Structure of the d(CGCGAATTCGCG)2 complex of the minor groove binding alkylating agent alkamin studied by mass spectrometry

Nitrogen mustard alkylating agents are important cancer drugs. Much interest has been focused on redirecting their covalent adducts from the N7 atoms of guanine in the major groove of DNA to the N3 atoms of adenine in the minor groove by attaching mustard groups to AT-selective minor groove binding ligands. Here we describe the use of electrospray ionization and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry to study the structure of the DNA complexes of two minor groove binding polybenzamide mustards, alkamin and alkamini; the former is a bis-half-mustard in which reactive groups are disposed at each end of the ligand, and the latter is its monofunctional analog. Alkamin is potently cytotoxic and active in experimental mouse tumor models, whereas alkamini is not. We have studied their interaction with the DNA dodecamer d(CGCGAATTCGCG)(2), designated A2T2, and we provide a detailed analysis of the observed DNA-ligand adduct ions and their fragmentation products. We find that alkamini alkylates A2T2 at guanine G4 and adenines A5 and A6 in a manner consistent with covalent attack on purine N3 atoms from the minor groove of the AT tract. Alkamin also forms monofunctional adducts at G4 and both adenines in which the second mustard arm is hydrolyzed but, in addition, forms a variety of interstrand cross-links between adenines A5/A6 and A5'/A6', an interstrand cross-link between G4 and A6', and an intrastrand cross-link between G4 and A6. We conclude that the marked cytotoxicity of alkamin and its experimental antitumor activity could be the consequence of its ability to cross-link cellular DNA at AT tract sequences.     

Base specificity for DNA interstrand cross-linking induced by anticancer agent bizelesin

Bizelesin is a promising novel anticancer agent which is known to alkylate N3 of adenine to induce DNA interstrand cross-links (ISC) within 5′-TAATTA and 5′-TAAAAAA. We have investigated the base specificity for DNA ISC induced by bizelesin using oligomers containing the cross-linkable sequence 5′-TAATTN, in which “N” was either A, C, G, or T. An analysis of denaturing polyacrylamide gel showed that bizelesin is able to induce DNA ISC in the duplex oligomer containing sequences 5′-TAATTA and 5′-TAATTG. The formation of interstrand cross-linking did not occur in the sequences 5′-TAATTC and 5′-TAATTT. DNA strand cleavage assay to determine the cross-linking site within 5′-TAATTG sequence showed that bizelesin alkylates guanine. These results demonstrate that bizelesin is able to induce DNA ISC at guanine but not at cytosine or thymine. In addition, guanine adducts have been found to be susceptible to DNA strand cleavage by exposure to hot piperidine. The extent of DNA strand cleavage, however, was not 100% efficient in either neutral pH buffer or hot piperidine.     

Synthesis and anti-tumour activity of the spatially-separated mustard bis-N,N'-[3-(N-(2-chloroethyl)-N-ethyl)amino-5-[N,N-dimethylamino)methy l)-aminophenyl]-1,4-benzenedicarboxamide, which alkylates DNA exclusively at adenines in the minor groove

The mustard derivative, bis-N,N'-[3-(N-(2-chloroethyl)-N-ethyl)amino-5- [N,N-dimethylamino)methyl)aminophenyl]-1,4-benzenedicarboxamide has been synthesized from 3-acetamido-5-nitrobenzoic acid in a 6-step procedure. This compound alkylates exclusively in the minor groove of DNA, at the N3 site of adenines occurring in sequences of runs of adenines and (to a small extent) at 5'-TA and 5'-AT sites. Gel electrophoresis studies and in vitro cytotoxicity assays against repair-deficient AA8 mutant cell lines show it has a high degree of DNA interstrand cross-linking ability.     

DNA minor groove alkylating agents

Recent work on a number of different classes of anticancer agents that alkylate DNA in the minor groove is reviewed. There has been much work with nitrogen mustards, where attachment of the mustard unit to carrier molecules can change the normal patterns of both regio- and sequence-selectivity, from reaction primarily at most guanine N7 sites in the major groove to a few adenine N3 sites at the 3'-end of poly(A/T) sequences in the minor groove. Carrier molecules discussed for mustards are intercalators, polypyrroles, polyimidazoles, bis(benzimidazoles), polybenzamides and anilinoquinolinium salts. In contrast, similar targeting of pyrrolizidine alkylators by a variety of carriers has little effect of their patterns of alkylation (at the 2-amino group of guanine). Recent work on the pyrrolobenzodiazepine and cyclopropaindolone classes of natural product minor groove binders is also reviewed.     

Development of distamycin-related DNA binding anticancer drugs

The relatively low therapeutic index of the clinically used alkylating agents is probably related to the fact that these compounds cause DNA damage in a relatively unspecific manner, mainly involving guanine-cytosine rich stretches of DNA present in virtually all genes, therefore inducing unselective growth inhibition and death, both in neoplastic and in highly proliferative normal tissues. These considerations explain why in the last twenty years there has been an increasing interest in the identification of compounds which can target DNA with a much higher degree of sequence specificity than that of conventional alkylators. Minor groove binders (MGBs) are one of the most widely studied class of alkylating agents characterised by a high level of sequence specificity. The prototype of this class of drugs is distamycin A which is an antiviral compound able to interact, non-covalently, in the minor groove of DNA in A-T rich regions. It is not cytotoxic against tumour cells and thus has been used as a carrier for targeting cytotoxic alkylating moieties in the minor groove of DNA. The benzoyl mustard derivative of distamycin A, tallimustine, was found to be able to alkylate the N₃ of adenine in the minor groove of DNA only in the target hexamer 5’-TTTTGA or 5’-TTTTAA. Tallimustine was investigated in the clinic and was not successful because it causes severe bone marrow toxicity. The screening of other distamycin derivatives, which maintain antitumour activity and exhibit much lower toxicity against human bone marrow cells than tallimustine led to the identification of brostallicin (PNU-166196) which is currently under early clinical investigation. Although MGBs which bind DNA in A-T rich regions have not fulfilled the expectations, it is too early to draw definitive conclusions on this class of compounds. The peculiar bone-marrow toxicity observed in the clinic both with tallimustine or with CC-1065 derivatives is not necessarily a feature of all MGBs, as indicated by recent evidence obtained with brostallicin and other structurally unrelated MGBs (e.g., ET-743).     

Cross-linking of the DNA repair protein Omicron6-alkylguanine DNA alkyltransferase to DNA in the presence of antitumor nitrogen mustards

The antitumor activity of chemotherapeutic nitrogen mustards including chlorambucil, cyclophosphamide, and melphalan is commonly attributed to their ability to induce DNA-DNA cross-links by consecutive alkylation of two nucleophilic sites within the DNA duplex. DNA-protein cross-linking by nitrogen mustards is not well characterized, probably because of its inherent complexity and the insufficient sensitivity of previous methodologies. If formed, DNA-protein conjugates are likely to contribute to both target and off-target cytotoxicity of nitrogen mustard drugs. Here, we show that the DNA repair protein, O (6)-alkylguanine DNA alkyltransferase (AGT), can be readily cross-linked to DNA in the presence of nitrogen mustards. Both chlorambucil and mechlorethamine induced the formation of covalent conjugates between (32)P-labeled double-stranded oligodeoxynucleotides and recombinant human AGT protein, which were detected by SDS-PAGE. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT that had been treated with the guanine half-mustards of chlorambucil or mechlorethamine revealed the ability of the protein to form either one or two cross-links to guanine. C145A AGT (a variant containing a single point mutation in the protein's active site) was found capable of forming a single guanine conjugate, while cross-linking was virtually abolished upon treatment of the C145A/C150S AGT double mutant with the guanine half-mustards. HPLC-ESI (+)-MS/MS sequencing of tryptic peptides obtained from the wild-type AGT protein that had been treated with nitrogen mustards in the presence of DNA confirmed that the cross-linking took place between the N7 position of guanine in DNA and two active site residues within the AGT protein (Cys (145) and Cys (150)). The exact chemical structures of AGT-DNA cross-links induced by chlorambucil and mechlorethamine were identified as N-(2-[ S-cysteinyl]ethyl)- N-(2-[guan-7-yl]ethyl)- p-aminophenylbuyric acid and N-(2-[ S-cysteinyl]ethyl)- N-(2-[guan-7-yl]ethyl)methylamine, respectively, based upon HPLC-MS/MS analysis of protein hydrolysates in parallel with the corresponding amino acid conjugates prepared synthetically. Mechlorethamine-induced AGT-DNA conjugates were isolated from protein extracts of AGT-expressing CHO cells but not control cells, demonstrating that nitrogen mustards can cross-link the AGT protein to DNA in the presence of other nuclear proteins. Because AGT is overexpressed in many tumor types, further investigations of the potential role of AGT-DNA cross-linking in the antitumor and mutagenic activity of antitumor nitrogen mustards are warranted.     

Structural elucidation of a novel DNA-DNA cross-link of 1,2,3,4-diepoxybutane

DNA-DNA cross-linking by 1,2,3,4-diepoxybutane (DEB) is considered the molecular basis for its potent cytotoxic and genotoxic effects. DEB reactions with DNA initially lead to N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine monoadducts, which can then alkylate neighboring DNA bases to form bifunctional lesions. We recently reported the structures of four regioisomeric guanine-adenine adducts of DEB involving the N7 position of guanine and the N1, N3, N6, and N7 positions of adenine (Park, S., et al. (2004) Chemical Research in Toxicology 17, 1638-1651). In the present work, a novel bifunctional DNA lesion of DEB was identified as 1-(hypoxanth-1-yl)-4-(guan-7-yl)-2,3-butanediol (N1HX-N7G-BD). An authentic standard of N1HX-N7G-BD was prepared and structurally characterized by proton NMR, UV, and mass spectrometry. HPLC-ESI-MS/MS analyses of acid hydrolysates of DEB-treated calf thymus DNA revealed a peak that had the same retention time, MS/MS fragmentation, and UV spectrum as the authentic standard of N1HX-N7G-BD. We propose that N1HX-N7G-BD is formed by the hydrolytic deamination of previously reported 1-(aden-1-yl)-4-(guan-7-yl)-2,3-butanediol. Although N1HX-N7G-BD adducts are less abundant in DEB-treated DNA than the corresponding guanine-guanine cross-links, they may play a role in the induction of both AT and GC base pair mutations.     

DNA minor groove alkylating agents structurally related to distamycin A

Analogues of naturally occurring antitumour agents, such us distamycin A, which bind in the minor groove of DNA, represent a new class of antineoplastic compounds currently under investigation. Distamycin Ahas attracted researchers’ attention not only for its biological activity, but also for its non-intercalative binding to the minor groove of double-stranded B-DNA, where it forms a strong reversible complex preferentially at the nucleotide sequences consisting of 4 - 5 adjacent AT base pairs. Distamycin has also been used as a DNA sequence-selective vehicle for the delivery of alkylating functions to DNA targets, leading to a sharp increase in cytotoxicity, in comparison to that of distamycin alone. In the last few years, several hybrid compounds, in which known antitumour derivatives or simple active moieties of known antitumour agents have been tethered to distamycin frames, have been designed, synthesised and tested. Several efforts have been made to modify the DNA sequence selectivity and stability of distamycin; structural modifications have been based on replacement of pyrrole by other heterocycles and/or benzoheterocycles resulting in a novel class of minor groove binding molecules called lexitropsins. The role of the amidino moiety has also been studied by substitution with various groups, including ionisable, acid or basic and non-ionisable groups. The synthesis of a hybrid derived from combining distamycin A and a naturally occurring alkylating agent structurally related to pyrrolo [2,1-c][1,4] benzodiazepine group, such as anthramycin and DC-81, has been also reported. Several classes of distamycin derivatives that have been reported in the published literature and in recent patent fillings have been described in this review article.     

Ecteinascidin 743: a minor groove alkylator that bends DNA toward the major groove.

The ecteinascidins (Ets), which are natural products derived from marine tunicates, exhibit potent antitumor activity. Of the numerous Ets isolated, Et 743 is presently being evaluated in phase II clinical trials. Et 743 binds in the minor groove of DNA and alkylates N2 of guanine. Although structurally similar to saframycin, which exhibits poor activity in cellular assays, Et 743 has shown good efficacy as an antitumor agent. In this study, DNA structural distortions induced by Et 743 were examined to provide insight into the molecular basis for the antitumor activity of Et 743. Electrophoretic mobility shifts of ligated oligomers containing site-directed adducts were used to examine the extent and direction of the Et 743-induced bend. Surprisingly, we find that Et 743 bends DNA toward the major groove, which is a unique feature among DNA-interactive agents that occupy the minor groove.     

Pyrazole analogues of the bispyrrolecarboxamide anti-tumour antibiotics: synthesis, DNA binding and anti-tumour properties.

Four bispyrazole compounds have been prepared as potentially more stable analogues of the DNA minor groove binding polypyrrole compounds netropsin and distamycin, which are susceptible to oxidative breakdown. These compounds bind less strongly to DNA, and show much lower specificity for binding to AT-rich DNA sequences in comparison with distamycin. N.m.r. studies show that two of these compounds cause a downfield shift of the DNA imino proton resonances on interaction with the oligonucleotide d(ATATATATAT)2, suggesting that these isomers can adopt low-energy conformations similar to that shown by distamycin in its DNA minor groove binding site. The benzoic acid mustard analogue of one of the minor groove binding bispyrazoles was prepared, and showed in vitro cytotoxicity comparable with that of the previously-reported distamycin mustard, but only a low level of activity in vivo.     

Biologically relevant oxidants cause bound proteins to readily oxidatively cross-link at Guanine

Oxidative DNA-protein cross-links have received less attention than other types of DNA damage and remain as one of the least understood types of oxidative lesion. A model system using ribonuclease A and a 27-nucleotide DNA was used to determine the propensity of oxidative cross-linking to occur in the presence of oxidants. Cross-link formation was examined using four different oxidation systems that generate singlet oxygen, superoxide, and metal-based Fenton reactions. It is shown that oxidative cross-linking occurs in yields ranging from 14% to a maximal yield of 61% in all oxidative systems when equivalent concentrations of DNA and protein are present. Because singlet oxygen is the most efficient oxidation system in generating DNA-protein cross-links, it was chosen for further analyses. Cross-linking occurred with single-stranded DNA binding protein and not with bovine serum albumin. Addition of salt lowered nonspecific binding affinity and lowered cross-link yield by up to 59%. The yield of cross-linking increased with increased ratios of protein compared with DNA. Cross-linking was highly dependent on the number of guanines in a DNA sequence. Loss of guanine content on the 27-nucleotide DNA led to nearly complete loss in cross-linking, while primer extension studies showed cross-links to predominantly occur at guanine base on a 100-nucleotide DNA. The chemical species generated were examined using two peptides derived from the ribonuclease A sequence, N-acetyl-AAAKF and N-acetyl-AYKTT, which were cross-linked to 2'-deoxyguanosine. The cross-link products were spiroiminodihydantoin, guanidinohydantoin, and tyrosyl-based adducts. Formation of tyrosine-based adducts may be competitive with the more well-studied lysine-based cross-links. We conclude that oxidative cross-links may be present at high levels in cells since the propensity to oxidatively cross-link is high and so much of the genomic DNA is coated with protein.     

Design, synthesis, and evaluation of a novel sequence-selective epoxide-containing DNA cross-linking agent based on the pyrrolo[2, 1-c][1,4]benzodiazepine system.

Synthetic routes have been investigated to prepare a novel C8-epoxide-functionalized pyrrolo[2,1-c][1,4]benzodiazepine 6 as a potential sequence-selective DNA cross-linking agent (Wilson et al. Tetrahedron Lett. 1995, 36, 6333-6336). A successful synthesis was accomplished via a 10-step route involving a pro-N10-Fmoc cleavage method that should have general applicability to other pyrrolobenzodiazepine (PBD) molecules containing acid- or nucleophile-sensitive groups. During the course of this work, a one-pot reductive cyclization procedure for the synthesis of PBD N10-C11 imines from nitro dimethyl acetals was also discovered, although this method results in C11a racemization which can reduce DNA binding affinity and cytotoxicity. The target epoxide 6 was shown by thermal denaturation studies to have a significantly higher DNA-binding affinity than the parent DC-81 (3) or the C8-propenoxy-PBD (15), which is structurally similar but lacks the epoxide moiety. The time course of effects upon thermal denaturation indicated a rapid initial binding phase followed by a slower phase consistent with the stepwise cross-linking of DNA observed for a difunctional agent. This was confirmed by an electrophoretic assay which demonstrated efficient induction of interstrand cross-links in plasmid DNA at concentrations >1 microM. Higher levels of interstrand cross-linking were observed at 24 h compared to 6 h incubation. A Taq polymerase stop assay indicated a preference for binding to guanine-rich sequences as predicted for bis-alkylation in the minor groove of DNA by epoxide and imine moieties. The pattern of stop sites could be partly rationalized by molecular modeling studies which suggested low-energy models to account for the observed binding behavior. The epoxide PBD 6 was shown to have significant cytotoxicity (45-60 nM) in the A2780, CH1, and CH1cis(R) human ovarian carcinoma cell lines and an IC(50) of 0.2 microM in A2780cis(R). The significant activity of 6 in the cisplatin-resistant CH1cis(R) cell line (IC(50) = 47 nM) gave a resistance factor of 0.8 compared to the parent cell line, demonstrating no cross-resistance with the major groove cross-linking agent cisplatin.     

DNA structural perturbation induced by the CPI-derived DNA interstrand cross-linker: molecular mechanisms for the sequence specific recognition

The highly potent cytotoxic DNA-DNA cross-linker consists of two cyclopropa[c]pyrrolo[3, 4-3]indol-4(5H)-ones indoles [(+)-CPI-I] joined by a bisamido pyrrole (abbreviated to "Pyrrole"). The Pyrrole is a synthetic analog of Bizelesin, which is currently in phase II clinical trials due to its excellent in vivo antitumor activity. The Pyrrole has 10 times more potent cytotoxicity than Bizelesin and mostly form DNA-DNA interstrand cross-links through the N3 of adenines spaced 7 bp apart. The Pyrrole requires a centrally positioned GC base pair for high cross-linking reactivity (i.e., 5'-T(A/T)2G(A/T)2A*-3'), while Bizelesin prefers purely AT-rich sequences (e.g., 5'-T(A/T)4 or 5A*-3', where T represents the cross-strand adenine alkylation and A* represents an adenine alkylation) (Park et al., 1996). In this study, the high-field 1H-NMR and rMD studies are conducted on the 11-mer DNA duplex adduct of the Pyrrole where the 5'-TTAGTTA*-3' sequence is cross-linked by the drug. A severe structural perturbation is observed in the intervening sequences of cross-linking site, while a normal B-DNA structure is maintained in the region next to the drug-modified adenines. Based upon these observations, we propose that the interplay between the bisamido pyrrole unit of the drug and central G/C base pair (hydrogen-bonding interactions) is involved in the process of cross-linking reaction, and sequence specificity is the outcome of those interactions. This study suggests a mechanism for the sequence specific cross-linking reaction of the Pyrrole, and provides a further insight to develop new DNA sequence selective and distortive cross-linking agents.     

Mitomycin dimers: polyfunctional cross-linkers of DNA

The three dimers 3, 4, and 5 of mitomycin C (MC), a natural antibiotic and cancer chemotherapeutic agent, were synthesized in which two MC molecules were linked with -(CH(2))(4)-, -(CH(2))(12)-, and -(CH(2))(3)N(CH(3))(CH(2))(3)- tethers, respectively. The dimeric mitomycins were designed to react as polyfunctional DNA alkylators, generating novel types of DNA damage. To test this design, their in vitro DNA alkylating and interstrand cross-linking (ICL) activities were studied in direct comparison with MC, which is itself an ICL agent. Evidence is presented that 3-5 multifunctionally alkylate and cross-link extracellular DNA and form DNA ICLs more efficiently than MC. Reductive activation, required for these activities, is catalyzed by the same reductases and chemical reductants that activate MC. Dimer 5, but not MC, cross-linked DNA under activation by low pH also. Sequence specificities of cross-linking of a 162-bp DNA fragment (tyrT DNA) by MC, 3, and 5 were determined using DPAGE. The dimers and MC cross-linked DNA with the same apparent CpG sequence specificity, but 5 exhibited much greater cross-linking efficacy than MC. Greatly enhanced regioselectivity of cross-linking to G.C rich regions by 5 relative to MC was observed, for which a mechanism unique to dimeric MCs is proposed. Covalent dG adducts of 5 with DNA were isolated and characterized by their UV and mass spectra. Tri- and tetrafunctional DNA adducts of 5 were detected. Although the dimers were generally less cytotoxic than MC, dimer 5 was highly and uniformly cytotoxic to all 60 human tumor cell cultures of the NCI screen. Its cytotoxicity to EMT6 tumor cells was enhanced under hypoxic conditions. These findings together verify the expected features of the MC dimers and warrant further study of the biological effects of dimer 5.     

Guanine-adenine DNA cross-linking by 1,2,3,4-diepoxybutane: potential basis for biological activity

1,2,3,4-Diepoxybutane (DEB) is a prominent carcinogenic metabolite of 1,3-butadiene (1,3-BD), an important industrial chemical and an environmental pollutant found in cigarette smoke and automobile exhaust. DEB is capable of inducing a variety of genotoxic effects, including point mutations, large deletions, and chromosomal aberrations. The mutagenicity and carcinogenicity of DEB are thought to result from its ability to form bifunctional DNA-DNA adducts by sequentially alkylating two nucleobases within the DNA double helix. We recently reported that DEB-induced DNA-DNA cross-linking leads to the formation of 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) adducts [Park, S., and Tretyakova, N. (2004) Structural characterization of the major DNA-DNA cross-link of 1,2,3,4-diepoxybutane. Chem. Res. Toxicol. 17 (2), 129-136]. However, guanine-guanine cross-linking by DEB cannot explain the development of A:T base pair mutations following exposure to DEB and 1,3-BD. In the present work, four asymmetrical DNA-DNA cross-links involving both adenine and guanine nucleobases were identified in double-stranded DNA treated with racemic DEB. These novel lesions were assigned the structures of 1-(aden-1-yl)-4-(guan-7-yl)-2,3-butanediol (N1A-N7G-BD), 1-(aden-3-yl)-4-(guan-7-yl)-2,3-butanediol (N3A-N7G-BD), 1-(aden-7-yl)-4-(guan-7-yl)-2,3-butanediol (N7A-N7G-BD), and 1-(aden-N6-yl)-4-(guan-7-yl)-2,3-butanediol (N6A-N7G-BD), based on the comparison of their MS/MS spectra, HPLC retention times, and UV spectra with those of the corresponding authentic standards prepared independently. Although guanine-adenine lesions are approximately 10 times less abundant in DEB-treated double-stranded DNA than the corresponding bis-N7G cross-links, N1A-N7G-BD and N6A-N7G-BD are more hydrolytically stable and, if formed in vivo, may accumulate in target tissues. HPLC-ESI-MS/MS analysis of guanine-adenine DEB cross-links induced in synthetic DNA duplexes 5'-(GGT)5, 5'-(GT)7G, and 5'-(GAA)5 (+-strand) demonstrate that G-A cross-linking by DEB produces primarily 1,3-interstrand N1A-N7G lesions. The formation of bifunctional guanine-adenine adducts is likely to contribute to AT base pair substitutions and deletion mutations following DEB exposure.     

Heterocyclic analogs of DNA minor groove alkylating agents

It is generally accepted that neoplastic transformation is related to genes alteration or oncogene activation. In particular, DNA minor groove binding drugs have been extensively studied through the years in order to influence the regulation of gene expression by means of specific interactions with DNA bases moieties. Pyrrolo[2,1-c],[1,4].benzodiazepines (PBDs), CC-1065 and distamycins are three classes of minor groove binders which showed interesting cytotoxicity profiles, refined through already reviewed processes of SAR studies. Among the modifications to the three families of antitumor compounds, heterocyclic substitutions have been extensively applied by many groups in order to either modify the reactivity profile or introduce extra interactions within the minor groove, thus changing the binding site or modulating the binding sequence. The updated material related to these modifications has been rationalised and ordered to offer an overview of the argument.     

Triazene compounds: mechanism of action and related DNA repair systems.

Triazene compounds of clinical interest (i.e. dacarbazine and temozolomide) are a group of alkylating agents with similar chemical, physical, antitumour and mutagenic properties. Their mechanism of action is mainly related to methylation of O(6)-guanine, mediated by methyldiazonium ion, a highly reactive derivative of the two compounds. The cytotoxic/mutagenic effects of these drugs are based on the presence of DNA O(6)-methylguanine adducts that generate base/base mismatches with cytosine and with thymine. These adducts lead to cell death, or if the cell survives, provoke somatic point mutations represented by C:G-->T:A transition in DNA helix. Triazene compounds have excellent pharmacokinetic properties and limited toxicity. Dacarbazine requires hepatic activation whereas temozolomide is spontaneously converted into active metabolite in aqueous solution at physiological pH. Moreover, temozolomide is fully active when administrated orally (100% bioavailability). The biological effects of triazene compounds and cell resistance to them depend on at least three DNA repair systems, (a) O(6)-alkylguanine-DNA-alkyltransferase, called also methyl-guanine methyl-transferase (MGMT); (b) mismatch repair (MMR), and (c) base excision repair (BER). MGMT is a small enzyme-like protein that removes small alkyl adducts from the O(6) position of DNA guanine through a stoichiometric and auto-inactivating reaction. This reaction consists in a covalent transfer of the alkyl group from the alkylated site in DNA to an internal cysteine residue of MGMT protein. High levels of MGMT are responsible for normal and tumour cell resistance to triazenes. Therefore, pre-treatment with MGMT inhibitors - i.e. O(6)-benzylguanine or O(6)-(4-bromotenyl)guanine (Lomeguatrib) - is followed by a great increase in the activity of triazenes against target cells expressing high MGMT levels. MMR is represented by a protein complex dedicated to the repair of biosynthetic errors generated during DNA replication. The MMR system recognizes base mismatches and insertion-deletion loops, cuts the nucleotide sequence containing the lesion, and restores the correct base sequence. Therefore, not only MGMT but also MMR is involved in target cell susceptibility to triazenes. However, the system does not suppress, but instead promotes the cytotoxic effects of triazenes. In fact, MMR is not able to repair the incorrect base pairing determined by treatment with triazenes and, according to a predominant hypothesis, it causes reiterated "futile" attempts of damage repair leading to the activation of cell cycle arrest and apoptosis. BER removes lesions due to cellular metabolism, or to physical or chemical agents. BER is able to repair N(7)-methylguanine and N(3)-methyladenine determined by treatment with triazenes. Therefore, triazene compounds can also kill tumour cells by a N(3)-methyladenine-mediated mechanism if BER activity is inhibited by chemical agents (i.e. PARP inhibitors). In conclusion, in selected cases, triazenes can represent a therapeutic alternative to treatment of neoplastic diseases including haematological malignancies. Moreover, the susceptibility of neoplastic cells to these compounds can be substantially increased through pharmacological modulation of the expression level and functional activity of DNA repair enzymes.     

Sequence specificity for DNA interstrand cross-linking induced by anticancer drug chlorambucil

Chlorambucil is known to alkylate primarily N7 of guanine and N3 of adenine to induce DNA monofunctional adducts and interstrand cross-links (ISC). We have investigated the sequence specificity for DNA ISC induced by chlorambucil using duplex oligomers containing a difined cross-linkable sequences 5′-A^*TT, 5′-G^*TT, or 5′-G^*CC in which asterisk indicates the potential cross-linking site and underlined base indicates the potential cross-linking site on the opposite strand. An analysis of 20% denaturing polyacrylamide gel electrophoresis showed that chlorambucil was able to induce DNA ISC in the duplex oligomers containing a sequence 5′-GCC. The formation of DNA ISC was not observed in the duplex oligomers containing sequences 5′-ATT or 5′-GTT. These results indicate that chlorambucil induces guanineguanine DNA ISC but not guanine-adenine or adenine-adenine DNA ISC. In addition, we have tested the ability of chlorambucil to induce DNA ISC within 5′-GNNC or 5′-GC sequences using duplex oligomers containing the sequence 5′-G⁴G³G²C. The result of DNA strand cleavage assay showed that DNA ISC was formed at the 5′-GGC sequence (an 1,3 cross-link, G₁-G₃) but not at 5′-GGGC (an 1,4 cross-link, G₁-G₄) or 5′-GC sequence (an 1,2 cross-link, G₁-G₂).