Roselyn Lipman

Electrostatic complexes of mitomycin C with nucleic acids and polyanions

Reductively activated mitomycin C exhibits strong, non-covalent electrostatic binding to polyanions such as polyvinylsulfate and polyphosphate. The protonated C-2 amino group generated by the reduction is most likely responsible for this type of interaction. At moderate drug and salt concentrations only covalent binding to nucleic acids is observable. This is shown to be guanine-specific in DNA for the first time, as well as in synthetic polyribo- and polydeoxyribonucleotides at 10--20 times higher binding levels than previously tested. At higher mitomycin C concentration, however, strong non-covalent electrostatic binding to nucleic acids also occurs, resulting in a binding ratio up to 1 mol drug bound per mol mononucleotide, although this non-specific binding is relatively inhibited compared to polyvinylsulfate. Salts also have an inhibitory effect on the non-specific binding to nucleic acids. A series of mitomycin derivatives were compared for their binding and cross-linking abilities using DNA as substrate, with the following results: (a) the presence of a basic nitrogen . funtion at C-2 promotes binding, both covalent and electrostatic, presumably by kinetically facilitating the approach between positively charged nitrogen and DNA. (b) The aziridine ring is the major covalent binding site, indispensable for crosslinking and determines the guanine-specificity of the binding.

The major mitomycin C-DNA monoadduct is cytotoxic but not mutagenic in Escherichia coli.

To determine the mutagenic and genotoxic properties of the major guanine N2-adduct formed by the antitumor drug mitomycin C, we have synthesized a decanucleotide, d(TTACG[MC]TATCT), containing the adduct, which was inserted into a gapped bacteriophage M13 genome. Analysis of the constructed genome indicated that 41% ligation of the adducted 10-mer occurred on both sides of the gap, whereas the control 10-mer ligated with 34% efficiency. After transfection of the adducted single-stranded M13 DNA into Escherichia coli, the adduct was found to be highly genotoxic. Viability of the adducted genome in a repair-competent strain was only 7%, which increased to 12% and 15% upon induction of SOS by irradiating the cells with 254-nm light at 20 and 50 J/m2, respectively. Even lower viability of 2%, 4.6%, and 0.2% was observed in uvrA, uvrB, and uvrC strains, respectively, which increased up to 10-fold with SOS. An examination of the surviving phage populations revealed that the adduct was not detectably mutagenic. No mutants from the repair-proficient strain were detected after analysis of more than 2500 progeny phage. Only 0.2% of the survivors were mutants in the uvrA strain. It is uncertain, however, if they were induced by the adduct, since all the mutants showed untargeted mutations. We conclude that the major guanine N2-adduct formed by mitomycin C is cytotoxic but not appreciably mutagenic in E. coli.

Isolation, identification, and assay of [3H]-porfiromycin adducts of EMT6 mouse mammary tumor cell DNA: effects of hypoxia and dicumarol on adduct patterns.

[3H]-(N-la-methyl) Porfiromycin (POR) was employed to detect and identify the radiolabeled mono- and bis-adducts formed in living EMT6 mouse mammary tumor cells under different conditions. To provide authentic standard adducts, calf-thymus DNA was treated with POR under reductive activation, then digested to nucleosides and POR-nucleoside adducts. The three major adducts formed were isolated by HPLC and authenticated. Two were mono-adducts, composed of deoxyguanosine linked at its N2-position to C-1 of POR and of 10-decarbamoyl POR. The third was a bis-adduct, in which POR was crosslinked to two deoxyguanosines at their N2-positions. DNA from [3H]-POR treated EMT6 cells was digested an analyzed by HPLC. DNA-associated label was located in thymidine and in two mono-adducts and one bis-adduct identical to those described above. Label in thymidine resulted from N-demethylation of POR and reincorporation of label into new thymidylate residues. Adducts were formed more abundantly in hypoxia than in air. In addition, the mono-adduct to crosslink ratios were different, approximately 1:1 and 2:1 for hypoxic and aerobic cells, respectively. The different patterns of alkylation in air and hypoxia may be related to the greater toxicity of POR in hypoxia. When cells were treated simultaneously with POR and dicumarol, adduct levels were lower, and a new, unknown adduct was observed primarily under hypoxia; these changes may be related to the altered toxicity of POR in the presence of dicumarol. The HPLC assay detected simultaneously the full array of stable mono- and bis-adducts in DNA with good sensitivity (greater than or equal to 2 x 10(6) adducts/nucleotide) and excellent reproducibility. This assay should be generally applicable to all cells and tissues when MC or POR with high specific radioactivity can be employed.

Synthesis of Guanine Derivatives Substituted in the O6-Position by Mitomycin C

Abstract Although adducts resulting from the reaction of mitomycin C (MC) and DNA have been characterized as guanine derivatives covalently linked to MC at the guanine N 2 -and/or O 6 -positions, lack of material and of protons on the quanine nucleus had led to difficulties in product characterization. Authentic O 6 -MC guanosine derivatives have been synthesized and comparison shows that N 2 -adducts and not O 6 -adducts are the major products from reactions of reduced MC with DNA.