Elevation of dCTP pools in xeroderma pigmentosum variant human fibroblasts alters the effects of DNA repair arrest by arabinofuranosyl cytosine

DNA excision repair inhibition by arabinofuranosyl cytosine (ara-C) or by ara-C/hydroxyurea (HU) was measured in log phase and confluent cultures of normal and xeroderma pigmentosium (XP)-variant human fibroblasts following insult by ultraviolet (UV) light (20 J/m²). Repair inhibition was determined by measuring the accumulation of DNA single-strand breaks/10⁸ daltons following cell culture exposure to ara-C or ara-C/HU in a series of 3 hr. pulses up ro 24 hr. after UV insult. Both normal and XP-variant derived cells showed a wide range of sensitivity to ara-C in log phase cells (0.2–9.4 breaks/10⁸ daltons DNA), although strand break accumulation was constant for each specific cell line. The same cells were more sensitive to ara-C/HU with a 2–14 fold increase in DNA strand breaks depending upon the cell line assayed. In confluent cultures of normal cells, maximum sensitivity to ara-C and ara-C/HU was achieved with similar levels of repair inhibition observed (16.1 and 16.5 breaks/10⁸ daltons, respectively). The same level of repair inhibition was observed in confulent XP-variants receiving ara-C/HU, but was reduced by 62–68% in cells treated with ara-C alone. Ara-C repair arrest was more rapidly reversed by competing concentrations of exogenous deoxycytidine (dCyd) in XP-variant compared to normal cells, especially in confluent cell cultures. In ara-C/HU treated cells, the level of dCyd reversal was reduced in the XP-variant when compared to cells exposed to ara-C alone. However, the same addition of HU had relatively little effect on dCyd reversal in normal cells. The measurements of dNTP levels indicate an elevated level of intracellular deoxycytosine triphosphate in XP-variant vs normal cells. The implications of these results are discussed as they relate to possible excision repair anomalies in the XP-variant.Abbreviations ara-C arabinofuranosul cytosine - dCTP deoxycytosine triphosphate - dCyd deoxycytidine - dNTP deoxynucleoside triphosphate - dT thymidine - HU hydroxyurea - XP xeroderma pigmentosium This research was sponsored jointly by the National Cancer Institute under Interagency Agreement #40-5-63, and the Office of Health and Environment Research, U. S. Department of Energy, under Contract W-7405-eng-26 with the Union Carbide Corporation.     

Application of arabinofuranosyl cytosine in the kinetic analysis and quantitation of DNA repair in human cells after ultraviolet irradiation.

We have developed a technique whereby 3-h pulses of arabinofuranosyl cytosine (ara-C) and hydroxyurea (HU) are used to analyze the kinetics of repair with time after ultraviolet irradiation in human fibroblasts. We demonstrate that this technique offers a significant improvement over existing repair assays in its ability to visualize between 57 and 100% of all sites undergoing repair in a given period of time. In addition, kinetic analyses of repair are more easily made and yield more information than techniques such as repair replication or unscheduled DNA synthesis. We have also examined the nature of the inhibition event by ara-C and have determined that repair breaks accumulate in the presence of ara-C and HU only up to a certain time beyond which no further breaks appear. The time needed to reach this saturation point depends on the number of sites undergoing repair during the treatment time. This observation is discussed with respect to a possible mechanism of excision repair inhibition by ara-C and HU.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VI. The correlation between UV-induced DNA lesions and chromosomal aberrations, and their modulations with inhibitors of DNA repair synthesis

The role of UV-induced DNA lesions and their repair in the formation of chromosomal aberrations in the xrs mutant cell lines xrs 5 and xrs 6 and their wild-type counterpart, CHO-K1 cells, were studied. The extent of induction of DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) due to UV irradiation in the presence or absence of 1-beta-D-arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) was determined using the alkaline and neutral elution methods. Results of these experiments were compared with the frequencies of induced chromosomal aberrations in UV-irradiated G1 cells treated under similar conditions. Xrs 6 cells showed a defect in their ability to perform the incision step of nucleotide repair after UV irradiation. Accumulation of breaks 2 h after UV irradiation in xrs 6 cells in the presence of HU and ara-C remained at the level of incision breaks estimated after 20 min, which was about 35% of that found in wild-type CHO-K1 cells. In UV-irradiated CHO-K1 and xrs 5 cells, more incision breaks were present after 2 h compared with 20 min post-treatment with ara-C, a further increase was evident when HU was added to the combined treatment. The level of incision breaks induced under these conditions in xrs 5 was about 80% of that observed in CHO-K1 cells. UV irradiation itself did not induce any detectable DNA strand breaks. Accumulation of SSBs in UV-irradiated cells post-treated with ara-C and HU coincides with the increase in the frequency of chromosomal aberrations. These data suggest that accumulated SSBs when converted to DSBs in G1 give rise to chromosome-type aberrations, whereas strand breaks persisting until S-phase result in chromatid-type aberrations. Xrs 6 appeared to be the first ionizing-radiation-sensitive mutant with a partial defect in the incision step of DNA repair of UV-induced damage.     

Effect of arabinosyl cytosine on the level of DNA polymerase and thymidine kinase activity in PHA-stimulated human tonsillar lymphocytes

Summary The effect of arabinosyl cytosine (ara-C) was studied on the uptake, phosphorylation and incorporation of ³H-thymidine in human tonsillar lymphocyte cultures is described along with its effect on the level of DNA polymerase and thymidine kinase activities induced by phytohaemagglutinin (PHA). Freshly isolated tonsillar lymphocytes are stimulated cells with a remarkably high activity of DNA polymerase a and thymidine kinase. During in vitro culture, these stimulated cells are transformed to the resting state with low DNA polymerase and thymidine kinase activity. However, a new DNA synthesising cycle can be induced by PHA with maximum at 48 h.10^–6 M ara-C inhibited the incorporation of ³H-thymidine by 90–95%. This inhibition may be reversed by rinsing the cells. The inhibition of the transport of ³H-thymidine seems to be only a consequence of the inhibitory effect of ara-C on the DNA polymerisation reaction, because at 10 °C, where DNA synthesis was arrested, ara-C does not influence the uptake and the phosphorylation of ³H-thymidine.Ara-C (10^–6 M) abolished also the PHA induced elevation of DNA polymerase a and thymidine kinase activities without influencing protein synthesis of the cell. This supports a coordinated regulation mechanism between DNA synthesis and the synthesis of enzymes involved in DNA replication.     

Cytosine arabinoside is a potent clastogen and does not affect the repair of X-ray-induced chromosome fragments in unstimulated human lymphocytes

The metabolic inhibitor of DNA synthesis cytosine arabinoside (ara-C) is known to induce chromosome aberrations in human lymphocytes. It has been recently argued, however, that there is no unequivocal evidence that ara-C can damage chromosomes directly. Therefore, the effect of ara-C on unstimulated human lymphocytes was examined directly by means of the premature chromosome condensation technique. In about 50% of the cells, ara-C effectively induced chromosome fragments, which did not show rejoining even after the chemical was washed out. These results suggest that a possible selection against damaged cells in their progress to mitosis could result in the low yields of ara-C-induced chromosome aberrations reported in the literature. The effect of ara-C on the repair of radiation-induced chromosome aberrations was also examined. Ara-C did not affect the rejoining of the chromosome fragments induced in unstimulated human lymphocytes by 6 Gy of X-rays.     

High frequencies of chromatid aberrations produced during G2 in human lymphocytes by very low doses (0.025-0.4 Gy) of X-rays in combination with inhibitors of DNA synthesis

Whole-blood cultures of human lymphocytes were exposed in the G2-phase (3.5 h before harvesting) to various doses of X-rays and post-treated for 3 h with inhibitors of DNA synthesis. The inhibitors used were 2'-deoxyadenosine (dAdo), hydroxyurea (HU) and 1-beta-D-arabinofuranosylcytosine (ara-C). To prevent deamination of dAdo by adenosine deaminase (ADA), the dAdo treatments were carried out in the presence of the ADA inhibitor coformycin. HU and Ara-C were used either alone or in combination. After the 3-h inhibitor treatments, the cultures were harvested and slides prepared and analyzed for chromatid aberrations in metaphase. When the inhibitors were used at concentrations high enough to cause marked chromosome damage by themselves, very low doses of X-rays (0.025-0.2 Gy) were sufficient to produce a dramatic increase in the frequency of chromatid aberrations. High frequencies of chromatid aberrations were also obtained when cultures that had received moderate doses of X-rays (0.4-0.8 Gy) were post-treated with low inhibitor concentrations that produce no or only a few aberrations by themselves.     

Effects of inhibitors of DNA synthesis on spontaneous and ultraviolet light-induced sister-chromatid exchanges in Chinese hamster cells

Effects of inhibitors of DNA synthesis on spontaneous and ultraviolet light (UV)-induced sister-chromatid exchanges (SCE) were examined in a Chinese hamster cell line, V79 B-1. The inhibitors used were hydroxyurea (HU), 1-beta-D-arabinofuranosylcytosine (ara-C), aphidicolin (APC), 2',3'-dideoxythymidine triphosphate (ddTTP), neocarzinostatin (NCS), novobiocin (NB) and cycloheximide (CHX). HU, ara-C, and APC increased spontaneous SCE frequency, and had a synergistic effect on UV-induced SCE frequency. DdTTP, NCS and NB failed to show any statistically significant effect on either spontaneous or UV-induced SCE frequencies, though NCS and NB did slightly increase both spontaneous and UV-induced SCE frequencies. On the contrary, CHX decreased spontaneous SCE frequency, and more drastically, also UV-induced SCE frequency. These results are interpreted with respect to the replicating fork of DNA, a structure postulated to be involved in the formation of spontaneous and UV-induced SCE. A new model for SCE formation is proposed.     

Induction of DNA ligase I by 1-beta-D-arabinosylcytosine and aphidicolin in MiaPaCa human pancreatic cancer cells

Exposure of MiaPaCa cells to 1-beta-D-arabinosylcytosine (ara-C) resulted in an increase in DNA ligase levels up to threefold compared to that in the untreated control cells, despite significant growth inhibition. Increased levels of DNA ligase I protein appear to correlate with the appearance of increased mRNA levels. The [(3)H]thymidine incorporation experiment and the biochemical assay of total polymerase activity revealed that an increase in DNA ligase I levels after treatment with ara-C was not accompanied by an increase of DNA synthesis or an increased presence of DNA polymerase activity inside cells. When cells resumed DNA synthesis after drug treatment, DNA ligase I levels began to drop, indicating that increased DNA ligase I is not required for DNA synthesis. An increase in DNA ligase I was also observed in cells treated with aphidicolin, another inhibitor of DNA synthesis that inhibits DNA polymerases without incorporating itself into DNA, indicating that an increase in DNA ligase I levels could be caused by the arrest of DNA replication by these agents. Interestingly, caffeine, which is a well-known inhibitor of DNA damage checkpoint kinases, abrogated the increase in DNA ligase I in MiaPaCa cells treated with ara-C and aphidicolin, suggesting that caffeine-sensitive kinases might be important mediators in the pathway leading to the increase in DNA ligase I levels in response to anticancer drugs, including ara-C and aphidicolin. We propose that ara-C and aphidicolin induce damage to the DNA strand by arresting DNA replication forks and subsequently increase DNA ligase I levels to facilitate repair of DNA damage.     

Secondary activities of diverse inhibitors potentiate the response of hamster embryo cultures to a mitotic stimulus

The reversal of the density dependent inhibition of replication (DDIR) of Syrian hamster embryo cultures by fresh medium containg 30-50% fetal bovine serum was poorly synchronized. There were two waves of DNA synthesis eight to nine hours apart, which by examination of autoradiograms of cultures pulse-labeled with 3H-thymidine, were found to involve 55% and 45% of the cell population. The second wave was found to be due to a subpopulation of epithelioid cells set in the predominantly fibroblastic cell cultures. Pretreatment of the DDIR cultures with hydroxyurea (HU) or arabinosyl cytosine (ara-C), followed by serum stimulation in the absence of the drugs, led to an enhancement of the synchrony. The effect increased with lengthening of the contact with antimetabolites, to a maximum after 20 hours' pre-exposure, and was in part due to the shortening of the GI phase of the epithelioid elements, and in part to increasing the synchrony of the fibroblastic cells. The resulting synchrony involved some 95% of the cells in simultaneous DNA synthesis after a median Gl period of 12 hours. The effect had no relationship to the role of HU and ara-C as specific inhibitors of DNA synthesis since the cultures were mitotically quiescent, and a similar enhanced response could be induced in DDIR cultures by prestimulation exposure lasting only two hours to cycloheximide (cyx), an inhibitor of protein synthesis. Pre-exposure of DDIR cultures to actinomycin D did not potentiate the cell response. A survey of the known secondary inhibitions caused by these three antimetabolites suggests that they all may cause deficiencies in the glycolipid or glycoprotein moieties of the cell surface. These observation provide a use ful, simple means of improving synchrony in these systems and may prove to be a useful probe for investigating the role of the cell surface in regulating cell replication.     

Deoxycytidine is salvaged not only into DNA but also into phospholipid precursors. II. Ara-C does not inhibit the later process in lymphoid cells

dCTP formed from exogenous deoxycytidine via the salvage pathways was previously shown to serve deoxyliponucleotide synthesis in lymphocytes (Spasokukotskaja et al, Biochem. Biophys. Res. Commun. (1988) 155, 923-929) and now in lymphoma cells. After treatment with 1-beta-D-arabino-furanosylcytosine (ara-C), much more araCTP as well as araCDP-choline was formed in lymphoma cells than in lymphocytes explaining the high sensitivity of lymphoma cells to this drug. Ara-C did not inhibit labeling of 5-3H-dCDP-choline from exogenous 5-3H-deoxycytidine while inhibiting DNA synthesis. Excess of exogenous ribocytidine diminished labeling of araCDP-choline, without any effect on dCDP-choline. These data suggest that araCDP-choline and dCDP-choline were synthesized from separate pools in these cells.     

Effect of cytosine arabinoside on viral-specific protein synthesis in cells infected with herpes simplex virus.

The relationship between viral DNA and protein synthesis during herpes simplex virus type 1 (HSV-1) replication in HeLa cells was examined. Treatment of infected cells with cytosine arabinoside (ara-C), which inhibited the synthesis of HSV-1 DNA beyond the level of detection, markedly affected the types and amounts of viral proteins made in the infected cell. Although early HSV-1 proteins were synthesized normally, there was a rapid decline in total viral protein synthesis beginning 3 to 4 h after infection. This is the time that viral DNA synthesis would normally have been initiated. ara-C also prevented the normal shift from early to late viral protein synthesis. Finally, it was shown that the effect of ara-C on late protein synthesis was dependent upon the time after infection that the drug was added. These results suggest that inhibition of progeny viral DNA synthesis by ara-C prevents the "turning on" of late HSV-1 protein synthesis but allows early translation to be "switched off."     

Modulation of human deoxycytidine kinase activity as a response to cellular stress induced by NaF.

Deoxycytidine kinase (dCK) is one of the key enzymes of deoxynucleoside salvage supplying resting lymphocytes with DNA precursors for synthesis and repair. The level of dCK activity is especially important in chemotherapy with the use of deoxynucleoside analogues like arabinosyl cytosine (Citarabid, ara-C), or 2-chloro-deoxyadenosine (Cladribine, CdA). Previous results showed that Cladribine treatment of human lymphocytes increased several fold the activity of dCK without increasing the amount of dCK protein itself (Sasvári-Székely, et al., 1998, Biochem. Pharmacol. 56, 1175), and a possible post-translational modification was suggested. This theory was further investigated using NaF as an inhibitor of protein phosphatases. It was shown that NaF treatment of cells elevated dCK activity while inhibiting DNA synthesis. The possible mechanism of dCK activation/inactivation induced by exposure of cell cultures to different agents is discussed.     

Cell cycle specificity of cytogenetic damage induced by 3,4-epoxy-1- butene.

3,4-epoxy-1-butene (EB), a primary metabolite of butadiene, is a direct-acting "S-dependent" genotoxicant that can induce sister chromatid exchanges (SCEs) and chromosome aberrations (CAs) in cycling cells in vitro. However, EB is almost inactive when splenic or peripheral blood lymphocytes are exposed at the G(0) stage of the cell cycle. To investigate whether repair of DNA lesions is responsible for the lack of cytogenetic responses seen after G(0) treatments, we used cytosine arabinoside (ara-C) to inhibit DNA polymerization during DNA repair. If enough repairable lesions are present, double-strand breaks should accumulate and form chromosome-type ("S-independent") deletions and exchanges. This is exactly what occurred. EB induced chromosome deletions and dicentrics at the first division following treatment, when the EB exposure was followed by ara-C. Without ara-C treatment, there was no induction of CAs. These experiments indicate that the relatively low levels of damage induced by EB in G(0) lymphocytes are removed by DNA repair prior to DNA synthesis and thus, before the production of SCEs or chromatid-type aberrations.     

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.     

Induction of alkaline-labile sites in DNA by benzo [a]pyrene and the repair of those lesions in cultured Chinese hamster cells

Formation of alkaline-labile sites in DNA by S9-activated benzo [a]pyrene (B [a]P) and the repair of those lesions were investigated using the technique of alkaline elution in cultured Chinese hamster V79 cells. When the cells were treated with B [a]P (1-5 micrograms/ml) there was negligible increase in DNA elution at pH 12.1 as compared to untreated controls. However, the elution of DNA increased at pH 12.6 with a concentration dependency, thereby indicating formation of alkaline-labile sites in DNA by B [a]P. After 4 h of repair incubation the elution of DNA at pH 12.6 of B [a]P (5 micrograms/ml) treated cells returned to the control levels. The half-life of alkaline-labile sites formed by B [a]P was approximately 1.5 h. Inhibitors of DNA-repair synthesis, hydroxyurea (HU) and 1-beta-D-arabinofuranosyl cytosine (ara-C) when added simultaneously with S9-activated B [a]P for 3 h showed an increase in elution of DNA at pH 12.1, indicating that a population of B [a]P-induced DNA lesions could be removed by a rapid DNA-repair process. These results indicate that at least two kinds of DNA lesions, repairable alkaline-labile sites and rapidly repairable DNA single-strand breaks, are detected after B [a]P treatment by the use of the alkaline elution procedure, by changing elution pH.     

Induction of alkaline-labile sites in DNA by benzo[a]pyrene and their repair of the lesions in cultured Chinese hamster cells

Formation of alkaline-labile sites in DNA by S9-activated benzo[a]pyrene (B[a]P) and the repair of those lesions were investigated using the technique of alkaline elution in cultured Chinese hamster V79 cells.When the cells were treated with B[a]P (1–5 μg/ml) there was negligible increase in DNA elution at pH 12.1 as compared to untreated controls. However, the elution of DNA increased at pH 12.6 with a concentration dependency, thereby indicating formation of alkaline-labile sites in DNA by B[a]P. After 4 h of repair incubation the elution of DNA at pH 12.6 of B[a]P (5 μg/ml) treated cells returned to the control lavels. The half-life of alkaline-labile sties formed by B[a]P was approximately 1.5 h. Inhibitors of DNA-repair synthesis, hydroxyrea (HU) and 1-β-arabinofuranosly cytosine (ara-C) when added simultaneously with S9-activated B[a]P for 3 h showed an increase in elution of DNA at pH 12.1 indicating that a population of B[a]P-induced DNA lesions could be removed by a rapid DNA-repair process.These results indicate that at least two kinds of DNA lesions, repairable alkaline-labile sites rapidly repairable DNA single-strand breaks, are detected after B[a]P treatment by the use of the alkaline elution procedure, by changing elution pH.     

The DNA repair inhibitors hydroxyurea and cytosine arabinoside enhance the sensitivity of the alkaline single-cell gel electrophoresis ('comet') assay in metabolically-competent MCL-5 cells.

We have found previously that the metabolically-competent human MCL-5 cell line did not appear to be usefully sensitive to the DNA-damaging effects of several carcinogens, as measured by the alkaline single-cell gel electrophoresis ('comet') assay. We therefore sought to increase its sensitivity by inhibiting DNA repair during exposure to test compounds, using 10 mM hydroxyurea (HU) and 1.8 mM cytosine arabinoside (ara-C), which inhibit DNA resynthesis during nucleotide excision repair. The following compounds were tested, using a 30-min exposure, in the absence or presence of HU/ara-C: 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4, 8-DiMeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-9H-pyrido[2,3-b]indole (A[alpha]C), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA[alpha]C), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), benzo[a]pyrene (B[a]P), 3-methylcholanthrene (3-MCA), 7, 12-dimethylbenz[a]anthracene (DMBA), 1-nitropyrene (1-NP), 2-nitrofluorene (2-NF), aniline, o-toluidine, benzene, lindane, bleomycin, cisplatin, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), sodium chromate, chromic chloride, and diethylstilboestrol (DES). We made the following observations. The background level of comet formation was reasonably constant over several months and was increased only slightly, but significantly, in the presence of the DNA-repair inhibitors. All compounds that induced comet formation did so without appreciable cytotoxicity as assessed by trypan blue exclusion. Of the compounds tested, the heterocyclic amines and polycyclic aromatic hydrocarbons (with the exceptions of PhIP and B[a]P) failed to induce convincing levels of comet formation in the absence of repair inhibitors. In their presence the heterocyclic amines tested induced comet formation (with the exception of 8-MeIQx), with widely differing potencies. 1-NP failed to elicit marked comet formation even in the presence of HU/ara-C. Aniline and o-toluidine produced significant levels of comet formation in the absence of HU/ara-C, but in their presence comet formation was markedly increased. Benzene, lindane, bleomycin, cisplatin, MNNG, sodium chromate and chromic chloride induced comet formation in the absence of HU/ara-C, but, with the exception of cisplatin, their presence enhanced comet formation. Neither sucrose nor DES elicited comet formation under the conditions used in this study. Many more agents need to be tested in order to determine how well the comet assay using MCL-5 cells (or modified versions of it) can distinguish genotoxins from non-genotoxins.     

1-β- -Arabinofuranosylcytosine stimulates thymidine incorporation into the DNA of contact-inhibited cells

In rapidly proliferating cells l-β- -arabinofuranosylcytosine (ara-C) is a potent inhibitor of DNA synthesis whose effect can be irreversible and consequently cytocidal. Whereas thymidine incorporation is greatly reduced in rapidly proliferating cells in the presence of ara-C, contact-inhibited cells, similarly treated, show increased thymidine incorporation by as much as 7-fold. This ara-C-induced stimulation appears to result from an influence on thymidine utilization rather than increased DNA synthesis.     

Mapping of apolipoprotein A-1 and ceruloplasmin genes on human, rat and mouse chromosomes by hybridization in situ with specific human DNA probes

In situ hybridization was carried out on metaphase-prometaphase chromosomes of PGA-stimulated lymphocytes and bone marrow cells obtained from laboratory rats and mice. Plasmid cloned sequences of human apolipoprotein A-1 (Apo A-1) and ceruloplasmin (CP) cDNA fragments have been used as specific probes labelled in nick-translation reaction with 3HdTTP and 3Hd ATP. The data of our study suggest that Apo A-1 is localized in 11q14-22, 9 A2-4 and 5q36 areas in men, mice and rats, respectively. The DNA sequences of human CP cDNA most probably occupy 3q23-25, 13q24-26 and 15q13-20 areas. Heterologous in situ hybridization of other species with DNA probes does not always give reliable results in gene mapping. Thus, the data of heterologous hybridization should be considered with caution.     

Reduced methyl group acceptance of 1-β-d-arabinofuranosylcytosine-containing DNA polymers

Previous studies have shown that 1-β-d-arabinofuranosylcytosine (ara-C) can induce differentiation of various malignant cells and that DNA methylation patterns become altered under ara-C treatment of those cells. The aim of this study was to investigate whether this influence on DNA methylation is caused by a direct effect of DNA-incorporated ara-C molecules on nuclear DNA methylase. For this reason, we constructed various ara-C-substituted DNA polymers and used them as substrates for highly purified eukaryotic DNA methylase isolated from murine P815 mastocytoma cells. The ara-C incorporation into DNA polymers was measured by either an ara-C-specific radioimmunoassay or by use of radioactive-labelled ara-C during the synthesis of those polymers. We found an inverse correlation between the level of ara-C substitution of the DNA polymers and their methyl group acceptance. Kinetic experiments performed with ara-C-modified DNA polymers pointed out that the mode of action of DNA methylase remains unaltered. DNA methylase is neither detached nor fixed at an ara-C site, but is somehow hindered in its enzymatic activity, probably by slowing down the walking mechanism. Hence, the previously observed hypermethylation of DNA of some eukaryotic cells, propagated in the presence of ara-C, is apparently not due to a direct effect of DNA-incorporated ara-C molecules on endogenous DNA methylase.