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.     

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.     

Expression of the SOS response following simultaneous treatment with methyl-nitrosoguanidine and mitomycin C in Escherichia coli

Simultaneous treatment of Escherichia coli cultures with methyl-nitrosoguanidine and mitomycin C induces recA-dependent inhibition of respiration but not inhibition of cell division. This pattern of SOS functions expression is the same as that is found following treatment with methyl-nitrosoguanidine alone and contrary to the pattern induced after mitomycin C addition. The same result is obtained when a culture of E. coli RecA441 (formerly tif) is shifted to 42 degrees C and treated simultaneously with methyl-nitrosoguanidine. The suppressor effect of this compound over the pattern of SOS functions expression induced by mitomycin C or high temperature in recA441 mutants is directly related to the increase in its dose. Moreover, the division temperature-sensitive mutant ftsA treated with methyl-nitrosoguanidine and high temperature does not show any decrease in its normal filamentous growth when cultured at 42 degrees C. This indicates that the effect of methyl-nitrosoguanidine on the recA-independent inhibition of cell division is not due to any indiscriminate effect of this compound over the division process. These results suggest that the specific kind of lesion caused in DNA is very important in determining which SOS function is induced.     

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。     

Genetic control of the protective effect of spermidine in Escherichia coli cells as affected by mitomycin C

The lethal action of mitomycin C and the effect of mutual treatment with mitomycin C and spermidine on Escherichia coli were studied. DNA repair in cells treated with mitomycin C was shown to have some differences, as compared to that of UV-induced pyrimidine dimers. The presence of the additive sbcB mutation increases the resistance of wild-type bacteria as well as of recBrecC and recF mutants to the lethal action of mytomicin C. Preliminary treatment of bacteria with spermidine increases resistance to the lethal action of the mutagen in wild-type bacteria as well as uvrB, recBrecC and sbcB strains. However, no such effect was observed in recF, recFsbcB and uvrE strains. The data suggest that the protective action of spermidine may be connected with stimulation of RecF-pathway of postreplication repair.     

Mitomycin C pharmacokinetics in patients with recurrent or metastatic colorectal carcinoma

The pharmacokinetics of mitomycin C as a single agent have been determined in 25 treatment courses given to 18 patients with recurrent or metastatic colorectal carcinoma using a high performance liquid chromatography (HPLC) assay to analyze plasma and urine samples. The plasma pharmacokinetics conformed to a two-compartment linear model in 21 of 25 courses monitored with a mean t1/2 lambda 1 of 9.8 +/- 1.2 (SEM) min and mean t1/2 lambda z of 64.1 +/- 8.9 (SEM) min. The large variation observed in t1/2 lambda z was not related to dose or treatment, but an interaction of these two factors approached significance (p = 0.057). Renal excretion in the 12 courses in which it was determined averaged only 2.3% of the total administered dose during the first 4 h monitored and no mitomycin C metabolites were detected in plasma or urine by the HPLC technique used. The most common toxicity, thrombocytopenia, did not correlate with t1/2 lambda z or the area under the curve. This may be due to a failure to monitor active metabolites of mitomycin C; other factors besides plasma drug concentrations that mediate toxicity towards marrow elements; or the small number of courses associated with thrombocytopenia (less than 100,000/mm3). Our study indicates that an interaction of drug dose and treatment course may be associated with increasing t1/2 lambda z; the renal clearance contributes a small component of mitomycin C elimination; metabolites of mitomycin C cannot be detected by the present HPLC technique; and routine monitoring of mitomycin C using present methods cannot be recommended for clinical use to predict toxicity.     

Bone marrow and lymphocyte cytogenetics of rhesus monkeys (Macaca mulatta) treated with the clastogen mitomycin C

Rhesus monkeys (Macaca mulatta) were used to determine their effectiveness as experimental animals for different cytogenetic tests with mitomycin C (MC). The micronucleus test (MNT) and/or chromosome analysis of blood and bone marrow were made before and/or after the treatment with mitomycin C. Thus, the controls data and treated data were obtained from the same animals. With the employed methology, the micronucleus test could not be performed on living animals. Less chromosomal damage was detected in the micronucleus test of post-mortem samples than in the chromosome analysis of bone marrow. No influence by the mutagen could be observed in lymphocyte chromosomes at any of the different times of analysis. In contrast to this, bone-marrow chromosomes seemed to be highly affected by mitomycin C at day 1, 2 and 3 after injection. However, before treatment and at day 14, 16 and 17 after treatment there was no visible increase in chromosomal aberration in bone marrow.     

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.     

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.     

Mitomycin C-induced acceleration of liver cell polyploidization in adult rat after partial hepatectomy

Mitomycin C, a DNA cross-linking agent, was administered for a week intraperitoneally to a normal 9-week-old rat in which hepatocyte proliferative activity had almost ceased. In the rat treated with mitomycin C, the number of polyploid cells with the DNA amounts more than 4C was not increased in comparison with that in the control rat without any treatment. However, the partial hepatectomy in the rat pretreated with mitomycin C provoked prominent hepatocyte polyploidization much greater in degree than that found in the hepatectomized rat without mitomycin C treatment. These results seem to indicate that the existence of cross-linking DNA damages is a latent factor necessary for the induction of abortive mitosis of a cell after completion of DNA synthesis, which results in the production of a mononuclear polyploid cell in one-step higher DNA class. Cross-linking DNA damages and DNA synthesis are the essential factors for the manifestation of polyploidization.     

Occurrence and Control of Sporadic Proliferation in Growth Arrested Swiss 3T3 Feeder Cells

Growth arrested Swiss mouse embryonic 3T3 cells are used as feeders to support the growth of epidermal keratinocytes and several other target cells. The 3T3 cells have been extensively subcultured owing to their popularity and wide distribution in the world and, as a consequence selective inclusion of variants is a strong possibility in them. Inadvertently selected variants expressing innate resistance to mitomycin C may continue to proliferate even after treatment with such growth arresting agents. The failure of growth arrest can lead to a serious risk of proliferative feeder contamination in target cell cultures. In this study, we passaged Swiss 3T3 cells (CCL-92, ATCC) by different seeding densities and incubation periods. We tested the resultant cultures for differences in anchorage-independent growth, resumption of proliferation after mitomycin C treatment and occurrence of proliferative feeder contaminants in an epidermal keratinocyte co-culture system. The study revealed subculture dependent differential responses. The cultures of a particular subculture procedure displayed unique cell size distribution and disintegrated completely in 6 weeks following mitomycin C treatment, but their repeated subculture resulted in feeder regrowth as late as 11 weeks after the growth arrest. In contrast, mitomycin C failed to inhibit cell proliferation in cultures of the other subculture schemes and also in a clone that was established from a transformation focus of super-confluent culture. The resultant proliferative feeder cells contaminated the keratinocyte cultures. The anchorage-independent growth appeared in late passages as compared with the expression of mitomycin C resistance in earlier passages. The feeder regrowth was prevented by identifying a safe subculture protocol that discouraged the inclusion of resistant variants. We advocate routine anchorage-independent growth assay and absolute confirmation of feeder disintegration to qualify feeder batches and caution on the use of fetal bovine serum.     

Characterization of cytotoxic and genotoxic effects of different compounds in CHO K5 cells with the comet assay (single-cell gel electrophoresis assay)

Different variants of the comet assay were used to study the genotoxic and cytotoxic properties of the following eight compounds: chloral hydrate, colchicine, hydroquinone, DL-menthol, mitomycin C, sodium iodoacetate, thimerosal and valinomycin. Colchicine, mitomycin C, sodium iodoacetate and thimerosal induced genotoxic effects. The other compounds were found to be inactive. The compounds were tested in the standard comet assay as well as in the all cell comet assay (recovery of floating cells after treatment), designed in our laboratory for adherently-growing cells. This latter procedure proved to be more adequate for the assessment of the cytotoxicity for some of the compounds tested (hydroquinone, DL-menthol, thimerosal, valinomycin). Colchicine was positive in the standard comet assay (3h treatment) and in the all cell comet assay (24h treatment). Sodium iodoacetate and thimerosal were positive in the standard and/or the all cell comet assay. Chloral hydrate, hydroquinone, sodium iodoacetate, mitomycin C and thimerosal were also tested in the modified comet assay using lysed cells. Mitomycin C and thimerosal showed effects in this assay, whereas sodium iodoacetate was inactive. This indicates that it does not induce direct DNA damage. Compounds that are known or suspected to form DNA-DNA cross-links or DNA-protein cross-links (chloral hydrate, hydroquinone, mitomycin C and thimerosal) were checked for their ability to reduce ethyl methanesulfonate (EMS)-induced DNA damage. This mode of action could be demonstrated for mitomycin C only.     

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.     

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.     

Effect of certain factors on the variability of strains of pertussis microbes

The population of degraded cells having stable changes in some phenotypical properties were isolated after subculturing some laboratory Bordetella pertussis strains in Bordet-Gengou culture medium and casein-charcoal agar with blood, treated with mitomycin C and allowed to proliferate in the spleen of mice injected intravenously with microbial suspensions. The characteristics indicative of cell degradation were the growth of large yellowish-white colonies appearing in 24 hours, the destruction of the agglutinogenic complex and toxic substances causing the atrophy of the spleen in mice, the increased capacity for active proliferation in the spleen. Electron-microscopic study revealed that the variants obtained by subculturing in culture media had the damaged membrane with the formation of cell-wall invaginations having rounded membrane-like formations on their surface, disappearing after treatment with mitomycin C; the treatment of the initial strains with mitomycin C resulted in cytoplasmic damage with the coagulation of the nucleoid.     

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.     

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.     

Non-random intrachromosomal distribution of chromatid aberrations induced by X-rays,alkylating agents and ethanol in Vicia faba

A reconstructed karyotype of Vicia faba with all chromosomes individually distinguishable was treated with triethylene melamine (TEM), cytostasan (CYT) (a new benzimidazol nitrogen mustard), mitomycin C (MI), ethanol (EA) and X-rays. The distribution within chromosomes of induced chromatid abberations was non-random for all agents. The number of segments involved in aberration clustering corresponded to the number of sites representing constitutive heterochromatin, or the regions immediately adjacent to these, as evidenced by the position of Giemsa marker bands. Which of these potential regions of aberration clustering reacted with preferential involvement in aberrations was, in part at least, dependent upon the inducing agent used. It is argued that this may be due to differences in the base composition and/or molecular conformation of heterochromatic regions. Unexpectedly, the distribution pattern of chromatid aberrations induced by mitomycin C was found to be different from those after treatment with the alkylating agents TEM and cytostasan although mitomycin C is assumed to induce aberrations via alkylation. If mitomycin C-induced aberrations are indeed due to alkylation, this indicates that different alkylating agents do not necessarily result in identical patterns of abberation clustering. The other two alkylating agents and ethanol resulted in similar patterns of preferential distribution of abberations. X-Ray induced chromatid aberrations also showed a non-random intrachromosomal distribution, but the clustering was less pronounced than after treatment with the chemical agents.     

Hydroxyl radical production by free and DNA-bound aminoquinone antibiotics and its role in DNA degradation. Electron spin resonance detection of hydroxyl radicals by spin trapping.

The reduced antitumor antibiotic mitomycin C in aqueous solution exposed to air gives a 36-line electron spin resonance spectrum of the semiquinone identified by computer simulation. Incubation of this radical with the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) gives the PBN.OH nitroxide radical identified by independent generation. This nitroxide radical is also formed from similar treatment of a DNA to which mitomycin C is covalently attached. Incubation of the semiquinone from mitomycin C, mitomycin B, or streptonigrin (SN) with catalase or with superoxide dismutase inhibits the generation of OH, implying the intermediacy of H2O2 and O2 in its formation. The formation of the spin-trapped nitroxide radical is similarly inhibited by EDTA, suggesting the intermediacy of trace metal ions in the generation of hydroxyl radicals from SN. The results are consistent with the generation by the aminoquinone antibiotics in vivo of OH. already implicated in the degradation of DNA.     

Evaluation of spermatogenic response of mice to the induction of mutations by combined treatment with X rays and antineoplastic drugs

Combined treatment with low doses of X-rays plus cyclophosphamide (0.25 Gy+25 mg/kg body weight) or X-rays plus mitomycin C (0.25 Gy+1.75 mg/kg body weight) did not induce significant dominant lethal effects in any stage of spermatogenesis when a parameter representing pre- and postimplantation loss, such as the decrease of live implants per female, was applied. After combined exposure to high doses of X-rays plus cyclophosphamide (1.00 Gy+100 mg/kg body weight) an increase of dominant lethal mutations (DLMs) was observed in differentiating spermatogonia, spermatids, and spermatozoa with the same parameter. Combined treatment with high doses of X-rays plus mitomycin C (1.00 Gy+5.25 mg/kg body weight) produced DLMs in differentiating spermatogonia and late spermatocytes. A calculation of enhanced risk was applied to the data of DLMs from the combined treatment regimen and was based on the proportion of dead implants (postimplantation loss only). Enhanced risk could be shown not only after high but also after low combined exposure to X-rays plus cyclophosphamide and X-rays plus mitomycin C. With low doses this enhanced risk was observed in spermatids for X-rays plus cyclophosphamide and in differentiating spermatogonia to early spermatocytes for X-rays plus mitomycin C.