Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA. (1/13)

The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduced and activated intracellularly, producing cytotoxic semiquinone anion radical and hydroquinone reduction intermediates. In vitro, MCRA protects DNA from cross-linking by the hydroquinone reduction intermediate of these mitomycins by oxidizing the hydroquinone back to the parent molecule; thus, MCRA acts as a hydroquinone oxidase. These findings suggest potential therapeutic applications for MCRA in the treatment of cancer with the mitomycins and imply that intrinsic or selected mitomycin C resistance in mammalian cells may not be due solely to decreased bioactivation, as has been hypothesized previously, but instead could involve an MCRA-like mechanism.  (+info)

Isolation and identification of urinary metabolites of porfiromycin in dogs and humans. (2/13)

Porfiromycin (PM), a bioreductive alkylating agent, is currently under development for the treatment of head and neck cancers as an adjunct to radiation therapy in phase III clinical trials. After i.v. administration of a single dose of PM to patients at 40 mg/m2, urinary metabolites were isolated by HPLC and identified by atmospheric pressure chemical ionization mass spectrometry. In dogs, [methyl-3H]PM was administered i.v. to three Beagle dogs at a single dose of 2 mg/kg. Urinary excretion of radioactivity and PM at different times was determined by liquid scintillation counting and by HPLC, respectively. An average of 48.0% of total radioactivity given to the dogs was cumulatively excreted in urine over a period of 7 days. Unchanged parent drug excreted in urine accounted for 10.8% of the administered dose over the same period of time. The results indicated that the majority of excreted dose in dog urine was in the form of metabolites. Three phase I and four phase II metabolites of PM were identified in human and dog urine. The phase I metabolites are 2-methylamino-7-aminomitosene, 1,2-cis and 1,2-trans-1-hydroxy-2-methylamino-7-aminomitosenes. The phase II metabolites are a pair of isomeric N-acetylcysteine S-conjugates and a pair of isomeric cysteine S-conjugates of mitosenes at the C-1 and C-10 positions. Most of the identified metabolites were confirmed by comparison with synthetic reference standards using HPLC and liquid chromatography/mass spectrometry (LC/MS). The identification of mercapturic acids and cysteine S-conjugates in urine indicates that the metabolism of PM may be through GSH conjugation.  (+info)

Mechanisms of resistance to etoposide and teniposide in acquired resistant human colon and lung carcinoma cell lines. (3/13)

Stable acquired resistance to etoposide (VP-16) or teniposide (VM-26) in HCT116 human colon carcinoma cells and A549 human lung adenocarcinoma cells, was previously obtained by weekly 1-h exposures to either drug (B. H. Long, Natl. Cancer Inst. Monogr., 4: 123-127, 1987). The purpose of this study was to identify possible mechanisms of resistance present in these cells by using human mdr1 and topoisomerase II DNA probes, antibodies to these gene products, and P4 phage unknotting assay for topoisomerase II activities. HCT116(VP)35 cells were 9-, 7-, and 6-fold resistant to VP-16, VM-26, and Adriamycin, respectively, and showed no cross-resistance to colchicine and actinomycin D. These cells had no differences in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels but displayed decreased levels of topoisomerase II mRNA and enzyme activity without any alteration of drug sensitivity displayed by the enzyme. HCT116(VM)34 cells were 5-, 7-, and 21-fold resistant to VP-16, VM-26, and Adriamycin; were cross-resistant to colchicine (7-fold) and actinomycin D (18-fold); and possessed a 9-fold increase in mdr1 mRNA and increased P-glycoprotein without evidence of mdr1 gene amplification. No alterations in topoisomerase II gene or mRNA levels, enzyme activity, or drug sensitivity were observed. A549(VP)28 and A549(VM)28 cells were 8-fold resistant to VP-16 and VM-26 and 3-fold resistant to Adriamycin. Both lines were not cross-resistant to colchicine or actinomycin D but were hypersensitive to cis-platinum. No alterations in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels, but lower topoisomerase II mRNA levels and decreased enzyme activities, were observed. Of the four acquired resistant cell lines, resistance is likely related to elevated mdr1 expression in one line and to decreased topoisomerase II expression in the other three lines.  (+info)

Potentiation of mitomycin C and porfiromycin antitumor activity in solid tumor models by recombinant human interleukin 1 alpha. (4/13)

The time- and dose-dependent effects of recombinant human interleukin 1 alpha (IL-1 alpha) on the antitumor activity of mitomycin C (MMC) and porfiromycin (PORF) were studied in RIF-1 and Panc02 solid tumor model systems. IL-1 alpha produced dose-dependent sensitization of clonogenic RIF-1 tumor cells to MMC in vivo. IL-1 alpha chemosensitization was highly schedule dependent, and the most efficacious schedules produced dose-modifying factors of 3.6 and 5.1 for MMC and PORF, respectively. More than additive clonogenic cell kill after IL-1 alpha-chemotherapy combinations reflected increased cellular sensitivity to MMC and PORF. The combinations also produced marked decreases in the yield of viable tumor cells, suggesting that the bioreductive drugs may have also potentiated the microvascular injury and ischemia produced by IL-1 alpha. Dexamethasone inhibited and ketoconazole, an inhibitor of corticosterone biosynthesis, enhanced IL-1 alpha-mediated chemosensitization in these models. IL-1 alpha mediated chemosensitization to MMC, and PORF was also demonstrated by tumor growth inhibition in the RIF-1 model and increased survival of mice in the spontaneously metastasizing Panc02 system. Chemosensitization of bone marrow spleen colony-forming units was not seen. IL-1 alpha (1000 units/ml) had no effect on MMC and PORF cytotoxicity in RIF-1 and PORF cell lines in vitro. The results indicate that the tumor-specific IL-1 alpha-induced pathophysiologies can sensitize solid tumors to agents which are preferentially activated, retained, and cytotoxic to cells under hypoxic conditions. Our results suggest that strategies combining bioreductively activated hypoxic cell cytotoxins and biological agents might offer efficacious alternatives or adjuvants to conventional combination approaches.  (+info)

Deficient activation by a human cell strain leads to mitomycin resistance under aerobic but not hypoxic conditions. (5/13)

Two non-transformed human skin fibroblast strains, GM38 and 3437T, were found to be more sensitive to the bioreductive alkylating agents mitomycin C (MMC) and porfiromycin (PM) under hypoxic compared to aerobic conditions. One of these strains, 3437T, was 6-7 times more resistant to these agents under aerobic exposure conditions, but was identical in sensitivity to the normal strain, GM38, under hypoxic conditions. Aerobic 3437T cells demonstrated no increased resistance to cisplatin compared to the normal strain, arguing against enhanced ability to repair DNA interstrand cross-links as the underlying explanation for the mitomycin resistance. The aerobic resistance of 3437T was not altered by dicumarol, an inhibitor of the enzyme DT-diaphorase which is believed to be involved in aerobic activation of MMC and PM. Dicumarol did increase the resistance of GM38, but not to the same level of resistance demonstrated by 3437T. These results suggest that the aerobic MMC and PM resistance of 3437T may arise, in part, from a deficiency in DT-diaphorase activity. The identical sensitivities under hypoxic conditions indicate that drug activation pathways operative in the absence of oxygen are similar in both the normal and 3437T cells.  (+info)

Modification of the metabolism and cytotoxicity of bioreductive alkylating agents by dicoumarol in aerobic and hypoxic murine tumor cells. (6/13)

We have demonstrated previously that dicoumarol (DIC) increased the generation of reactive metabolites from mitomycin C (MC) in EMT6 cells under hypoxic conditions in vitro. This increased reaction rate was associated with an increased toxicity of MC to hypoxic EMT6 cells. In contrast, aerobic cells treated with DIC in vitro were protected from MC toxicity. We now demonstrate that DIC sensitizes EMT6 cells to two MC analogues, porfiromycin (POR) and the 7-N-dimethylaminomethylene analogue of mitomycin C (BMY-25282), in hypoxia and protects cells from these agents in air, despite the fact that POR is preferentially toxic to hypoxic cells and BMY-25282 is preferentially toxic to aerobic cells. In contrast, DIC increases menadione cytotoxicity in both air and hypoxia and has no effect on the cytotoxicity of Adriamycin. We have also shown previously that the preferential toxicity of POR to hypoxic cells is associated with an increased rate of drug uptake. In the present study, DIC had no measurable effect on the uptake of [3H]POR but increased the extent of efflux of this agent. MC-induced DNA cross-links, which have been proposed as the lesions responsible for the lethality of MC, are decreased by DIC in air and increased by DIC in hypoxia, in concert with the observed modifications of MC cytotoxicity by DIC. However, in aerobic cells treated with DIC and MC, the decrease in DNA interstrand cross-links is not directly associated with a decrease in cytotoxicity. L1210 cells, which have no measurable quinone reductase activity, demonstrate increased toxicity when treated with DIC and MC in hypoxia, as observed with EMT6 cells. Unlike EMT6 cells, however, L1210 cells are not protected by DIC from MC toxicity in air. Taken together, these findings suggest that DIC is altering the intracellular metabolism of MC and that quinone reductase or another, unidentified, enzyme sensitive to DIC may be involved in activating MC to a toxic product in aerobic EMT6 cells.  (+info)

Mechanism of transport and intracellular binding of porfiromycin in HCT 116 human colon carcinoma cells. (7/13)

The mechanism of uptake and efflux of porfiromycin (PFM) by HCT 116 human colon carcinoma cells or freshly obtained human RBC was investigated. The time course of uptake of radioactivity upon exposure of HCT 116 cells to [14C]PFM showed one fast and one slow phase of linear increase. The initial phase of PFM uptake was not saturable with external drug concentrations from 2 to 100 microM. PFM accumulation was temperature dependent with a temperature coefficient (Q10 24-37 degrees C) of 2.3 +/- 0.3. PFM uptake was not affected either by individual inhibitors such as 1 mM 2,4-dinitrophenol, sodium azide, iodoacetic acid, ouabain, 0.02 mM oligomycin, p-hydroxylmercuribenzoate, 0.2 mM N-ethylmaleimide, or by combinations of inhibitors. PFM uptake did not demonstrate competitive inhibition by unlabeled PFM and mitomycin C. Efflux of cellular radioactivity was not affected by the above mentioned inhibitors or by verapamil, diltiazem, or trifluoperazine. Only aliphatic alcohols accelerated the initial influx rate. The RBC, however, only exhibited the initial fast accumulation of [14C]PFM, and all the 14C accumulated by RBC was exchangeable. These data demonstrate that the uptake and the efflux of PFM in HCT 116 cells and RBC comprise a passive diffusion process.  (+info)

Cytotoxicity and DNA lesions produced by mitomycin C and porfiromycin in hypoxic and aerobic EMT6 and Chinese hamster ovary cells. (8/13)

Solid neoplasms may contain deficient or poorly functional vascular beds, a property that leads to the formation of hypoxic tumor cells, which form a therapeutically resistant cell population within the tumor that is difficult to eradicate by ionizing irradiation and most existing chemotherapeutic agents. As an approach to the therapeutic attack of hypoxic cells, we have measured the cytotoxicity and DNA lesions produced by the bioreductive alkylating agents mitomycin C and porfiromycin, two structurally similar antibiotics, in oxygen-deficient and aerobic cells. Mitomycin C and porfiromycin were preferentially cytotoxic to hypoxic EMT6 cells in culture, with porfiromycin producing a greater differential kill of hypoxic EMT6 cells relative to their oxygenated counterparts than did mitomycin C. Chinese hamster ovary cells were more resistant to these quinone antibiotics; although in this cell line, porfiromycin was significantly more cytotoxic to hypoxic cells than to aerobic cells, and the degree of oxygenation did not affect the toxicity of mitomycin C. Alkaline elution methodology was utilized to study the formation of DNA single-strand breaks and DNA interstrand cross-links produced by mitomycin C and porfiromycin in both EMT6 and Chinese hamster ovary cells. A negligible quantity of DNA single-strand breaks and DNA interstrand cross-links were produced in hypoxic and aerobic Chinese hamster ovary cells by exposure to mitomycin C or porfiromycin, a finding consistent with the considerably lower sensitivity of this cell line to these agents. In EMT6 tumor cells, no single-strand breaks appeared to be produced by these antitumor antibiotics under both hypoxic and aerobic conditions; however, a significant number of DNA interstrand cross-links were formed in this cell line following drug treatment, with substantially more DNA interstrand cross-linking being produced under hypoxic conditions. Mitomycin C and porfiromycin caused the same amount of cross-linking under conditions of oxygen deficiency; however, mitomycin C produced considerably more DNA cross-linking than did porfiromycin in oxygenated cells. DNA interstrand cross-links were observed in hypoxic EMT6 cells throughout a 24-h period following removal of mitomycin C and porfiromycin, with a decrease in DNA interstrand cross-links observed at 24 h. An increase in DNA interstrand cross-links occurred in aerobic EMT6 cells treated with mitomycin C and porfiromycin at 6 h after drug removal, with a decrease in these lesions being observed by 24 h, suggesting that the rate of formation of the cross-links may be slower and the removal of cross-links more rapid under aerobic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)  (+info)