(CANCER RESEARCH 50, 4692-4697, August 1, I990|

Decreased NADPH:Cytochrome P-450 Reductase Activity and Impaired Drug

Activation in a Mammalian Cell Line Resistant to Mitomycin C underAerobic but not Hypoxie Conditions1

Paul R. Mohan. Michael I. Walton, Craig N. Robson,2 Judith Godden, Ian J. Stratford, Paul Workman,Adrian L. Harris,2 and Ian D. Hickson2-3

Department of Clinical Oncology. Medical School, University of Newcastle upon Tyne. NE2 4HH ¡P.R. H., C. N. R.. A. L. H., I. D. H.J; MRC Clinical Oncology andRadiotherapeutics Unit, Medical Research Council Centre Hills Road, Cambridge [M. I. W., P. H'.]; and Medical Research Council, Radiohiology Unit, Chillón,Didcot,

Oxon /J. G., I. J. S.J, United Kingdom

ABSTRACT

Mitomycin C (MMC) is regarded as the prototype bioreductive alkyl-ating agent in clinical use. To elucidate the biochemical basis of MMCresistance, we isolated a drug resistant derivative (designated CHO-MMC) of a Chinese hamster ovary cell line (CHO-K1) by exposure toprogressively higher concentrations of MMC. CHO-MMC cells exhibited a 17-fold increase in resistance to MMC and were 33-fold cross-resistant to the monofunctional derivative, decarbamoyl mitomycin C. Incontrast, CHO-MMC cells showed only a 2-fold level of resistance toBMY 25282, a more easily activated analogue of MMC, and exhibitedparental sensitivity to MMC under radiobiologically hypoxic conditions.CHO-MMC cells showed no increased resistance to a range of DNAdamaging agents including several other alkylating agents (e.g., mel-phalan and methyl methanesulfonate). Cross-resistance to drugs associated with the multidrug resistant phenotype (e.g., Adriamycin and vin-cristine) was present only at very low levels.

Using a specific high performance liquid Chromatograph) technique,we examined the rates of reduction of MMC and BMY 25282 in cellextracts from CHO-K1 and CHO-MMC cells under both aerobic (air)and hypoxic (N2) conditions. Reduction rates for both drugs were at least30-fold faster under nitrogen than in air. Metabolism of MMC wasundetectable in air but was readily detectable under nitrogen and was 2-3-fold slower in CHO-MMC' cell extracts than in CHO-K1 cell extracts.

Although BMY 25282 was more readily reduced under nitrogen, nodifference was detected between extracts from CHO-K1 or CHO-MMCcells in the rate of reduction of BMY 25282 under either air or nitrogen.The activity of NADPH:cytochrome P-450 (cytochrome c) reducíase,anenzyme implicated in the bioreductive activation of MMC, was 3-4-foldlower in CHO-MMC cells than in the parental line. These findingssuggest that the resistance of CHO-MMC cells to MMC under aerobicconditions may be due to impaired metabolic activation of the drug as aresult of a decrease in NADPH:cytochrome P-450 reducíaseactivity.This supports the view that decreased bioreductive enzyme activity maybe a significant mechanism for acquired resistance to MMC in tumorcells in vivoand that more readily activated analogues may be potentiallyuseful in overcoming this specific form of resistance.

INTRODUCTIONMMC4 is regarded as the prototype bioreductive drug in

clinical use due to its requirement for activation of cytotoxicmetabolites by reductive enzymes. It is one of the few chemo-therapeutic drugs to show responses in colorectal and gastric

Received 10/2/89; revised 2/13/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' Work supported by the North of England Cancer Research Campaign and

the Medical Research Council.1Present address: The Imperial Cancer Research Fund. Institute of Molecular

Medicine, John Radcliffe Hospital, Headington. Oxford, OX3 9DU, UnitedKingdom.

3To whom requests for reprints should be addressed.'The abbreviations used are: MMC, mitomycin C; DCMMC, decarbamoyl

mitomycin C; BCNU, bischloroethylnitrosourea; c/í-platinum, cis-plati-num(II)diamminedichloride; VP16, etoposide: CHO, Chinese hamster ovary;PBS, phosphate-buffered saline; HPLC, high performance liquid chromatogra-Phy.

carcinoma (1). It also has activity in squamous lung cancer (2)and in breast cancer patients pretreated with several otheranticancer drugs including anthracyclines (3, 4). MMC hasbeen shown to be preferentially toxic to hypoxic tumor cellsboth in vitro and in vivo (5, 6). Hypoxic tumor cells are resistantto conventional radiotherapy as well as to several chemothera-peutic agents (5, 7, 8); thus MMC is potentially a usefulchemotherapeutic agent under these conditions. AlthoughMMC has been shown to have activity against a broad range ofmalignancies, these responses are frequently limited to duration, possibly due to the development of tumor resistance

Following bioreductive activation, MMC can produce a number of cytotoxic lesions in DNA including intrastrand andinterstrand cross-links and monoadducts (9, 10). Recent studieshave identified the bis adduct formed by reduction dependentreaction at the C-l and C-10 positions of MMC with neighboring N-2 guanine atoms in DNA (11, 12). Studies using liverhomogenates or cell extracts (particularly the microsomal andnuclear subcellular fractions) have shown that bioreductiveactivation of MMC to an alkylating species requires the presence of reducing equivalents and the absence of molecularoxygen (13, 14). Using purified enzymes, it has been demonstrated that either NADPH:cytochrome P-450 reducíase orxanthine oxidase can activate MMC into a reactive speciescapable of alkylating purified DNA in vitro (11, 12, 15-17).Reaction of the one-electron reduction product with oxygenleads to the generation of Superoxide and associated oxygenradicals ( 18). Thus, toxicity to cells treated under hypoxic versusaerobic conditions (5) will depend upon the relative efficienciesof the bioreductive alkylation pathway against the oxygen radical formation pathway and upon the ability of the cell torespond to these different insults. It is likely, therefore, thatMMC resistance may arise in cells through a decreased abilityto bioactivate the drug.

Here, we describe the isolation of a mammalian cell linemade resistant to MMC by sequential exposure of CHO-K1cells to increasing drug concentrations. We show that thesecells exhibit a decreased capacity to activate MMC comparedto the parental cell line and that this is associated with a lowerNADPHrcytochrome P-450 reducíaseactivily in ihe resistantcells. The resistance of CHO-MMC' cells is eliminated underhypoxia. Furthermore, CHO-MMC' cells exhibil near wild lype

sensitivity to the MMC derivalive BMY 25282 (which has alower oxidalion-reduclion polenlial than MMC) and activateBMY 25282 with the same efficiency as do CHO-K1 cells.

MATERIALS AND METHODS

Cell Culture and Media. Parental CHO-K1 cells and the mitomycinC resistant derivative, CHO-MMC', were maintained in Ham's F-10

medium (Northumbria Biologicals) supplemented with 5% fetal calfserum, 5% newborn calf serum, 3 mM glutamine. and antibiotics

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MITOMYCIN C RESISTANCE AND DRUG ACTIVATION

(penicillin, 100 units/ml; streptomycin, 100 Mg/ml; nystatin, 50 units/ml). Cells were grown as monolayers in tissue culture flasks or Petridishes at 37°Cunder 5% CO2/95% air.

Isolation of Drug Resistant Cell Lines. The drug resistant CHO-MMCr cell line was isolated by growing CHO-K1 cells in complete

medium containing MMC (up to 250 ng/ml). Cells were exposed toMMC for 2-3 days, cultured in the drug free medium for 24 h andthen reexposed to a higher concentration of MMC. This pattern wasrepeated for 3 months after which potential MMC resistant cells werecloned and expanded into bulk cultures. A clonal isolate, designatedCHO-MMO, was taken for further study.

Isolation of Cell Hybrids. To generate cell hybrids, one or the otherof the dominant selectable markers gpt or neo was introduced into eachcell line by transfection. The resulting mycophenolic acid and G4I8resistant transfectants were mixed 1:1 and fused using PEG 6000.Hybrids were selected in medium containing both mycophenolic acidand G418. Flow cytometry was used to confirm that hybrid cells weretetraploid.

Drug Treatments. Drugs were generally purchased from Sigma, theexceptions being: MMC, Kyowa Hakko Kogyo; BCNU, a gift from Dr.Narayanan, National Cancer Institute, Bethesda, MD; and BMY25282, a gift from Dr. Bradner, Bristol Myers, Syracuse, NY.

For most drugs, storage and preparation were as described previously(19, 20) with the following additions. Melphalan and DCMMC wereprepared freshly in H2O, while VP 16 was dissolved in dimethyl sulf-oxide and stored at 4°Cprior to use.

Survival Studies. Survival curves under aerobic conditions were performed as described previously (19).

In general, cells were exposed to drug for 24 h before being washedtwice with PBS and returned to fresh growth medium. The exceptionswere BCNU and cis-platinum, for which 0.5- and 2-h exposures, respectively, were used. Cells were exposed to VP 16 and ethyl methane-sulfonate for 1 h.

For comparison of the toxicity of MMC under aerobic or hypoxicconditions, CHO-K1 or CHO-MMC cells were placed into 100-ml fiatglass bottles (5 x 10s cells/bottle) and allowed to attach and grow for

24 h prior to exposure to various concentrations of MMC (in 5 mlgrowth medium) for 3 h at 37°C.Aerobic exposures were made by

equilibrating cells with air plus 5% CO2. Alternatively, hypoxia wasinduced by flowing N2 plus 5% CO2 at 500 ml/min over the surface ofthe cell monolayer for the duration of the exposure to MMC. Thisprocedure induces "radiobiological" hypoxia within 60 min of commencement of deaeration (21).' Further, this method has been used to

demonstrate the differential hypoxic toxicity of a range of bioreductiveagents (22-24). After treatment medium containing drug was removed,the cells were trypsinized, washed, and plated for colony formation.

Plating efficiency of untreated cells was always >70%.Treatment with UV or X-rays was as described previously (19).Following drug or radiation exposure, cells were incubated for up to

14 days at 37°Cuntil visible colonies developed. These were fixed in

methanohacetic acid (3:1), stained with crystal violet (400 Mg/ml), andcounted. Colonies containing more than 50 cells were counted assurvivors.

The drug concentration required to reduce the cell survival to 37%of the control represents the average dose required to kill a cell. Thesevalues were estimated directly from survival curves.

Preparation of Cell Extracts. Exponentially growing cells werewashed with PBS and harvested with a cell scraper in 10 ml of PBS(lacking Ca2+ and Mg2+) supplemented with 0.02% EDTA. The cells

were centrifuged at 2000 rpm and the resulting pellets were resuspendedin 10 volumes of ice cold 100 m\i sodium phosphate buffer (pH 7.4).This suspension was disrupted by sonication with an MSE 150 Wultrasonic disintegrator (3 x 10 s at 8-10 Mmpeak to peak amplitude,with cooling on ice for 15 s between each disruption). The sonicatedpreparations were centrifuged in an Eppendorf microfuge for 5 min at4°C,and the supernatants were stored in aliquots at -70°C. Cell

disruption was confirmed by microscopic examination.Reductive Metabolism of MMC and BMY 25282. The reductive

metabolism of MMC and BMY 25282 was carried out under air or N2

' I. J. Stratford el al., unpublished data.

using specially adapted 25-ml conical flasks as described in detailelsewhere (25). Each incubation contained 0.9 niM NADH andNADPH, cell cytosol (1-9 mg/ml protein), 50 MMdrug, and 100 mMsodium phosphate buffer (pH 7.4) in a final volume of 1.5 or 2 ml.Flasks were preincubated under air or N2 for 5-6 min and the reactionwas started by the addition of drug in dimethyl sulfoxide (20-30 n\).This period of preincubation under N2 produced a level of O2 < 10ppm and there was no lag phase in the enzyme reaction progress curves.Extended preincubations under N2 produced no further effect on therates of drug metabolism. Incubations were carried out at 37°Cwith

shaking (150 oscillations/min) for up to 60 min in N2 and 90 min inair. Aliquots (100 p\) of the reaction mixture were removed and addedto 2 volumes of methanol on dry ice. Samples were centrifuged for 5min at 4°Cand the supernatant was analyzed directly by HPLC for

loss of drug and appearance of metabolites. Reaction rates were derivedfrom progress curves before 50% substrate loss had occurred. Threeindependent cytosol preparations were examined for reducíaseactivitywith MMC and BMY 25282 as substrates.

HPLC Analyses. Loss of MMC and BMY 25282 was determinedusing reverse-phase HPLC, which also allowed the formation of bio-reductive metabolites to be monitored. Chromatography was carriedout on equipment and columns supplied by Waters (Milford, MA). Theequipment consisted of an M-45 chromatography pump, a model 71OBAutomated Sample processor (WISP), a model 490 variable wavelengthdetector, and a Z-module fitted with a Guard-Pak precolumn. Separations were carried out on Waters Rad-Pack reverse-phase ^Bondapak

octadecylsilane (Ci8) columns (8 mm x 10 cm; 10 ^m beads). Themobile phase consisted of either 35% methanol (for MMC) or 27%acetonitrile (for BMY 25282) in 20 mM sodium phosphate buffer, pH7.4. Elution was isocratic at 3 and 1.5 ml/min for MMC and BMY25282, respectively. Peak identity was confirmed by cochromatographywith authentic standards and spectral properties, and quantitation wasby peak height reference with known calibration standards. Using aninjection volume of 20 n\ the lower limit of detection of MMC andBMY 25282 was 0.2-0.3 Mg/ml.

Enzyme Activities. NADPH:cytochrome P-450 reducíaseactivily incell extracts (0.3-1.8 mg/ml protein) was assayed by monitoring cyto-chrome e reduclion al 500 nm on a Konlron Uvicon 860 speclropho-tometer, using a molar extinclion coefficient of 21.1 mM/cm (26).

DT-diaphorase activity in cell extracts was assayed by measuring theeffects of the inhibitor dicumarol (10 MM)on the reduction of menadione(10 MM)with cylochrome c (77 MM)as lerminal eleclron acceplor (27).

Enzyme aclivity in extracts from both parental and resistant cellswas linear with protein over the range used. Protein concentralionswere assayed by the method of Lowry et al. (28), using bovine serumalbumin as a standard.

Statistics. Levels of significance were calculated using a Student ttest.

RESULTS

Cell Survival Studies. Following the selection procedure described in "Materials and Methods," a clonal population of

MMC resistant cells was recovered and designated CHO-MMCr. These cells were kept in continuous culture and main

tained in drug free medium for 6 months without loss of MMCresistance. Survival curves for parental CHO-K 1 and CHO-MMCr cells following a 24-h exposure to MMC are shown in

Fig. 1. A comparison of values for drug concentration requiredto reduce the cell survival to 37% of the control shows thatCHO-MMCr cells are 17-fold more resistant to MMC than the

parental line (Table 1). A similar degree of MMC resistancewas seen in CHO MMC' cells following a 3-h drug exposure(data not shown). Table 1 also shows the cross-resistance pattern of CHO-MMCr cells to a range of other DNA damaging

agents and antitumor drugs. Although exhibiting a high levelof resistance to DCMMC, the monofunctional derivative ofMMC, CHO-MMCr cells show no cross-resistance to various

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MITOMYCIN C RESISTANCE AND DRUG ACTIVATION

MMC(..9 ml)

Fig. 1. Survival of CHO-K1 and CHO-MMC cells following a 24-h exposureto MMC. CHO-K1 (•);CHO-MMC' (•).Points, mean of 3 independent exper

iments. Bars, SE.

Table I OJ7 values for CHO-KI and CHO-MMC' cells following exposure to

DNA damaging agents and anticancer drugsValues were obtained from survival curves constructed using pooled data from

at least 3 independent experiments.

DrugMMC

(ng/ml)DCMMC(ng/ml)*BMY

25282(ng/ml)Melphalanlug/ml)Methyl

methanesulfonate(^g/ml)EMS(xg/ml)c/s-Platinum

(»nml)BCNU(¿ig/ml)Vincristine(ng/ml)Adriamycin(ng/ml)Actinomycin

D(ng/ml)VPlo^g/ml)X-rays

(rads)UVlight (J/mz)CHO-KI12098280.8713351.413.390070951.786006.8D,,CHO-MMC'20803200600.75943501.6712.824001101055.56006.8Resistance

factor*17332.10.91.31.01.21.02.71.61.13.11.01.0

' D,7 CHO-MMC'/D,7 CHO-KI.* Based on !>,.,,.

other alkylating agents such as methyl methanesulfonate, ethylmethanesulfonate, melphalan, and BCNU. There is also nocross-resistance to UV light, X-irradiation, or m-platinum.However, CHO-MMC' cells exhibit a low level of resistance to

vincristine, VP 16, and Adriamycin (Table 1), drugs associatedwith the multidrug resistant phenotype.

Fig. 2 shows survival curves for CHO-KI and CHO-MMC'

cells against BMY 25282 (an analogue of MMC with a lowerreduction potential). On a molar basis, BMY 25282 is 3 timesmore toxic than MMC to CHO-KI cells. CHO-MMC' are onlyabout 2-fold resistant to BMY 25282 compared to CHO-KIcells.

Comparing hypoxic versus aerobic conditions, parental CHO-Kl cells show no difference in sensitivity to MMC (Table 2).However, CHO-MMC' cells are approximately 12-fold more

sensitive to MMC under hypoxic conditions than under aerobicconditions (Table 2), such that they exhibit no resistance toMMC relative to CHO-KI cells under hypoxic conditions.

Survival of Cell Hybrids. CHO-MMC'/CHO-MMC tetra-

ploid cell hybrids exhibit a similar degree of resistance to MMCto that seen with diploid CHO-MMC' cells (Table 3). However,when fused with parental CHO-KI cells, the resulting CHO-Kl/CHO-MMO cell hybrid is only 2-fold more resistant toMMC than the self-cross CHO-KI/CHO-KI hybrid.

HPLC Analyses of MMC and BMY 25282 Metabolism. Todemonstrate the metabolites of MMC and BMY 25282 pro-

BMY 25282 (ng ml)

Fig. 2. Survival of CHO-KI and CHO-MMC' cells following a 24-h exposureto BMY 25282. CHO-KI (•);CHO-MMC' (•).Points, mean of 3 independent

experiments. Bars, SE.

Table 2 DJ7 values for CHO-KI and CHO-MMC' cells following a 3-h exposure

to MMC under aerobic or hypoxic conditionsValues were obtained from survival curves constructed using pooled data from

six independent experiments.

D,,(«g/ml)Cell

lineCHO-KI

CHO-MMC1Air0.7 9.5N20.7 0.8Resistance

factor1.0

11.9

Table 3 Drug resistance in CHO cell hybridsResistance factor"

Drug CHO-MMC/CHO-MMC'CHO-KI/CHO-

MMC'

MMCAdriamycin

172

* Resistance factors were calculated from D37values for the hybrids relative tothose of the parental CHO-KI/CHO-KI hybrid. Values were obtained frompooled data from 4 independent experiments.

duced under hypoxic and aerobic conditions, a series of incubations were carried out with cell extracts and the reactionproducts were separated by HPLC. The HPLC chromatogramsin Fig. 3, A and A, correspond to a sample removed from a 60-min incubation of MMC with an extract from CHO-KI cellsunder N2, monitored at 365 and 313 nm, respectively. Thesingle peak at 365 nm represents MMC (Fig. 3/1). At 313 nm(Fig. 3Ä), both MMC (Peak 2) and its reduced metabolites(Peaks 1 and 3) can be seen. Fig. 3, C and D, shows similarchromatograms from a 12-min incubation of BMY 25282 witha CHO-MMC' cell extract under N2, and monitored at 390 and

330 nm, respectively. The large peak at 390 nm (Fig. 3C)represents BMY 25282 (Peak 6) and the minor one (Peak 5) adegradation product also seen in the absence of enzyme. Thepeaks at 330 nm in Fig. 3D represent BMY 25282 (Peak 6),the degradation product (Peak 5) and a reduction product (Peak4). In all cases, loss of MMC was accompanied by the appearance of metabolite peaks. There was no detectable loss of MMCor BMY 25282 under N2 in boiled extracts from either cell line.

Rates of Metabolism of MMC and BMY 25282. No detectableloss of MMC occurred under aerobic conditions after 90 minincubation in the presence of 16 mg of cell protein (i.e., theactivity was <10 pmol/min/mg protein in air). By contrastMMC loss was readily detected in both CHO-MMCr and CHO-

KI cell extracts under N2. However, the rate of MMC metabolism by CHO-MMCr extracts was approximately 2-3-foldslower than the rate in CHO-KI extracts (Table 4; Fig. 4). In

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MITOMYCIN C RESISTANCE AND DRUG ACTIVATION

0.011-1

IOIOco

0-

0.003-1

con

0.003-1

E

OO»PO

0J

B

0.025

EeOco

02468 02468

Time (min)Fig. 3. Reverse-phase HPLC chromatograms of MMC and BMY 25282 and

their reduced metabolites from anaerobic incubations. (.1) Chromatogram of a60-min sample of an anaerobic incubation mixture containing MMC (Peak 2)and (H) chromatogram of the same sample ¡is.I showing MMC (Peak 2) and tworeduced metabolites (Peaks 1 and 3). (C) Chromatogram of a 12-min aliquot ofan anaerobic incubation of BMY 25282 which contains a degradation product(Peak 5) and parent drug (Peak 6). (D) Chromatogram of the same sample as Ccontaining BMY 25282 (Peak 6) and the degradation product (Peak 5) but, inaddition, a reduced metabolite (Peak 4). Chromatography conditions as describedin "Materials and Methods."

Table 4 Kales of bioreductive activation of MMC by CHO-KI and CHO-MMC'

cell extracts in air versus nitrogenValues shown are the mean ±SE for i determinations from three independent

cell extracts with duplicate determinations for each preparation.

Rate of reduction(nmol/min/mg protein)

CellextractCHO-KI

CHO-MMC'Air<0.009ND"N,0.273

±0.028(n = 6)

0.1 23 ±0.03*(n = 6)

* ND, not determined.* Significantly different from CHO-KI cell extracts (P < 0.01).

contrast to MMC, BMY 25282 reduction was also readilydetectable in air in both CHO-KI and CHO-MMC' cell extracts(Table 5). BMY 25282 reduction was about 10-fold faster thanfor MMC under N2 (Tables 4 and 5). The rate of loss of BMY25282 in CHO-KI cell extracts under aerobic conditions was30-fold slower than in N2 (Table 5). However, there was nosignificant difference in BMY 25282 metabolism between extracts from parental and resistant lines, under either air or N2(Table 5; Fig. 4).

NADPH:Cytochrome P-450 ReducíaseActivity. CHO-MMC'cell extracts showed a consistent 3- to 4-fold decrease inNADPH:cytochrome P-450 reducíaseactivily as compared lo

Fig. 4. Progress curves for the reductive metabolism of MMC in N¡(A) andBMY 25282 in N2 (A) by CHO-KI (•)and CHO-MMC' (•)cell extracts. Forreaction conditions, see "Materials and Methods."

Table 5 Rate of bioreductive activation of BMY 25282 by CHO-K1 and CHO-MMC' celt extracts in air versus nitrogen

Results shown are the mean ±SE of n determinations from 3 independent cellextracts.

Rate of reduction(nmol/min/mg protein)

Cell extract Air NI

CHO-KI

CHOMMC'

0.10 ±0.01

0.10 ±0.02°

3.34 ±0.48

3.00 ±0.24°(1 = 4)

" Not significantly different from values for parental cells under either condi

tion (P> 0.5).

Table 6 NADPH.-cytochrome P-450 reducíaseactivity in CHO-KI and CHO-MMC' cell extracts

Values shown are the mean ±SE for n determinations from 3 independentcell extracts.

CellextractCHO-KI

CHO-MMC'Rate

of cytochrome c reduction(nmol/min/mgprotein)3.

17 ±0.23(n = 16)

0.93 ±0.11°(1 = 17)

" (P< 0.001) significantly different from value for the CHO-KI cell line.

parental cell extracts (Table 6). The identity of this enzyme wasconfirmed by incubation with an inhibitory polyclonal goatanti-rat antibody to NADPH:cytochrome P-450 reducíase.

DT-Diaphorase Activity. DT-diaphorase activities in CHO-KI and CHO-MMCr extracts were not significantly different(P> 0.05) at 13.0 ±2.6 (n = 6) and 10.9 ±2.0 (n = 5) nmol/min/mg protein, respectively.

DISCUSSION

We have isolated a CHO cell line (CHO-MMC) whichdisplays enhanced resistance under aerobic conditions to thebioreductive alkylating agent MMC. CHO-MMC' cells are also

highly resistant to DCMMC, the monofunctional derivative ofMMC, but show no cross-resistance to a range of other alkylating agents, to m-platinum or to X- and UV irradiation.

In some cases, acquired resistance to MMC has been shownto accompany the MDR phenotype, although the level of MMCresistance in these cells is usually far lower than that seen withanthracyclines or Vinca alkaloids (29-31). MMC has also beenused as the challenging agent in the selection of a mouse MDRcell line (32). CHO-MMC' cells do show a very low level cross-

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resistance to Adriamycin, VP 16, and vincristine, drugs involvedin the MDR phenotype. It seems unlikely, however, that theMDR mechanism is involved in resistance to MMC in this cellline for two reasons: (a) the resistant cells do not overexpressmdr specific mRNA or P-glycoprotein (although a degree of<2-fold cannot be excluded); (b) the calcium channel blockerverapamil does not reverse resistance to MMC in this cell line.6

The most abundant lesion produced by activated MMC isthe monofunctional alkylation of DNA (12, 33). However,MMC is believed to exert a more potent cytotoxic effect throughthe formation of a bis adduct which causes intra- and interstrandcross-linking of DNA strands (9, 12). For these reactions to beachieved, MMC requires bioreductive activation. This involvesreduction of the quinone moiety with subsequent loss of themethoxy group, resulting in the opening of the aziridine ringwith the generation of reactive alkylating sites at C-l, C-10,and possibly C-7 positions of MMC (12, 34). The C-2 and C-10 positions of reduced MMC react with the N-2 position oftwo adjacent deoxyguanosines and the resulting bis adduct fitsinto the minor groove with minimal distortion of the DNAstructure (11, 12).

The importance of one-electron versus two-electron reductiveactivation of MMC remains to be clarified. However, one-electron reduction is considered sufficient for bioreductive DNAalkylation under hypoxic conditions (12,35, 36) but in air leadsto the generation of oxygen radicals as a consequence of thereaction of oxygen with the semiquinone or its radical anión.In some systems, but not all, the formation of oxygen radicalsappears to contribute to the cytotoxicity of MMC in air (18,37-39).

It has been demonstrated that purified rat liverNADPH:cylochrome P-450 reducíaseand xanthine oxidase cancatalyze the one-electron reduction of MMC into a reactivespecies (12, 18, 36) and both enzymes may have importantbioactivation roles in mammalian cells (16). We have shownthat extracts of CHO-MMC' cells exhibit a 2-3-fold lower

capacity for MMC reductive metabolism compared to parentalCHO-K1 preparations. We have further shown that CHO-MMC' cell extracts exhibit a 3-4-fold decrease in the activityof NADPH:cytochrome P-450 reducíase.Taken together, thesedata would suggest lhal Ihe mechanism of resistance to MMCexhibiled by CHO-MMCr cells under aerobic condilions is

ihrough Ihe failure to efficiently bioaclivale Ihe drug due lodecreased aclivity or quantity of Ihis bioactivating enzyme.

When CHO-MMCr cells are fused with the parental line, the

resulting hybrid exhibits almost wild type sensilivity to MMC.Preliminary evidence indicates that this CHO-Kl/CHO-MMCrhybrid has normal NADPH:cytochrome P-450 reducíaseactivity.7 This would suggest that the recessive nature of the resistance mechanism to MMC in CHO-MMC' cells is in keeping

with a deficiency in this enzyme.The much greater degree of resistance seen under aerobic as

against hypoxic conditions suggests that the lower activity ofNADPHxytochrome P-450 reducíasein the resistant line results in an impaired production of toxic oxygen radicals in air.Although NADPH:cylochrome P-450 reduction is known tobioactivate MMC to a DNA reactive species, it appears thatthe decreased activity of this enzyme and impaired metabolismof MMC in CHO-MMC' cells does not lead to greatly reduced

sensitivity under hypoxic conditions.DT-diaphorase is known to calalyze Ihe obligate two-eleclron

reduction of quiñones(27). Under hypoxia, this enzyme may

6 Unpublished data.7 Unpublished observations.

protecl againsl quinone toxicity by decreasing the concentrationavailable for one-eleclron reduction to the semiquinone freeradical (40, 41). A similar mechanism has been proposed forMMC (16). However, no difference in DT-diaphorase aclivilywas present in CHO-K1 or CHO-MMC' cell extracts. In addi

tion, recent work has shown that MMC does not appear to bea substrate bul is an inhibitor of human and ral DT-diaphorase,although this may not be true for the hamster enzyme (42, 43).

A series of CHO cell lines resislant to MMC in air wereisolated by Dulhanty et ai. (44). Various mechanisms weresuggested including deficient drug activation by DT-diaphorase.A similar proposal was made to explain the intrinsic resistanceto MMC of fibroblasts from a cancer prone family (45). However, in both cases the involvement of DT-diaphorase wasimplicated only indirectly by the effect of dicumarol on cellsurvival, and dicumarol is known to exert additional biochemical effects (see Ref. 43). Failure to efficiently activate MMC,as measured by spectrophotometric disappearance, has beensuggested as the mechanism of induced resistance to MMC ina human colon carcinoma cell line (46). This cell line exhibitedonly a very low degree of cross-resistance to the MMC analogue, BMY 25282 (46, 47). However, none of the potentialactivating mechanisms was studied. BMY 25282 has an amidegroup substituted at position 7 of the quinone ring of MMCresulting in a lower reduction potential (46). This facililalesmore rapid reduclion (41). We have shown that CHO-MMC'cells show a very low level of cross-resistance to BMY 25282.Analysis of the rates of bioreductive metabolism of BMY 25282in CHO-K1 and CHO-MMC' cell extracts showed that this

analogue is activated at least 100 times faster than MMC underaerobic conditions. In addition, under N2 the rate of bioreductive activation is enhanced at least 30-fold for both drugs inCHO-K1 and CHO-MMC' cell extracts alike. In contrast to

MMC, BMY 25282 metabolism proceeded with equal efficiency in parent and resistant cell lines, under both air and N2.This is in line with the enhanced potency of BMY 25282 relativeto MMC under aerobic conditions and is consistent with thecomparative lack of differential toxicity seen with BMY 25282in parental and resistant cells.

Previous studies have shown that whereas NADPHrcyto-chrome P-450 reducíaseand xanthine oxidase are Ihe principalenzymes involved in MMC aclivalion, the mitochondrial re-duclases and NADPH:cylochrome P-450 reducíasewere mosteffeclive at catalyzing the activation of BMY 25282 (18). Thusit seems likely thai Ihe residual NADPH:cylochrome P-450reducíaseaclivily, logether with the mitochondrial reductases,may be sufficienl lo activate BMY 25282 as effeclively in CHO-MMC' cells as in parenlal CHO-K1 cells.

In addilion lo Ihe CHO-MMC' line described here, we haveisolated a multidrug resistanl cell line which shows amplifica-lion of Ihe mdr genes and which exhibils exlreme resistance toMMC. This MMC resislance is partially reversible by verapamil(and therefore probably includes an mdr dependent component), bul NADPH:cylochrome P-450 reducíaseaclivity is similar to thai in Ihe parenlal cell line.6 This suggesls lhal resislance

lo MMC may be mediated by at least 2 different mechanismsand ihus a variety of stralegies may be required lo circumvenlMMC resistance in vivo.

The present work emphasizes the potenlial importance ofenzymatic bioreductive activalion as one possible mechanismof in vivo resistance to MMC. This is the first reporl in whicha cell line wilh induced resistance to MMC has been showndirectly to exhibit an altered aclivily of a specific enzymeimplicaled in ils bioreduclive activation. In the future il may be

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MITOMYCIN C RESISTANCE AND DRUG ACTIVATION

possible by measuring the activity of this and other activatingenzymes to select those tumors most suited to receive a particular bioreductive drug therapy (48). In addition, cell lines suchas CHO-MMC may be useful in screening for MMC analoguesthat are more readily activated, possibly by alternative mechanisms. Such drugs may have a role in the treatment of humantumors which are resistant as a result of inefficient bioreductiveactivation.

ACKNOWLEDGMENTS

We thank Dr. Narayanan, National Cancer Institute, and Dr. Brad-ner, Bristol Myers, for the kind gift of drugs. We also thank LizClemson for typing the manuscript.

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1990;50:4692-4697. Cancer Res   Paul R. Hoban, Michael I. Walton, Craig N. Robson, et al.   Mitomycin C under Aerobic but not Hypoxic ConditionsImpaired Drug Activation in a Mammalian Cell Line Resistant to Decreased NADPH:Cytochrome P-450 Reductase Activity and

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