[CANCER RESEARCH 50, 7789-7792, December 15, 1990]
Activation of Mitomycin C by NADPH:Cytochrome P-450 ReducÃ-ase1
H. Frances J. Bligh, Agnieszka Bartoszek,2 Craig N. Robson, Ian D. Hickson, Charles B. Kasper, Jean D. Beggs, and
C. Roland Wolf3
Department of Molecular Biology, Kings Buildings, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom [H. F. J. B., J. D. B.J; Imperial Cancer Research
Fund, Molecular Pharmacology Group, Department of Biochemistry, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom [A. B., C. R. W.j; Imperial Cancer
Research Fund, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom [C. N. R., I. D. H.J; and McArdle Laboratory
for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706 [C. B. K.J
ABSTRACT
Mitomycin C is an alkylating agent used in cancer chemotherapy that
shows some specificity towards hypoxic cells. The therapeutic effects of
this compound are thought to result from its metabolic activation by
enzymes such as NADPH:cytochrome P-450 reducÃ-ase.In a previous
report we described a Chinese hamster ovary cell line resistant to
mitomycin C, which had a decreased NADPH:cytochrome P-450 reductase activity coupled with a lower rate of mitomycin C metabolism. In
order to provide further evidence that the lower reducÃ-aseactivity is a
factor in the resistance mechanism, we incorporated NADPH:cytochronte
P-450 reducÃ-aseinto cytotoxicity assays and showed that it significantly
sensitizes cells to mitomycin C. Also, the difference in drug sensitivity
between the wild-type and drug-resistant Chinese hamster ovary cells
was no longer observed. In addition to these studies, we expressed a rat
liver NADPH:cytochrome P-450 reducÃ-asecDNA in a Salmonella typhimurium strain, LR5000. The bacteria expressing the rat NADPH:
cytochrome P-450 reducÃ-aseshowed increased sensitivily to mitomycin
C when incubated with Ihis compound under aerobic condilions. However,
under hypoxic condilions increased sensitivity was not observed. This
parallels Ihe previous finding wilh milomycin ( -resistant Chinese ham
ster ovary cells. These dala provide direct evidence for Ihe role of
NADPH:cylochrome P-450 reducÃ-asein Ihe cyloloxic aclion of Ihis
milomycin C under aerobic bui noi hypoxic condilions and suggesl thai
reduced levels of Ihis enzyme can lead lo drug resislance. P-450 reducÃ-ase
expressed in S. typhimurium may provide a valuable tool for evaluating
the role of Ihis enzyme in Ihe loxicily of drugs activaled Ihrough a one
electron reduction palhway.
quinone free radical generated by one electron reduction reacts
with molecular oxygen to generate Superoxide, and as a conse
quence the MMC then reverts to the parent molecule. The
recovery of unmetabolized drug and the proteclive effect of
oxygen radical scavengers against MMC cytotoxicity under
aerobic conditions support this reaction pathway (2, 11). These
data provide circumstantial evidence that the products gener
ated, e.g., by P-450 reductase-mediated one electron reduction,
are cytotoxic. However, this has not been formally shown to be
the case.
Recently a CHO cell line (CHO-MMC') was established,
which exhibited a 17-fold resistance to MMC under aerobic
conditions but had the same sensitivity as the parental cell line
in the absence of oxygen (8). The reduced level of MMCinduced cytotoxicity in CHO-MMC' appeared to be due to a
INTRODUCTION
reduced rate of metabolic activation. This possibility was sub
stantiated by the finding that the drug-resistant cell line con
tained lower cytochrome P-450 reducÃ-aseactivity. In order to
show unequivocally that P-450 reducÃ-ase activates MMC to
cytotoxic products and to determine whether the change in P450 reducÃ-asemay be directly involved in the resistance mech
anism, we have added P-450 reducÃ-aseexogenously in cytotox
icity assays. In addition, we have expressed rat liver P-450
reducÃ-ase in Salmonella typhimurium and have shown that
these cells become significantly more sensitive to MMC-induced toxicity. This effect is only observed under aerobic con
ditions, which parallels the findings obtained using mammalian
cells.
In order to exert its cytotoxic effects, the anticancer antibiotic
MMC4 appears to require reductive activation to products that
MATERIALS
either bind covalently to DNA or induce the formation of DNA
cross-links (1-3). Several enzymes have been implicated in this
activation pathway including xanthine oxidase, DT diaphorase,
NADPHxytochrome
P-450 reducÃ-ase, and mitochondrial
NADPH reducÃ-ase(4-6). The cytotoxic effect of MMC has
been shown to be often greater in cells grown under hypoxic
conditions (7, 8). Hypoxia is considered to be an important
factor in the treatment of solid tumors with ionizing radiation
(9). Enhanced toxicity in the absence of oxygen has been
attributed to the preferential activation to reactive metabolites
under these conditions (10). Under aerobic conditions the semiReceived6/11/90;accepted9/10/90.
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 in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by funds from the Science and Engineering Research
Council and the Imperial Cancer Research Fund. J. D. B. holds a Royal Society
E.P.A. Cephalosporin Fund Senior Research Fellowship.
1On leave from the Department of Pharmaceutical Technology and Biochem
istry, The Technical University of Gdansk, Poland.
3To whom requests for reprints should be addressed, at Imperial Cancer
Research Fund, Hugh Robson Building, George Square, Edinburgh EH8 9XD,
United Kingdom.
4 The abbreviations used are: MMC, mitomycin C; P-450 reducÃ-ase,
NADPH:cytochrome P-450 reducÃ-ase;CHO, Chinese hamster ovary; cDNA,
complementary DNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
AND METHODS
Escherìchiacoli strain MM294 (end A, thi A, hsd R) was used to
propagate recombinant plasmid DNA. 5. typhimurium strain LR5000
[met A, met B, Irp B, leu, val (unstable), sir (rps L) hsd LT, hsd SA, hsd
SB] was used to express P-450 reductase cDNA for cytoloxicity lesls.
The mammalian cell lines used were Ihe wild lype, Chinese hamster
ovary cell line (CHO-K1), the mitomycin C-resistanl variant (CHOMMC1) (5), and Ihe human mammary carcinoma cell line, MCF-7.
CHO-K1 and CHO-MMC' cells were cultured as described previously
(8). MCF-7 cells were cultured in RPMI 1640 (Gibco) supplemenled
wilh 10% felal calf serum, 100 units ml"1 penicillin, and 100 fig/ml
slreplomycin. All chemicals were obtained from Sigma Chemical Com
pany. Reslriction enzymes and T4 DNA ligase were obtained from
Boehringer-Mannheim Corporation.
Cytoloxicily Assays. MCF-7 cells (7-9 x 103/well) were plaled out
in 180 fil of medium in 96-well microtiler plates and left al 37'C for
16 h to adhere. Cells were ihen treated for 3 h at 37°Cwith appropriate
concentrations of MMC plus NADPH plus P-450 reductase all added
in a volume of 20 n\. The total volume of culture medium was 200 n\.
Final concentrations were NADPH, 1 HIM,and P-450 reductase, 250
units/ml. In control experimenls, either NADPH, P-450 reductase, or
MMC was omitted. After 3 h, the medium was replaced and the cells
allowed to grow for 72 h. Cell survival was then assessed using the
MTT assay as described previously (12). In experiments using CHO
cells, the cells were plated out in 30-mm l'etri dishes at a density of
250-1000 cells/plate and left for 4 h at 37'C to adhere. The medium
7789
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P-450 REDUCTASE AND MITOMYCIN C RESISTANCE
Bam HI
I
of molten (42°C)LB agar (0.5% w/v) along wilh 100 ti\ of an appro
Bam HI
priate diluì
¡onof MMC. This was ihen mixed and poured on top of a
Luria broth agar plate containing am pici11in (100 /^g/ml). Each dose
was performed in triplicale. Plates were incubated at 37 °Cfor 24-48 h
P450 reducÃ-ase cDNA
unlil colonies were visible. In anaerobic experiments, plates were incu
bated in an airtight BBL GasPak jar and the oxygen purged to a redox
potential of -200 mV according to the manufacturer's instructions.
RESULTS
Fig. 1. Construction of pJLFl. The P-450 reducÃ-asecDNA was ligated into
the BamHl site of pJLASOS placing the endogenous ATG within the optimum
distances for efficient translation of transcripts produced by the PLPR promoter
system.
was then removed, replaced by 1 ml of phosphate-buffered saline
containing 1 mM NADPH, 250 units of P-450 reducÃ-ase, and an
appropriate dose of MMC. The plates were then incubaled at 37°Cfor
3 h. The medium was then removed, ihe cells were washed Iwice wilh
phosphate-buffered saline and following ihe addilion of fresh medium
the cells were incubated for 12-14 d. Cell survival was determined by
colony counting as described previously (5).
Enzyme Purification. P-450 reducÃ-asewas isolated from rat liver
microsomes by affinity chromatography as described previously (13).
On the basis of Coomassie staining following sodium dodecyl sulfatepolyacrylamide gel electrophoresis, P-450 reducÃ-asewas judged to be
essentially pure.
Plasmid Construction. Bacterial transformations, plasmid DNA prep
arations, and ligations were performed using standard melhods (14).
pJLASOS, a baclerial expression veclor lhat contains the heat-inducible
PRPL tandem promoter (15), was used to create pJLFl (Fig. 1). A
BamHl fragment conlaining the rat P-450 reducÃ-asecDNA (13) was
ligaled into the unique BamHl site of pJLASOS, such lhal Ihe ATG of
ihe P-450 reducÃ-asegene was the optimal dislance from ihe ribosome
binding site of the PL promoter for efficient gene expression (15).
Expression Studies. Rat P-450 reducÃ-asewas expressed in LR5000/
pJLFl by growing 50-ml cultures to ^6oo = 0.1 at 30°Cand incubating
for 20 min at 42°Cand ihen al 37°Cfor 1-3 h. Baclerial cells were
harvested by centrifugation and resuspended in 0.1 M Tris-HCl (pH
7.5), 0.9% (w/v) NaCl conlaining 1 mM phenylmelhylsulfonyl fluoride,
1 mM dithiothreitol (Buffer A). To obtain cell extracts, cell suspensions
were sonicated for 3 x 10 s on ice and centrifuged at 3,000 rpm for 2
min in an Eppendorf microcenlrifuge lo remove unbroken cells. For
fraclionation, the supernalant was centrifuged at 10,000 x g for 10 min
and the resulting supernatant centrifuged at 100,000 x g for 1 h lo give
the soluble fraction and a membrane pelici. The pellet was resuspended
in Buffer A using a hand-held homogenizer. Determination of protein
concentration, sodium dodecyl sulfale-polyacrylamide gel electropho
resis using 7.5% (w/v) acrylamide gel, and Western blot analysis were
performed as described previously (16-18). Western blots were devel
oped using a polyclonal goat antibody lo ral P-450 reducÃ-aseand a
peroxidase-conjugated rabbit anti-goat IgG Vectastain color detection
kil (Veclor Laboralories, Brelton, Peterborough, United Kingdom).
Cytochrome c reducÃ-aseassays were performed using the method of
Vermillion and Coon (19).
S. typhimurium Cytotoxicity Assays. Cytoloxicity assays were per
formed on S. typhimurium transformed with pJLFl or pJLASOS, cells
grown to an Atoa value of approximately 0.1, indicated at 42°Cfor 20
min, and then incubated at 37°Cfor 3 h. Cultures were diluted with
sterile distilled water and 100-200 cells in 100 p\ were added to 3 ml
Initial experiments to establish the ability of P-450 reducÃ-ase
to activate MMC to cytotoxic products were performed using
MCF-7 cells and exogenously added reducÃ-ase enzyme. The
presence of P-450 reducÃ-asemarkedly polenliated MMC loxicily with the dose required to kill 50% of Ihe cells changing
approximalely 6-fold from 0.12 lo 0.02 Mg/ml (Fig. 2). P-450
reducÃ-asein the absence of MMC was also slightly loxic bul
resulled in less lhan a 10% loss in viabilily. On the basis of Ihe
above finding, similar experimenls were carried oui on CHOKl cells and Iheir milomycin C-resislant derivative CHOMMCr (Fig. 3). Consislenl wilh previous findings, Ihe resislanl
cell line required a 30-fold higher dose of MMC lo kill 50% of
Ihe cells than Ihe parenlal line. The addition of P-450 reducÃ-ase
lo the assay system again resulted in a very marked increase in
Ihe sensilivily of both cell lines lo Ihis compound (Fig. 3), but
with a much greater effecl being observed wilh ihe CHO-MMCr
cells (approximalely 100-fold) lhan wilh Ihe CHO-K1 cells (5-
o.o
0.1
0.2
0.3
0.4
0.5
MMC
(u.g/ml)
Fig. 2. Potentiation of mitomycin C toxicity by P-450 reducÃ-ase.MCF-7 cells
were treated with MMC in the presence or absence of active P-450 reductase and
assayed for survival using the MTT assay (see "Materials and Methods"). Cells
treated with MMC alone (D) and cells treated with MMC in the presence of P450 reductase and NADPH (•).Survival is expressed as percentage of an
equivalent control containing no MMC. Bars, SD calculated from a minimum of
three independent experiments.
.001
.01
.1
1
10
100
MMC
(M-g/ml)
Fig. 3. Effect of P-450 reductase and MMC toxicity towards CHO-K1 and
CHO-MMC'. CHO-K1 + MMC only (A), CHO-MMC + MMC only (A), CHOKl + MMC + NADPH -I-P-450 reductase (O), CHO-MMC' + MMC + NADPH
+ P-450 reductase (•).Survival is expressed as percentage survival in comparison
to the equivalent control containing no MMC. liars, SD calculated from a
minimum of three independent experiments.
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P-450 REDUCTASE AND MITOMYCIN C RESISTANCE
PÃœLA505
have been noted for P-450 reducÃ-aseexpressed in E. coli (20).
Fractionation of cell extracts showed immunologically detect
able protein in both the cytosolic and membrane fractions,
although the M, 78,000 band was confined to the membrane
fraction (data not shown). The enzyme in both fractions was
shown to be active by measuring the reduction of cytochrome
c. The activity measured in whole cell homogenates was 21.0 ±
6 (SD) nmol cytochrome c reduced/min/mg total protein com
pared with control cells (transfected with the parent vector
alone) of 6.1 ±1.0 nmol/min/mg. The activity due to the
reducÃ-aseof 15.0 nmol/min/mg tolal cellular protein is not
very different from thai measured in rodenl hepalocyles.
Having established Ihe presence of aclive P-450 reducÃ-asein
LRSOOO/pJLFl, this strain was used lo assess cyloloxicily of
MMC. In three independent experiments, LRSOOO/pJLFl
showed a significant increase in sensitivily lo MMC compared
wilh LR5000/pJLA505
under aerobic condilions (Fig. 5a).
Because il has been reporled lhal hypoxic condilions potentiate
MMC loxicily, these experiments were repealed in Ihe absence
of oxygen. In Ihese experimenls, no significant difference beiween LRSOOO/pJLFl and LR5000/pJLA505 was found (Fig.
50). In these latter experimenls, Ihe expression of Ihe reducÃ-ase
under anaerobic condilions was confirmed by Weslern blol
analysis (noi shown).
pJLFI
178
[66
Fig. 4. Expression of P-450 reducÃ-asein S. typhimurium. Cells were trans
formed with pJLASOS (control) or pJLFI (containing the P-450 reducÃ-asecDNA)
plasmiti* and the cells heated at 42°Cfor 20 min. Following incubation at 37°C
for 3 h, cells were harvested and whole-cell homogenates prepared. Samples were
then analyzed by Western blot analysis as described in "Materials and Methods."
Track pJLASOS, 40 /ig total cell protein from cells transformed with pJLASOS;
Track pJLFI, 40 ¿igof protein from cells transformed with pJLFI. Arrows,
expressed protein of M, 78,000 with the same mobility as an authentic rat P-450
reducÃ-asestandard and the protein of M, 66,000.
10-fold). When either NADPH or P-450 reducÃ-asewas omitted
from the incubation mixlure, no polenlialion of MMC loxicily
was observed wilh any of Ihe cell lines tested, demonstraling
lhal aclive P-450 reducÃ-aseis required to observe Ihis effecl. In
the presence of P-450 reducÃ-ase,Ihe two CHO cell lines had
very similar sensitivily to MMC. In these experimenls, however,
the addilion of exogenous P-450 reducÃ-ase combined with
NADPH resulled in a decrease in cell viabilily lo 15-30% (dala
not shown). This effecl was only observed if bolh NADPH and
P-450 reducÃ-asewere present in Ihe assay mixlure. The mech
anism of Ihis effect, however, is unclear.
In order to establish Ihe effecls of P-450 reducÃ-aseexpressed
inlracellularly on MMC loxicily, Ihe P-450 reducÃ-asecDNA
was expressed in S. typhimurium and Ihe resullanl cells assayed
for cyloloxicily changes. S. typhimurium was chosen because
cyloloxicily and mulagenicily lesling is well established in ihis
organism. Plasmid pJLASOS carries Iwo tandem promoters (PL
and PR) from phage X, which are repressed at 30°Cby Ihe
product of the cl857 gene. However, al 37-42°C, Ihe lemperalure-sensilive ci gene produci is inaclive and iranscription is
initialed. Cells transformed with pJLFI were irealed to induce
P-450 reducÃ-aseexpression (see "Materials and Methods"), and
whole cell extracts were analyzed for P-450 reducÃ-aseproduc
tion by Weslern blot analysis (Fig. 4). Maximal reducÃ-aselevels
were obtained after 3 h growih al 37°C.Weslern blot analysis
demonstraled Ihe presence of a prolein wilh Ihe same molecular
size as Ihe authentic reducÃ-asestandard (Mr 78,000) in cells
containing pJLFI. However, in addilion a second band of M,
66,000 was also delecled (Fig. 4). Similar breakdown producÃ-s
DISCUSSION
In ihis report, we provide direct evidence thai the metabolism
of mitomycin C by cylochrome P-450 reducÃ-aseincreases Ihe
cyloloxicily of Ihis compound. This finding is consislenl wilh
Ihe reports thai cell lines made resislanl lo MMC have a
reduced capacity to actÃ-valeIhe drug lo cyloloxic producÃ-sand
a
3
U)
if
6
8
(ng/plate)
100
(A
20-
02468
MMC
(ng/plate)
Fig. 5. Effect of P-450 reducÃ-aseexpression on sensitivity of S. typhimurium
to mitomycin C. a, dose-response curve for 5. typhimurium treated with MMC
aerobically. D, LR5000/pJLA505; •,LRSOOO/pJLFl. Bars, SD for one experi
ment. Similar results were obtained from three independent experiments. /'. doseresponse curve for 5. typhimurium treated with MMC anaerobically. D, LR5000/
pJLASOS; •.LRSOOO/pJLFl. Bars, SD for one experiment. Similar results were
obtained from three independent experiments.
7791
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P-450 REDUCTASE AND MITOMYCIN C RESISTANCE
that free radical scavengers inhibit its cytotoxic effects (8, 21).
In the case of the CHO-MMC cell line, the reduced rate of
activation has been attributed to reduced cytochrome P-450
reducÃ-aseactivity (8). We have preliminary Western blot data
that indicate that the lower activity is due to a reduced level of
the enzyme in the drug-resistant cells.5 The addition of P-450
reducÃ-asereversed the resistance observed in the MMC-resistant cell line. There are two explanations for this effect. The
exogenously added reducÃ-ase could be compensating for the
reduced rale of MMC aclivalion in MMC and Iherefore in
crease Ihe level of DNA binding lo ihe same as lhal found in
Ihe wild-lype cells. Alternatively, the activated MMC could be
acting extracellularly by alkylating Ihe cell membrane. In Ihis
case, Ihe mechanisms lhal confer drug resislance in MMCr
would noi come inlo play. To dale, ihere is lillle evidence lo
indicale lhal Ihe mechanism of MMC loxicily is Ihrough ils
interact Um wilh cell membranes, Iherefore Ihe former possibil
ity appears lo be mosl likely. If Ihis is Ihe case, il would support
the evidence lhal neilher an increased rale of DNA repair nor
MMC deloxificalion is involved in Ihe resislance exhibited by
the MMC cell line (8).
No difference in MMC sensilivily was observed belween
CHO-K1 and CHO-MMCr when Ihe cells were incubaled under
anaerobic condilions (8). The same finding was noted here wilh
baclerial cells expressing P-450 reducÃ-ase.This observalion is
difficult lo explain unless differenl enzymes play Ihe predominanl role in MMC aclivalion under aerobic or hypoxic condi
lions. Il is feasible lhal Ihe enzymes involved in MMC aclivation under hypoxic condilions include milochondrial reduclases, which in Ihe absence of molecular oxygen are diverted
from Ihe respiralory palhway. This would explain Ihe enhanced
MMC loxicily observed in some hypoxic mammalian cells (10).
It cannol be ruled oui, however, lhal Ihe mechanism of aclion
of MMC is complelely changed under hypoxic condilions and
no longer involves a one-eleclron reduclion process. The imporlanl conclusion from these observations is lhal P-450 re
ducÃ-aseonly appears lo mediale MMC loxicily in aerobic cells.
Cylochrome P-450 reducÃ-ase has been implicaled in Ihe
melabolic aclivalion of a wide variely of compounds, including
anlicancer drugs, loxins, and mulagens. The expression of Ihis
enzyme in S. typhimurium, which is roulinely used in mulagenicily lests (22), will allow its involvement in bolh Ihe cyloloxicily and genoloxicily of chemicals lo be evalualed. Sludies of
Ihis nalure have been reported by Walanabe et al. (23), who
observed a marked increase in Ihe mutation rale induced by
nilroarenes in baclerial slrains expressing nilroreduclase and
acelyllransferase genes.
ACKNOWLEDGMENTS
Strain LR5000 was a gift from Professor C. Higgins, Imperial Cancer
Research Fund, Oxford, United Kingdom.
1 H. F. J. Bligh, A. Bartoszek, C. N. Robson, I. D. Hickson, C. B. Kasper, J.
D. Beggs, and C. R. Wolf, unpublished observations.
REFERENCES
1. Iyer, V. N., and Szybalski, W. A molecular mechanism of mitomycin C
action: linking of complementary DNA strands. Proc. Nati. Acad. Sci. USA,
50: 355-362, 1963.
2. Tomasz, M., and Lipman, R. Reductive metabolism and alkylating activity
of mitomycin C induced by rat liver microsomes. Biochemistry, 20: 50565061, 1981.
3. Tomasz, M., Lipman, R., Chowdary, D., Pawlak, J., Verdine, G. L., and
Nakagishi, K. Isolation and structure of a covalent cross-link adduci between
mitomycin C and DNA. Science (Washington DC), 235: 1204-1208, 1987.
4. Keyes, S. R., Facasso, P. M., Heimbrook, D. C., Rockwell, S., Sligar, S., and
Sartorelli, A. C. Role of NADPH: cytochrome c reducÃ-aseand DT-diaphorase
in the biotransformalion of milomycin C. Cancer Res., 44:5638-5643,1984.
5. Workman, P., Walton, M. I., Powis, G., and Schlager, J. J. DT-diaphorase
questionable role in milomycin C resislance bul a largel for novel bioreductive
drugs? Br. J. Cancer, 60:800-802, 1989.
6. Buller, J., and Hoey, B. M. Are reduced quiñonesinvolved in Ihe anlilumor
aclivily of drugs? Br. J. Cancer, 55:53-59, 1987.
7. Raulh, A. M., Mohindra, J. K., and Tannock, I. F. Aclivily of milomycin C
for aerobic and hypoxic cells in vitro and in vivo. Cancer Res., 43: 41544158, 1983.
8. Hoban, P. R., Wallon, M. I., Robson, C. N., Godden, J., Slralford, I. J.,
Workman, P., Harris, A. I,., and Hickson, I. D. Milomycin C resislance
under aerobic bui noi hypoxic conditions in a mammalian cell line: associalion wilh impaired drug aclivalion and decreased NADPH: cylochrome P450 reducÃ-aseaclivily. Cancer Res., 50: 4692-4697, 1990.
9. Sarlorelli, A. C. Therapeutic ¡mackof hypoxic cells of solid lumors: presi
denlial address. Cancer Res., 48: 775-778, 1988.
10. Kennedy, K. A., Rockwell, S., and Sartorelli, A. C. Preferential aclivation of
milomycin C lo cyloloxic metabolites by hypoxic lumor cells. Cancer Res.,
40: 2356-2360, 1980.
11. Doroshow, J. H. Role of hydrogen peroxide and hydroxyl radical formation
in Ihe killing of Ehrlich lumor cells by anticancer quiñones.Proc. Nail. Acad.
Sci. USA, «3:4514-4518, 1986.
12. farnik-had. J., DeGraff, W. G., Gazdar, A. F., Minna, J. D., and Mitchell.
J. B. Evaluation of a telrazolium-based semiaulomaled colorimetrie assay:
assessment of chemosensilivily lesling. Cancer Res., 47: 936-942, 1987.
13. Yasukochi, Y., and Maslers, B. S. S. Some properties of a delergent solubilized NADPH cylochrome (cylochrome P450) reducÃ-asepurified by biospecific affinity chromatography. J. Biol. Chem., 251: 5337-5344, 1976.
14. Manialis, T., Frilsch, E. F., and Sambrook, J. Molecular Cloning: A Labo
ratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Press, 1982.
15. Schauder, B., Blocker, H., Frank, R., and McCarthy, J. E. G. Inducible
expression veclors incorporaling the Escherichia coli alpE Iranslalional ini
liât
ion region. Gene, 52: 279-283, 1987.
16. Bradford, M. M. Rapid and sensilive method of quanlilation of microgram
quantities of protein ulilizing principle of prolein dye binding. Anal.
Biochem.. 72: 248-256, 1976.
17. Lacrimili. U. K. Cleavage of structural proteins during Ihe assembly of Ihe
head of bacleriophage T4. Nalure (Lond.), 227: 680-685, 1970.
18. Towbin, H., Staehelin, T., and Gordon, J. Eleclrophoretic Iransfer of proteins
from polyacrylamide gels lo nitrocellulose sheels: procedure and some appli
cations. Proc. Nail. Acad. Sci. USA, 76:4350-4354, 1979.
19. Vermillion, J. L., and Coon, M. J. Purified liver microsoma! NADPHcylochrome P450 reducÃ-ase.J. Biol. Chem., 253: 2694-2704, 1978.
20. Porter, T. D., Wilson, T. E., and Kasper, C. B. Expression of a functional
78,000 dalton mammalian flavoprolein, NADPH-cytochrome P450 oxidoreduclase, in Escherichia coli. Arch. Biochem. Biophys., 254:353-367,1987.
21. Dusre, L., Rajagopalan, S., Eliol, H. M., Covey, J. M., and Sinha, B. K.
DNA interslrand cross-link and free radical formation in a human multidrugresislanl cell line from mitomycin C and ils analogues. Cancer Res., 50:648652, 1990.
22. Ames, B. N., McCann, J., and Yamasuki, E. Melhods for delecling carcino
gens and mulagens with Ihe .SVi/mo«i'//<i/mammalianmicrosome mutagenicity assay. Mulal. Res., 31: 347-364, 1975.
23. Walanabe, M., Nohmi, T., and Ishidale, M. New lesler slrains of Salmonella
typhimurium highly sensilive lo mulagenic nitroarenes. Biochem. Biophys.
Res. Commun., 147: 974-979, 1987.
7792
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1990 American Association for Cancer Research.
Activation of Mitomycin C by NADPH:Cytochrome P-450
Reductase
H. Frances J. Bligh, Agnieszka Bartoszek, Craig N. Robson, et al.
Cancer Res 1990;50:7789-7792.
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