Effects of nitrosoureas on human DNA polymerase activities from acute and chronic granulocytic leukemia cells.

In vitro DNA synthesis by isolated cytoplasmic DNA polymerases of human leukemic cells was found to be inhibited by 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methyl-cyclohexyl)-1-nitrosourea. 2-Chloroethyl isocyanate and cyclohexyl isocyanate, the decomposition products of 1,3-bis(2-chloroethyl)-1-nitrosourea and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, respectively, are as effective as their parent nitrosoureas in inhibiting the enzyme activity. Preincubation studies indicated that these compounds inhibit DNA synthesis primarily by altering the enzyme DNA polymerases without significantly affecting the DNA template activities.     

Inactivation of glutathione reductase by 2-chloroethyl nitrosourea-derived isocyanates.

The specific inactivation of yeast glutathione reductase (GSSG-reductase) by 2-chloroethyl isocyanate and cyclohexyl isocyanate derived from their respective 2-chloroethyl nitrosoureas has been demonstrated. Titration of the enzyme with 2-chloroethyl isocyanate or [¹⁴C] labeling with 1-(2-chloroethyl)-3-(1-¹⁴C-cyclohexyl)-1-nitrosourea or 1,3-bis (2-¹⁴C-chloroethyl)-1-nitrosourea resulted in near stoichiometric inactivation and/or covalent labeling of the enzyme. In addition to 1,3-bis (2-chloroethyl)-1-nitrosourea and 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea, several other 2-chloroethyl nitrosoureas were capable of inactivation of not only purified GSSG-reductase, but also the activity of this enzyme in cell-free extracts of murine lymphoma L5178Y ascites tumor cells and murine bone marrow.     

The sensitivities of SV40-transformed human fibroblasts to monofunctional and DNA-crosslinking alkylating agents.

4 repair-deficient (Mer-) and 2 repair-proficient (Mer+) lines of SV40-transformed human fibroblasts were assayed for colony-forming ability after treatment with MNNG, methyl methanesulfonate (MMS), 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), and 1-(2-chloroethyl)-3-(2-hydroxyethyl)-1-nitrosourea (HECNU). The sensitivities to MMS, BCNU and HECNU of these SV40-transformed lines were similar to those of comparably treated human tumor cells observed previously. However, unlike human tumor lines, whose post-MNNG survival is strongly dependent upon Mer phenotype, SV40-transformed lines showed a lack of dependence of post-MNNG colony-forming ability on Mer phenotype. No differences in glutathione levels that might explain these differences were detected. The amounts of SV40-specific DNA and RNA among the lines were found to vary widely, but no correlation with Mer phenotype was found.     

Mutations in the alpha8 loop of human APE1 alter binding and cleavage of DNA containing an abasic site

Recent crystallographic studies reveal loops in human AP endonuclease 1 (APE1) that interact with the major and minor grooves of DNA containing apurinic/apyrimidinic (AP) sites. These loops are postulated to stabilize the DNA helix and the flipped out AP residue. The loop alpha8 interacts with the major groove on the 3' side of the AP site. To determine the essentiality of the amino acids that constitute the alpha8 loop, we created a mutant library containing random nucleotides at codons 222-229 that, in wild-type APE1, specify the sequence NPKGNKKN. Upon expression of the library (2 x 10(6) different clones) in Escherichia coli and multiple rounds of selection with the alkylating agent methyl-methane sulfonate (MMS), we obtained approximately 2 x 10(5) active mutants that complemented the MMS sensitivity of AP endonuclease-deficient E. coli. DNA sequencing showed that active mutants tolerated amino acid substitutions at all eight randomized positions. Basic and uncharged polar amino acids together comprised the majority of substitutions, reflecting the positively charged, polar character of the wild-type loop. Asn-222, Asn-226, and Asn-229 exhibited the least mutability, consistent with x-ray data showing that each asparagine contacts a DNA phosphate. Substitutions at residues 226-229, located nearer to the AP site, that reduced basicity or hydrogen bonding potential, increased Km 2- to 6-fold and decreased AP site binding; substitutions at residues 222-225 exhibited lesser effects. This initial mutational analysis of the alpha8 loop supports and extends the conclusion of crystallographic studies that the loop is important for binding of AP.DNA and AP site incision.     

The Saccharomyces cerevisiae ETH1 gene, an inducible homolog of exonuclease III that provides resistance to DNA-damaging agents and limits spontaneous mutagenesis

The recently sequenced Saccharomyces cerevisiae genome was searched for a gene with homology to the gene encoding the major human AP endonuclease, a component of the highly conserved DNA base excision repair pathway. An open reading frame was found to encode a putative protein (34% identical to the Schizosaccharomyces pombe eth1(+) [open reading frame SPBC3D6.10] gene product) with a 347-residue segment homologous to the exonuclease III family of AP endonucleases. Synthesis of mRNA from ETH1 in wild-type cells was induced sixfold relative to that in untreated cells after exposure to the alkylating agent methyl methanesulfonate (MMS). To investigate the function of ETH1, deletions of the open reading frame were made in a wild-type strain and a strain deficient in the known yeast AP endonuclease encoded by APN1. eth1 strains were not more sensitive to killing by MMS, hydrogen peroxide, or phleomycin D1, whereas apn1 strains were approximately 3-fold more sensitive to MMS and approximately 10-fold more sensitive to hydrogen peroxide than was the wild type. Double-mutant strains (apn1 eth1) were approximately 15-fold more sensitive to MMS and approximately 2- to 3-fold more sensitive to hydrogen peroxide and phleomycin D1 than were apn1 strains. Elimination of ETH1 in apn1 strains also increased spontaneous mutation rates 9- or 31-fold compared to the wild type as determined by reversion to adenine or lysine prototrophy, respectively. Transformation of apn1 eth1 cells with an expression vector containing ETH1 reversed the hypersensitivity to MMS and limited the rate of spontaneous mutagenesis. Expression of ETH1 in a dut-1 xthA3 Escherichia coli strain demonstrated that the gene product functionally complements the missing AP endonuclease activity. Thus, in apn1 cells where the major AP endonuclease activity is missing, ETH1 offers an alternate capacity for repair of spontaneous or induced damage to DNA that is normally repaired by Apn1 protein.     

Cell killing by various monofunctional alkylating agents in Chinese hamster ovary cells

Cell killing by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), and methyl methanesulfonate (MMS) was measured in Chinese hamster ovary (CHO) cells using the colony-formation assay. Cell killing by these agents was determined in exponentially growing asynchronous cells, in synchronous cells as a function of cell-cycle position and in nondividing cells. Distinct differences in the cytotoxic effect of the 4 alkylating agents were found in respect to dose-response, cell cycle phase-sensitivity and growth state. MNNG and MNU showed the same biphasic dose-survival relationship in exponentially growing cells, with an initial steep decline followed by a shallow component. The shallow component disappeared in growth-arrested cells. MNNG and MNU differed, however, in the cell-cycle age response. No cell-cycle phase difference was seen with MNNG, whereas cells in G1 seemed more sensitive to MNU than cells in S phase. MMS and ENU both showed shouldered dose-response curves for exponentially growing asynchronous cells, and the same cell-cycle pattern for synchronous cultures with cells in early S phase being the most sensitive. However, survival of nondividing cells versus dividing cells was reduced much more by MMS than by ENU. Caffeine, which interferes with the regulation of DNA synthesis and is known to modify cell killing by DNA-damaging agents, enhanced cell killing by all agents. It is concluded that there must be a number of factors which contribute to cell killing by monofunctional alkylating agents, and that besides alkylation of DNA reaction with other cellular macromolecules should be considered.     

Role of O6-alkylguanine-DNA alkyltransferase in the resistance of mouse spermatogenic cells to O6-alkylating agents

The O(6)-alkylguanine-DNA alkyltransferase inactivator O(6)-benzylguanine was administered to BALB/c mice either alone or before exposure to 1,3-bis(2-chloroethyl)-1-nitrosourea to study the role of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase in the protection of the testis against anti-cancer O(6)-alkylating agents. Exposure of the mice to 1, 3-bis(2-chloroethyl)-1-nitrosourea or O(6)-benzylguanine alone did not produce any marked testicular toxicity at the times studied. Testicular O(6)-alkylguanine-DNA alkyltransferase concentrations were assayed between 0 and 240 min after O(6)-benzylguanine treatment and were shown to be > 95% depleted 15 min after treatment with O(6)-benzylguanine and remained at > 95% at all the times assayed. Histological examination, the reduction in testicular mass and the induction of spermatogenic cell apoptosis showed that this depletion significantly potentiated 1, 3-bis(2-chloroethyl)-1-nitrosourea-induced testicular damage after treatment. Major histological damage was apparent 42 days after treatment, demonstrating that the stem spermatogonia were significantly affected by the combination. These results demonstrate that O(6)-alkylguanine-DNA alkyltransferase plays a significant role in protecting the spermatogenic cells from damage caused by DNA alkylation and indicate that the observed toxicity may result from damage to stem spermatogonia.     

Modulating effect of thiol-disulfide status on [14C]aminopyrine accumulation in the isolated parietal cell

Thiol-oxidizing agents were found to stimulate [14C] aminopyrine accumulation, a reliable index of acid secretory function of isolated canine parietal cells. Glutathione is the predominant intracellular free thiol; thus, its oxidation status largely determines the thiol-disulfide status of the cell by thiol-disulfide interchange reactions. Three agents which alter glutathione oxidation status by different mechanisms were applied to parietal cells in vitro to investigate whether enhanced formation of GSSG alters acid secretory function. The agents studied were diamide (which nonenzymatically oxidizes GSH to GSSG), tert-butyl hydroperoxide (an organic peroxide specifically reduced by glutathione peroxidase, thereby generating GSSG for GSH), and 1,3-bis(2-chloroethyl)-1-nitrosourea (an inhibitor of NADPH:GSSG reductase, which presumably allows the accumulation of GSSG). Each of these agents stimulated aminopyrine accumulation in a dose-dependent fashion. Simple depletion of GSH by diethyl maleate or 2-cyclohexene-1-one did not stimulate aminopyrine accumulation. Likewise, enhanced aminopyrine accumulation occurred at diamide concentrations which did not cause significant depletion of total cellular glutathione. The thiol-reducing agent, dithiothreitol, prevented enhanced aminopyrine accumulation by 1,3-bis(2-chloroethyl)-1-nitrosourea and tert-butyl hydroperoxide. These observations support the hypothesis that thiol-disulfide interchange reactions involving GSSG modulate the acid secretory function of the isolated parietal cell.     

The effectiveness of the O(6)-alkylguanine-DNA alkyltransferase encoded by the ogt(ST) gene from S. typhimurium in protection against alkylating drugs, resistance to O(6)-benzylguanine and sensitisation to dibromoalkane genotoxicity

Here we demonstrate that the Ogt(ST) from Salmonella typhimurium is a highly efficient O(6)-alkylguanine-DNA alkyltransferase (AGT) in affording protection against antitumour chloroethylating drugs (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU)). In addition, Ogt(ST) is refractory to O(6)-benzylguanine (BG) inactivation and its expression provides only minor sensitisation to genotoxicity by environmental dibromoalkanes (DBE). No other of the assayed bacterial or human AGTs displayed such advantageous properties for chemoprotective gene therapy strategy. Our observations indicate that the Ogt(ST) AGT might be, under some circumstances, of potential use to improve cancer chemotherapy. At least, its properties may provide further insight into the design of human AGT variants that could be expressed in normal or tumour cells to provide either protection or ablation.     

Suppression of oxidative phosphorylation in mouse embryonic fibroblast cells deficient in apurinic/apyrimidinic endonuclease

The mammalian apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is an essential DNA repair/gene regulatory protein. Decrease of APE1 in cells by inducible shRNA knockdown or by conditional gene knockout caused apoptosis. Here we succeeded in establishing a unique mouse embryonic fibroblast (MEF) line expressing APE1 at a level far lower than those achieved with shRNA knockdown. The cells, named MEF^la (MEF^lowAPE1), were hypersensitive to methyl methanesulfonate (MMS), and showed little activity for repairing AP-sites and MMS induced DNA damage. While these results were consistent with the essential role of APE1 in repair of AP sites, the MEF^la cells grew normally and the basal activation of poly(ADP-ribose) polymerases in MEF^la was lower than that in the wild-type MEF (MEF^wt), indicating the low DNA damage stress in MEF^la under the normal growth condition. Oxidative phosphorylation activity in MEF^la was lower than in MEF^wt, while the glycolysis rates in MEF^la were higher than in MEF^wt. In addition, we observed decreased intracellular oxidative stress in MEF^la. These results suggest that cells with low APE1 reversibly suppress mitochondrial respiration and thereby reduce DNA damage stress and increases the cell viability.     

Relation between the changes in the structure and synthesis of DNA in the cells of mouse leukemia L1210 induced by 1-methyl-1-nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea

The damage of DNA structure and synthesis in murine leukemia L1210 cells upon single administration in therapeutic doses of antitumour agents of N-nitrosourea type, such as 1-methyl-1-nitrosourea (MNU) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was studied. MNU and BCNU were characterized by stronger inhibitory effects on de novo DNA synthesis compared to additional pathway of DNA synthesis in leukemia L1210 cells in vivo. Centrifugation in alkaline sucrose density gradients of L1210 cell lysates has revealed persistent single-strand breaks and alkaline-labile sites in newly replicated DNA. Parental DNA structure was more stable to damaging drug effects than that of newly replicated DNA. The results are consistent with our previous data on the differences in the mechanisms of MNU and BCNU action and the absence of complete cross resistance between the drugs.     

Involvement of two endonuclease III homologs in the base excision repair pathway for the processing of DNA alkylation damage in Saccharomyces cerevisiae

DNA base excision repair (BER) is initiated by DNA glycosylases that recognize and remove damaged bases. The phosphate backbone adjacent to the resulting apurinic/apyrimidinic (AP) site is then cleaved by an AP endonuclease or glycosylase-associated AP lyase to invoke subsequent BER steps. We have used a genetic approach in Saccharomyces cerevisiae to determine whether or not AP sites are blocks to DNA replication and the biological consequences if AP sites persist in the genome. We previously reported that yeast cells deficient in the two AP endonucleases (apn1 apn2 double mutant) are extremely sensitive to killing by a model DNA alkylating agent methyl methanesulfonate (MMS) and that this sensitivity can be reduced by deleting the MAG1 3-methyladenine DNA glycosylase gene. Here we report that in the absence of the AP endonucleases, deletion of two Escherichia coli endonuclease III homologs, NTG1 and NTG2, partially suppresses MMS-induced killing, which indicates that the AP lyase products are deleterious unless they are further processed by an AP endonuclease. The severe MMS sensitivity seen in AP endonuclease deficient strains can also be rescued by treatment of cells with the AP lyase inhibitor methoxyamine, which suggests that the product of AP lyase action on an AP site is indeed an extremely toxic lesion. In addition to the AP endonuclease interactions, deletion of NTG1 and NTG2 enhances the mag1 mutant sensitivity to MMS, whereas overexpression of MAG1 in either the ntg1 or ntg2 mutant severely affects cell growth. These results help to delineate alkylation base lesion flow within the BER pathway.     

Oxidative cell injury in the killing of cultured hepatocytes by allyl alcohol

The killing of cultured hepatocytes by allyl alcohol depended on the metabolism of this hepatotoxin by alcohol dehydrogenase to the reactive electrophile, acrolein. An inhibitor of alcohol dehydrogenase, pyrazole, prevented both the toxicity of allyl alcohol and the rapid depletion of GSH. Treatment of the hepatocytes with a ferric iron chelator, deferoxamine, or an antioxidant, N,N'-diphenyl-p-phenylenediamine (DPPD), prevented the cell killing but not the metabolism of allyl alcohol and the resulting depletion of GSH. Inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) sensitized the hepatocytes to allyl alcohol, an effect that was not attributable to the reduction in GSH with BCNU. The cell killing with allyl alcohol was preceded by the peroxidation of cellular lipids as evidence by an accumulation of malondialdehyde in the cultures. Deferoxamine and DPPD prevented the lipid peroxidation in parallel with their protection from the cell killing. These data indicate that acrolein produces an abrupt depletion of GSH that is followed by lipid peroxidation and cell death. Such oxidative cell injury is suggested to result from the inability to detoxify endogenous hydrogen peroxide and the ensuing iron-dependent formation of a potent oxidizing species. Oxidative cell injury more consistently accounts for the hepatotoxicity of allyl alcohol than does the covalent binding of acrolein to cellular macromolecules.     

Identification and Characterization of Inhibitors of Human Apurinic/apyrimidinic Endonuclease APE1

APE1 is the major nuclease for excising abasic (AP) sites and particular 3′-obstructive termini from DNA, and is an integral participant in the base excision repair (BER) pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5-fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC¹²⁸⁰), a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays – a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen – and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals.     

Deletion of the MAG1 DNA glycosylase gene suppresses alkylation-induced killing and mutagenesis in yeast cells lacking AP endonucleases.

DNA base excision repair (BER) is initiated by DNA glycosylases that recognize and remove damaged bases. The phosphate backbone adjacent to the resulting apurinic/apyrimidinic (AP) site is then cleaved by an AP endonuclease or glycosylase-associated AP lyase to invoke subsequent BER steps. We have used a genetic approach in Saccharomyces cerevisiae to address whether AP sites are blocks to DNA replication and the biological consequences if AP sites persist in the genome. We found that yeast cells deficient in the two AP endonucleases (apn1 apn2 double mutant) are extremely sensitive to killing by methyl methanesulfonate (MMS), a model DNA alkylating agent. Interestingly, this sensitivity can be reduced up to 2500-fold by deleting the MAG1 3-methyladenine DNA glycosylase gene, suggesting that Mag1 not only removes lethal base lesions, but also benign lesions and possibly normal bases, and that the resulting AP sites are highly toxic to the cells. This rescuing effect appears to be specific for DNA alkylation damage, since the mag1 mutation reduces killing effects of two other DNA alkylating agents, but does not alter the sensitivity of apn cells to killing by UV, gamma-ray or H(2)O(2). Our mutagenesis assays indicate that nearly half of spontaneous and almost all MMS-induced mutations in the AP endonuclease-deficient cells are due to Mag1 DNA glycosylase activity. Although the DNA replication apparatus appears to be incapable of replicating past AP sites, Polzeta-mediated translesion synthesis is able to bypass AP sites, and accounts for all spontaneous and MMS-induced mutagenesis in the AP endonuclease-deficient cells. These results allow us to delineate base lesion flow within the BER pathway and link AP sites to other DNA damage repair and tolerance pathways.     

The effects of X rays on BCNU-induced DNA crosslinking

We have used the technique of alkaline elution to study DNA interstrand crosslinking in 9L rat brain tumor cells treated with combinations of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and X rays. Irradiation with doses as low as 50 rad of X rays immediately or 6 hr after a 1-hr treatment with 60, 80, or 100 microM BCNU produced a significant increase in BCNU-induced DNA interstrand crosslinking. If cells were irradiated before BCNU treatment, the amount of crosslinking was not affected compared with BCNU alone. Cell survival experiments using 600 rad of X rays and 1-hr treatments with 0-30 microM BCNU were also performed. As found in the crosslinking studies, irradiation immediately or 6 hr after the BCNU treatment produced enhanced cell kill, but irradiation 6 hr before BCNU treatment did not produce enhanced cell kill. Therefore, the X-ray-mediated increase in BCNU-induced DNA interstrand crosslinking may be the mechanism through which cell kill is increased by combination treatment with the agents.     

Enhancement of repair replication in mammalian cells by hydroxyurea

The effect of hydroxyurea on DNA repair replication has been studied in Chinese hamster ovary cells. Mitotic cells were treated with UV irradiation, methyl methanesulfonate or nitrogen mustard and incuated in the presence of each of the 4 [³H]deoxyribonucleosides plus BrdUrd and FdUrd for 2 h. The amount of repair replication was quantitated on CsCl gradients and similar values were obtained for each nucleoside. In all cases addition of HU during the incubation period increased these values approximately 2-fold. Following MMS treatment, pool sizes for each of the nucleosides were estimated by varying the amount of exogenously supplied nucleoside. They were found to be insensitive to the addition of HU and it is concluded that the increased incorporation of [³H]deoxyribonucleosides in the presence of HU reflects an increased amount of repair replication.