Membrane transport,sulfhydryl levels and DNA cross-linking in Chinese hamster ovary cell mutants sensitive and resistant to melphalan

The mechanism of resistance to the alkylating agent melphalan was investigated in drug-sensitive and -resistant mutants of Chinese hamster ovary cells. Melphalan-resistant cells (Mel^R6), selected by a single exposure to melphalan, were 4.5-fold more resistant to drug than sensitive AUXB1 parental cells. Colchicine-resistant cells (CH^RC5), which are cross-resistant to melphalan, were 15-fold more resistant than wild type cells. The kinetic parameters for drug influx were not significantly different in sensitive and resistant cells. The steady-state drug level in both Mel^R6 and CH^RC5 cells was approximately 25 and 35% lower respectively than that in sensitive cells and this difference was accounted for by a more rapid rate of drug efflux from the resistant mutants. However, the level of drug resistance could not be explained entirely by this difference in drug transport.Sulfyddryl group levels were elevated in both Mel^R6 and CH^RC5 cells relative to sensitive cells and these differences were statistically significant (P < 0.001). Furthermore, DNA interstrand cross-link formation was significantly lower in resistant cells than in sensitive cells. A similar rate of repair of DNA interstrand cross-links was observed in sensitive and resistant cells with the possible exception of a slower rate of repair in Mel^R6 cells. A higher level of DNA-protein cross-link activity, which may represent a mechanism for drug inactivation was observed in Mel^R6 cells.These studies suggest that resistance to melphalan in Mel^R6 and CH^RC5 Chinese hamster ovary cell mutants is multifactorial involving lowered steady-state drug levels, enhanced drug efflux, elevated levels of sulfhydryl groups and decreased DNA interstrand cross-linking.     

Relative toxicities of DNA cross-links and monoadducts: new insights from studies of decarbamoyl mitomycin C and mitomycin C

Mitomycin C (MC), a cytotoxic anticancer drug and bifunctional DNA DNA alkylating agent, induces cross-linking of the complementary strands of DNA. The DNA interstrand cross-links (ICLs) are thought to be the critical cytotoxic lesions produced by MC. Decarbamoyl mitomycin C (DMC) has been regarded as a monofunctional mitomycin, incapable of causing ICLs. Paradoxically, DMC is slightly more toxic than MC to hypoxic EMT6 mouse mammary tumor cells as well as to CHO cells. To resolve this paradox, EMT6 cells were treated with MC or DMC under hypoxia at equimolar concentrations and the resulting DNA adducts were analyzed using HPLC and UV detection. MC treatment generated both intrastrand and interstrand cross-link adducts and four monoadducts, as shown previously. DMC generated two stereoisomeric monoadducts and two stereoisomeric ICL adducts, all of which were structurally characterized; one was identical with that formed with MC, the other was new and unique to DMC. Overall, adduct frequencies were strikingly higher (20-30-fold) with DMC than with MC. Although DMC monoadducts greatly exceeded DMC cross-link adducts ( approximately 10:1 ratio), the latter were equal or higher in number than the cross-link adducts from MC. DMC displayed a much higher monoadduct:cross-link ratio than MC. The similar cytotoxicities of the two drug show a correlation with their similar DNA cross-link adduct frequencies, but not with their total adduct or monoadduct frequencies. This provides specific experimental evidence that the ICLs rather than the monoadducts are critical factors in the cell death induced by MC. In vitro, overall alkylation of calf thymus DNA by DMC was much less efficient than by MC. Nevertheless, ICLs formed with DMC were clearly detectable. The chemical pathway of the cross-linking was shown to be analogous to that occurring with MC. These results also suggest that the differential sensitivity of Fanconi's Anemia cells to MC and DMC is related to factors other than a selective defect in cross-link repair.     

A novel trinuclear platinum complex overcomes cisplatin resistance in an osteosarcoma cell system

Multinuclear platinum compounds have been designed to circumvent the cellular resistance to conventional platinum-based drugs. In an attempt to examine the cellular basis of the preclinical antitumor efficacy of a novel multinuclear platinum compound (BBR 3464) in the treatment of cisplatin-resistant tumors, we have performed a comparative study of cisplatin and BBR 3464 in a human osteosarcoma cell line (U2-OS) and in an in vitro selected cisplatin-resistant subline (U2-OS/Pt). A marked increase of cytotoxic potency of BBR 3464 in comparison with cisplatin in U2-OS cells and a complete lack of cross-resistance in U2-OS/Pt cells were found. A detailed analysis of the cisplatin-resistant phenotype indicated that it was associated with reduced cisplatin accumulation, reduced interstrand cross-link (ICL) formation and DNA platination, microsatellite instability, and reduced expression of the DNA mismatch repair protein PMS2. Despite BBR 3464 charge and molecular size, in U2-OS and U2-OS/Pt cells, BBR 3464 accumulation and DNA-bound platinum were much higher than those observed for cisplatin. In contrast, the frequency of ICLs after exposure to BBR 3464 was very low. The time course of ICL formation after drug removal revealed a low persistence of these types of DNA lesions induced by BBR 3464, in contrast to an increase of DNA lesions induced by cisplatin, suggesting that components of the DNA repair pathway handle the two types of DNA lesions differently. The cellular response of HCT116 mismatch repair-deficient cells was consistent with a lack of influence of mismatch repair status on BBR 3464 cytotoxicity. Because BBR 3464 produces high levels of lesions different from ICLs, likely including intra-strand cross-links and monoadducts, the ability of the triplatinum complex to overcome cisplatin resistance appears to be related to a different mechanism of DNA interaction (formation of different types of drug-induced DNA lesions) as compared with conventional mononuclear complexes rather than the ability to overcome specific cellular alterations.     

Does the antitumor cyclopropylpyrroloindole antibiotic CC-1065 cross-link DNA in tumor cells?

We have found that a cyclopropylpyrroloindole antibiotic, compound CC-1065 (benzo[1,2-b:4,3-b']dipyrrole-3(2H)-carboxamide, 7-[[1, 6-dihydro-4-hydroxy-5-methoxy-7-[(4,5,8, 8a-tetrahydro-7-methyl-4-oxocyclopropan[c]pyrrolo[3, 2-e]indol-2(1H)-yl)carbonyl]benzo[1,2-b:4, 3-b']dipyrrol-3(2H)-yl]-carbonyl]-1,6-dihydro-4-hydroxy-5-methoxy, (7bR,8aS)), forms interstrand DNA cross-links of an apparently covalent nature in HeLa S(3) cells. This compound induced interstrand cross-links at concentrations ranging from 0.1 to 1 nM/3 hr in whole cells, but these cross-links were absent or marginally low when the drug was added to cell lysates with inactivated cellular enzymes or isolated nuclei, which suggests that metabolic activation of the drug is a necessary step for DNA cross-linking to occur. In contrast, an analog of CC-1065, Bizelesin, which forms DNA-DNA cross-links by direct alkylation, induced interstrand DNA cross-links in both whole cells and in cell lysates. Interestingly, a demethoxy analog of CC-1065, Adozelesin, did not induce DNA cross-links under the same conditions. CC-1065 was found to be extremely potent in terms of concentrations required to cross-link DNA of tumor cells, and this may be related to its remarkable cytotoxic activity.     

In vitro pharmacokinetics and pharmacodynamics of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)

The relationship between treatment efficacy and the pharmacokinetics (PK) and pharmacodynamics (PD) of anticancer drugs is poorly defined. 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is an alkylating agent used in the treatment of brain and other forms of cancer. It is postulated that BCNU kills cells by forming DNA interstrand cross-links. The present study was undertaken to characterize the PK and PD of BCNU in mouse L1210 cells. L1210 cells were exposed to BCNU (0-160 microM) and analyzed for intracellular BCNU concentrations, DNA interstrand cross-links, cell cycle phase, and cytotoxicity. The half-life of BCNU in cells was approximately 40 min. The maximum reduction of mitochondrial enzyme activity (maximum cell death) achieved within 24 hr after exposure to BCNU was concentration-dependent and could be described by a Hill equation. At lower concentrations, the area under the DNA interstrand cross-link-time curve linearly correlated with the maximum cell death and the area under the BCNU concentration-time curve. BCNU induced cell accumulation in the G(2)/M phase of the cell cycle, which continued even after apparent completion of cross-link repair. Loss of membrane permeability was minimal (approximately 2%) during the first 24 hr. Thereafter, cells died exponentially over the next 9 days, primarily by necrosis. In conclusion, while cytotoxicity was concentration-dependent, an indirect relationship was found among the time-course of BCNU concentrations, DNA interstrand cross-links, and cell death. Because of the disparity between the time-scale of PK and PD, focusing only on the early events may provide limited information about the process of anticancer drug-induced cell death.     

Near-UV photolysis of 2-methyl-1,4-naphthoquinone-DNA duplexes: characterization of reversible and stable interstrand cross-links between quinone and adenine moieties

Near-UV photolysis of 2-methyl-1,4-naphthoquinone (MQ, menadione) tethered DNA induces initial charge transfer followed by either transport of the damage to G or the formation of interstrand cross-links between MQ and DNA bases. In this work, the products responsible for interstrand cross-links have been characterized by mass spectrometry, NMR, and comparison with model compounds. Three major products were formed in the photolysis of MQ-DNA duplexes. Two of the products (isomers) have a cross-link between C2 of a 2,3-saturated MQ moiety and N6 of a dAdo moiety. These products readily convert back to MQ and dAdo upon heating in neutral solution, and thus, they lead to reversible cross-links in MQ-DNA duplexes. The third product has a cross-link between C3 of a 2,3-unsaturated MQ moiety and N6 of an dAdo moiety. This product was stable in neutral solution. The formation of MQ to A cross-links in DNA may be explained by the coupling of MQ radicals that arise from the protonation of MQ radical anions, together with adenin-N6-yl radicals that arise from the deprotonation of A radical cations.     

The role of DNA repair in nitrogen mustard drug resistance

The nitrogen mustards are an important class of DNA cross-linking agents, which are utilized in the treatment of many types of cancer. Unfortunately, resistance often develops in the treatment of patients and the tumor either never responds to or becomes refractory to these agents. Resistance to the nitrogen mustards in murine and human tumor cells has been reported to be secondary to alterations in (i) the transport of these agents, (ii) their reactivity, (iii) apoptosis and (iv) altered DNA repair activity. In the present review, we will discuss the role of DNA repair in nitrogen mustard resistance in cancer. The nitrogen mustards' lethality is based on the induction of DNA interstrand cross-links (ICLs). Two DNA repair pathways are known to be involved in removal of ICLs: non-homologous DNA end-joining (NHEJ) and Rad51-related homologous recombinational repair (HRR). The reports discussed here lead us to hypothesize that low NHEJ activity defines a hypersensitive state, while high NHEJ activity, along with increased HRR activity, contributes to the resistant state in chronic lymphocytic leukemia. Studies on human epithelial tumor cell lines suggest that HRR rather than NHEJ plays a role in nitrogen mustard sensitivity.     

The formation of DNA interstrand cross-links by a novel bis-[Pt2Cl4(diminazene aceturate)2]Cl4.4H2O complex inhibits the B to Z transition

We present data demonstrating that the cytotoxic compound [Pt2Cl4(diminazene aceturate)2]Cl4.4H2O (Pt-berenil) circumvents cisplatin resistance in ovarian carcinoma cells. The analysis of the interaction of Pt-berenil with linear and supercoiled DNA indicates that this compound induces the formation of a large number of covalent interstrand cross-links on DNA and that this number is significantly higher than that produced by cis-diamminedichloroplatinum(II) (cis-DDP). Renaturation experiments, interstrand cross-link assays, and electron microscopy indicate that the kinetics of DNA interstrand cross-link formation caused by Pt-berenil binding is faster than that caused by cis-DDP at similar levels of platinum bound to DNA. Furthermore, the number of DNA interstrand cross-links in Pt-berenil-DNA complexes is influenced by supercoiling. Circular dichroism experiments show that Pt-berenil strongly inhibits the B-DNA-to-Z-DNA transition of poly(dG-m5 dC). poly(dG-m5dC) at salt concentrations (3 mM MgCl2) at which the native methylated polynucleotide readily adopts the Z-DNA conformation, which suggests that the induction of interstrand cross-links by Pt-berenil inhibits the Z-DNA transition. On the basis of these results, we propose that bis(platinum) compounds with structure similar to Pt-berenil may act as blockers of DNA conformational changes and may also display activity in cisplatin-resistant cells.     

A comparative analysis of drug-induced DNA effects in a nitrogen mustard resistant cell line expressing sensitivity to nitrosoureas

In the Walker 256 rat mammary carcinoma cell line, WR, resistance to nitrogen mustards (NM) is accompanied by collateral sensitivity to chloroethylnitrosoureas (CENUs). DNA-interstrand cross-links, DNA-protein cross-links, and sister chromatid exchange (SCE) induction were assayed in WR and the parent cell line (WS) after treatment with nitrogen mustard (HN2), phosphoramide mustard (PM), chlorozotocin (CLZ) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). Treatment of cells with HN2 caused extensive levels of cross-links, approximately 50% of which were DNA-interstrand, equal in both WR and WS, whereas PM caused no detectable cross-links in either cell line. CLZ induced low levels of DNA-interstrand cross-links, similar in WR and WS, but no DNA-interstrand cross-links could be detected in either cell line after treatment with CCNU. Both CLZ and CCNU induced low levels of DNA-protein cross-links in both cell lines, though higher in WR than WS. There was no difference in the rate of removal of HN2-induced DNA-interstrand or DNA-protein cross-links or total CLZ-induced cross-links by the two cell lines, suggesting that differential repair was not relevant to the expression of resistance. Both HN2 and PM caused more SCEs in WS than in WR, whereas CLZ and CCNU induced more SCEs in WR. Thus, NM-induced SCEs were related to cell killing but not cross-linking, whilst CENU-induced SCEs were related to cell killing and DNA-protein but not DNA-interstrand cross-links. Furthermore, the collateral sensitivity of WR cells to CENUs was not due to the differential induction of DNA-interstrand cross-links or repair of total cross-links, or repair of total cross-links, although higher levels of DNA-protein cross-links occurred in WR, and these may be either a cause or a consequence of increased susceptibility of these cells to CENUs. Presumably NMs and CENUs have several distinct and separate macromolecular targets which result in differential cell killing. It is concluded that a range of lesions occurred after treatment of WR and WS cells with either NMs or CENUs and that, in these cell lines, there is no simple correlation between drug-induced cross-linking, SCE induction and cytotoxicity.     

DNA interactions of new antitumor platinum complexes with trans geometry activated by a 2-metylbutylamine or sec-butylamine ligand

The global modification of mammalian and plasmid DNAs by novel platinum compounds, trans-[PtCl(2)(NH(3))(Am)], where Am=2 -methylbutylamine or sec-butylamine was investigated in cell-free media using various biochemical and biophysical methods. These modifications were analyzed in the context of the activity of these new compounds in several tumor cell lines including those resistant to antitumor cis-diamminedichloroplatinum(II) (cisplatin). The results showed that the replacement of one amine group by 2-methylbutylamine or sec-butylamine ligand in clinically ineffective trans-diamminedichloroplatinum(II) (transplatin) resulted in a radical enhancement of its activity in tumor cell lines so that they are more cytotoxic than cisplatin and exhibited significant antitumor activity including activity in cisplatin-resistant tumor cells. Importantly, this replacement also markedly altered DNA binding mode of transplatin and reduced the efficiency of repair systems to remove the adducts of the new analogues from DNA. The results support the view that one strategy to activate trans geometry in bifunctional platinum(II) compounds including circumvention of resistance to cisplatin may consist in a chemical modification of the ineffective transplatin which results in an increased efficiency to form DNA interstrand cross-links.     

Induction of alkylator (melphalan) resistance in HL60 cells is accompanied by increased levels of topoisomerase II expression and function.

Human leukemic HL60 cells were selected for resistance to alkylating agents by stepwise exposure to increasing concentrations of L-phenylalanine mustard (melphalan). The resulting resistant cell line (R-HL60) was 4-fold resistant (melphalan IC50 value, 27.84 +/- 4.2 microM) to melphalan compared with parental HL60 cells (melphalan IC50 value, 6.9 +/- 1.78 microM). Nuclear extracts from R-HL60 cells possess a approximately 4-fold increase in DNA topoisomerase II activity compared with parental HL60 cells. As determined using Western blot analysis, the level of topoisomerase IIalpha protein expressed in R-HL60 cells was approximately 3-fold that of parental HL60 cells. However, there were no differences observed in the level of topoisomerase IIbeta protein, in the topoisomerase I activity, or in the level of topoisomerase I protein expression comparing the two cell lines. R-HL60 cells were 5-fold more sensitive than parental HL60 cells to the cytotoxic effect of the topoisomerase II inhibitor doxorubicin. The sensitivity to the cytotoxic effects of the topoisomerase I inhibitor camptothecin did not differ in R-HL60 and parental HL60 cell lines. Preincubation with doxorubicin significantly increased melphalan-induced interstrand DNA cross-link formation and cytotoxicity in R-HL60 cells compared with the parental HL60 cells. The affinity of topoisomerase II for UV-irradiated cross-linked HL60 DNA was increased by approximately 2.5-fold compared with that of HL60 native DNA. The affinity of topoisomerase II for both UV-irradiated (cross-linked) and native DNA was significantly decreased after doxorubicin pretreatment. Elevated topoisomerase II activity and the increased affinity of topoisomerase II for cross-linked DNA in melphalan-resistant cells seems to contribute to alkylator resistance by changing DNA topology, thereby facilitating DNA repair.     

上调NDRG1表达降低奥沙利铂耐药结肠癌细胞的存活率

目的观察上调NDRG1表达对结肠癌细胞及奥沙利铂耐药结肠癌细胞的毒性作用并探讨其机制。方法以重组真核表达质粒pEGFP-NDRG1-N3转染HCT 116及奥沙利铂耐药的OHP-HCT 116结肠癌细胞,以实时定量(qRT)-PCR法和Western blot法检测转染效率。MTT法检测奥沙利铂对不同结肠癌细胞的毒性及验证OHP-HCT 116细胞的耐药性。给予奥沙利铂干预转染pEGFP-NDRG1-N3的HCT 116细胞及OHP-HCT 116细胞,MTT法检测细胞存活率,流式细胞术检测细胞凋亡率,Western blot法检测凋亡蛋白Bcl-2及p53蛋白水平变化。结果不同浓度奥沙利铂干预下的3株结肠癌细胞中HCT 116细胞对奥沙利铂最敏感,OHP-HCT 116细胞对奥沙利铂耐药得到验证。以不同浓度梯度奥沙利铂干扰转染pEGFP-NDRG1-N3的HCT 116细胞及OHP-HCT 116细胞,与MOCK及siNC组比较,在HCT 116细胞及OHP-HCT 116细胞,转染pEGFP-NDRG1-N3细胞的存活率下降(P<0.05),凋亡率升高(P<0.05),Bcl-2表达降低(P<0.05),p53表达升高(P<0.05)。结论上调NDRG1表达通过调控p53表达改善结肠癌细胞奥沙利铂耐药,提高化疗敏感性。     

Nuclear and mitochondrial distribution of organoamidoplatinum(II) lesions in cisplatin-sensitive and -resistant adenocarcinoma cells

The DNA binding pattern of the organoamidoplatinum(II) compound 1a is of considerable interest because of its known activity against cisplatin-resistant cells. The activity of 1a appears to be due at least in part to a greater cellular uptake than cisplatin into cisplatin-resistant cells, but little is known of the DNA reactions of the organoamidoplatinum(II) compounds. In this study the level of DNA cross-linking and total DNA lesions formed by 1 a were measured by gene-specific Southern hybridization cross-linking assays and by quantitative PCR in cisplatin-sensitive (2008) and in cisplatin-resistant 2008/R human adenocarcinoma cell lines. The surprising result was that the major difference between cisplatin and 1a was that the number of interstrand cross-links induced by 1a were approximately 5-fold greater than that induced by cisplatin in the nuclear (but not mitochondrial) DNA of resistant cells, even though the total number of lesions were essentially the same in both sensitive and resistant cells. This result suggests that the extent of interstrand cross-linking is a critical determinant of the cellular response to 1a and that the enhanced uptake of 1a into resistant cells results in this elevated level of cross-linking, leading to good activity of 1a against cisplatin-resistant cells. It remains unclear as to why 1a exhibits such selective damage to nuclear DNA, and insight into the molecular aspects of this selectivity will provide new opportunities for the further development of new platinum-based agents with activity against cisplatin-resistant cells.     

Kinetics of reaction of cis-diamminedichloroplatinum(II) with DNA

A method based on cation exchange chromatography was developed to determine the adducts formed in the reaction of cis-diamminedichloroplatinum(II) (cis-Pt) with DNA. DNA was incubated with various concentrations of cis-Pt for various periods of time, ethanol precipitated, and enzymatically digested to nucleosides and Pt-containing oligonucleotides. The unmodified nucleosides were separated from the positively charged intra- and interstrand cis Pt adducts with a weak cation exchanger, CM-Sephadex C-25, and the adducts were further purified by HPLC. The main adduct was shown to be an intrastrand cross-link of cis-Pt bound to the N-7 atoms of two neighboring guanines. The minor adducts were intra- and interstrand cross-links of cis-Pt with adenine and guanine and an interstrand cross-link of cis-Pt with two guanines. At low levels of DNA-modification (cis-Pt:nucleotide = 1:50-1:1000) the intrastrand cross-link of cis-Pt with two guanines consisted of 60-70% of the total platination of DNA. At higher levels of DNA-modification (greater than 1:20), the amount of undigested products increased, indicating shielding of DNA by cis-Pt from nucleolytic enzymes.     

Quantitative effects on c-Jun N-terminal protein kinase signaling determine synergistic interaction of cisplatin and 17-allylamino-17-demethoxygeldanamycin in colon cancer cell lines

We investigated the effects of cisplatin and the hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) in combination in a panel of human colon adenocarcinoma cell lines that differ in their p53 and mismatch repair status. Analysis of cytotoxicity after combined treatment revealed additive effects of cisplatin and 17-AAG in the HCT 116, DLD1, and SW480 cell lines and antagonism in HT-29 cells. Clonogenic assays demonstrated antagonism in HT-29, an additive effect in SW480, and synergism in HCT 116 and DLD1 cell lines. Analysis of signaling pathways revealed that cisplatin-induced activation of c-Jun N-terminal kinase (JNK) was fully blocked by 17-AAG in HT-29 and SW480 cells, whereas in HCT 116 and DLD1 cells it was inhibited only partially. The activation of caspases was also more pronounced in DLD1 and HCT 116 cell lines. These data suggested that a minimal level of apoptotic signaling through JNK was required for synergism with this combination. To test this hypothesis, we used the specific JNK inhibitor SP600125; when JNK was inhibited pharmacologically in HCT 116 and DLD1 cells, they demonstrated increased survival in clonogenic assays. Alternatively, sustained activation of JNK pathway led to an increase of the cytotoxicity of the cisplatin/17-AAG combination in HT-29 cells. Taken together, these data suggest that the synergistic interaction of this combination in colon cancer cell lines depends on the effect exerted by 17-AAG on cisplatin-induced signaling through JNK and associated pathways leading to cell death. An implication of that finding is that quantitative effects of signaling inhibitors may be critical for their ability to reverse cisplatin resistance.     

Transplatin is cytotoxic when photoactivated: enhanced formation of DNA cross-links

It is well-known that although cisplatin, [cis-[PtCl2(NH3)2], is an anticancer drug, its isomer transplatin is not cytotoxic. Here we show that transplatin is almost as cytotoxic as cisplatin when treated cells (human keratinocytes HaCaT and ovarian cancer A2780 cells) are irradiated with UVA light (50 min, 1.77 mW cm-2). Chemical studies show that light activates both chloride ligands of transplatin, and experiments on pSP73 plasmid DNA and a 23 base-pair DNA duplex show that irradiation can greatly enhance formation of interstrand cross-links and of DNA-protein cross-links (which are not formed in the dark). Comet assays showed that UVA irradiation of transplatin-treated cells resulted in an increased inhibition of H2O2-induced DNA migration, supporting the conclusion that the cytotoxicity of photoactivated transplatin is mainly due to formation of DNA interstrand and DNA-protein cross-links.     

Role of mismatch repair and MGMT in response to anticancer therapies

Tumor resistance to cytotoxic chemotherapy drugs and their toxicity to normal cells are major clinical obstacles to anticancer therapy effectiveness. Alterations in various DNA repair pathways play a key role in the development of both mechanisms of drug resistance and toxicity. Since deregulation of the DNA damage response and alterations in DNA repair pathways are relatively common in human cancer, the knowledge of these alterations in cancer cells would be an important predictive factor for the clinical response to chemotherapy and a useful guide in designing an appropriate therapeutic strategy. This review is focused on the mismatch repair (MMR) pathway and the O(6)-methylguanine-DNA-methyltransferase (MGMT) repair protein. In particular, we examine how inactivation of these DNA repair mechanisms might affect the response of tumor cells to chemotherapy, with a special emphasis on agents inducing methylation and oxidative DNA damage and interstrand DNA cross-links (ICLs). In addition, we provide novel experimental evidence indicating that MMR is required for efficient repair of ICLs via stabilization of RAD51 containing repair intermediates. Finally, we discuss possible emerging therapeutical strategies for treating MMR-defective tumors.     

DNA repair mechanisms are involved in the hypoxia-dependent toxicity of NLCQ-1 (NSC 709257) and its synergistic interaction with alkylating agents

BACKGROUND: The hypoxia selective cytotoxin NLCQ-1 significantly potentiates the antitumor effect of several alkylating agents in vitro and in vivo. Synergy in mice requires administration of NLCQ-1 ca. 1 h before the alkylating agent, a fact that may be related to an in vitro hypoxic pretreatment effect. Since NLCQ-1 targets DNA through weak intercalation, the induction of DNA lesions upon reductive metabolism may be a reasonable mechanism that predisposes cells to further damage by a subsequent alkylating agent. MATERIALS AND METHODS: To indirectly identify such lesions, cell lines defective in specific DNA repair genes (EM9 and UV41) and their repair-proficient parental AA8, were exposed to NLCQ-1 +/-L-PAM/cisDDP under hypoxic/aerobic conditions and appropriate administration routes, and assessed for clonogenicity. Selected comparisons with tirapazamine (TPZ) were also performed. RESULTS: EM9 cells, which lack the functional XRCC1 gene involved in base excision repair, and thus are unable to efficiently repair DNA single-strand breaks (ssbs), were 3.7-fold and 4.5-fold more sensitive to NLCQ-1 and TPZ, respectively, than the parental AA8 cells, under hypoxic conditions. UV41 cells, which are defective in repairing DNA interstrand cross-links (ERCC4/XPF), were 4.5-fold more sensitive than AA8 cells to NLCQ-1. In potentiation studies with melphalan or cisplatin, synergy was observed in AA8 cells but not in EM9 or UV41 cells, with either NLCQ-I or TPZ. CONCLUSION: These results suggest that NLCQ-1 is involved in the formation of DNA ssbs and interstrand crosslinks under hypoxic conditions. The synergistic interaction of NLCQ-1 with L-PAM or cisDDP is probably due to an enhancement in the L-PAM/cisDDP-induced DNA interstrand cross-links, possibly as a result of an inhibited repair mechanism of these lesions.     

Synthesis and characterization of DNA containing an N1-2'-deoxyinosine-ethyl-N3-thymidine interstrand cross-link: a structural mimic of the cross-link formed by 1,3-bis-(2-chloroethyl)-1-nitrosourea

Interstrand cross-links, which are generated by chemotherapeutic treatment with bis-alkylating agents, exert their therapeutic effect by connecting the nucleobases of adjacent DNA strands together and represent some of the most threatening forms of damage suffered by genomic DNA. However, one of the reasons for treatment failure using these agents is due to enhanced repair of this DNA damage. The pursuit of understanding the repair of interstrand cross-links by repair systems has necessitated the synthesis of sufficient quantities of such damaged DNA. We report the synthesis of a site-specific interstrand cross-linked duplex containing an ethylene-bridged N (1)-2'-deoxyinosine- N (3)-thymidine base pair prepared by solution and solid-phase synthesis as a mimic for the lesion formed by the therapeutic agent 1,3-bis-(2-chloroethyl)-1-nitrosourea using both a phosphoramidite and a bis-phosphoramidite approach. UV thermal denaturation experiments revealed that this cross-linked duplex was stabilized by 52 degrees C relative to the noncross-linked control, and circular dichroism studies indicated little deviation from a B-form structure compared to a duplex that contained a G-C base pair at the same position. Molecular models of the cross-linked duplex that were geometry optimized using the AMBER forcefield also suggest that this lesion induces minimal distortion in B-form DNA. This modified oligonucleotide will be useful for studies related to the investigation of interstrand cross-linked DNA repair.