Reaction of acid-activated mitomycin C with calf thymus DNA and model guanines: elucidation of the base-catalyzed degradation of N7-alkylguanine nucleosides

Mitomycin C (MC, 1) forms covalent adducts under acidic activating conditions (pH approximately 4) with deoxyguanosine, d(GpC), and guanine residues of calf thymus DNA. In the case of deoxyguanosine, five adducts arise from a common precursor, N7-(2' beta, 7'-diaminomitosen-1'-yl)-2'-deoxyguanosine (10a; not isolated), which hydrolyzes spontaneously via two pathways: scission of the glycosidic bond to form N7-(2' beta, 7'-diaminomitosen-1' alpha-yl)guanine (5) and its 1' beta-isomer (6) and imidazolium ring opening to generate three 2,6-diamino-4-hydroxy-5-(N-formyl-2' beta, 7'-diaminomitosen-1' beta-yl)pyrimidine (FAPyr) derivatives that are substituted at N6 by isomeric 2'-deoxyribose units [i.e., 1' beta-furanose (7), 1' alpha-furanose (8), and 1' beta-pyranose (9)]. The structures of 5-9 were determined by spectroscopic methods. The same five adducts were obtained from d(GpC), but only the guanine adducts 5 and 6 were formed in DNA. Adducts 7-9 interconvert during high-performance liquid chromatography (HPLC). The unexpected isomerization of the deoxyribose moiety of the initially formed 1' beta-furanose adduct 7 to those of 8 and 9 occurs upon imidazolium ring opening, as discerned by the course of imidazolium cleavage of the simple models N7-ethyl- and N7-methylguanosine and N7-methyl-2'-deoxyguanosine. All ring-opened N7-alkylguanosine derivatives studied here exist as a mixture of distinct N-formyl rotamers, manifested by multiple interconverting peaks on HPLC and in the 1H NMR spectra. In the UV spectra of such derivatives, a new and diagnostic maximum at 218 nm (at pH 7) is observed. Acid-activated MC is found to alkylate preferentially the Gua-N7 position in deoxyguanosine or d(GpC), in contrast to reductively activated MC, which preferentially alkylates the Gua-N2 position. This finding is explained by the different electronic structures of acid- and reduction-activated MC. In DNA, the N7 specificity of acid-activated MC is partially offset by steric factors.     

Alkylation of calf thymus DNA with reductively activated mitomycin C

Mitomycin C (MMC) was catalytically reduced in the presence of a nucleotide or calf thymus DNA. Reaction with 5'-guanylic acid (5'-GMP) gave 1,2-cis-2, 7-diamino-1-(5'-guanylyl) mitosene. Reaction with calf thymus DNA gave modified DNA, which on enzymatic hydrolysis gave two alkylated 5'-deoxyguanylic acid (MG-1 and MG-2) and an alkylated 5'-deoxyadenylic acid (MA). This is the first example of isolation of nucleotides from DNA modified by MMC.     

Inhibition of DNA cross-linking by mitomycin C by peroxidase-mediated oxidation of mitomycin C hydroquinone.

Mitomycin C requires reductive activation to cross-link DNA and express anticancer activity. Reduction of mitomycin C (40 microm) by sodium borohydride (200 microm) in 20 mm Tris-HCl, 1 mm EDTA at 37 degrees C, pH 7.4, gives a 50-60% yield of the reactive intermediate mitomycin C hydroquinone. The hydroquinone decays with first order kinetics or pseudo first order kinetics with a t(12) of approximately 15 s under these conditions. The cross-linking of T7 DNA in this system followed matching kinetics, with the conversion of mitomycin C hydroquinone to leuco-aziridinomitosene appearing to be the rate-determining step. Several peroxidases were found to oxidize mitomycin C hydroquinone to mitomycin C and to block DNA cross-linking to various degrees. Concentrations of the various peroxidases that largely blocked DNA cross-linking, regenerated 10-70% mitomycin C from the reduced material. Thus, significant quantities of products other than mitomycin C were produced by the peroxidase-mediated oxidation of mitomycin C hydroquinone or products derived therefrom. Variations in the sensitivity of cells to mitomycin C have been attributed to differing levels of activating enzymes, export pumps, and DNA repair. Mitomycin C hydroquinone-oxidizing enzymes give rise to a new mechanism by which oxic/hypoxic toxicity differentials and resistance can occur.     

Sequence-specific DNA damage induced by reduced mitomycin C and 7-N-(p-hydroxyphenyl)mitomycin C.

Mitomycin C reduced with sodium borohydride induced the DNA damage at deoxyguanosines preferentially in dinucleotide sequence G-T. The DNA damage produced strand breaks when subsequently heated. The DNA damage scarcely occurred when the end-labeled DNA was preincubated with ethidium bromide or actinomycin D before the addition of mitomycin C and the reducing agent. Fully reduced mitomycin C did not induce the DNA damage. The mitomycin C-inducing DNA damage seems to require the intercalation of the partially reduced mitomycin C of short life time, probably semiquinone radical, between DNA base pairs. The inhibitory effects of sodium chloride and radical scavengers suggested that the requirement of the covalent bond formation of mitomycin C to DNA and the involvement of oxygen radicals in the DNA damage. 7-N-(p-hydroxyphenyl)mitomycin C, which is reported to show a higher antitumor activity and a lower toxicity than mitomycin C, was readily reduced with dithiothreitol and induced the sequence-specific DNA damage, whereas mitomycin C was not.     

Interactions of the high-mobility-group-like Ceratitis capitata C1 proteins with DNA

We have studied the interactions of the high-mobility-group-like proteins (C1a1, C1a2 and C1b) from the fruit fly Ceratitis capitata with DNA. Nitrocellulose filter binding assays, thermal denaturation studies and spectrofluorimetry of the complexes revealed the existence of specific and nonspecific interactions. Thermal denaturation curves showed that the three proteins stabilized the DNA, thus suggesting a preferential binding to double-stranded DNA. The calculation of the thermodynamic parameters of the interactions showed that the nonspecific bindings were characterized by low association constants (Ka) with values ranging from 2.7 X 10(4) M-1 to 2.0 X 10(6) M-1. Also, the cooperativity of these interactions was relatively high (cooperativity factor, w, values ranging over 20-35), and the number of nucleotides involved was low (1-3 base pairs). On the other hand, the existence of specific interactions between C1 proteins and DNA was suggested by two facts: the retention of C. capitata [3H]DNA in nitrocellulose filters was only a low percentage of total input DNA and there was a marked size dependence of the binding (25% retention of a 40-kb DNA and only 3% retention with a DNA of 1 kb). The specific bindings had higher Ka values than the nonspecific ones, and they also were cooperative. Some differences were observed between C1b and the C1a proteins about the way they interact with C. capitata DNA.     

Mechanism of monofunctional and bifunctional alkylation of DNA by mitomycin C

The relative amounts of monofunctional and bifunctional alkylation products of DNA with mitomycin C (MC) depend on whether one or both masked alkylating functions of MC are activated reductively; adduct 8 is the result of one function and adducts 7 and 9, formed as a pair, are the result of both functions being activated [Tomasz, M., Lipman, R., Chowdary, C., Pawlak, J., Verdine, G. L., & Nakanishi, K. (1987) Science (Washington, D.C.) 235, 1204-1208]. To determine the mechanism governing this differential reactivity of MC with DNA, MC-Micrococcus luteus DNA complexes formed under varying conditions in vitro were digested to nucleosides and adducts. Adduct distribution, analyzed by high-performance liquid chromatography, served as the measure of monofunctional and bifunctional activation. H2/PtO2 and xanthine oxidase/reduced nicotinamide adenine dinucleotide (NADH) activated MC mostly monofunctionally, and Na2S2O4 activated the drug bifunctionally under comparable conditions. Excess MC selectively suppressed, but excess PtO2 selectively promoted, bifunctional activation by H2/PtO2; excess xanthine oxidase and/or NADH also had promoting effects. O2 tested in the Na2S2O4 system was inhibitory. 10-Decarbamoyl-MC acted strictly monofunctionally under all conditions. Monoadducts bound to DNA were converted to bis adducts upon rereduction. A mechanism with the following features was derived: (i) Activation of MC at C-1 and C-10 is sequential (C-1 first). (ii) A one-time reduction is sufficient for both. (iii) Activation of the second function may be selectively inhibited by kinetic factors or O2. (iv) 7 and 9 are coproducts of bifunctional activation; their ratio depends on the DNA base sequence. (v) Activation of the second function involves an iminium intermediate. Direct applications to the action of MC in vivo are discussed.     

Synthesis of a major mitomycin C DNA adduct via a triaminomitosene

We report here the synthesis of two amino precursors for the production of mitomycin C and 10-decarbamoylmitomycin C DNA adducts with opposite stereochemistry at C-1. The triamino mitosene precursors were synthesized in 5 steps from mitomycin C. In addition synthesis of the major mitomycin C-DNA adduct has been accomplished via coupling of a triaminomitosene with 2-fluoro-O⁶-(2-p-nitrophenylethyl)deoxyinosine followed by deprotection at the N² and O⁶ positions.     

Sequence specificity of heat-labile sites in DNA induced by mitomycin C

The sequence specificity of the mitomycin C-DNA interaction was directly determined by using DNA sequencing techniques and by using 3'- or 5'-end-labeled DNA fragments of defined sequence as substrates. Mitomycin C reduced with sodium borohydride induced heat-labile sites in DNA preferentially at specific sequences. The heat-labile sites were induced most preferentially at the dinucleotide sequence G-T ( especially Pu G-T), which was determined by scanning autoradiograms with a microdensitometer after gel electrophoresis. DNA was cleaved at the 3' side of deoxyguanosines and of some deoxyadenosines by heat treatment. Oligonucleotides produced by heat treatment after reaction with reduced mitomycin C contained phosphoryl groups at the 5' termini. The 3' termini seemed not to have simple structures, judging from their electrophoretic mobilities. Oxygen radicals such as singlet oxygen and hydroxyl radical were possibly involved in the induction of heat-labile sites.     

Recognition of specific DNA sequences by mitomycin C for alkylation.

Synthetic oligodeoxyribonucleotides were reacted with mitomycin C (MC) under conditions which restricted MC to monofunctional alkylating activity. The yields of monofunctional alkylation of oligonucleotides with variable sequence were determined by enzymatic digestion of the reaction mixture to unreacted nucleosides and the product of alkylation, a MC-deoxyguanosine adduct (2), followed by quantitative analysis by HPLC. The relative yields of 2 reflected relative monoalkylation reactivities. They were compared in a series of oligonucleotides having the sequence 5'-NGN' in which the 5'-base was varied while the 3'-base was kept constant as T. Under Na2S2O4 activation conditions a striking enhancement of the yield was observed at the 5'-CG sequence: 36%, compared to 2% at 5'-AG and 4.1% at 5'-TG. The 5'-GG sequence also showed enhanced reactivity although to a lesser extent (14.7%). The enhancements were specific to the duplex state of the oligonucleotides. Using NADPH:cytochrome c reductase as the reducing agent gave similar results. MC activated by acidic pH also displayed 5'-CG alkylation specificity. 10-Decarbamoyl-MC activated by Na2S2O4 showed the same 5'-CG specificity as MC. Replacement of deoxyguanosine by deoxyinosine in the opposite strand at a 5'-CG site abolished the enhancement of alkylation. Such replacement at a 5'-GG site had a similar effect. It was found that the base 3' to the guanine had only a relatively modest modulating effect on the enhanced reactivity of the G at the 5'-CG sequence. This 3'-base effect appeared to be independent of the 5'-base of the 5'-NGN' triplet. The order of reactivity is 3'-(C greater than T greater than A).(ABSTRACT TRUNCATED AT 250 WORDS)     

Recognition between mitomycin C and specific DNA sequences for cross-link formation

An extensive series of oligodeoxyribonucleotides was reacted with reductively activated mitomycin C (MC), and the resulting cross-linked drug-oligonucleotide complexes were isolated by reverse-phase HPLC and characterized by nucleoside and MC-nucleoside adduct analysis. HPLC also served for assay of the yield of cross-linked oligonucleotides. AT-rich duplex oligonucleotides, containing a single central CG.CG, gave high yields of cross-links between the two guanines while those having GC.GC, instead, gave none. In another series, the central sequences CGC.GCG and CGC.ICG both yielded 50% cross-link while CGC.GCI was completely resistant. Cross-linking was conducted also in two steps: Oligonucleotides substituted monofunctionally by MC at guanine at either a CG or GC sequence were annealed with their complementary strands followed by reductive reactivation of the bound MC to form a cross-link. The CG oligomers were cross-linked quantitatively while the GC ones were again resistant. These results show unambiguously that the MC cross-link is absolutely specific to the CG.CG duplex sequence, confirming our previous finding [Chawla, A.K., Lipman, R., & Tomasz, M. (1987) in Structure and Expression, Volume 2: DNA and Its Drug Complexes (Sarma, R.H., & Sarma, M.H., Eds.) Adenine Press, Guilderland, NY]. Evidence is presented that this specificity is due to the specific orientation of the monofunctionally attached MC in the minor groove. Superimposed on the CG.CG requirement, a four-base-pair sequence preference was observed at PuCGPyr.PuCGPyr sequences. This suggests that the guanine N2 atom of GpPyr is more reactive toward the drug than that of GpPu, due to the favorable effect of the negative dipole of the O2 of the Pyr on the reaction; in accordance, GpT was more reactive than GpC.(ABSTRACT TRUNCATED AT 250 WORDS)     

The miRNA aberrant expression dependence on DNA methylation in HeLa cells treated with mitomycin C

The dependence of expression of miRNAs and their precursors (pre-miRNAs) on the DNA methylation level in HeLa cells 8 days after mitomycin C treatment was studied. A massive parallel DNA sequencing method was applied to analyze miRNA expression. 5-Azacytidine (DNA methylation inhibitor) was added to the medium 6 days after mutagenic agent exposure. The results indicated that the change in expression for some mature miRNAs (39 of 61) was accompanied by the change in the expression of their pre-miRNAs, while there were no significant changes in the expression of pre-miRNA for other mature miRNAs (22 of 61). The aberrant expression was maintained by 8 of 61 mature miRNAs and 6 of 55 pre-miRNAs in the induced HeLa cells after 5-azacytidine treatment. In addition, the expression of more than 90% of miRNAs, which indicated a significant change in expression after mitomycin C treatment, does not depend or depends slightly on the DNA methylation level in HeLa cells without mitomycin C treatment. The results suggest that mitomycin C induces aberrant DNA methylation which affects maintenance of changes in the miRNA expression in cell generations after mutagen treatment.     

A comparison of intraperitoneal injection and oral gavage in the micronucleus test with mitomycin C in mice

Intraperitoneal (i.p.) injection and oral (p.o.) gavage were evaluated in the mouse micronucleus test with mitomycin C (MMC). The tests were carried out in 2 laboratories with the MS/Ae and CD-1 mouse strains. On the basis of a small-scale acute toxicity study and a pilot experiment, the full-scale micronucleus test was performed with a 24-h sampling time at doses of 1, 2, 4, and 8 mg/kg for both treatment routes. In both strains, a clear positive dose-response relation was shown by both routes. Although the frequency of micronucleated polychromatic erythrocytes (MNPCEs) was higher with i.p. on a mg/kg basis, this tendency was reversed when dose was expressed as a percentage of the LD50.     

Interactions of antitumor triazoloacridinones with DNA

Triazoloacridinones (TA) are a new group of potent antitumor compounds, from which the most active derivative, C-1305, has been selected for extended preclinical trials. This study investigated the mechanism of TA binding to DNA. Initially, for selected six TA derivatives differing in chemical structures as well as cytotoxicity and antitumor activity, the capability of noncovalent DNA binding was analyzed. We showed that all triazoloacridinones studied stabilized the DNA duplex at a low-concentration buffer but not at a salt concentration corresponding to that in cells. DNA viscometric studies suggested that intercalation to DNA did not play a major role in the mechanism of the cytotoxic action of TA. Studies involving cultured cells revealed that triazoloacridinone C-1305 after previous metabolic activation induced the formation of interstrand crosslinks in DNA of some tumor and fibroblast cells in a dose dependent manner. However, the detection of crosslink formation was possible only when the activity of topoisomerase II in cells was lowered. Furthermore, it was impossible to validate the relevance of the ability to crosslink DNA to biological activity of TA derivatives.     

Interactions of DNA with giant liposomes

DNA interactions with the bilayers of cationic liposomes were studied using a novel model experiment: DNAs were locally injected by a micropipette to a part of a giant unilamellar vesicle. The resulting phenomena were directly observed in optical microscope. Giant unilamellar vesicles (GUVs), about 100 microm in diameter, made of phosphatidylcholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine, were obtained by electroformation. The effects of DNAs of different length were tested: (i) 'short' DNAs-oligonucleotide 21b, and calf thymus 250 bp; (ii) 'long' DNAs-plasmid DNAs in super coil or liner form (between 2.7 and 8.0 kbp). DNAs were injected native, as well as marked with the fluorescent dye Hoechst. The resulting membrane topology transformations were monitored in phase contrast, while the DNA distribution was followed in fluorescence. DNA-induced endocytosis was observed due to the DNA/lipid membrane local interactions for all DNAs tested. Some of the DNA in the formed complex was associated with the induced endosomes, and some of it remained spread over the 'mother' GUV membrane for all DNAs tested, except for the longest one--the linear plasmid of 8 kbp. The last remained at the 'mother' GUV membrane and was not transported with the induced endosomes to the internal GUV space. Possible mechanisms for DNA/lipid membrane interaction were suggested. One of them involves DNA encapsulation within an inverted micelle included in the lipid membrane. The model observations could help in understanding events associated with interaction of DNA with biological membranes, as well as cationic liposomes/DNA complexes formation in gene transfer processes.     

Trabeculectomy with mitomycin C in glaucoma associated with uveitis

The patients with uveitic glaucoma are at high risk for failure following drainage surgery because of young age of these patients, preoperative long-term control of inflammation and postoperative complications. Twenty-two trabeculectomies performed in 22 patients with uveitic glaucoma were retrospectively evaluated to analyze the effect of intraoperative application of mitomycin C (MMC). Success rates, postoperative levels of intraocular pressure (IOP) and postoperative complications were studied. After a mean follow-up of 10.6 months (range, 5-28 months), 15 patients (68.2%) achieved IOP of 21mmHg or less without antiglaucoma medications. There were statistically significant reduction in IOP postoperatively during the period studied (p < 0.001). Early postoperative complications included chorioidal detachment (9.1%), shallow anterior chamber (9.1%), hyphema (13.6%), macular edema (4.5%) and raised IOP (27.3%). Late postoperative complications included exacerbation of uveitis (4.5%), macular edema (4.5%), cataract (22.7%) and raised IOP (31.8%). The eyes with raised IOP needed additional antiglaucoma medication. The results of this retrospective and uncontrolled study suggest that intraoperative application of MMC may be a good option for enhancement of short-term trabeculectomy success rates in patients with uveitic glaucoma.