Effects of formamidoxime on macromolecular synthesis in regenerating liver and hepatomas

Formamidoxime caused an inhibition of [³H]thymidine incorporation into DNA in regenerating liver and transplanted hepatomas of different growth rates when administered by i.p. injection to rats. A dose level of formamidoxime (500 mg/kg body weight) which caused at least a 75% inhibition of DNA synthesis in these tissues had little or no effect on the incorporation of [³H]orotate into total RNA. After administration of formamidoxime there was no significant effect on amino acid nitrogen concentration in the tissues. The incorporation of ³H-labeled amino acids into acid-soluble material, cytoplasmic proteins and acid-insoluble nuclear proteins were either unaffected or showed only small changes after treatment of rats with the drug. In regenerating rat liver and Morris hepatomas 7787 and 7777, formamidoxime caused an inhibition of incorporation of ³H-labeled amino acids into both lysine-rich and arginine-rich histones. In the host livers of rats bearing the transplanted hepatomas, histone synthesis was less affected. The data indicated that formamidoxime causes inhibitory effects which are similar in nature and extent to those previously shown for the structurally related compound, hydroxyurea, in the regenerating rat liver and demonstrated that these effects can also be observed in liver tumors.     

Actions of exogenous histones and other proteins on [3H]-thymidine incorporation into DNA of Novikoff hepatoma cells.

The effects of exogenous proteins on the incorporation of [3H]-thymidine into DNA was studied in Novikoff hepatoma ascites cells incubated in Eagle's minimal essential medium. A liver cytosol fraction (8 mg protein/ml) caused approximately 80% inhibition of isotope incorporation. The inhibitory activity of cytosol fractions from Morris hepatomas 9618A2, 5123C, and 20 were inversely related to their growth rate. Under conditions in which there appeared to be a density dependent inhibition of growth, a mean 10-20% stimulation of isotope incorporation was observed after addition of total calf thymus histones and individual fractions in the concentration range of 100-400 microgram/ml. In experiments with lower cell concentrations, a 60% or greater increase in [3H]-thymidine incorporation could be obtained with total calf thymus histone and with F1 and arginine-rich histones from rat liver. At concentrations of 1-2 mg/ml, histones inhibited DNA synthesis. Bovine serum albumin had little effect on DNA synthesis. Polylysine caused an 80-90% inhibition at a concentration of 1 mg/ml, but stimulatory effects were detected under certain conditions at 10 microgram/ml. The results suggest critical dependence on the ratio of cell and exogenous protein concentration in the action of proteins on DNA synthesis.     

Absolute rates of cholesterol synthesis in extrahepatic tissues measured with 3H-labeled water and 14C-labeled substrates.

This study was undertaken to develop techniques for measuring absolute rates of sterol synthesis in extrahepatic tissues in vitro and to estimate the magnitude of the errors inherent in the use of various 14C-labeled substrates for such measurements. Initial studies showed that significant errors were introduced when rates of synthesis were estimated using [3H]water since about 20 nmol of water were bound to each mg of tissue cholesterol isolated as the digitonide. This source of error could be eliminated by subtracting apparent incorporation rates obtained at 0 degrees C from those obtained at 37 degrees C or by regenerating and drying the free sterol. In a second set of experiments, the H/C incorporation ratio in cholesterol was determined in the liver by measuring the absolute rates of hydrogen and acetyl CoA flux into sterols. The ratio of 0.69 +/- 0.03 was found to be independent of the rate of hepatic cholesterol synthesis, the rate of hepatic acetyl CoA generation, or the source of the acetyl CoA. In a third set of studies, rates of incorporation of [3H]water or 14C-labeled acetate, octanoate, and glucose into digitonin-precipitable sterols were simultaneously measured in nine different extrahepatic tissues. Assuming that the H/C ratio measured in the liver also applied to these tissues, the [3H]water incorporation rates were multipled by the reciprocal of the H/C ratio to give the absolute rates of sterol synthesis in each tissue. When these were compared to the incorporation rates determined with the 14C-labeled substrates the magnitude of the errors in the rates of sterol synthesis obtained with these substrates in each tissue could be assessed. Only [14C]octanoate gave synthesis rates approaching 100% of those obtained with [3H]water and this occurred only in the intestine and kidney; in the other extrahepatic tissues this substrate gave rates of 6--66+ of the absolute rates. Rates of [14C]acetate incorporation in sterols varied from 4 to 62% of the [3H]water incorporation rates while those obtained with [14C]glucose were only 2--88% of the true rates. These studies document the large and highly variable errors inherent in estimating rates of sterol synthesis in extrahepatic tissues using 14C-labeled substrates under in vitro conditions.     

Effects of N-methyl-N-Nitrosourethane on DNA synthesis in the guinea pig pancreas

The effects of N-methyl-N-nitrosourethane (NMUT) on pancreatic DNA synthesis were investigated at sequential intervals following gavage of Hartley guinea pigs with a single dose of 30 mg/kg. There was a highly significant stimulation of DNA synthesis, as evidenced by increased incorporation of [³H] methyl-thymidine ([³H] TdR), throughout the whole pancreas and particularly in the duodenal segment, at 4 h following NMUT administration, thereafter, DNA synthesis declined sharply up to 24 h, and then recovered gradually to control levels from 24–96 h. DNA synthesis stimulated by NMUT was suppressed by hydroxyurea (HU), and hence is likely to represent replicative, rather than repair, synthesis.     

Unscheduled DNA synthesis in rat pleural mesothelial cells treated with mineral fibres

Unscheduled DNA synthesis (UDS) was studied in confluent rat pleural mesothelial cells (RPMCs) arrested in G0/G1 with hydroxyurea (HU) and treated with various fibre types, i.e., chrysotile, crocidolite or attapulgite. In addition, the effects of UV light and of benzo[a]pyrene were determined as references. Using autoradiography after [3H]thymidine incorporation ([3H]dThd), RPMCs treated with 4 micrograms/cm2 of chrysotile fibres exhibited a low but significant enhancement of net grains compared to untreated cells. Treatment with higher doses of chrysotile was not possible because of the impairment of microscopic observation due to the presence of the fibres. Using liquid scintillation counting, RPMCs treated with chrysotile or crocidolite showed a significant dose-dependent increase in [3H]dThd incorporation compared to untreated cells. In contrast, attapulgite did not enhance [3H]dThd incorporation compared to untreated cells. Treatment of RPMCs with 1, 2 or 4 micrograms/ml of benzo[a]pyrene resulted in a significant increase in [3H]dThd incorporation. In order to discount a possible role of S cells in the augmentation of [3H]dThd incorporation, despite the presence of 5 mM HU, S cells were counted by autoradiography. Results indicated that the percentage of S cells was similar in asbestos-treated and untreated cultures. Stimulation of the S phase also seems unlikely because treatment of RPMCs with asbestos fibres in the absence of HU resulted in a reduction of [3H]dThd incorporation attributed to an impairment of the S phase by the fibres. 1-4 micrograms/ml benzo[a]pyrene or 10-50 J/m2 UV light resulted in an approximate doubling of [3H]dThd incorporation. The effects of inhibitors of DNA repair were determined in chrysotile-treated RPMCs. [3H]dThd incorporation was inhibited by cytosine arabinoside and nalidixic acid. These results show that asbestos produces UDS in RPMCs.     

Inhibitory effect of hydroxyurea on [3-H]leucine incorporation in human peripheral lymphocytes.

Hydroxyurea (HU), generally considered to be a specific inhibiter of DNA synthesis, has an inhibitory effect on the incorporation of TCA-precipitable [3-H]leucine in peripheral lymphocytes. This action is not secondary to the inhibition of DNA synthesis since incorporation of [3-H]leucine is unaffected when DNA synthesis is inhibited by 5-fluorodeoxyuridine (FUdR); it does not appear to be directly related to inhibition of RNA synthesis; and it is not mediated at the level of translation since HU has no effect on protein synthesis in rabbit reticulocytes. The relevance of these findings to the use of HU as a DNA inhibitor is discussed.     

Incorporation of 2-fluorohistidine in murine protein in vivo.

The tissue distribution and time course of incorporation into acid insoluble (bound) and acid soluble (free) fractions of [3H]2-fluorohistidine is compared to that of U[14C]Histidine in mouse tissues in vivo. The cycloheximide-sensitive incorporation of 2-FHis is between 9 and 17 percent of that of His. Unlike [14C]His a major fraction, approximately 90% at 72 hrs, of isotope derived from [3H]2-FHis remains in tissues for a prolonged period in an acid soluble form. The excretion of isotope derived from [14C]His (T1/2 = 5 hr) is more rapid than from [3H]2-FHis (T1/2 = 11.4 hrs). 2-FHis, at doses from 100 to 250 mg/kg produce a reversible inhibition of growth in mice.     

Inhibition of nucleic acid synthesis in Ehrlich tumor cells by periodate-oxidized adenosine and adenylic acid

Periodate-oxidized adenosine and AMP were inhibitory to both RNA and DNA synthesis in Ehrlich tumor cells in culture. With periodate-oxidized adenosine, the inhibition of RNA synthesis paralleled the inhibition of DNA synthesis. Periodate-oxidized AMP, however, was more inhibitory to DNA synthesis than to RNA synthesis. With both compounds, there was a decrease in the conversion of [¹⁴C]cytidine nucleotides to [¹⁴C]deoxycytidine nucleotides in the acid-soluble pool. The borohy-dride-reduced trialcohol derivative of the periodate-oxidized adenosine compound was not inhibitory to DNA or RNA synthesis in the tumor cells. The incorporation of [³H]uridine into 28S and 18S ribosomal RNA was inhibited by both periodate-oxidized adenosine and AMP, but the incorporation of [³H]uridine in 45S, 5S, and 4S RNA was essentially unaffected by these compounds. Periodate-oxidized adenosine inhibited Ehrlich tumor cell growth in vivo.     

Comparison of the continuous rat hepatoma cell line 2sFou with primary rat hepatocyte cultures for the induction of DNA repair synthesis by nitrosamines, benzo[a]pyrene and hydroxyurea

We have examined the suitability of the continuous rat hepatoma cell line 2sFou for testing the genotoxicity of chemicals in comparison with that of primary rat hepatocyte cultures (HPC). The capacity of the cells for metabolic activation was assessed by measuring induction of DNA-repair synthesis and inhibition of replicative DNA synthesis by the test compounds dimethylnitrosamine (DMN), diethylnitrosamine (DEN), hydroxyurea (HU) and benzo[a]pyrene (BaP), which are substrates for major hepatic and extrahepatic forms of cytochrome P-450 dependent monooxygenases. The cellular capacity for DNA-repair synthesis was assessed using UV-light as a DNA-damaging agent. Repair-specific incorporation of [3H]deoxycytidine (3H-dCyd) caused by UV-light was higher in 2sFou cells than in HPC. In contrast, background repair incorporation of 3H-dCyd in 2sFou cells was only 1/3 that found in HPC. All the test agents induced DNA repair and inhibited DNA synthesis in both 2sFou cells and HPC. The two nitrosamines were more effective in HPC than in 2sFou cells. HU and BaP affected DNA repair and DNA synthesis in the two cell systems at a similar range of concentrations. In general, DNA repair in the 2sFou cells increased near linearly with the concentrations of the test compounds. The data indicate that 2sFou cells are capable of activating hepatotropic pro-mutagens/carcinogens such as dialkylnitrosamines, and are sensitive indicators of DNA damage. In contrast, BaP, a non-hepatotoxic compound, caused only little DNA repair in these cells. Thus, continuously growing cells, such as 2sFou, show a qualitatively similar response to genotoxic chemicals as HPC and offer a potential alternative to HPC for genotoxicity testing.     

Influence of chlorambucil, a bifunctional alkylating agent, on the histone variant biosynthesis of HEp-2 cells

The effect of chlorambucil on the synthesis of histone variants of a cancer cell line HEp-2 is analysed and compared to that of nontreated and hydroxyurea treated cells. Cell proteins were labelled with [14C]lysine and [14C]arginine and histone variants resolved by one- or two-dimensional electrophoresis. Chlorambucil shows no significant decrease in total protein synthesis but shows a significant decrease in histone biosynthesis. It does not selectively inhibit the synthesis of the S-phase variants, i.e., H2A.1, H2A.2, H3.2 or the G1/G2 phase (basal) histone variants, i.e., H2A.Z, H2A.X and H3.3. On the contrary, hydroxyurea treated cells, which also show no significant decrease in amino acid incorporation into total cellular protein but do exhibit a significant inhibition of histone biosynthesis, show a selective inhibition of the synthesis of S-phase variants, but have no effect on the synthesis of basal histone variants. On the basis of histone variants being synthesized in the presence of chlorambucil, it is shown that although chlorambucil shows a specificity for histone synthesis inhibition it has a general action over the whole variant complement and is not coupled to S-phase synthesis in a way typical for DNA synthesis inhibiting drugs.     

Cell-Cycle-Dependent and -Independent Damage to Rat Haemopoiesis By Hydroxyurea

The generally accepted cell-killing effect of hydroxyurea (HU) on S-phase cells, as well as its potential to arrest cells at the G₁/S boundary, hardly explain its benefit for application in human chronic myelogenous leukaemia. Studies were therefore performed in rat haemopoiesis in order to quantify the cell-killing effect on various phases of the cell cycle. For this purpose, the [³H]thymidine ([³H]TdR) labelling index and the specific activity of [³H]TdR in the DNA-synthesizing fraction of cells were determined after a non-cytoreductive dose of 25 mg/kg HU, as well as a medium cytoreductive dose of 100 mg/kg. Furthermore, flow cytometric DNA histograms and absolute as well as differential cell counts of femoral bone marrow were performed after 100 mg/kg HU. The results indicate a predominant cell kill in G₁ encompassing almost all 2c cells in the proliferative pool, while the S-phase fraction is not even reduced to half its initial value. the specific activity of [³H]TdR in cells synthesizing DNA, as well as the labelling index after HU show an initial dip and a tendency to recovery, as has been observed in many other cell systems. Instead of a complete restoration, however, there is a second depression of these parameters lasting for at least one cell cycle. the results are interpreted as a partly cell-cycle-dependent and partly independent action of HU in this cell system. the independent component may be attributed to the repeatedly described direct interference of HU with DNA. In rat haemopoiesis, therefore, this direct effect of HU on the DNA strands appears to be much more pronounced than in cell-culture systems and other mammalian tissues. In view of these findings, some caution should be taken in using HU for the determination of the S-phase fraction by way of a suicide experiment.     

In vitro DNA and RNA synthesis by human platelets.

In vitro incorporation of [Me-3H] thymidine and [5-3H] uridine into human platelets was demonstrated. Thymidine incorporation was inhibited by three specific inhibitors of DNA synthesis: hydroxyurea, cytosine arabinoside and daunomycin. The effect was dose-dependent. Uridine uptake by platelets was found to be inhibited by specific inhibitors of RNA synthesis such as actinomycin D, rifampicin and vincristine, the effect of actinomycin D being dose dependent. The drug also led to a time-dependent inhibition of protein synthesis when preincubated with platelets. The platelet RNA profile on polyacrylamide gel was demonstrated to be similar to that of embryonic mouse erythroblast RNA. Synthesis of all three fractions, 28 S, 18 S and 4 S, was inhibited by actinomycin D. These findings show that human platelets are capable of DNA and RNA synthesis, and that these activities play a role in controlling protein synthesis in these cells. Detectable amounts of DNA have been found in whole human platelets, and in isolated mitochondria derived from these cells. Isolated platelet mitochondria incorporated [3H] thymidine and [3H] uridine into their macromolecules. These activities were inhibited by daunomycin and by both rifampicin and actinomycin D, respectively. These results support the assumption that DNA and RNA synthesis found in intact cell preparations takes place most probably in platelet mitochondria.     

DNA repair synthesis in guinea pig pancreatic slices following in vitro exposure to N-nitrosomethylurethane.

The incorporation of thymidine into DNA in the presence of hydroxyurea (HU) by guinea pig pancreatic slices following exposure to N-nitrosomethylurethane (NMUT) was used to follow DNA repair synthesis. HU was used to suppress normal replicative DNA synthesis. Slices from the duodenal segment of the pancreas were exposed for periods of 15 to 90 min to NMUT at concentrations of 2 to 20 mM, then incubated in tritiated thymidine ([H3]-TdR) free of carcinogen, and radioactivity in DNA was determined. NMUT induced a a dose- and time-dependent increase in HU-insensitive thymidine incorporation. This stimulated incorporation, which could be attributed to repair synthesis, occurred immediately following the treatment and was largely complete within 3 h.     

Inhibition of thymidylate synthase by hydroxyurea in rapidly proliferating P815 mastocytoma cells

The incorporation of [¹⁴C]deoxycytidine, [³H]deoxyuridine, and [³H]thymidine, respectively into pyrimidine bases of DNA has been measured in rapidly proliferating P815 mouse mastocytoma cells in the presence of hydroxyurea. The incorporation of [¹⁴C]deoxycytidine-derived radioactivity into DNA cytosines is increased when compared to the incorporation into DNA thymines. The [³H]deoxyuridine-derived radioactivity is incorporated solely into DNA thymines and this incorporation is inhibited by hydroxyurea in a dose-dependent manner. This suggests an inhibitory effect of hydroxyurea on the thymidylate synthase which was proved in experiments in which the conversion of deoxyuridine monophosphate into deoxythymidine monophosphate catalysed by a crude enzyme preparation from P815 cells was inhibited in the presence of hydroxyurea. Enzymatic DNA methylation as measured by the conversion of incorporated [¹⁴C]deoxycytidine into 5-methylcytosines was not affected by hydroxyurea.     

Selective inhibition of proteoglycan and hyaluronate synthesis in chondrocyte cultures by cyclofenil diphenol, a non-steroidal weak oestrogen.

Cyclofenil diphenol, a weak non-steroidal oestrogen, binds to albumin. In the presence of concentrations of albumin just sufficient to keep cyclofenil diphenol in solution, the compound inhibited the synthesis of [35S]proteoglycans, [3H]glycoproteins, [3H]hyaluronate and [3H]proteins in primary cultures of chondrocytes from the Swarm rat chondrosarcoma in a dose-dependent manner. When excess albumin was present, conditions were found (90 micrograms of cyclofenil diphenol and 4 mg of albumin per ml of culture medium) which completely inhibited [35S]proteoglycan and [3H]hyaluronate synthesis but had little effect on [3H]protein or [3H]glycoprotein synthesis. The time of onset of inhibition of [35S]proteoglycan synthesis by cyclofenil diphenol was very rapid (t1/2 less than 25 min) and incompatible with an action mediated through suppression of proteoglycan core protein synthesis. Cyclofenil diphenol inhibited the synthesis of [35S]chondroitin sulphate chains onto p-nitrophenyl beta-D-xyloside in the cultures. Cyclofenil diphenol had little effect on the secretion from chondrocytes of [35S]proteoglycans synthesized immediately prior to treatment. Chondrocyte cultures treated with cyclofenil diphenol recovered their biosynthetic activities almost completely within 3 h of removing the compound from the culture medium. Cyclofenil diphenol had a similar inhibitory action on the synthesis of [35S]proteoglycans in secondary cultures of human dermal fibroblasts from both normal subjects and patients with systemic sclerosis. It is proposed that cyclofenil diphenol inhibits the synthesis of [35S]proteoglycans by interfering with the formation of the glycosaminoglycan side chains of these molecules in the Golgi apparatus of cells. The action may be due to disturbance of Golgi membrane organization by the compound.     

Dose-dependent cytokinetic changes following 1-beta-D-arabinofuranosylcytosine and hydroxyurea in L1210 and S-180 in vivo.

DNA synthesis inhibition and recovery in L1210 and S-180 ascites tumors following 1-beta-D-arabinofuranosylcytosine (Ara-C) and hydroxyurea (HU) were measured autoradiographically as a basis for optimizing drug schedules. Tumor bearing mice, 10(6) cells day 0, were treated on day 4 with 20, 200 or 2000 mg/kg Ara-C or 50, 300 or 1800 mg/kg HU. At various intervals following drug, [3H]thymidine was administered i.p. and mice were killed 1 hr later. Tumor cells were analyzed for labeling index (LI) and grain count (GC) to determine the percentage of cells in S phase and the distribution of DNA synthesis rates among the labeled cells, respectively. Following each dose of HU, DNA synthesis was inhibited completely. Recovery of LI was rapid and approached control values by 6 hr. Following each dose of Ara-C, DNA synthesis was inhibited completely for at least 6 hr. Recovery of LI was first noted 6 hr following 20 mg/kg Ara-C and 9 hr following 200 mg/kg. Following both doses the LI reached 100% of the control value by 26 hr. GC analysis indicated that following Ara-C treatment, DNA synthesis was reinitiated first with cells with low GC from 6 to 12 hr followed by cells with increasing GC from 12 to 20 hr. The labeling intensity reached control values by 20 hr and an 'overshoot' occurred by 26 hr. These data suggest that the recovery of DNA synthesis rate is a gradual process. Survival data for mice receiving two doses of Ara-C indicated that the optimal interval for retreatment following the lower dose of Ara-C occurred by 6 hr as compared to 12--16 hr for the higher dose. These times coincided in both instances with recovery of LI to 33--50% of control values. Early recovery of LI may be the best method currently available for estimating the optimal time for retreatment with an S phase specific drug.     

Action of Miracil D and related compounds on histone synthesis and phosphorylation in regenerating liver

The effect of Miracil D and hycanthone on ³H-amino acid incorporation into histones was studied under conditions known to cause a greater than 90% inhibition of thymidine incorporation into DNA of regenerating rat liver. A dose level of 50 mg of either drug per kg body weight administered 8 h after partial hepatectomy caused an approximate 50% inhibition of amino acid incorporation into fl, f2b and combined f2a plus f3 histone in 24-h regenerating liver. There was little or no effect on amino acid nitrogen concentration or incorporation of ³H-amino acid into the acid-soluble fraction, cytoplasmic proteins or acid-insoluble nuclear proteins. Under the same conditions, Miracil D caused a 65% inhibition of ³²P incorporation into lysirierich f1 histone whereas a structurally related compound, GE-99, did not have a significant inhibitory effect on this parameter nor on [³H]thymidine incorporation into DNA. Temporal studies with hycanthone revealed a suppression of the increased phosphorylation of fl histone in regenerating rat liver without influencing the phosphorylation of other histones. The data support the concept of coordinated control of DNA synthesis and phosphorylation of fl histone.     

DNA nucleotide excision-repair synthesis is independent of perturbations of deoxynucleoside triphosphate pool size

We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of [3H]TTP from [3H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the [3H]TTP pool was formed within 30 min of the addition of [3H]thymidine and remained relatively constant for the next 6 h. Addition of 2-10 mM hydroxyurea (HU) caused a gradual 2-4-fold increase in the [3H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the [3H]TTP pool size in cells exposed to 20 J/m2 and unirradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8-10-fold reduction in the size of the [3H]TTP pool relative to the initial studies. In the UV excision-repair studies the presence of hydroxyurea did not alter the specific activity of [3H] thymidine 5'-monophosphate incorporated into parental DNA due to repair replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair synthesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the [3H]TTP pool.     

Differential and kinetic effects of cell cycle inhibitors on neoplastic and primary astrocytes

Alterations in cell cycle regulation underlie the unrestricted growth of neoplastic astrocytes. Chemotherapeutic interventions of gliomas have poor prognostic outcomes due to drug resistance and drug toxicity. Here, we examined the in vitro growth kinetics of C6 glioma (C6G) cells and primary astrocytes and their responses to 2 phase-specific inhibitors, lovastatin and hydroxyurea. C6G cells demonstrated a shorter G₁ phase and an earlier peak of DNA synthesis in S phase than primary astrocytes. As C6G cells and primary astrocytes re-entered the cell cycle in the presence of lovastatin or hydroxyurea, they exhibited different sensitivities to the inhibitory effects of these agents, as measured by [³H]-thymidine incorporation. Compared to primary astrocytes, C6G cells were more sensitive to lovastatin, but less sensitive to hydroxyurea. Studies using 2 different paradigms of exposure uncovered dramatic differences in the kinetics of DNA synthesis inhibition by these 2 agents in C6G cells and primary astrocytes. One notable difference was the ability of C6G cells to more easily recover from the inhibitory effects of hydroxyurea following short exposure. Our results provide insight into C6 glioma drug resistance as well as the inhibitory effects of these 2 phase-specific inhibitors and their chemotherapeutic potential.     

Disturbances in DNA precursor metabolism associated with exposure to an inhibitor of poly(ADP-ribose) synthetase

3-Aminobenzamide (3AB) is widely used as an inhibitor of poly(ADP-ribose) synthetase to study the effect of protein ribosylation on various cellular processes, but the specificity of its inhibition has not been demonstrated. We found that 3AB has a wide range of effects on DNA precursor metabolism, as determined by high-performance liquid chromatographic separation of deoxynucleosides derived from enzymatic digestion of cellular DNA. 3AB (10-20 mM) significantly reduced cell growth in human lymphoblastoid cells. Furthermore, the incorporation of [3H]deoxycytidine into DNA was significantly enhanced relative to incorporation of [3H]deoxythymidine, [3H]deoxyguanosine, and [3H]deoxyadenosine. Incorporation of fragments of [3H]glucose into the pyrimidine fraction of DNA was significantly inhibited relative to incorporation into the purine fraction. At only 1 mM, 3AB had a major inhibitory effect on the incorporation of the methyl group from [3H]methionine into deoxyguanosine, deoxyadenosine, and deoxycytidine, with 50% inhibition into deoxyguanosine and deoxyadenosine and 90% inhibition into deoxycytidine. The specificity of 3AB inhibition to poly(ADP-ribose) synthetase is therefore doubtful in view of this variety of metabolic effects, involving pyrimidine synthesis and de novo synthesis via the one-carbon pool.