Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA

Belcourt, Michael F.; Penketh, Philip G.; Hodnick, William F.; Johnson, David A.; Sherman, David H.; Rockwell, Sara; Sartorelli, Alan C.

doi:10.1073/pnas.96.18.10489

Cited by 24 publications

(24 citation statements)

References 17 publications

(21 reference statements)

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“…The existence of enzymes capable of oxidizing MCH 2 back to the MC prodrug or other inactive forms gives rise to another possible mechanism by which resistance to the mitomycin antibiotics and toxicity differentials between oxygenated and hypoxic tumor cells could arise. Recently, work from our laboratory has shown that MCRA expressed in CHO-K1/dhfr Ϫ cells conferred profound resistance to MC under aerobic conditions only, resulting in a phenotype with an extreme oxic/hypoxic differential (14). High levels of resistance only under aerobic conditions resembles that produced in cell lines selected aerobically for MC resistance (see Ref.…”

Section: Discussionmentioning

confidence: 99%

“…DNA Cross-linking and Nicking-DNA cross-linking kinetics and the extent of DNA cross-linking were determined using a DNA renaturation assay (14,19). The assay is based upon the observation that, under conditions of neutral pH, upon snap cooling thermally denatured covalently cross-linked T7 DNA rapidly renatures since the strands are held in register, yielding a highly fluorescent complex with H33258, whereas T7 DNA containing no cross-links does not.…”

Section: Methodsmentioning

confidence: 99%

“…Expression of the cDNA for the bacterial resistance protein MCRA in CHO-K1/dhfr Ϫ cells resulted in profound resistance to MC in these cells under aerobic conditions, with little change in sensitivity to MC under hypoxia (14). The marked resistance to MC under aerobic conditions observed in MCRA-expressing CHO-K1/dhfr Ϫ cells resembles that produced in cell lines selected for resistance to MC under aerobic conditions (14). This finding suggests that a mechanism of resistance based upon the oxidation of MCH 2 by a functional homologue of the MCRA protein may be operative.…”

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confidence: 99%

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confidence: 99%

“…One of these genes, mcrA, codes for a 54-kDa protein (MCRA) that appears to be a mitomycin C hydroquinone oxidase (13,14). Expression of the cDNA for the bacterial resistance protein MCRA in CHO-K1/dhfr Ϫ cells resulted in profound resistance to MC in these cells under aerobic conditions, with little change in sensitivity to MC under hypoxia (14). The marked resistance to MC under aerobic conditions observed in MCRA-expressing CHO-K1/dhfr Ϫ cells resembles that produced in cell lines selected for resistance to MC under aerobic conditions (14).…”

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Inhibition of DNA Cross-linking by Mitomycin C by Peroxidase-mediated Oxidation of Mitomycin C Hydroquinone

Penketh¹,

Hodnick²,

Belcourt³

et al. 2001

Journal of Biological Chemistry

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Mitomycin C requires reductive activation to crosslink DNA and express anticancer activity. Reduction of mitomycin C (40 M) by sodium borohydride (200 M) in 20 mM Tris-HCl, 1 mM EDTA at 37°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 t1 ⁄2 of ϳ15 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 crosslinking, 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.The mitomycin antibiotics, produced by various Streptomyces, give rise to electrophiles capable of cross-linking DNA following either one-electron or two-electron reduction by enzymatic or chemical systems (1). The one-electron reduction product, the mitomycin C semiquinone radical anion (MC . ), 1 reacts with molecular oxygen (itself a stable diradical) at close to the diffusion controlled rate (10 9 to 10 10 M Ϫ1 s Ϫ1 ) to give the parent mitomycin C (MC) quinone and superoxide (O 2 . ) (2).Since very few cross-links are required to give rise to a lethal event (3), the regeneration of MC and the production of a superoxide anion, a species of low toxicity relative to a DNA cross-link, represents a detoxification step. Under physiological oxygen concentrations, the half-life of MC . would be expected to be less than 0.1 ms. Therefore, in the presence of physiological concentrations of oxygen, only an extremely small proportion of the MC . produced could be involved in the direct alkylation of biomolecules, and the yields of DNA cross-links via this pathway would be negligible. The two-electron reduced species, mitomycin C hydroquinone (MCH 2 ), does not react rapidly with oxygen and, therefore, cross-links DNA in a manner largely independent of the concentration of oxygen. Thus, the degree of initial DNA damage by MC under aerobic conditions almost exclusively depends upon a two-electron reduction to MCH 2 . Under very low oxygen concentrations, greater cellular damage would be expected, reflecting the rate of production of both MCH 2 and MC . . There is also evidence that most of the damage resulting from the production of MC . under hypoxic conditions is due to disproportionation (4) or further reductio...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Inhibition of DNA Cross-linking by Mitomycin C by Peroxidase-mediated Oxidation of Mitomycin C Hydroquinone

Penketh¹,

Hodnick²,

Belcourt³

et al. 2001

Journal of Biological Chemistry

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Antitumor Natural Products

Mitscher

Dutta

2003

Burger's Medicinal Chemistry and Drug Discovery

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The literature associated with major naturally occurring antitumor agents and related substances is reviewed and illustrated. Covered are dactinomycin, bleomycin, mitomycin, plicamycin, anthracyclines (daunorubicin, doxorubicin, epirubicin, idarubicin, and valrubicin), camptothecins (topotecan and irinotecan), idopodophyllotoxins (etoposide and teniposide), taxus diterpenes (docetaxel and paclitaxel), and vinca dimeric alkaloids (vinblastine, vincristine, and vinorelbine).

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Natural Products as Cytotoxic Agents

Cutler

2010

Burger's Medicinal Chemistry and Drug Discovery

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In the first decade of the twenty‐first century, the occurrence of cancers has increased not only in First World countries but also in those countries considered to have a lower standard of living, according to a world cancer report issued in 2008 for the IARC Nonserial Publication by Boyle and Levin. So widespread is cancer, in its various forms, that most adults have either had family members or had friends who have been afflicted by the disease. Projected figures for cancers indicate, in the aforementioned publication, that 12 million cancer cases were diagnosed in 2008 alone and that the number of cancer victims has doubled since 1978. Coupled to the psychological impact that a diagnosis has on an individual is the cost of treatment that must also be considered. Again, according to the National Cancer Institute (December 2008) these costs are estimated to be US$147 billion by 2020. An article, published in USA Today , claims that new cancer cases in 2008 were projected to be 40,480. And the cost for elderly Medicare patients will be US$21.1 billion for 5 years of treatment for the period 2004–2009. Sadly, one in eight patients with advanced stages of cancer will turn down the offer of recommended care because of costs. While cases may have been misdiagnosed in the twentieth century, the sophisticated techniques available today give a clearer graph of the incidence of the disease. With advanced diagnostics, there has been a concomitant advance in anticancer chemotherapy.

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Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA

Cited by 24 publications

References 17 publications

Inhibition of DNA Cross-linking by Mitomycin C by Peroxidase-mediated Oxidation of Mitomycin C Hydroquinone

Inhibition of DNA Cross-linking by Mitomycin C by Peroxidase-mediated Oxidation of Mitomycin C Hydroquinone

Antitumor Natural Products

Natural Products as Cytotoxic Agents

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