. increased activity of hepatic ADH and decreased activity of gastric ADH. Ethanol had no inducing effect on hepatic ADH in either male or female rats. 2-ME and 2-EE caused an increase in the activity of hepatic ADH in male and castrated male rats only. The present data demonstrate a different expression of ADH isoenzymes in male and female rats, and a sex-dependent induction of ADH isoenzymes. The different possible regulatory mechanisms for the different ADH isoenzymes require further investigation.

Increased concentrations of methylated 6-mercaptopurine metabolites and 6-thioguanine nucleotides in human leukemic cells in vitro by methotrexate.

The effect of methotrexate (MTX) on 6-mercaptopurine (6-MP) metabolism was studied in four human leukemic cell lines in vitro. CCRF-CEM, WI-L2, TBJ, and HL-60 all expressed thiopurine methyltransferase (TPMT) activity. The cells were grown in horse serum-supplemented RPMI 1640 medium to which was added 4 microM of 6-MP or 4 microM of 6-MP and 20 nM of MTX. The presence of MTX resulted in a 2.1-, 1.7-, 2.4- and 8-fold increase in the concentrations of methylmercaptopurine ribonucleotides (MMPRP) in CEM, WI-L2, TBJ, and HL-60 cells, respectively (P < 0.0008). The concentrations of 6-thioguanine nucleotides (6 TGN) increased 1.9-, 1.4-, 2.4- and 1.9-fold in the same cell lines (P < 0.02). The four cell lines differed with respect to the effect of MTX on the consumption of 6-MP from the medium; CEM consumed more 6-MP and WI-L2 less 6-MP from media containing MTX than from media containing 6-MP only (P = 0.005 and 0.02, respectively). MTX did not affect the consumption of 6-MP by TBJ cells (P = 0.17). Media in which HL-60 cells had been grown did not contain detectable amounts of 6-MP at the end of the experiment. The simultaneous increase in methylated 6-MP metabolites and 6-TGN represents a possible explanation for the synergism of MTX and 6-MP; however, the clinical importance of increased MMPRP remains to be elucidated.

Differential cell cycle perturbation by transmethylation inhibitors.

Cell cycle distribution of HL-60 cells was studied by flow cytometry after incubation with the transmethylation inhibitors 3-deaza-(+/-)-aristeromycin (c3 Ari) and 3-deazaadenosine (c3 Ado). Cells were incubated with the drugs (25 microM) for two cell doublings in control cells (36 hr). The presence of c3 Ari caused a dose-dependent, reversible G2 + M arrest, whereas c3 Ado-treated cells accumulated in G0/G1. The G2 + M arrest was also found in NIH/3T3 cells incubated for 36 hr with 25 microM c3 Ari, but not in U937 and K562 cells. Possible mechanisms for the described effects of c3 Ari are discussed from the perspective that inhibition of S-adenosyl homocysteine hydrolase, and subsequent inhibition of transmethylation reactions, at present is the only known site of action of c3 Ari.

Cell death initiated by 3-deazaadenosine in HL-60 cells is apoptosis and is partially inhibited by homocysteine.

Cell death initiated by the adenosine analog 3-deazaadenosine (c3 Ado) was studied in human promyelocytic leukemia HL-60 cells. A rapid decrease in cell number was seen after 4-hr exposure to 50-100 microM c3 Ado. The dominating mode of cell death was apoptosis as demonstrated by condensation and fragmentation of the nucleus, formation of apoptotic bodies and endonucleolytic degradation of DNA. Four hour treatment with 100 microM c3 Ado resulted in a reduction of early S-phase cells, and appearance of cells with a lower DNA and protein content than that of the G1 population. Whereas 25 and 50 microM c3 Ado only initiated apoptosis in S-phase cells, 75 and 100 microM c3 Ado also initiated apoptosis in G1- and G2 + M-phase cells, suggesting different mechanisms for cell death at different concentrations. Apoptosis initiated by 100 microM c3 Ado was completely inhibited by 1 mM ZnCl2. Addition of homocysteine thiolactone (Hcy) partly inhibited cell death by c3 Ado. Light microscopic examination of cultures treated with 100 microM c3 Ado and 1 mM Hcy showed nuclear condensation and fragmentation consistent with the first stage in apoptosis, however, only a minor formation of apoptotic bodies took place in these cultures compared to that observed in cultures treated with 100 microM c3 Ado alone. The modifying action of Hcy on c3 Ado initiated apoptosis in HL-60 cells and this suggests that c3 Ado and 3-deazaadenosylhomocysteine (c3 AdoHcy) interact with different targets during initiation and progression of cell death in this cell line.

Interactions with the protein binding of 7-hydroxy-methotrexate in human serum in vitro.

7-Hydroxy-methotrexate (7-OH-MTX), the major extracellular methotrexate (MTX) metabolite, is 90-95% bound in human serum, with albumin (HSA) as the major binding protein. Reports of an interaction with concomitantly administered non-steroidal antiinflammatory drugs (NSAIDs) during MTX therapy led us to investigate whether these compounds could reduce the binding of 7-OH-MTX in vitro. Equilibrium dialysis experiments demonstrated that naproxen and indomethacin concentration dependently reduced the binding of 1 microM 7-OH-MTX. After ingestion of 1000 mg naproxen, per cent unbound 7-OH-MTX in sera from volunteers increased 2-3-fold in vitro, positively correlated to naproxen concentrations (P less than 0.00015). In addition, etacrynic acid, bilirubin, sulphamethizole and acetylsalicylic acid displaced 7-OH-MTX from its binding protein(s) in a competitive manner. The data suggest that 7-OH-MTX interacts with several exogenous and endogenous substances associated with HSA in human serum. Displacement of 7-OH-MTX from HSA may contribute to the interaction between NSAIDs and MTX.

Growth, differentiation and the beta-adrenergic signal system of HL-60 cells. Characterization in a medium with insulin as the only added protein.

The purpose of the present investigation was to define experimental conditions for studies on growth, differentiation and the beta-adrenergic signal system of HL-60 cells. The cell medium was completely devoid of added proteins and hormones other than insulin. The HL-60 cell was able to grow and differentiate in this medium. The spontaneous differentiation along the granulocytic pathway after 72 hr, as assessed by the Nitro Blue tetrazolium test, increased by 400% compared to the serum supplemented medium, but the response to 1 microM retinoic acid was equal in the two media. Induction of monocytic differentiation by 0.16 microM phorbol-13-acetate-12-myristate, as determined by surface adherence after 24 hr, was lower in the absence than in the presence of serum. cAMP levels were elevated in response to (-)-isoproterenol. The EC50 was 0.36 +/- 0.01 microM (mean +/- SE, N = 3). The beta-adrenergic ligand 3H-CGP 12177 was specifically bound to 1 single class of binding sites (Kd: 0.15 +/- 0.04 nM, Bmax: 2220 +/- 150, mean +/- SE, N = 3). These data are comparable to our previously reported findings in serum supplemented medium. The present data show that HL-60 cells are able to grow and differentiate in the absence of serum proteins and hormones other than insulin. Under the present experimental conditions, these cells possessed functional beta-adrenergic receptors.

The effect of vindesine on methotrexate hydroxylation in the rat.

The effect of vindesine (VDS) on methotrexate (MTX) disposition was studied in bile-drained rats administered VDS prior to [3H]MTX, and in isolated rat hepatocytes and rat liver homogenate concomitantly incubated with MTX and VDS at 37 degrees. In vivo, 7-hydroxylation was reduced by 0.65 mg/kg VDS. In VDS-treated animals, biliary recovery of the MTX dose (50 mg/kg) as 7-hydroxymethotrexate (7-OH-MTX) (1.75 +/- 0.2%, mean +/- SEM) was significantly reduced compared to controls (2.83 +/- 0.57%). In vitro, hydroxylation of MTX (10-200 microM) in hepatocytes was reduced by 14.3 and 66.4% (means) at 12.5 and 100 microM VDS, respectively. With increasing VDS concentrations up to 100 microM, a reduction in intracellular MTX accumulation could account for the decreased MTX hydroxylation. Experiments in a cell free system gave no evidence of inhibition of 7-OH-MTX formation by VDS. In vitro MTX transport studies demonstrated that VDS inhibited the hepatocellular influx of MTX, as (1) the accumulation of MTX corresponded inversely to increasing VDS concentrations and (2) the MTX efflux was not increased by VDS. The apparent Ki for VDS inhibition of MTX influx was 57 microM. We suggest that VDS, by reducing the 7-OH-MTX formation in liver cells, may have implications for combination chemotherapy regimens which include MTX.

Is there an association between an increase in c-myc RNA steady state levels and c-myc methylation in HL-60 cells treated with 3-deaza-(+/-)-aristeromycin, an indirect inhibitor of methylation?

Alteration in gene expression of the proto-oncogene c-myc in HL-60 cells is associated with differentiation of these cells. We have studied the steady state levels of c-myc transcripts, the levels of transmethylation metabolites S-adenosylmethionine and S-adenosyl-homocysteine and the methylation pattern of the c-myc gene after treatment of HL-60 cells with the transmethylation inhibitor and granulocytic inducer, 3-deaza-(+/-)-aristeromycin. A transient increase in c-myc RNA levels after 45 min of drug exposure was observed which was accompanied by changes in the ratio of transmethylation metabolites in both whole cells and nuclei. The changes in transmethylation metabolites in whole cells, although compatible with levels frequently associated with hypomethylation of cellular components, caused no changes in methylation of c-myc DNA sequences of the HL-60 cells as detected by HpaII or MspI digestion and Southern blotting.