Influence of O6-methylguanine on DNA damage and cytotoxicity of temozolomide in L1210 mouse leukemia sensitive and resistant to chloroethylnitrosoureas.

Temozolomide is a new anticancer agent which in the early clinical investigation has shown promising antitumor activity. It decomposes spontaneously to the active metabolite of DTIC (MTIC). Temozolomide is more cytotoxic against L1210 than against a subline L1210/BCNU, resistant to chloroethylnitrosoureas. Using [methyl-3H] temozolomide we found that after 1 h exposure the amount of O6-methylguanine (O6mGua) was twice as high in L1210 than in L1210/BCNU whereas the amount of N7 mGua was approximately the same in the two cell lines. O6-alkylguanine DNA alkyltransferase (AT) levels were higher in L1210/BCNU than in L1210, supporting the view that the resistance to methyltriazenes is probably related to the efficient repair of O6mGua in L1210/BCNU. Exposure of L1210/BCNU cells to 0.4 mM O6mGua for 24 h resulted in a depletion of AT and in a higher temozolomide-induced cytotoxicity. In the sensitive cell line L1210, temozolomide activity was not potentiated by O6mGua pretreatment. Moreover, in L1210/BCNU, O6mGua increased DNA single-strand breaks caused by temozolomide, suggesting that O6-guanine alkylation induces an excision repair mechanism in cells depleted in AT.     

Modulation of nitrosourea resistance in human colon cancer by O6-methylguanine.

Human colon cancer is resistant to a variety of alkylating agents including the nitrosoureas. To specifically evaluate nitrosourea resistance, we studied the role of O6-alkylguanine-DNA alkyltransferase (alkyltransferase) which is known to repair nitrosourea-induced cytotoxic DNA damage. Alkyltransferase activity varied over a similar wide range in 25 colon cancer biopsies and 14 colon cancer cell lines but the activity was not correlated with differentiation status, Dukes' classification or in vitro growth characteristics. 1,3-Bis-(2-chloroethyl)-1-nitrosourea (BCNU) resistance and alkyltransferase activity were highly correlated (R2 = 0.929, P less than 0.001) in 7 different colon cancer cell lines, suggesting that the alkyltransferase is an important component of nitrosourea resistance in colon cancer cells. In the BCNU-resistant, high alkyltransferase VACO 6 cell line, inactivation of the alkyltransferase by O6-methylguanine caused a proportional decrease in the BCNU IC50, consistent with that predicted by the regression line. Enzyme inactivation was also associated with a marked increase in DNA cross-link formation. Because alkyltransferase correlates with BCNU resistance in colon cancer, and resistance can be reversed by inactivating the protein, the alkyltransferase may have an important role in nitrosourea resistance in human colon cancer cells. These data provide the rationale for clinical trials in colon cancer with biochemical modulators of the alkyltransferase to increase the therapeutic response to nitrosoureas.     

S-alkylthiolation of O6-methylguanine-DNA-methyltransferase (MGMT) to sensitize cancer cells to anticancer therapy

O6-methylguanine DNA methyltransferase/O6-alkylguanine DNA alkyltransferase (MGMT/AGT) removes alkyl adducts from the O6-position of guanine in DNA. Expression of MGMT in human cancers has been associated with resistance to therapies using alkylating agents. MGMT promoter methylation regulates its expression and response to alkylating agents. A combination of O6-benzylguanine-based inhibitors of MGMT with alkylating agents improved the efficacy. However, this is associated with enhanced cytotoxicity and the induction of GC to AT transition mutations presumably also in progenitor/stem cells. A few recent studies have described analogs of O6-benzylguanine targeting defined pathways of cancer cells that can be used to improve the selectivity of O6-benzylguanine-based inhibitors for cancer cells. Therefore, MGMT inhibitor targeting represents a reliable strategy for improving cancer therapy with alkylating agents.     

Strategies to Improve the Killing of Tumors Using Temozolomide: Targeting the DNA Repair Protein MGMT

Alkylating agents such as temozolomide (TMZ) are effective anticancer drugs for treating a variety of solid tumors including melanoma, glioma, and astrocytoma. TMZ exerts its effects mainly via the mutagenic product O6-methylguanine, a cytotoxic DNA lesion. This damage may be repaired by the DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT), a key player in the resistance of cancers to TMZ. Several strategies are presently being pursued to improve the killing of tumor cells by TMZ, with inhibition of MGMT being the most promising. In this review, we provide an overview of recent advances in this field.     

Mammalian cells expressing Escherichia coli O6-alkylguanine-DNA alkyltransferases are hypersensitive to dibromoalkanes.

The effect of expression of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, on the growth inhibitory effects of the dibromoalkanes (DBA) dibromomethane (DBM) and dibromoethane (DBE) was determined in Chinese hamster lung fibroblasts transfected with and expressing high levels of the Escherichia coli alkyltransferase (ATase) genes. These included the ogt gene and complete or truncated versions of the E. coli ada gene encoding either O6-alkylguanine (O6-alkG) or alkylphosphotriester (alkPT) ATase activities. The functional activity of the ATase in these cells was demonstrated by in vitro assay of cell extracts using 3H-methylated DNA as a substrate, and by the protection they provided against the growth inhibitory effects of methylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU) and the chloroethylating agent 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU). However, cells expressing the full length or the O6-alkG ATase region, but not the alkPT ATase region, of Ada were found to be more sensitive to the growth inhibitory effects of the DBA; Ogt expression sensitized cells to DBM but not significantly to DBE. Addition of DBA to cell extracts depleted O6-alkG ATase activity on the methylated DNA substrate, but had no effect on alkPT ATase activity. This suggests that ATase-mediated sensitization of the intact cells may be related to the inactivation of the ATase protein. Addition to the cell culture medium of GSH or buthionine sulfoximine in attempts to augment or deplete cellular levels of GSH had no marked effect on the ATase-mediated sensitization to DBA. This suggests that rather than GSH-mediated DNA damage, the effect may be mediated by a DNA adduct caused by the oxidative metabolic pathway. These observations indicate that expression of ATase may have a detrimental effect on cellular sensitivity to environmentally relevant alkylating agents.     

The therapeutic potential of O6-alkylguanine DNA alkyltransferase inhibitors

Resistance to O(6-)alkylating agents can be overcome by depletion of the DNA repair protein, O(6)-alkylguanine DNA alkyltransferase. Inhibitors of this protein act as pseudosubstrates and, so far, O(6)-benzylguanine and lomeguatrib have been tested in clinical trials. Inherently non-toxic, optimum doses for protein depletion have been established for both agents. Myelosuppression of alkylating agents is significantly enhanced when used in combination with these agents, necessitating significant reductions in standard doses. Consequently, no improvement in efficacy is seen. Strategies to limit myelotoxicity are complex and will be very difficult to apply clinically. O(6)-alkylguanine DNA alkyltransferase inhibition may also potentiate the toxicity of other agents such as cyclophosphamide and irinotecan. Other mechanisms of DNA repair are also important and drugs targeting some of these systems are in early phase clinical trials.     

Inhibition of DNA repair as a means of increasing the antitumor activity of DNA reactive agents

Chemotherapeutic alkylnitrosoureas (BCNU, CCNU, streptozotocin) and alkyltriazenes (DTIC, temozolomide) produce a cytotoxic lesion at the O(6)-position of guanine. The DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase removes damage from the O(6)-position in a single-step mechanism without co-factors. There is extensive evidence that this protein is one of the most important factors contributing to alkylnitrosourea and alkyltriazene treatment failure. There is an inverse correlation between the level of this protein and the sensitivity of cells to the cytotoxic effects of O(6)-alkylating agents. Attempts have been made to modulate AGT activity using anti-sense technology, methylating agents, O(6)-alkylguanines, and O(6)-benzylguanine analogs. O(6)-Benzylguanine and its analogs are clearly the most potent direct inactivators of the AGT protein. The mechanism involves O(6)-benzylguanine acting as a low-molecular weight substrate with transfer of the benzyl group to the cysteine residue within the active site of the repair protein. Pretreatment of cells with non-toxic doses of O(6)-benzylguanine results in an increase in the sensitivity to O(6)-alkylating agents. Animal studies revealed that the therapeutic index of BCNU increased when administered in combination with O(6)-benzylguanine. This drug is currently in phase I clinical trials. Evidence from animal studies indicates that myelosuppression may be the dose-limiting toxicity, thus, efforts are aimed at improving the therapeutic index by the stable expression of O(6)-benzylguanine-resistant AGT proteins into targeted normal tissue such as bone marrow. The successful modulation of alkyltransferases brings on an exciting new era for alkylnitrosoureas and alkyltriazenes.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin modulates the induction of DNA strand breaks and poly(ADP-ribose) polymerase-1 activation by 17beta-estradiol in human breast carcinoma cells through alteration of CYP1A1 and CYP1B1 expression

The primary purpose of this research is to investigate the effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) pretreatment and estrogen receptors-alpha (ER alpha) status on the induction of DNA damage by 17beta-estradiol (E 2) in human ER alpha(+)/MCF-7 and ER alpha(-)/MDA-MB-231 breast cancer cells. Results indicated that E 2 (0.1-100 nM) alone induced significant increases in cytotoxic response, reactive oxygen species (ROS) generation, and glutathione depletion in MDA-MB-231 cells but not in MCF-7 cells. At noncytotoxic concentrations, E 2 induced dose-related reduction in intracellular NAD(P)H in MDA-MB-231 cells through decreases in intracellular NAD (+) mediated by poly(ADP-ribose) polymerase-1 (PARP-1) activation as determined by detection of the presence of polymers of ADP-ribose-modified PARP-1 using Western blotting. Further investigation using the single-cell gel electrophoresis (Comet) assay confirmed that the PARP-1 activation induced by estrogen in MDA-MB-231 was due to increases in the number of DNA strand breaks. This evidence indicates that E 2 induces decreases in intracellular NAD(P)H and NAD (+) in MDA-MB-231 cells through PARP-1 activation mediated by the formation of DNA strand breaks. Further investigation indicated that the cytotoxic and DNA-damaging effects induced by E 2 in MDA-MB-231 cells were completely blocked by pretreatment of TCDD (10 nM for 72 h). In contrast, with TCDD pretreatment, significant increases in cytotoxic response, ROS generation, glutathione (GSH) depletion, DNA strand breaks, and PARP-1 activation were detected in MCF-7 cells exposed to E 2. We demonstrated that TCDD modulated the differential induction of DNA damage by estrogen in MDA-MB-231 and MCF-7 cells primarily through the inducibility of cytochrome P450 1A1 and 1B1 expression. Overall, this evidence suggests that TCDD is capable of inducing imbalances in the expression of enzymes responsible for the bioactivation of estrogen leading to the subsequent accumulation of DNA damage and initiation of DNA repair in MDA-MB-231 and MCF-7 cells. Furthermore, we confirmed that ER alpha plays a protective role in modulating the induction of DNA damage by E 2 in human breast cancer cells.     

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%.     

Poly(ADP-ribose) polymerase-independent potentiation of nitrosourea cytotoxicity by 3-aminobenzamide in human malignant glioma cells

Poly(ADP-ribose) polymerase is a zinc-finger DNA-binding protein that detects specifically DNA strand breaks generated by genotoxic agents and is thought to be involved in DNA repair. Here, we examined the effects of 3-aminobenzamide, a poly(ADP-ribose) polymerase inhibitor, on the chemosensitivity of human malignant glioma cells. 3-Aminobenzamide selectively potentiated the cytotoxicity of the nitrosoureas, nimustine, carmustine and lomustine in 10 of 12 human malignant glioma cell lines. In contrast, 3-aminobenzamide did not modulate the cytotoxic effects of doxorubicine, teniposide, vincristine, camptothecin or cytarabine. The nitrosoureas did not induce poly(ADP-ribose) polymerase activity in the glioma cells. Ectopic expression of truncated poly(ADP-ribose) polymerase containing the poly(ADP-ribose) polymerase DNA-binding domain, which acts as a dominant-negative mutant, in LN-18 or LN-229 cells did not alter the 3-aminobenzamide effect on nitrosourea-mediated cytotoxicity. Thus, 3-aminobenzamide may target another nicotinamide adenine dinucleotide (NAD)-requiring enzyme, but not poly(ADP-ribose) polymerase, when enhancing nitrosourea cytotoxicity in human malignant glioma cells. Carmustine cytotoxicity was associated with a G2/M arrest. Coexposure to carmustine and 3-aminobenzamide overcame this G2/M arrest in T98G cells, which are sensitized to carmustine by 3-aminobenzamide, but not in U251MG cells, which are refractory to 3-aminobenzamide-mediated sensitization to carmustine. Thus, 3-aminobenzamide-mediated sensitization to carmustine cytotoxicity may result from interference with the stable G2/M arrest response to carmustine in human glioma cells.     

The use of cultured primary bovine colon epithelial cells as a screening model to detect genotoxic effects of heterocyclic aromatic amines in the comet assay

Isolated epithelial cells from the bovine colon were maintained in dividing monolayer cultures and used as a model system for colon tissue in in vitro toxicological studies. The cytotoxic effects of the heterocyclic aromatic amines 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhiP) were investigated in these cells and IC(50) values were determined by inhibition of neutral red uptake into the cultured cells. Although PhiP was not cytotoxic up to concentrations of 500 microM, IQ was cytotoxic above 300 microM. The induction of DNA strand breaks in cultured bovine epithelial colon cells was determined using the alkaline single-cell gel electrophoresis (Comet assay) technique, and subsequently, the DNA damage was used as a determinant of genotoxic effects of the heterocyclic aromatic amines in order to establish this system for detection of adverse effects of chemicals in a model system for the colon. In the absence of an external enzymatic metabolizing system (S9 mix) both amines did not induce DNA strand breaks. When S9 mix was used, PhiP induced DNA strand breaks above 10 microM whereas IQ did not show any significant effect at 300 microM. This cell culture system was found to be a useful screening system for testing of compounds that are considered to affect colonic tissue.     

DNA damage-triggered apoptosis: critical role of DNA repair, double-strand breaks, cell proliferation and signaling

Genotoxic DNA damaging agents may activate both membrane death receptors and the endogenous mitochondrial damage pathway leading to cell death via apoptosis. Here, apoptotic responses in cells exhibiting a defect in various DNA repair pathways such as alkyltransferase, base excision repair, nucleotide excision repair and mismatch repair are reviewed. The HSVTk/ganciclovir and VZV/BVDU suicide system will also be discussed. Data are available to show that critical DNA damage triggers apoptosis in a DNA replication dependent way by activating the mitochondrial damage pathway in fibroblasts. It is proposed that DNA double-strand breaks (DSBs) are common ultimate apoptosis-triggering lesions arising from primary DNA lesions during DNA replication. Thus, DNA replication is a necessary component in DNA damage-triggered apoptosis, at least in fibroblasts treated with genotoxins not inducing DSBs themselves. For methylating agents inducing O(6)-methylguanine, an additional requirement is mismatch repair provoking DSB formation that triggers Bcl-2 decline and caspase-9/-3 activation. This occurs independent of p53 since most of the repair deficient cell lines under study were mutated for p53. Moreover, p53 knockout fibroblasts are more sensitive to methylating agents and UV light than p53 wt cells, suggesting p53 to play a protective rather than a pro-apoptotic role in this cell system, probably by its involvement in DNA repair. However, for lymphoblastoid cells p53 wt variants are more sensitive to DNA damage indicating that p53 participates in apoptotic signaling in a cell type-specific fashion. The role of topoisomerase II inhibitors and c-Fos/AP-1 in apoptosis will also be discussed.     

Detection of poly(ADP-ribose) synthetase activity in alveolar macrophages of rats exposed to nitrogen dioxide and ozone

The toxic effects of nitrogen dioxide (NO2) and ozone (O3) are mediated through the formation of free radicals, which can cause DNA strand breaks. The present study demonstrates that exposure to NO2 and O3 causes a stimulation of poly(ADP-ribose) (polyADPR) synthetase in alveolar macrophages of rats. Three-month-old male Sprague-Dawley rats, specific pathogen free, were exposed to either 1.2 ppm NO2 or 0.3 ppm O3 alone or a combination of these 2 oxidants continuously for 3 days. The control group was exposed to filtered room air. To evaluate whether exposure to these two oxidants (NO2 and O3) caused DNA damage to lung cells, the activity of polyADPR synthetase was measured. Cellular DNA repair is dependent upon the formation of poly(ADP-ribose) polymerase, which is catalyzed by polyADPR synthetase. PolyADPR synthetase is known to be activated in response of DNA damage. The results showed that the enzyme activity was stimulated after exposure to O3 or exposure to NO2 + O3. Ozone exposure caused a 25% increase in the enzyme activity as compared to the control. Combined exposure to NO2 + O3 showed a 53% increase in the enzyme activity. These results were statistically significant as compared to the control and NO(2) exposure groups. Other parameters such as total cell count, cell viability, and differential cell count were also determined. The stimulation of polyADPR synthetase activity after O3 exposure or NO2 + O3 exposure reflects a response to lung cellular DNA repair, which may be used as an indicator for assessing DNA damage caused by oxidant injury.     

Poly (ADP-ribose) polymerase, nitric oxide and cell death.

Poly (ADP-ribose) polymerase (PARP) is a nuclear enzyme that is activated by DNA strand breaks to participate in DNA repair. Excessive activation of PARP, however, can deplete tissue stores of nicotinamide adenine dinucleotide (NAD), the PARP substrate which, with the resultant depletion of ATP, leads to cell death. In many cases of CNS damage, for example vascular stroke, nitric oxide release is a key stimulus to DNA damage and PARP activation. In conditions as diverse as focal cerebral ischaemia, myocardial infarction and toxin-induced diabetes, PARP inhibitors and PARP gene deletion afford dramatic protection from tissue damage. Accordingly, PARP inhibitors could provide novel therapeutic approaches in a wide range of clinical disorders.     

Inactivation of O(6)-alkylguanine-DNA alkyltransferase by folate esters of O(6)-benzyl-2'-deoxyguanosine and of O(6)-[4-(hydroxymethyl)benzyl]guanine

O6-Alkylguanine-DNA alkyltransferase (alkyltransferase) provides an important source of resistance to some cancer chemotherapeutic alkylating agents. Folate ester derivatives of O6-benzyl-2'-deoxyguanosine and of O6-[4-(hydroxymethyl)benzyl]guanine were synthesized and tested for their ability to inactivate human alkyltransferase. Inactivation of alkyltransferase by the gamma-folate ester of O6-[4-(hydroxymethyl)benzyl]guanine was similar to that of the parent base. The gamma-folate esters of O6-benzyl-2'-deoxyguanosine were more potent alkyltransferase inactivators than the parent nucleoside. The 3'-ester was considerably more potent than the 5'-ester and was more than an order of magnitude more active than O6-benzylguanine, which is currently in clinical trials to enhance therapy with alkylating agents. They were also able to sensitize human tumor cells to killing by 1,3-bis(2-chloroethyl)-1-nitrosourea, with O6-benzyl-3'-O-(gamma-folyl)-2'-deoxyguanosine being most active. These compounds provide a new class of highly water-soluble alkyltransferase inactivators and form the basis to construct more tumor-specific and potent compounds targeting this DNA repair protein.     

Poly(ADPR)polymerase inhibition and apoptosis induction in cDDP-treated human carcinoma cell lines

Poly(ADPR)polymerases' (PARPs) inhibitors potentiate the cytotoxic effects of chemotherapeutic agents like alkylating compounds and TOPO I poisons, while their action in combination with cisplatin still needs investigation. In fact, one of the earliest responses to DNA single- or double-strand breaks is the synthesis of poly(ADP-ribose) (PAR) by PARPs; these enzymes are components of DNA repair machineries and substrates of caspases. Cisplatin (cDDP) yields intra- and inter-strand DNA cross-links and several proteins that recognise cDDP-induced DNA damage, such as p53, are also targets of poly(ADP-ribosyl)ation. We compared the effects of treatments with cDDP and the PARPs inhibitor PJ34 in p53 mutated carcinoma cell lines (HeLa, KB, HT29) that exhibited differential sensitivities to the drugs, in terms of cell growth inhibition and onset of apoptosis. In cDDP-resistant HT29 cells we determined: (i) PJ34 potentiation of cDDP-induced cell growth inhibition; (ii) an increment of PARP-1 automodification following cDDP treatment. In cDDP-sensitive HeLa cells, we found that the drug induced apoptotic cell death associated with caspase-dependent PARP-1 proteolysis.