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.     

Analysis of a parallel branch in the mitomycin biosynthetic pathway involving the mitN-encoded aziridine N-methyltransferase

Mitomycin C is a natural product with potent alkylating activity, and it is an important anticancer drug and antibiotic. mitN, one of three genes with high similarity to methyltransferases, is located within the mitomycin biosynthetic gene cluster. An inframe deletion in mitN of the mitomycin biosynthetic pathway was generated in Streptomyces lavendulae to produce the DHS5373 mutant strain. Investigation of DHS5373 revealed continued production of mitomycin A and mitomycin C in addition to the accumulation of a new mitomycin analog, 9-epi-mitomycin C. The mitN gene was overexpressed in Escherichia coli, and the histidine-tagged protein (MitN) was purified to homogeneity. Reaction of 9-epi-mitomycin C with MitN in the presence of S-adenosylmethionine yielded mitomycin E showing that the enzyme functions as an aziridine N-methyltransferase. Likewise, MitN was also shown to convert mitomycin A to mitomycin F under the same reaction conditions. We conclude that MitN plays an important role in a parallel biosynthetic pathway leading to the subclass of mitomycins with 9alpha-stereochemistry but is not involved directly in the biosynthesis of mitomycins A and C.     

Bioreductive activation of mitomycin C by DT-diaphorase.

The role of DT-diaphorase (DTD, EC 1.6.99.2) in the bioreductive activation of mitomycin C was examined using purified rat hepatic DTD. The formation of adducts with reduced glutathione (GSH), binding of [3H]mitomycin C to DNA, and mitomycin C-induced DNA interstrand cross-linking were used as indicators of bioactivation. Mitomycin C was metabolized by DTD in a pH-dependent manner with increasing amounts of metabolism observed as the pH was decreased from 7.8 to 5.8. The major metabolite observed during DTD-mediated reduction of mitomycin C was 2,7-diaminomitosene. GSH adduct formation, binding of [3H]mitomycin C and mitomycin C-induced DNA interstrand cross-linking were observed during DTD-mediated metabolism. In agreement with the pH dependence of metabolism, increased bioactivation was observed at lower pH values. Temporal studies and experiments using authentic material showed that 2,7-diaminomitosene could be further metabolized by DTD resulting in the formation of mitosene adducts with GSH. DNA cross-linking during either chemical (sodium borohydride) or enzymatic (DTD) mediated reduction of mitomycin C could be observed at pH 7.4, but it increased as the pH was decreased to 5.8, showing the critical role of pH in the cross-linking process. These data provide unequivocal evidence that the obligate two-electron reductase DTD can bioactivate mitomycin C to reactive species which can form adducts with GSH and DNA and induce DNA cross-linking. The use of mitomycin C may be a viable approach to the therapy of tumors high in DTD activity, particularly when combined with strategies to lower tumor pH.     

Reductive activation of mitomycins A and C by vitamin C

The anticancer drug mitomycin C produces cytotoxic effects after being converted to a highly reactive bis-electrophile by a reductive activation, a reaction that a number of 1-electron or 2-electron oxidoreductase enzymes can perform in cells. Several reports in the literature indicate that ascorbic acid can modulate the cytotoxic effects of mitomycin C, either potentiating or inhibiting its effects. As ascorbic acid is a reducing agent that is known to be able to reduce quinones, it could be possible that the observed modulatory effects are a consequence of a direct redox reduction between mitomycin C and ascorbate. To determine if this is the case, the reaction between mitomycin C and ascorbate was studied using UV/Vis spectroscopy and LC/MS. We also studied the reaction of ascorbate with mitomycin A, a highly toxic member of the mitomycin family with a higher redox potential than mitomycin C. We found that ascorbate is capable to reduce mitomycin A efficiently, but it reduces mitomycin C rather inefficiently. The mechanisms of activation have been elucidated based on the kinetics of the reduction and on the analysis of the mitosene derivatives formed after the reaction. We found that the activation occurs by the interplay of three different mechanisms that contribute differently, depending on the pH of the reaction. As the reduction of mitomycin C by ascorbate is rather inefficiently at physiologically relevant pH values we conclude that the modulatory effect of ascorbate on the cytotoxicity of mitomycin C is not the result of a direct redox reaction and therefore this modulation must be the consequence of other biochemical mechanisms.     

Action of mitomycin C on the variability of Actinomyces hygroscopicus in forming a proteolytic complex of hygrolytin enzymes and the antibiotic, hygromycin B]

The lethal and mutagenic effect of mitomycin C in doses of 10 and 15 micrograms/ml on the spores and 24-hour culture of Act. hygroscopicus, strain O878 producing hygrolytin, a proteolytic enzyme and hygromycin B, an antibiotic was studied. It was found that mitomycin C had a high lethal effect on the organism. The lethal effect of the antibiotic depended on the stage of the culture development, mitomycin C dose and exposure time. The 24-hour culture was most sensitive to the effect of mitomycin in a dose of 50 micrograms/ml. Exposure to mitomycin increased the actinomycete variation with respect to the colony morphology and induction of new morphological mutations. Exposure of strain O878 to mitomycin C significantly increased the culture variation with respect to the quantitative features of production of the hygrolytin proteolytic enzyme complex and hygromycin B. The character of the strain induced variation with respect to the features studied was different which indicated the absence of correlation between them. The use of mitomycin C proved to be promising in selection of Act. hygroscopicus with a purpose of increasing the culture proteolytic and antibiotic activity.     

Reductive activation of mitomycin C and mitomycin C metabolites catalyzed by NADPH-cytochrome P-450 reductase and xanthine oxidase

Under anaerobic conditions and with proper electron donors, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and xanthine oxidase (EC 1.2.3.2) similarly reductively metabolized mitomycin C. Reversed phase high performance liquid chromatography was used to separate, detect, and isolate several metabolites. Three metabolites were identified by mass spectrometry and thin layer chromatography as 1,2-cis- and trans-2,7-diamino-1-hydroxymitosene and 2,7-diaminomitosene. Three metabolites were phosphate-dependent, and two of them were identified to be 1,2-cis- and trans-2,7-diaminomitosene 1-phosphate. The amounts of the five identified metabolites generated during the reduction of mitomycin C varied with pH and nucleophile concentration. At pH 6.5, 2,7-diaminomitosene was essentially the only metabolite formed, whereas from pH 6.8 to 8.0, trans- and cis-2,7-diamino-1-hydroxymitosene increased in quantity as 2,7-diaminomitosene decreased. The disappearance of mitomycin C and the production of metabolites were enzyme and mitomycin C concentration-dependent. Substrate saturation was not reached for either enzyme up to 5 mM mitomycin C. Electron paramagnetic resonance studies demonstrated the formation of mitomycin C radical anion as an intermediate during enzymatic activation. Our results indicate that either enzyme catalyzed the initial activation of mitomycin C to a radical anion intermediate. Subsequent spontaneous reactions, including the elimination of methanol and the opening of the aziridine ring, generate one active center at C-1 which facilitates nucleophilic attack. Simultaneous generation of two reactive centers was not observed. All five primary metabolites were metabolized further by either flavoenzyme. The secondary metabolites exhibited similar changes in their absorbance spectra and were unlike the primary metabolites, suggesting that a second alkylating center other than C-1 was generated during secondary activation. We propose that secondary activation of monofunctionally bound mitomycin C is probably a main route for the bifunctional binding of mitomycin C to macromolecules and that the cytotoxic actions of mitomycin C result from multiple metabolic activations and reactions.     

Modification of the sensitivity of CHO cells to mitomycin C by dibutyryl cyclic AMP

The survival of CHO cells exposed to mitomycin C was decreased three times that of the cells treated with 1 mM dibutyryl cyclic AMP before mitomycin C treatment, as compared to the absence of treatment with this cyclic nucleotide. The sensitization effect began at 3-4 hours after the start of pre-treatment, reached a maximum at around 10 hours and continued to be effective. Post-treatment with the cyclic nucleotide for more than 12 hours increased the survival of CHO cells exposed to mitomycin C.     

头状轮生链霉菌中丝裂霉素C抗性基因的克隆及功能研究

头状轮生链霉菌(\%Streptoverticillium caespitosus\%)ATCC27422是抗肿瘤药物丝裂霉素的主要产生菌,实验通过诱变筛选获得不产生丝裂霉素同时对丝裂霉素C敏感的阻断变种S6,并以它为受体宿主,以质粒pIJ699为载体,建立野生型头状轮生链霉菌菌株ATCC27422的基因库。采用鸟枪法克隆技术,从库中筛选获得含有丝裂霉素C抗性基因的62kb外源片段的克隆子。将含此外源片段的质粒pLX5导入变铅青链霉菌(\%Streptomyces lividans\%)获得表达。并且首次成功地运用电穿孔法将pLX5导入野生型菌株中,使其对丝裂霉素C的抗性大幅度提高：最低抑制浓度(MIC)由原来的200μg/mL上升至1000μg/mL以上。摇瓶发酵实验表明：单位菌量的ATCC27422(pLX5)的丝裂霉素产量高于野生菌株ATCC27422,因此丝裂霉素C抗性与产量之间存在一定的相关性。     

Sensitivity of mitomycin C and nitrogen mustard crosslinks to extreme alkaline conditions

DNA-DNA crosslinks in cells treated with mitomycin C, nitrogen mustard, or decarbamoyl mitomycin C were measured in alkaline isopycnic gradients as a function of pH. Crosslinks from cells treated with mitomycin C and nitrogen mustard, which react with DNA purines, could be detected at pH 12.5 but not at pH 14. No crosslinks from cells treated with decarbamoyl mitomycin C were detected at either pH. Previous studies with cells exposed to psoralen derivatives plus 360 nm light, which produce DNA-DNA crosslinks with pyrimidines, demonstrated stable crosslinks at pH 14. These studies indicate that DNA-DNA crosslinks involving DNA purines are much less stable at high pH than those involving pyrimidines, and that methods involving exposure to extreme alkaline conditions may give inaccurate information for some agents.     

Isolation of Chinese hamster cells hyposensitive to ethyl methanesulfonate and their characterization in induction and antagonization of sister-chromatid exchanges

Dominant lethal tests were performed on female mice injected intraperitoneally with cyclophosphamide (200 mg/kg) or with mitomycin C (0.2 or 5 mg/kg) at the preovulatory stage of oogenesis. Complementary experiments were undertaken to clarify the results obtained. Embryo culture showed that sterility found after treatment with cyclophosphamide or with the high dose of mitomycin C was the reflection of true dominant lethal effects. Mortality after cyclophosphamide treatment occurred predominantly at the 2- and 3-cell stages, while it was reported in all preimplantation stages after treatment with the high dose of mitomycin C. Embryos treated with the low dose of mitomycin C developed normally to the blastocyst stage, confirming the absence of preimplantation effects found with this dose in the dominant lethal test. Cytogenetic analysis of female pronuclei at the first cleavage division were performed after mating treated females with males homozygous for one Robertsonian translocation. This method allowed one to distinguish easily the female pronuclei from the male ones, which exhibited one translocated 'marker' chromosome. After treatment with cyclophosphamide, most female pronuclei showed multiple chromatid exchanges or shattering of the entire genome. After treatment with the high dose of mitomycin C, various types of premature chromosome condensation were found, and they were often accompanied by important interchromosome associations. After treatment with the low dose of mitomycin C, no structural chromosome aberrations were found, and the number of numerical anomalies was not significantly different from that found in control embryos. These last results suggest that the increase in rate of postimplantation loss obtained in the dominant lethal test with the low dose of mitomycin C was not due to clastogenic effects of this compound in the female germ cells, but rather to indirect effects on the maternal organism.     

Possible mechanism of congenital malformations induced by exposure of mouse preimplantation embryos to mitomycin C

ICR mice were treated intraperitoneally with mitomycin C at 5 mg/kg on day 3 of gestation. On day 18 of gestation, fetuses of treated dams were inspected for external, skeletal and visceral malformations. At 6 or 12 hr after mitomycin C treatment, the blastocysts were obtained from the uteri of treated dams and the degenerated cells within inner cell mass (ICM) and trophectoderm (TE) tissues were examined microscopically. On day 5, 8, 11, or 18 of gestation, the uteri of treated dams were obtained and those including embryos/fetuses and placentae were examined histologically. Finally, on each of gestational days 5-14, the blood of the treated dams was collected and the hematological parameters determined. Pre- and postimplantation losses in the dams treated with mitomycin C were significantly increased; increased frequency of abdominal wall defects and lumbar ribs in term fetuses, decreased fetal weight, and increased placental weight were noted as well. No significant increase in visceral malformations was found in term fetuses treated with mitomycin C. Frequency of degenerated cells within ICM and TE of blastocysts from dams treated with mitomycin C was significantly increased as compared with the controls. In dams treated with mitomycin C, decidua developed insufficiently and the trophoblast giant cell layer was not separated from the uterine lumen by maternal components; hemorrhage from the denuded trophoblast giant cell layer into the uterine lumen was noted. The number of erythrocytes, as well as hemoglobin concentration, hematocrit, and the percentage of reticulocytes in blood of dams treated with mitomycin C were significantly lower from days 6-12 of gestation, as compared with controls. The results of the present study showed that an increase in number of degenerated cells within blastocysts results in preimplantation loss and both maternal and embryonic hypoxia during major organogenesis results in postimplantation loss and congenital fetal malformations.     

丝裂霉素C抗性基因（mcr）的克隆及其高效表达

头状链轮丝菌（Ｓｔｒｅｐｔｏｖｅｒｔｉｃｉｌｌｉｕｍ ｃａｅｓｐｉｔｏｓｕｍ）ＡＴＣＣ２７４２２是抗肿瘤药物丝裂霉素Ｃ的主要产生菌。为了研究丝裂霉素Ｃ抗性的分子机制,实验通过鸟枪法克隆技术,从库中筛选得含有丝裂霉素Ｃ抗性基因（ｍｃｒ）的６．６ｋｂ外源片段的克隆子,对此外源片段进行一系列亚克隆,将丝裂霉素Ｃ抗性基因定位在３．１ｋｂ的片段中。序列分析的结果表明,此３．１ｋｂ外源片段中存在一长度为１３４     

Preparation of antigastric cancer monoclonal antibody MGb2-mitomycin C conjugate with improved antitumor activity

In the present study, an antigastric cancer monoclonal antibody, MGb2, was chosen to prepare antibody-mitomycin C conjugate with dextran T-40 as intermediary. Up to 20 molecules of mitomycin C were specifically bound per molecule of antibody, without significantly impairing the antigen-binding capacity of the antibody and the pharmacological activity of mitomycin C. The conjugate showed selective cytotoxicity upon human gastric cancer cell line SGC-7901 in vitro. Radioimmunoimaging and biodistribution studies indicated that, after conjugation with mitomycin C via dextran T-40 as intermediary, the tumor localization capacity of the antibody was well-retained. When tested in nude mice inoculated with human gastric carcinoma GAII in bilateral subrenal capsules, intraperitoneal injection of the conjugate twice a week for 3 weeks at the dose of 1 mg/kg of drug gave a tumor inhibitory rate of 152.29%, the result being far better than that of free mitomycin C or an irrelevant conjugate. A similar result was found in another nude mouse model of human gastric carcinoma SGC-7901. Meanwhile, after conjugation with antibody, the toxicity of mitomycin C on tested animals was significantly reduced.     

cea-kil operon of the ColE1 plasmid.

We isolated a series of Tn5 transposon insertion mutants and chemically induced mutants with mutations in the region of the ColE1 plasmid that includes the cea (colicin) and imm (immunity) genes. Bacterial cells harboring each of the mutant plasmids were tested for their response to the colicin-inducing agent mitomycin C. All insertion mutations within the cea gene failed to bring about cell killing after mitomycin C treatment. A cea- amber mutation exerted a polar effect on killing by mitomycin C. Two insertions beyond the cea gene but within or near the imm gene also prevented the lethal response to mitomycin C. These findings suggest the presence in the ColE1 plasmid of an operon containing the cea and kil genes whose product is needed for mitomycin C-induced lethality. Bacteria carrying ColE1 plasmids with Tn5 inserted within the cea gene produced serologically cross-reacting fragments of the colicin E1 molecule, the lengths of which were proportional to the distance between the insertion and the promoter end of the cea gene.     

The apoptotic effect of intercalating agents on HPV-negative cervical cancer C-33A cells

Summary. Cervical cancer is one of the leading causes of female cancer death worldwide with about 500,000 deaths per year. Both mitomycin C and cisplatin are alkylating agents, which bind and intercalate DNA, and thus used as anti-cancer drugs. In these studies, we focused on investigating the apoptotic effects of intercalating agents on HPV-negative cervical cancer C-33A cells. Accordingly, C-33A cells were treated with carboplatin, mitomycin C or cisplatin. Cell cycle analysis revealed that treatment with mitomycin C and cisplatin but not with carboplatin resulted in apoptosis. Both mitomycin C and cisplatin induced apoptosis in C-33A cells via caspase-8 and -3 processing in a Fas/FasL-dependent manner and also suppressed IL-18 expression, while they down-regulated IκB expression and up-regulated p65 expression. These results suggest that both mitomycin C and cisplatin induce apoptosis, not only via the caspase-8 and -3 dependent Fas/FasL pathway, but also via the regulation of NF-κB activity and IL-18 expression in HPV-negative cervical cancer C-33A cells.     

敲减BLM解旋酶表达增强前列腺癌PC3细胞对丝裂霉素C的敏感性

丝裂霉素C是一种广谱抗肿瘤抗生素,对多种癌症有抗癌作用,其作用原理可使细胞的DNA发生链间交联,引起DNA双链断裂,阻碍DNA的复制,从而抑制肿瘤细胞分裂。临床上主要用于胃癌、肠癌、肝癌及胰腺癌等消化道癌方面的治疗。本文研究丝裂霉素C对转染人BLM解旋酶基因（shRNA载体）前后前列腺癌PC3细胞活性的影响。使用前期成功构建的干扰载体转染PC3细胞,在转染48 h后加药,通过荧光定量PCR、MTT法、Transwell小室实验、细胞划痕实验、流式细胞术,分别检测加药12、24、36 h BLM基因的表达量、PC3细胞增殖能力、侵袭能力、迁移能力及凋亡情况的变化。结果显示,敲减BLM基因表达后的PC3细胞相对于正常PC3细胞其增殖能力、侵袭能力和迁移能力能显著被丝裂霉素C抑制,且丝裂霉素C能显著促进其细胞的凋亡,说明BLM基因低表达的前列腺癌细胞对丝裂霉素C更敏感。研究结果为丝裂霉素C在前列腺癌的临床治疗上奠定了理论基础。     

Comparative Study of the Events Associated with Colicin Induction

Colicinogenic factors ColI and ColV, which have been shown to behave as sex factors, could not be induced with mitomycin C. In contrast, the ColE(1), ColE(2), and ColE(3) factors, which do not exhibit any fertility factor characteristics, are inducible by this agent. The induced production of colicins E(1), E(2), and E(3) was accompanied by a loss in viability at a concentration of mitomycin C which was bacteriostatic to noncolicinogenic cells or to cells carrying the ColV or ColI factors. The loss in viability accompanying the mitomycin C induction of the ColE(1), ColE(2), or ColE(3) factors also occurred when colicin synthesis was blocked by chloramphenicol or amino acid starvation. However, chloramphenicol was able to block the loss of viability of a recipient cell after mitomycin C induction of a newly acquired Col factor if the antibiotic was present throughout the mating period. No detectable internal colicin or colicin precursor could be demonstrated during the lag period prior to the appearance of colicin outside the cell 20 to 30 min after the addition of mitomycin C. If chloramphenicol was present during the lag period following the addition of mitomycin C, colicin synthesis began immediately after the removal of these antibiotics. The synthesis of tryptophan synthetase and induced beta-galactosidase proceeded normally throughout the lag period and well into the period of colicin production. Regulation of beta-galactosidase synthesis did not seem to be profoundly affected during the lag period subsequent to mitomycin C addition. Induced colicin synthesis, like bacterial or induced prophage protein synthesis, was subject to inhibition by virulent phage infection.     

Fanconi anaemia cells are not uniformly deficient in unhooking of DNA interstrand crosslinks,induced by mitomycin C or 8-methoxypsoralen plus UVA

Summary Fanconi anaemia (FA) cells are extremely sensitive to crosslinking agents, e. g. mitomycin C, but only moderately sensitive to trimethylpsoralen plus UVA. Evidence has been reported suggesting that there is a deficient DNA crosslink repair mechanism in FA cells, but others failed to confirm this conclusion using other methods and other crosslinking agents. We reinvestigated the mitomycin C and 8-methoxypsoralen crosslink repair in FA cells with a high sensitivity to mitomycin C. Although an essentially similar methodology was used to that previously described, no difference between the control and FA cell strains was observed, neither for mitomycin C- nor for 8-methoxypsoralen-induced crosslinks.     

In vitro tumor cell killing by peritoneal macrophages from mitomycin C-treated rats

Summary The local cellular response induced by intraperitoneal injection of mitomycin C was examined in terms of cell-mediated cytotoxicity for tumor cells. An in vitro cytolysis assay involving ¹²⁵I-iododeoxyuridine-labeled tumor target cells revealed that treatment of normal ACI/N rats (200 g) with a single intraperitoneal injection of mitomycin C (50, 100, or 200 g) induced tumoricidal macrophages in the peritoneal cavity. The tumoricidal activity was dependent on the dose of mitomycin C injected and it was detectable as early as 1 day after the intraperitoneal injection of mitomycin C. In addition to the increased tumoricidal activity, the functional activities of the peritoneal macrophages were found to be increased with respect both to uptake of 2-deoxy-d-glucose and to phagocytosis of latex beads. Additional experiments excluded the possibility that the tumor cell cytolysis was the result of direct cytotoxicity by mitomycin C that might have been incorporated in the peritoneal macrophages or of nutrient depletion in the medium during the cytolysis assay. Furthermore, endotoxin contamination of the mitomycin C, which might have produced the activated macrophages, was not detected. The mechanism by which mitomycin C injected intraperitoneally induced the tumoricidal macrophages locally remains uncertain; however, it is possible also in clinical situations.