Incorporation of two deoxycytidine oxidation products into cellular DNA.

The oxidation of cytosine in DNA by free radicals and other oxidants leads to an assortment of products including pyrimidine ring 5,6-saturated, 5,6-unsaturated, contraction, and fragmentation products. The formation of these products in cellular DNA may explain in part the preponderance of C to T transitions induced spontaneously and by H2O2 or ionizing radiation. Our studies have focused on the biological effects of two major 5,6-unsaturated oxidation products of cytosine: 5- hydroxycytosine and 5-hydroxyuracil. In the present work, we have attempted to study the repair of these two lesions by specifically incorporating them into cellular DNA upon incubation of cells with 5-hydroxy-2'deoxycytidine and 5-hydroxy-2'-deoxyuridine. Incubation of mouse L1210 cells with 250 M 5-hydroxy-2'-deoxycytidine led to the incorporation of this lesion to a level 20 times higher (43 lesions/10(5) cytosines) than base-line levels; however, there was no evidence for its repair following a 15-h chase. In contrast, we did not observe any significant incorporation of 5-hydroxy-2'-deoxyuridine into the DNA of L1210 cells but did observe an unidentified product, presumably an oxidation product. This unidentified pyrimidine was incorporated at a very high level (about 2000 lesions/10(5) cytosine residues) and then partially repaired in chase experiments.

A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-dependent eukaryotic DNA-binding protein that specifically recognizes DNA strand breaks produced by various genotoxic agents. To study the biological function of this enzyme, we have established stable HeLa cell lines that constitutively produce the 46-kDa DNA-binding domain of human PARP (PARP-DBD), leading to the trans-dominant inhibition of resident PARP activity. As a control, a cell line was constructed, producing a point-mutated version of the DBD, which has no affinity for DNA in vitro. Expression of the PARP-DBD had only a slight effect on undamaged cells but had drastic consequences for cells treated with genotoxic agents. Exposure of cell lines expressing the wild-type (wt) or the mutated PARP-DBD, with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in an increase in their doubling time, a G2 + M accumulation, and a marked reduction in cell survival. However, UVC irradiation had no preferential effect on the cell growth or viability of cell lines expressing the PARP-DBD. These PARP-DBD-expressing cells treated with MNNG presented the characteristic nucleosomal DNA ladder, one of the hallmarks of cell death by apoptosis. Moreover, these cells exhibited chromosomal instability as demonstrated by higher frequencies of both spontaneous and MNNG-induced sister chromatid exchanges. Surprisingly, the line producing the mutated DBD had the same behavior as those producing the wt DBD, indicating that the mechanism of action of the dominant-negative mutant involves more than its DNA-binding function. Altogether, these results strongly suggest that PARP is an element of the G2 checkpoint in mammalian cells.

DNA strand scission and base release photosensitized by metallo-phthalocyanines.

Photoirradiation of aqueous solutions of DNA in the presence of Al- or Zn-tetrasulphonated phthalocyanines (AIPcS4 and ZnPcS4) causes formation of strand breaks and liberation of nucleobases. The effect of added D2O, which enhances singlet oxygen (1O2) lifetime, radical scavengers including alcohols and the spin-trap DMPO, as well as superoxide dismutase, indicates that both singlet oxygen (1O2) and free radicals contribute to the production of strand breaks. However, in the case of base release, only free radicals, such as the hydroxyl radical (.OH), appear to be involved in the degradation process. Detection of the characteristic free-radical oxidation products of deoxyribose provides evidence that .OH are involved in the photosensitized DNA damage. EPR and spin trapping data suggest that superoxide (O2.-) is the most likely precursor of .OH and a Fenton-type mechanism is proposed for their formation.

DNA polymerase delta mediates excision repair in growing cells damaged with ultraviolet radiation.

In confluent, stationary phase cells, an aphidicolin-sensitive DNA polymerase mediates UV-induced excision repair, but the situation in growing cells is still controversial. The sensitivity of repair synthesis to aphidicolin, an inhibitor of DNA polymerases alpha and delta, was determined in growth phase and confluent normal human fibroblasts (AG1518) using several techniques. Repair synthesis in confluent cells was always inhibited by aphidicolin, no matter which measurement technique was used. However, the inhibition of repair synthesis in growth-phase cells by aphidicolin was only detectable when techniques unaffected by changes in nucleotide metabolism were used. We conclude that UV-induced repair synthesis in growing cells is actually aphidicolin sensitive, but that this inhibition can be obscured by changes in nucleotide metabolism. Employing butylphenyl-deoxyguanosine triphosphate, a potent inhibitor of polymerase alpha and a weak inhibitor of delta, we have obtained evidence that polymerase delta is responsible for repair synthesis in growth-phase cells following UV irradiation.

Involvement of DNA polymerase delta in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation.

DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase delta as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, we describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, alpha or delta. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase alpha several hundred times more strongly than it inhibits DNA polymerase delta. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase delta. It appears that repair synthesis at late times after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase delta.

Inhibition of repair patch ligation by an inhibitor of poly(ADP-ribose) synthesis in normal human fibroblasts damaged with ultraviolet radiation.

The effect of inhibiting poly(ADP-ribose) synthesis on DNA excision repair following UV irradiation of cultured normal human fibroblasts was determined under conditions which did not perturb NAD+ concentration. Following UV irradiation, there was a transient increase in DNA strand breaks to a maximum of 800 rad eq of breaks 30 min after damage. 3-Aminobenzamide (5 mM) caused a 50% increase in the maximum number of DNA single strand breaks following damage but did not prevent the decline in strand breaks which normally occurs within the first hour after damage. Addition of 3-aminobenzamide several hours after damage, when most of the strand breaks had disappeared, caused a reaccumulation of strand breaks. 3-Aminobenzamide inhibited ligation of repair patches, as measured by exonuclease III, following damage by UV radiation and the magnitude of the inhibition was sufficient to account for the increases in strand breaks caused by 3-aminobenzamide. UV radiation alone did not lower NAD+ concentrations; however, when the repair synthesis step was inhibited by aphidicolin and hydroxyurea, the number of single strand breaks increased and the NAD+ concentration fell to 11%. 3-Aminobenzamide inhibited this depletion of NAD+ by 80%.

Effects of 6-aminonicotinamide on cell growth, poly(ADP-ribose) synthesis and nucleotide metabolism.

The purpose of this study was to determine if the cytotoxic effects of 6-aminonicotinamide are solely the result of an inhibition of poly(ADP-ribose) synthesis. The effects of 6-aminonicotinamide on cell growth, poly(ADP-ribose) synthesis and nucleotide concentrations were compared with the effect of 3-aminobenzamide, a more potent inhibitor of poly(ADP-ribose) synthesis. The growth of L1210 cells was not inhibited by 1 mM 3-aminobenzamide and was only slightly inhibited by 5 mM 3-aminobenzamide even though poly(ADP-ribose) synthesis, as measured by the N-methyl N-nitrosourea induced depletion of NAD+, was inhibited substantially. In contrast, 6-aminonicotinamide was found to be a potent inhibitor of L1210 and CHO cell growth. A 5 mM concentration of 3-aminobenzamide had no effect on purine and pyrimidine ribonucleotide concentrations or on the ATP to ADP ratio, but it did cause a slight elevation of NAD+. 6-Aminonicotinamide at 0.01 mM caused a depletion of purine and pyrimidine nucleotides and NAD+ as well as a reduction in the ATP to ADP ratio. 6-Aminonicotinamide at 1 mM caused a substantial inhibition of purine nucleotide synthesis from [14C] glycine but did not stimulate ATP breakdown. We conclude that inhibition of poly(ADP-ribose) synthesis caused little growth inhibition in itself and that the effects of 6-amininicotinamide on nucleotide metabolism were sufficient to produce an inhibition of both cell growth and DNA repair.

Specificity of inhibitors of poly(ADP-ribose) synthesis. Effects on nucleotide metabolism in cultured cells.

The effects of inhibitors of poly(ADP-ribose) synthesis on cell growth and several parameters of nucleotide metabolism have been determined. At concentrations which produced similar inhibitions of poly(ADP-ribose) synthesis, 3-acetylaminobenzamide (1 mM) had no effect on L1210 cell growth, 3-aminobenzamide (5mM) was slightly inhibitory and 3-methoxybenzamide (5 mM) was a potent inhibitor of growth. During a 2-h incubation, none of the inhibitors affected ribo- or deoxyribonucleotide concentrations in cells treated with or without N-methyl-N-nitrosourea; however, N-methyl-N-nitrosourea treatment reduced dCTP concentrations by 50%. During a 24-hr incubation, 3-aminobenzamide and 3-acetylaminobenzamide did not lower ribonucleotide concentrations in cells grown with either undialyzed or dialyzed serum. In contrast, 3-methoxybenzamide caused a depletion of UTP in cells grown with undialyzed serum and caused a depletion of all purine and pyrimidine ribonucleotides in cells grown with dialyzed serum. 3-Aminobenzamide and 3-acetylaminobenzamide had no effect on the conversion of hypoxanthine to ATP and GTP but did slightly inhibit incorporation of formate into ATP and GTP. 3-Methoxybenzamide inhibited incorporation of both hypoxanthine and formate into purine ribonucleotides. 3-Aminobenzamide, 3-acetylaminobenzamide, and 3-methoxybenzamide all inhibited glycine incorporation into ATP and GTP and reduced both the incorporation of thymidine into DNA and the apparent specific activity of the dTTP pool. We conclude that inhibition of poly(ADP-ribose) synthesis causes little or no growth inhibition and has no effect on purine or pyrimidine nucleotide synthesis de novo. The effect of all the inhibitors on glycine and formate metabolism may be related to an inhibition of ADP-ribose synthesis or may be a secondary effect of the inhibitors. The growth inhibition and the reduction in nucleotide concentration caused by 3-methoxybenzamide are apparently secondary effects of this drug and may result from an inhibition of phosphoribosyl pyrophosphate synthesis.

Methylation of deoxycytidine incorporated by excision-repair synthesis of DNA.

Methylation of deoxycytidine incorporated by DNA excision-repair was studied in human diploid fibroblasts following damage with ultraviolet radiation, N-methyl-N-nitrosourea, or N-acetoxy-2-acetylaminofluorene. In confluent, nondividing cells, methylation in repair patches induced by all three agents is slow and incomplete. Whereas after DNA replication in logarithmic-phase cultures a steady state level of 3.4% 5-methylcytosine is reached in less than 2 hr after cells are labeled with 6- 3H-deoxycytidine, following ultraviolet-stimulated repair synthesis in confluent cells it takes about 3 days to reach a level of approximately 2.0% 5-methylcytosine in the repair patch. In cells from cultures in logarithmic-phase growth, 5-methylcytosine formation in ultraviolet-induced repair patches occurs faster and to a greater extent, reaching a level of approximately 2.7% in 10-20 hr. Preexisting hypomethylated repair patches in confluent cells are methylated further when the cells are stimulated to divide; however, the repair patch may still not be fully methylated before cell division occurs. Thus DNA damage and repair may lead to heritable loss of methylation at some sites.

The effect of anoxia on anthracycline-induced DNA damage in the RPMI-6410 human lymphoblastoid cell line.

The alkaline elution procedure developed by Kohn and co-workers was used with the RPMI-6410 cultured human lymphoblastoid cell line to examine the hypothesis that anthracycline-induced DNA strand scission is mediated by oxygen- or superoxide-derived free radicals. Hypoxia was induced by gassing with nitrogen containing 5% carbon dioxide and less than 4 ppm oxygen. Alkaline elution studies showed hypoxia was induced, as the oxygen enhancement ratios for DNA strand breaks was 2.4 and 2.6 for the 250 R +/- oxygen and the 500 R +/- oxygen (1 R = 2.58 x 10(-4) C/kg) experiments, respectively. The pattern of adriamycin-induced DNA strand breaks and cross-linking was not affected by hypoxia with 1-h adriamycin exposures between 0.05 and 1.0 microgram/ml. Similarly, 1-h exposures of N-trifluoroacetyladriamycin-14-valerate at 3 or 10 micrograms/mL gave essentially identical alkaline elution profiles in the presence or absence of oxygen. These results do not support the hypothesis that oxygen-derived radicals play a primary role in anthracycline-induced DNA strand breakage.

N-trifluoroacetyladriamycin-14-valerate and adriamycin induced DNA damage in the RPMI-6410 human lymphoblastoid cell line.

The adriamycin- and N-trifluoroacetyladriamycin-14-valerate (AD-32) induced DNA cross-linking and breakage in human RPMI-6410 cells was compared using the alkaline elution technique of Kohn and co-workers. At comparable growth-inhibitory concentrations both adriamycin and AD-32 caused DNA cross-linking. Treatment with proteinase-K showed this cross-linking to be mainly DNA-protein in character. Proteinase-K treatment also revealed that both drugs caused either single-strand DNA breaks or increased alkaline sensitivity. With adriamycin the degree of cross-linking and breakage was dose related over the range studied (0.05 - 0.4 micron/mL), whereas with AD-32 there appeared to be a saturation of both effects at concentrations in excess of 3 micron/mL. With both drugs the extent of cross-linking and breakage was maximal at the end of the drug exposure. This work suggests that AD-32 or some metabolite of its binds to DNA and this binding leads to DNA damage that is similar to that caused by adriamycin. These AD-32 results are somewhat surprising in light of earlier model studies showing that AD-32 does not bind to isolated DNA.

L-phenylalanine mustard (melphalan) uptake and cross-linking in the RPMI 6410 human lymphoblastoid cell line.

L-Phenylalanine mustard (melphalan) induced a time- and concentration-dependent arrest of cycling RPMI 6410 cells in the G2 phase of the cell cycle as evidenced by flow cytofluorometry. A melphalan exposure of 1 microgram/ml for 1 hr caused a temporary G2 blockage which was overcome by 48 hr. Higher concentrations or longer exposures lead to irreversible blockages. Melphalan caused DNA cross-linking which was monitored by the alkaline elution method. The cross-linking was shown to be between DNA and protein. The degree of DNA cross-linking increased for approximately 4 hr after a 1-hr drug exposure of 1 microgram/ml. At 36 to 48 hr after the drug exposure, the cells overcame the G2 block and were dividing. The DNA cross-links have apparently been repaired as they are no longer detected by alkaline elution. The extent of melphalan cross-linking was dependent on both drug dosage and exposure time. Using a culture medium lacking amino acids, it was shown that melphalan uptake into RPMI 6410 cells was inhibited by leucine, isoleucine, or glutamine. The increased uptake of melphalan and the increased cross-linking in amino acid-deficient media were reduced by readdition of the aforementioned amino acids.

Deoxyadenosine metabolism and toxicity in cultured L5178Y cells.

The growth of cultured L5178Y cells is inhibited by relatively low concentrations fo deoxyadenosine in the presence of deoxycoformycin, an inhibitor of adenosine deaminase. Cell viability is reduced, presumably as a consequence of the induced state of unbalanced growth which is characterized by inhibition in DNA synthesis, accumulation of cells in G1 or early S phase, a continuation in RNA synthesis, and increasing cell volume. The intracellular concentrations of purine and pyrimidine ribonucleoside phosphates remain essentially unchanged. The significant changes in the intracellular deoxynucleoside triphosphate pools are an increase in deoxyadenosine triphosphate and a decrease in deoxycytidine triphosphate.