Association between serotonin transporter availability and overall rating scores of quality of life in healthy volunteers.

Depression and impaired quality of life (QOL) are frequently observed in patients suffering from a variety of diseases. In addition, it has been reported that an enhanced degradation of the serotonin precursor tryptophan may contribute to QOL deterioration in some diseases. However, it is unclear whether the correlation between the QOL scores and the central serotonergic tone is only mediated by the severity of either the depression symptoms or the physical illness itself. The present study examined the relationship between serotonin transporter (SERT) availability and life quality as measured by the World Health Organization Quality of Life brief version questionnaire (WHO-QOL) in healthy participants in order to exclude the influence of depressive mood and disease. The SERT availability in the midbrain was approximated using SPECT with [(123)I] ADAM ligand in fifty-eight healthy volunteers. The overall rating sub scores of the WHO-QOL correlated positively with serotonin transporter availability in the males. Central serotoninergic activity may play a role in the overall rating scores of the WHO-QOL.

Mapping DNA adducts of mitomycin C and decarbamoyl mitomycin C in cell lines using liquid chromatography/ electrospray tandem mass spectrometry.

The antitumor antibiotic and cancer chemotherapeutic agent mitomycin C (MC) alkylates and crosslinks DNA, forming six major MC-deoxyguanosine adducts of known structures in vitro and in vivo. Two of these adducts are derived from 2,7-diaminomitosene (2,7-DAM), a nontoxic reductive metabolite of MC formed in cells in situ. Several methods have been used for the analysis of MC-DNA adducts in the past; however, a need exists for a safer, more comprehensive and direct assay of the six-adduct complex. Development of an assay, based on mass spectrometry, is described. DNA from EMT6 mouse mammary tumor cells, Fanconi Anemia-A fibroblasts, normal human fibroblasts, and MCF-7 human breast cancer cells was isolated after MC or 10-decarbamoyl mitomycin C (DMC) treatment of the cells, digested to nucleosides, and submitted to liquid chromatography electrospray-tandem mass spectrometry. Two fragments of each parent ion were monitored ("multiple reaction monitoring"). Identification and quantitative analysis were based on a standard mixture of six adducts, the preparation of which is described here in detail. The lower limit of detection of adducts is estimated as 0.25 pmol. Three initial applications of this method are reported as follows: (i) differential kinetics of adduct repair in EMT6 cells, (ii) analysis of adducts in MC- or DMC-treated Fanconi Anemia cells, and (iii) comparison of the adducts generated by treatment of MCF-7 breast cancer cells with MC and DMC. Notable results are the following: Repair removal of the DNA interstrand cross-link and of the two adducts of 2,7-DAM is relatively slow; both MC and DMC generate DNA interstrand cross-links in human fibroblasts, Fanconi Anemia-A fibroblasts, and MCF-7 cells as well as EMT6 cells; and DMC shows a stereochemical preference of linkage to the guanine-2-amino group opposite from that of MC.

NRH:quinone oxidoreductase 2 (NQO2) catalyzes metabolic activation of quinones and anti-tumor drugs.

NRH:quinone oxidoreductase 2 (NQO2) is a cytosolic flavoprotein that utilizes NRH as electron donor. The present studies investigate the role of NQO2 in metabolic detoxification/activation of quinones and quinone based anti-tumor drugs. Chinese hamster ovary (CHO) cells stably overexpressing cDNA derived mouse NQO2 and mouse keratinocytes from DMBA-induced skin tumors in wild-type and NQO2-null mice were generated. The CHO cells overexpressing NQO2 and mouse keratinocytes expressing or deficient in NQO2 were treated with varying concentrations of mitomycin C (MMC), CB1954, MMC analog BMY25067, EO9, menadione and BP-3,6-quinone, in the absence and presence of NRH. The cytotoxicity of the drugs was evaluated by colony formation. The CHO cells overexpressing higher levels of mouse NQO2 showed significantly increased cytotoxicity to menadione, BP-3,6-quinone and to the anti-tumor drugs MMC and CB1954 when compared to CHO cells expressing endogenous NQO2. The cytotoxicity increased in presence of NRH. Similar results were also observed with BMY25067 and EO9 treatments, but to a lesser extent. The results on keratinocytes deficient in NQO2 supported the data from CHO cells. The inclusion of NRH had no effect on cytotoxicity of quinones and drugs in keratinocytes deficient in NQO2. Mouse NQO2 protein was expressed in bacteria, purified and used to study the role of NQO2 in MMC-induced DNA cross-linking. Bacterially expressed and purified NQO2 efficiently catalyzed MMC activation that led to DNA cross-linking. These results concluded that NQO2 plays a significant role in the metabolic activation of both quinones and anti-tumor drugs leading to cytotoxicity and cell death.

Bioreductive metabolism of mitomycin C in EMT6 mouse mammary tumor cells: cytotoxic and non-cytotoxic pathways, leading to different types of DNA adducts. The effect of dicumarol.

The six DNA adducts formed in EMT6 mouse mammary tumor cells upon treatment with mitomycin C (MC) fall into two groups: (1) four guanine adducts of MC and (2) two guanine adducts derived from 2,7-diaminomitosene (2,7-DAM), the major reductive metabolite of MC. The two groups of adducts were proposed to originate from two pathways arising from reductive activation of MC: (a) direct alkylation of DNA and (b) formation of 2,7-DAM, which then alkylates DNA. The aim of this study was to test the validity of this proposal and to evaluate the significance of alkylation of DNA by 2,7-DAM. Treatment of the cells with 2,7-DAM itself yielded the same 2,7-DAM-guanine adducts as treatment with MC; however, 2,7-DAM was approximately 100-fold less cytotoxic than MC. The uptake and efflux of 2,7-DAM by EMT6 cells was comparable to that of MC, but 2,7-DAM alkylated DNA with higher efficiency than MC. These results validate the two proposed pathways and show that formation of 2,7-DAM-DNA adducts in MC-treated cells represents a relatively non-toxic pathway of reductive metabolism of MC. A selective stimulatory effect of dicumarol (DIC) on 2,7-DAM-DNA adduct formation in EMT6 cells treated with MC was also investigated. DIC had no effect on alkylation by MC in cell-free systems, nor did it have significant effects on adduct formation or cell survival for cells treated with 2,7-DAM. It is proposed that in the cell DIC stimulates a reductase enzyme located at subcellular sites where the activated MC species has no direct access to DNA and therefore is diverted into the non-cytotoxic pathway, which leads to the formation of 2,7-DAM and its adducts.

C5 cytosine methylation at CpG sites enhances sequence selectivity of mitomycin C-DNA bonding.

We have established that UvrABC nuclease is equally efficient in cutting mitomycin C (MC)-DNA monoadducts formed at different sequences and that the degree of UvrABC cutting represents the extent of drug-DNA bonding. Using this method we determined the effect of C5 cytosine methylation on the DNA monoalkylation by MC and the related analogues N-methyl-7-methoxyaziridinomitosene (MS-NMA) and 10-decarbamoylmitomycin C (DC-MC). We have found that C5 cytosine methylation at CpG sites greatly enhances MC and MS-NMA DNA adduct formation at those sites while reducing adduct formation at non-CpG sequences. In contrast, although DC-MC DNA bonding at CpG sites is greatly enhanced by CpG methylation, its bonding at non-CpG sequences is not appreciably affected. These cumulative results suggest that C5 cytosine methylation at CpG sites enhances sequence selectivity of drug-DNA bonding. We propose that the methylation pattern and status (hypo- or hypermethylation) of genomic DNA may determine the cells' susceptibility to MC and its analogues, and these effects may, in turn, play a crucial role in the antitumor activities of the drugs.

Mitomycin resistance in Streptomyces lavendulae includes a novel drug-binding-protein-dependent export system.

Sequence analysis of Streptomyces lavendulae NRRL 2564 chromosomal DNA adjacent to the mitomycin resistance locus mrd (encoding a previously described mitomycin-binding protein [P. Sheldon, D. A. Johnson, P. R. August, H.-W. Liu, and D. H. Sherman, J. Bacteriol. 179:1796-1804, 1997]) revealed a putative mitomycin C (MC) transport gene (mct) encoding a hydrophobic polypeptide that has significant amino acid sequence similarity with several actinomycete antibiotic export proteins. Disruption of mct by insertional inactivation resulted in an S. lavendulae mutant strain that was considerably more sensitive to MC. Expression of mct in Escherichia coli conferred a fivefold increase in cellular resistance to MC, led to the synthesis of a membrane-associated protein, and correlated with reduced intracellular accumulation of the drug. Coexpression of mct and mrd in E. coli resulted in a 150-fold increase in resistance, as well as reduced intracellular accumulation of MC. Taken together, these data provide evidence that MRD and Mct function as components of a novel drug export system specific to the mitomycins.

Mitomycin C linked to DNA minor groove binding agents: synthesis, reductive activation, DNA binding and cross-linking properties and in vitro antitumor activity.

Mitomycin C (MC) is a natural cytotoxic agent used in clinical anticancer chemotherapy. Its antitumor target appears to be DNA. Upon bioreductive activation MC alkylates and cross-links DNA. MC derivatives were synthesized in which MC was linked to DNA minor groove binding agents, analogous to netropsin and distamycin. One, two and three N-methylpyrrole carboxamide units were conjugated with MC by a (CH2)5-tether to the 7-amino group of MC (11, 12 and 13, respectively). In contrast to MC 11, 12 and 13 displayed non-covalent affinity to DNA. Their bioreductive activation by NADPH-cytochrome c reductase proceeded as fast as that of MC. Metabolites arising from reductive and low-pH activation were characterized and found to be analogous to those of MC. DNA cross-linking activities were weak and decreased with an increasing number of N-methylpyrrole carboxamide units linked with the mitomycin molecule. No adducts were formed with calf thymus DNA in detectable amounts. In vitro antitumor activities of 11-13 were determined using the NCI in vitro antitumor screen. The conjugates 11-13 are growth inhibitory; however, their activities are 1.5-2 orders of magnitude lower than that of MC. COMPARE analysis indicates that the mechanism of the action of 11 and 12 correlates moderately with MC but negatively with distamycin. Conjugate 13 correlates neither with MC nor with distamycin. The results suggest that the basic cause of the observed low activity of the MC-minor groove binder conjugates is the fast irreversible decay of the activated MC, competing effectively with the slow drug delivery to CpG sites, required for the alkylation.

Exploring the mechanistic aspects of mitomycin antibiotic bioactivation in Chinese hamster ovary cells overexpressing NADPH:cytochrome C (P-450) reductase and DT-diaphorase.

We have directly demonstrated the involvement of human NADPH: cytochrome c (P-450) reductase in the aerobic/hypoxic differential toxicity of mitomycin C and porfiromycin in living cells by varying only this enzyme in a transfected cell line. In the same manner, we have implicated rat DT-diaphorase in the aerobic and hypoxic activation of mitomycin C, but found only a minor role for this enzyme in the aerobic activation of porfiromycin. DT-Diaphorase does not cause the production of an aerobic/hypoxic differential toxicity by mitomycin C, but rather activates this agent through an oxygen insensitive pathway. The evidence suggests that DT-diaphorase activates mitomycin C more effectively than porfiromycin, with porfiromycin being preferentially activated through a one-electron reductive pathway. The therapeutic potential of mitomycin antibiotics in the treatment of cancer can be envisioned to be enhanced for those tumors containing elevated levels of the bioreductive enzymes. However, cytogenetic heterogeneity within the tumor cell population and the various environmental factors which impact on bioreductive enzyme function, including pH and oxygen tension, may subvert this approach. Moreover, if high tumor levels of a drug activating enzyme reflect high levels in the normal tissues of the patient, normal tissue damage may also be enhanced with possibly no improvement in the therapeutic ratio. Approaches utilizing gene therapy, whereby a specific bioreductive catalyst is introduced into the tumor cell population via a targeting vehicle to activate a particular prodrug, may be more effective in that not only will the prodrug of choice be specifically activated in the tumor, but the source of the catalyst, be it bacterial, rodent, or human, will not be important. In fact, in the case of DT-diaphorase and mitomycin C, the rat form of the enzyme could be advantageous because it is more effective in activating mitomycin C than is the human form of this enzyme. Assuming targeted gene delivery to malignant cells, a non-host enzyme which is more effective at activating mitomycin C than the analogous host enzyme might also result in less drug activation in normal tissue and, hence, less normal tissue toxicity.

Formation of a major DNA adduct of the mitomycin metabolite 2,7-diaminomitosene in EMT6 mouse mammary tumor cells treated with mitomycin C.

Treatment of EMT6 mouse mammary tumor cells with [3H]mitomycin C (MC) results in the formation of six major DNA adducts, as described earlier using an HPLC assay of 3H-labeled products of enzymatic hydrolysis of DNA isolated from MC-treated cells. Four of these adducts were identified as monofunctional and bifunctional guanine-N2 adducts in the minor groove of DNA. In order to establish relationships between individual types of MC-DNA adducts and biological responses it is necessary to identify all of the adducts formed in cells. To this end we have now identified a predominant, previously unknown adduct formed in MC-treated EMT6 cells as a derivative not of MC, but of 2,7-diaminomitosene (2,7-DAM), the major bioreductive metabolite of MC. Rigorous proof demonstrates that it is a DNA major groove, guanine-N7 adduct of 2,7-DAM, linked at C-10 to DNA. The adduct is relatively stable at ambient temperature, but is readily depurinated upon heating. Its isolation from MC-treated cells indicates that MC is reductively metabolized to 2,7-DAM, which then undergoes further reductive activation to alkylate DNA, along with the parent MC. Low MC:DNA ratios were identified as a critical factor promoting 2,7-DAM adduct formation in an in vitro model calf thymus DNA/ MC/reductase model system, as well as in MC-treated EMT6 cells. The 2,7-DAM-guanine-N7 DNA adduct appears to be relatively noncytotoxic, as indicated by the dramatically lower cytotoxicity of 2,7-DAM in comparison with MC in EMT6 cells. Like MC, 2,7-DAM exhibited slightly greater cytotoxicity to cells treated under hypoxic as compared to aerobic conditions. However, 2,7-DAM was markedly less cytotoxic than MC under both aerobic and hypoxic conditions. Thus, metabolic reduction of MC to 2,7-DAM represents a detoxification process. The differential effects of MC-DNA and 2,7-DAM-DNA adducts support the concept that specific structural features of the DNA damage may play a critical role in the cytotoxic response to a DNA-targeted chemotherapeutic agent.

Mitosene-DNA adducts. Characterization of two major DNA monoadducts formed by 1,10-bis(acetoxy)-7-methoxymitosene upon reductive activation.

Reductive activation of racemic 1,10-bis(acetoxy)-7-methoxymitosene WV15 in the presence of DNA, followed by enzymatic digestion and HPLC analysis, revealed the formation of various DNA adducts. Reduction is a necessary event for adduct formation to occur. This reductive activation was performed under hypoxic conditions in various ways: (1) chemically, using a 2-fold excess of sodium dithionite (Na2S2O4), (2) enzymatically using NADH-cytochrome c reductase, (3) electrochemically on a mercury pool working electrode, and (4) catalytically, using a H2/PtO2 system. Five different mitosene-DNA adducts were detected. These adducts were also present when poly(dG-dC) was used instead of DNA, but were absent with poly(dA-dT). All were shown to be adducts of guanine. Reduction of 1, 10-dihydroxymitosene WV14 in the presence of DNA did not result in detectable adduct formation, demonstrating the importance of good leaving groups for efficient adduct formation by these mitosenes. Finally, two of the adducts were isolated and their structures elucidated, using mass spectrometry, 1H NMR and circular dichroism (CD). The structures were assigned as the diastereoisomers N2-(1"-n-hydroxymitosen-10"-yl), 2'-deoxyguanosine (n = alpha or beta). These type of adducts, in which the mitosene C-10 is covalently bonded to the N-2 of a guanosylic group, are different from the well-known mitomycin C 2'-deoxyguanosine monoadducts, that is linked via the mitomycin C C-1 position, demonstrating that the order of reactivity of the C-1 and C-10 in these mitosenes is reversed, as compared to mitomycin C. The 7-methoxy substituent of WV15 is a likely factor causing this switch. Evidence is presented that the 7-substituent of mitosenes also influences their DNA alkylation site. Adducts 4 and 5 represent the first isolated and structurally characterized covalent adducts of DNA and a synthetic mitosene.

The mitomycin bioreductive antitumor agents: cross-linking and alkylation of DNA as the molecular basis of their activity.

This review focuses on the chemical and enzymatic aspects of the reductive activation of mitomycin C, its disulfide analogs KW-2149 and BMS-181174, and, in less detail, FR66979 and FR900482, newly discovered antitumor antibiotics related to mitomycins. Furthermore, structural aspects of DNA damage induced by these drugs in vitro and in vivo are described, including the chemical and conformational characteristics of DNA interstrand and intrastrand cross-links and monofunctional alkylation products, with emphasis on DNA adducts of mitomycin C. The DNA sequence specificity of the damage and its mechanism is reviewed. The relationship between the chemical and structural properties of the DNA damage on the one hand, and the antitumor and other biological activities of the mitomycins on the other, is discussed.

Chirality of a 1,10-bisacetoxymitosene compound. Impact on reductive activation, DNA interstrand cross-linking and antitumour activity.

The absolute configuration at the C-1 position of a 1,10-bisacetoxymitosene (WV15) appears to be important for enzymatic reduction, DNA interstrand cross-linking and in vitro antitumour activity of this compound. DNA cross-linking by the (-)-(S)-enantiomer of WV15 upon reduction with sodium dithionite (Na2S2O4) was more efficient than cross-linking by the (+)-(R)-enantiomer. Also, following enzymatic two-electron reduction by DT-diaphorase or one-electron reduction by xanthine oxidase, (-)-(S)-WV15 was more efficient in DNA cross-linking than (+)-(R)-WV15. However, the difference in cross-linking efficiency was less than upon chemical reduction, and in the case of enzymatic reduction that higher amount of DNA cross-links formed by (-)-(S)-WV15 can be explained by more efficient enzymatic activation of this enantiomer as compared to (+)-(R)-WV15. The enantiomeric preference upon chemical reduction can be explained by a second chemical reduction of DNA-bound WV15, which presumably does not occur upon enzymatic reduction. (-)-(S)-WV15 appeared to be more active than its (+)-(R) counterpart in A204 and L1210 tumour cell lines, with (+)-(R)/(-)-(S) toxicity ratios as high as 200 and 68, respectively. In Chinese hamster V79 cell lines, toxicity of the enantiomers was measured under oxic and hypoxic conditions. The oxic/hypoxic toxicity ratios of (+)-(R)-and (-)-(S)-WV15 in the Chinese hamster V79 cell line were 5.5 and 2.4, respectively. These different oxic/hypoxic toxicity ratios may indicate that different reducing enzymes are involved in the activation of the enantiomers. Generally, in biological systems, different activities of (+)-(R)- and (-)-(S)-WV15 appear not to be caused by different intrinsic cross-linking capacities of the enantiomers, but by more efficient enzymatic activation of (-)-(S)-WV15, as compared to (+)-(R)-WV15. The (-)-(S)-enantiomer of WV15 appears to be more active both in in vitro tumour models and in DNA cross-linking assays, and therefore the absolute configuration of mitosenes is indicated to be important for the antitumour activity of these compounds.

Noncovalent binding of a mitomycin C metabolite, 2,7-diaminomitosene, to duplex DNA.

The major metabolite of mitomycin C, 2,7-diaminomitosene (DAM), interacts noncovalently with DNA. This was supported by ultraviolet-visible spectrum changes upon mixing with DNA and ethidium bromide displacement from DNA, measured as fluorescence changes. Moreover, DAM bound to DNA sufficiently strongly to hold DNA in a double stranded conformation under denaturing gel electrophoresis conditions commonly used to measure mitomycin C cross-links. These data show that generation of DAM and interaction with DNA represent a potential additional mechanism of DNA damage induced by mitomycin C.

Reduction of antitumour mitosenes in non-aqueous and aqueous environment. An electron spin resonance and cyclic voltammetry study.

Chemical reduction of mitosenes under aerobic conditions in DMSO showed characteristic ESR signals of the mitosene derived semiquinone free radicals. However, these signals diminished strongly upon addition of water to the reaction mixture, indicating a short lifetime of the mitosene semiquinone free radicals under aqueous conditions. In addition, enzymatic one-electron reduction of these mitosenes with either xanthine oxidase or purified NADPH cytochrome P450 reductase under anaerobic conditions showed no signals of the mitosene semiquinone free radicals. Subsequent cyclic voltammetry measurements demonstrated facilitation of the further one-electron reduction of the mitosene semiquinone free radicals in the presence of water in comparison with non-aqueous conditions. The present results strongly suggest that in the presence of water relatively stable hydroquinones are formed upon reduction of mitosenes. Consequently, the steady state concentrations of mitosene semiquinone free radicals will be lowered substantially in aqueous environment. Thus under physiological conditions, two-electron reduction and formation of the mitosene hydroquinone might be important in processes leading to DNA alkylation by these mitosenes.

Binding of 2,7-diaminomitosene to DNA: model for the precovalent recognition of DNA by activated mitomycin C.

Mitomycin C (MC), mitomycin A, porfiromycin, BMY-25067, and BMY-25287, antitumor antibiotics collectively termed "mitosanes", were found to have no appreciable binding affinity to various natural and synthetic DNAs, as tested by UV spectrophotometry and equilibrium dialysis. Further tests of DNA binding applied to MC including thermal melting measurements, displacement of ethidium fluorescence, and unwinding of closed circular DNA were similarly negative. In contrast, 2,7-diaminomitosene (2,7-DAM), a major end product of the reductive activation of MC, binds to the same series of DNAs by all of these criteria. In the presence of DNA its UV absorbance at the 313 nm maximum decreased and underwent a slight red shift. This effect was used for determining DNA binding constants (Kb) by the spectrophotometric titration method. At pH 6.0 the Kbs of three natural DNAs with varying GC content, as well as poly(dA-dT).poly(dA-dT), and poly(dG-dC).poly(dG-dC), were all in the range of (1.2-5.3) x 10(4) (M nucleotide)-1, with no apparent specificity of binding. Poly(dG-m5dC).poly(dG-m5dC) displayed a slightly higher Kb ((7.5-8.4) x 10(4)). Binding of other, closely related mitosenes was tested to calf thymus DNA by equilibrium dialysis. Neither the presence of a 1-OH substituent, removal of the 10-carbamoyl group, nor methylation of the 2-amino group modifies the binding affinity of the mitosenes significantly. The 1-phosphate substituent abolishes binding. The binding of 2,7-DAM to DNA increased with decreasing pH and decreasing ionic strength. It was determined that 2,7-DAM is protonated at the 2-amino group with a pKa = 7.55, and this correlated well with the observed pH dependence of the binding, indicating that the binding affinity has a strong electrostatic component. This was confirmed by the finding that the extrapolated Kb to 1 M Na+ concentration diminishes to only 10% of the value of Kb at 0.01 M Na+ concentration. Viscosity tests showed conclusively that 2,7-DAM intercalates in DNA, in a nonspecific manner. DNA binding by 2,7-DAM is shown to be a close model of the binding of the reduced activated form of MC, previously characterized indirectly [Teng, S. P., Woodson, S. A., and Crothers, D. M. (1989) Biochemistry 28, 3901-3907]. The nonspecific precovalent binding of the active form may serve in the cell to concentrate the drug at its critical target, DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclopropamitosenes: novel bioreductive anticancer agents--mechanism of action and enzymic reduction.

The mechanism of action of cyclopropamitosenes, novel bioreductive anticancer agents, has been investigated using a unique combination of chemical and biochemical techniques. The compounds 4 function as reductively activated alkylating agents under chemical reducing conditions, and the biochemical experiments establish that the methoxy- and aziridinyl-derivatives 4a and 4b behave differently upon bioreduction.

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.

DT-diaphorase activity and mitomycin C sensitivity in non-transformed cell strains derived from members of a cancer-prone family.

Non-transformed skin fibroblasts derived from five members of a cancer-prone family and three unrelated healthy volunteers were assayed for their levels of activity of the quinone reductase DT-diaphorase and for their sensitivity to the antitumor quinone mitomycin C (MMC). Previous studies of skin fibroblasts derived from one afflicted member of this family (3437T) demonstrated increased resistance to MMC under aerobic exposure conditions and a reduced level of DT-diaphorase. In the present study 3437T cells and a cell strain derived from another afflicted member of the cancer-prone family were found to be hyperresistant to the cytotoxic effects of MMC, and demonstrated negligible DT-diaphorase activity (30 +/- 10 nmol/min/mg protein). Cell strains derived from the three other family members demonstrated intermediate DT-diaphorase activity (400-800 nmol/min/mg protein). Enzyme activities of 1800-6000 nmol/min/mg protein were measured in the three control cell strains. A protein that was reactive with a rabbit polyclonal antibody raised against rat DT-diaphorase and corresponded to the known mol. wt of DT-diaphorase was clearly evident in the three control cell strains, but absent in the two MMC-hyperresistant cell strains. This protein was present in intermediate amounts in the remaining members of the cancer-prone family. Southern analysis of DNA isolated from all eight cell strains and restricted with EcoRI demonstrated the presence of a DNA sequence of approximately 15 kb which hybridized to a rat DT-diaphorase cDNA probe. Northern analysis revealed the presence of an RNA species approximately 1200 bp in size, consistent with that for a human DT-diaphorase mRNA, in all cell strains derived from family members. A post-transcriptional defect would, therefore, appear to be responsible for the decreased enzyme activity observed in the resistant cell strains. These results suggest a role for DT-diaphorase in MMC bioactivation and that reduced levels of the protein may be causally related to the cancer-prone tendency of this family.

The role of NAD(P)H: quinone reductase (EC 1.6.99.2, DT-diaphorase) in the reductive bioactivation of the novel indoloquinone antitumor agent EO9.

EO9 [3-hydroxymethyl-5-aziridinyl-1-methyl-2-(H-indole-4, 7-indione)-propenol] is a novel indoloquinone structurally related to mitomycin C, a quinone anticancer drug that requires reductive bioactivation. NAD(P)H: (quinone-acceptor) oxidoreductase (quinone reductase, DT-diaphorase, EC 1.6.99.2) is an obligate 2-electron donating enzyme that can reduce a variety of quinones resulting either in bioactivation or bioprotection. Using quinone reductase (QR) preparations from rat Walker 256 mammary tumor cells and human HT29 colon carcinoma cells, we have characterized the role of this enzyme in EO9 reductive metabolism. QR activity was assayed under optimal conditions by following cytochrome c reduction at 550 nm in the presence of enzyme, quinone substrate, NADH, and bovine albumin, and confirmed by loss of EO9 absorbance at 550 nm. Both the rat and human tumor cell enzymes catalyzed reduction of the benchmark quinone menadione with a similar Km of 1.4-3.1 microM, although the Vmax was 7 to 8-fold lower for the human preparation. EO9 was readily reduced by the rat Walker QR. The mean Km was about 5-fold higher than for menadione at around 15 microM and the Vmax was 6-fold lower at around 2.5 mumol of cytochrome c reduced mg-1 of protein. EO9 was also metabolized by QR from HT29 human colon carcinoma cells but rather less efficiently than by the rat tumor enzyme. For example, the rate was 6-fold lower than that for the Walker tumor enzyme at 100 microM substrate concentration after correcting for the 7- to 8-fold difference in specific activity for the two preparations.(ABSTRACT TRUNCATED AT 250 WORDS)