A DNA cross-link adduct of the antitumor agent mitomycin C (MC) to DNA has been isolated and characterized; the results provide direct proof for bifunctional alkylation of DNA by MC. Exposure of MC to Micrococcus luteus DNA under reductive conditions and subsequent nuclease digestion yielded adducts formed between MC and deoxyguanosine residues. In addition to the two known monoadducts, a bisadduct was obtained. Reductive MC activation with Na2S2O4 (sodium dithionite) leads to exclusive bifunctional alkylation. The structure of the bisadduct was determined by spectroscopic methods that included proton magnetic resonance, differential Fourier transform infrared spectroscopy, and circular dichroism. Formation of the same bisadduct in vivo was demonstrated upon injection of rats with MC. Computer-generated models of the bisadduct that was incorporated into the center of the duplex B-DNA decamer d(CGTACGTACG)2 indicated that the bisadduct fit snugly into the minor groove with minimal distortion of DNA structure. A mechanistic analysis of the factors that govern monofunctional and bifunctional adduct formation is presented.

The wild-type p53 gene product plays an important role in the control of cell proliferation, differentiation, and survival. Altered function is frequently associated with changes in p53 stability. We have studied the role of the ubiquitination pathway in the degradation of p53, utilizing a temperature-sensitive mutant, ts2O, derived from the mouse cell line BALB/c 3T3. We found that wild-type p53 accumulates markedly because of decreased breakdown when cells are shifted to the restrictive temperature. Introduction of sequences encoding the human ubiquitin-activating enzyme El corrects the temperature sensitivity defect in ts2O and prevents accumulation of p53. The data therefore strongly indicate that wild-type p53 is degraded intracellularly by the ubiquitinmediated proteolytic pathway.The nuclear phosphoprotein p53 is a negative regulator of cell proliferation and transformation (2,11,14). In many established cell lines and human cancers, the gene is mutated. Such mutations commonly result in a p53 which has increased stability and markedly altered function and have been the subject of several studies (30,31,38,57). Genetic instability indicated by gene amplification and impaired control of the G1-S transition in the cell cycle are exhibited by cells homozygous for p53 mutations (reviewed in reference 23). A role for p53 in transcriptional control is shown by sequence-specific DNA binding (4,15,26,28). Other studies have shown that wild-type p53 can affect expression of several genes and that specific binding of p53 to transcription factors, TATA-binding protein, and CCAAT-binding factor results in the altered transcription from the test promoters (1,51). An association between mdm2 protein and p53 has been established, and the mdm2 gene is known to undergo amplification in murine transformed cells and many human sarcomas (13,40,42). Transgenic mice with a complete loss of p53 protein develop normally but have a high frequency of tumors postnatally (10). Humans heterozygous for germ line p53 mutations have a high susceptibility to carcinogenesis (34).The mechanism by which p53 is regulated intracellularly is not completely understood, although it is widely accepted that turnover of the protein is an important aspect. Normally, p53 is a short-lived protein with a half-life of about 30 min (16,43,46). DNA tumor viruses express gene products which bind the normal p53 and alter its properties. In the case of human papillomavirus type 16, for example, the half-life of p53 is decreased in complexes with E6 and p53 (50), whereas complexes with simian virus 40 (SV40) large T protein and adenovirus ElB p55 result in an increased half-life of p53 (46,49). Mutations in SV40 large T protein which affect binding of p53 decrease the efficiency of SV40-mediated transformation of susceptible cells (32,35,56,63 (50) have shown that E6-p53 complexes generated in vitro are more rapidly degraded in reticulocyte extracts and that ubiquitinated intermediates can be detected. Under their experimental conditions, however, free p53 wa...

The antitumor antibiotic mitomycin C is shown to form a covalent complex with calf thymus DNA under anaerobic conditions in the presence of either NADPH cytochrome c reductase/NADPH, xanthine oxidase/NADH, or the chemical reducing system H2/PtO2. Digestion of the complex with DNase I/snake venom diesterase/alkaline phosphatase yields a single mitomycin deoxyguanosine adduct as the major DNA alkylation product, identified as N2-(2",3,7"-diaminomitosen-l"a-yl) 2'-deoxyguanosine (Structure 2). Two minor adducts, 2-5% each of the total adduct pool, are isolated and identified as the 1"j8 stereoisomer of 2 (Structure 3), and 10"-decarbamoyl-2 (Structure 7). The same results were obtained with M13 DNA and poly(dG-dC) poly(dG-dC); however, in the latter case, a minor adduct apparently possessing two deoxyguanosine and one mitomycin unit is isolated. Digestion of the covalent mitomycin-calf thymus DNA complex with nuclease P1 yields four dinucleotide adducts, all of which consist of 2 linked at its 3' end to each of the four possible 5' nucleotides (A, T, G, and C). Upon treatment of each dinucleotide adduct with snake venom diesterase/alkaline phosphatase, 2 is released along with the corresponding free nucleoside. In apparent conflict with the present results, previous reports from another laboratory have indicated that modification of calf thymus DNA by mitomycin C under conditions identical to those described here result in the isolation of three mitomycin C mononucleotide adducts possessing linkages of the drug to N2 and 06 of guanine and N6 of adenine. Evidence is shown suggesting that the latter adducts are actually three of the above four dinucleotide derivatives of 2 obtained independently by us and, thus, all of them in fact possess an identical N2-mitosenylguanine adduct moiety. Model-building studies indicate an excellent fit of the guanine N2-linked drug molecule inside the minor groove of B-DNA with no appreciable distortion of the DNA structure.Mitomycin C (MC; Structure 1), a potent antibiotic and clinically used antitumor agent, is known to interact covalently with DNA in vivo and in vitro. This is manifested by the reversible melting behavior of MC-exposed DNA, attributed to formation of covalent crosslinks between the complementary strands (1) and by covalent association of the ultraviolet chromophore of MC to DNA (2). These processes require reduction of MC into a transiently activated form, which is thought to occur in cells and can be mimicked easily in vitro chemically or enzymatically (1). The cytotoxicity of MC is most likely a direct result of DNA alkylation, as indicated by the parallels in biological activity of MC with a number of known "DNA damaging agents": selective inhibition of DNA replication (1) and sister chromatid exchange (4), and cross-resistance or cross-hypersensitivity of bacterial (1, 5) and mammalian (6) cells to UV light and MC. The molecular nature of the covalent interactions between MC and DNA has remained elusive, mainly because of the difficulty of isolating low...

Oligodeoxyribonucleotides cross-linked by reductively activated mitomycin C (MC) were prepared and purified for the first time. The cross-linked products were structurally characterized by nucleoside and MC-nucleoside adduct analysis. Optimal conditions were established for the cross-linking reaction, resulting in high yields, typically in the 20-50% range. Nuclease digests of the cross-linked oligonucleotides yielded the same bifunctional MC-deoxyguanosine adduct as that previously isolated from DNA exposed to MC in vitro and in vivo [Tomasz et al. (1987) Science 235, 1204]. The cross-linked oligonucleotides displayed broad thermal melting profiles, greatly increased Tm, and complex circular dichroism spectra. Phosphodiester linkages at the cross-link were resistant to spleen exonuclease, nuclease P1, and TaqI and ClaI restriction endonucleases; snake venom diesterase action was uninhibited. The cross-links are stable to heat at neutral pH but are removed by treatment in hot piperidine or by the reducing agents Na2S2O4 and dithiothreitol. Mechanisms are proposed for these reactions. These studies define optimal methods for introducing mitomycin cross-links into DNA fragments at a specific site, providing a versatile tool to study the effects of the MC cross-links on DNA structure and function.