Download Antioxidant and Xenobiotic-metabolizing Enzyme Gene Expression

(CANCER RESEARCH 50. 1397-1402, March 1, 1990]
Antioxidant and Xenobiotic-metabolizing
resistant MCF-7 Breast Cancer Cells'
Enzyme Gene Expression in Doxorubicin-
Steven A. Akinan, Gerald Forrest, Fong-Fong Chu, R. Steven Esworthy, and James H. Doroshow2
Department of Medical Oncology and Therapeutics Research and Division of Biology, City of Hope National Medical Center, Duarte, California 91010
2.5.1.18) (7) and UDP-glucuronyl transferase (EC 2.4.1.17) (6,
8), are increased in activity and particular group I enzymes,
We investigated the expression of the genes for several antioxidant
such as aryl hydrocarbon hydroxylase (EC 1.14.14.1), are de
and xenobiotic-detoxifying enzymes in the multidrug-resistant variant of
creased in activity in these cells (6, 9).
the human breast cancer cell line MCF-7, MCF-7/Dox. MCF-7/Dox is
Changes in antioxidant enzyme levels in MCF-7/Dox cells,
greater than 500-fold resistant to doxorubicin by donogenic assay. En
including
an increase in glutathione peroxidase (EC 1.11.1.9;
zyme activity determinations in the cytoplasmic compartment of MCFGSH:H2O2
oxidoreductase) (10, 11) and Superoxide dismutase
7/Dox revealed a 25-fold increase in glutathione peroxidase level com
(4) (EC 1.15.1.1; Superoxide Superoxide oxidoreductase), have
pared to the parent line (mean ±SD, 10 ±2.8 versus 0.4 ±0.24 nmol/
min/mg; P < 0.005). The activity of the other major hydrogen peroxidealso been described. Sinha et al. (10) inferred that increased
detoxifying enzyme, catatase, was diminished in MCF-7/Dox (2.0 ±0.4
glutathione peroxidase activity was an important determinant
versus 4.8 ±1.4 «imol/min/rag;P < 0.025 compared to MCF-7). Superof resistance to doxorubicin in MCF-7/Dox cells because the
oxide disunitasi- activity did not differ between the two cell lines. The
capacity for doxorubicin-stimulated
hydroxyl radical produc
specific activity of the xenobiotic-detoxifying enzyme DT-diaphorase was tion was diminished in this cell line, probably as a consequence
4-fold lower in MCF-7/Dox compared to MCF-7 (DT-diaphorase, 117 ± of a glutathione peroxidase-mediated decrease in the level of
45 rcrsH.v509 ±123 nmol/min/mg; P < 0.005). Daunorubicinol-producing
intracellular H2O2 after doxorubicin treatment. Kramer et al.
carbonyl reducÃ-aseactivity was equal in the two lines. Northern blot
(12)
have strengthened this inference by demonstrating that
analysis demonstrated a 0.9-kilobase band of glutathione peroxidase
depletion of the intracellular reduced glutathione pool by bumRNA in MCF-7/Dox; no glutathione peroxidase mRNA was detected
thionine sulfoximine partially restores doxorubicin sensitivity
in MCF-7. A 2.4-kilobase catalase and 0.7- and 1.4-kilobase Superoxide
in MCF-7/Dox and that combining glutathione depletion with
dismutase mRNAs were detectable in MCF-7/Dox and MCF-7. When
gp-170 blockade by verapamil completely overcomes doxorub
normalized to 28S RNA, no difference in the mRNA levels of catalase
and Superoxide dismutase in MCF-7/Dox and MCF-7 could be deter
icin resistance. Because increased glutathione peroxidase activ
mined. DT-diaphorase mRNAs of 1.4 and 2.7 kilobases were found in ity is an important feature of doxorubicin resistance in this cell
both MCF-7/Dox and MCF-7 cells. A 1.2-kilobase mRNA homologous
line and because it has not been determined whether the ob
to the putative carbonyl reducÃ-asecDNA was also easily detectable in
served increase in glutathione peroxidase activity is associated
both MCF-7 and MCF-7/Dox. The amount of mRNA for both xenobioticwith enhanced gene expression, we studied the expression of
detoxifying enzymes was decreased 2- to 4-fold in the doxorubicinresistant cells. Southern blot analysis of l'xtÃ--and Afs/7l-reslricled gen- the gene coding for selenium-dependent glutathione peroxidase,
omic DNA revealed no evidence for amplification or rearrangement of as well as the other antioxidant enzymes Superoxide dismutase
and catalase in MCF-7/Dox cells. Our data demonstrate that
the glutathione peroxidase gene. These results indicate that, in addition
to the previously described overexpression of anionic glutathione 5the mRNA for the selenium-dependent glutathione peroxidase
transferase in MCF-7/Dox cells, an augmented glutathione peroxidase
is overexpressed in this doxorubicin-resistant cell line, through
mRNA level is the major alteration in antioxidant and xenobiotic-detox
a mechanism which does not involve gene amplification.
ifying enzyme expression that could contribute to doxorubicin insensitivThe role of xenobiotic-metabolizing enzymes in mediating
ity in these multidrug-resistant breast cancer cells.
doxorubicin toxicity is controversial and not well established.
Doxorubicin is converted to its metabolite doxorubicinol by
reduction of the C9 carbonyl group by aldo-keto reductases,
INTRODUCTION
particularly the pH 6 optimum reducÃ-asetermed carbonyl reThe clinically useful antineoplastic agent doxorubicin is a ductase (13-16). It has been proposed that decreased enzyme
multifunctional molecule, with several possible mechanisms of activity could diminish doxorubicin sensitivity (17). Carbonyl
cytotoxicity (1-5). Therefore, it is not unreasonable to expect
reducÃ-ase activily can be delecled in leukemic blasts from
that tumor cell resistance to doxorubicin may be complex,
patients (18-21), and in some studies, both in leukemic patients
involving multiple and perhaps coincident mechanisms. The and cell lines, enzyme activity has been correlated with sensitiv
doxorubicin-resistant variant of the human breast cancer cell ity to daunorubicin (18, 19). However, recently. Goren et al.
line MCF-7, designated MCF-7/Dox, is an excellent example
(22) developed myeloblast-Chinese hamster ovary cell hybrids
of this point. Although this cell line expresses an increased
with marked variations in carbonyl reducÃ-aseactivily, wilhoul
amount of the multidrug resistance-associated membrane pro
observing variations in daunorubicin sensitivity. Given the con
tein gp-170 (6, 7), it also contains significant alterations in flicting data on the role of carbonyl reducÃ-ase in mediating
certain enzymatic activities which may contribute significantly
doxorubicin resistance, assessment of the level of expression of
to doxorubicin resistance. Several group II xenobiotic-metabothis gene by MCF-7/Dox was of greal interest to us.
lizing enzymes, including anionic glutathione 5-transferase (EC
DT-diaphorase (EC 1.6.99.2; NAD(P)H:quinone oxidoreduclase) is anolher xenobiolic-melabolizing enzyme which can
Received 8/21/89; revised 11/17/89; accepted 11/29/89.
provide prolection against quinone drug-stimulaled free radical
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
produclion (23). Daunorubicin has recenlly been observed lo
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
induce DT-diaphorase activity in neonatal rat myocytes (24).
' This study was supported by Grants CA 31788 and CA 33572 from the
National Cancer Institute.
However, Wallin (25) has reported that doxorubicin is not a
J To whom requests for reprints should be addressed, at Department of Medical
substrate for purified hepatic DT-diaphorase. Furthermore,
Oncology and Therapeutics Research, City of Hope National Medical Center.
Goren et al. (22) did not observe any significanl varialion in
1500 E. Duarte Road, Duarte, CA 91010.
1397
ABSTRACT
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1990 American Association for Cancer Research.
ANTIOXIDANT
ENZYME GENE EXPRESSION
the sensitivity to doxorubicin of cell hybrids with enhanced DTdiaphorase activity. Thus, the importance of DT-diaphorase as
a mediator of doxorubicin toxicity remains to be determined.
For this reason, we also examined the expression of DTdiaphorase in conjunction with daunorubicin reducÃ-aseand the
antioxidant enzymes in both MCF-7 and MCF-7/Dox human
breast carcinoma cells.
MATERIALS
AND METHODS
Cell Culture
MCF-7 and MCF-7/Dox cells were obtained as a generous gift from
Dr. Kenneth Cowan, National Cancer Institute (Bethesda, MD). Both
the parent line and the multidrug-resistant subline were maintained in
monolayer culture as previously described (26). MCF-7/Dox cells were
routinely grown in the presence of 10 //M doxorubicin; however, all
experiments using the MCF-7/Dox line were performed with cells that
had been passed for I week in the absence of doxorubicin. Cytotoxicity
to doxorubicin in MCF-7 and MCF-7/Dox cells was assessed by
clonogenic assay as previously described (26).
Probes
All of the probes used in this report were cloned in our laboratory,
except for chicken fi-actin, which was purchased from the American
Type Culture Collection. The Superoxide dismutase and catalase (EC
1.11.1.6; H2O2:H2O2 oxidoreductase) cDNAs were isolated from a
human liver cDNA library obtained from Clontech (Clontech Labora
tories, Inc., Palo Alto, CA). The glutathione peroxidase cDNA was
isolated from a human hepatocellular carcinoma (Hep G-2) cDNA
library. DT-diaphorase cDNA was isolated from a rat liver cDNA
library. Human carbonyl reducÃ-asecDNA was isolated from a breast
cancer cDNA library. Positive clones were identified by hybridization
with "P-end-labeled synthetic oligonucleotides oblained either from
previously published sequences, in the case of Cu-Zn Superoxide dis
mutase, catalase, and glutathione peroxidase, or from amino acid data,
in the cases of DT-diaphorase and carbonyl reducÃ-ase.All of these
cDNA probes were subcloned as EcoR\ fragments into the vector
Bluescript (Stralegene Cloning Systems, La Jolla, CA) and partially
sequenced by the dideoxy technique (27), using a Sequenase kit (United
States Biochemicals, Cleveland, OH). Sequences obtained corre
sponded to those previously published, confirming their identities (2832). The cDNA obtained for carbonyl reducÃ-asecoded for a sequence
which corresponded to the sequence oblained from peptide fragments
of purified human liver carbonyl reducÃ-ase,confirming its identity.'
All probes were isolated for labeling as EcoRl fragments from the
Bluescript vector and included the 3' untranslated region plus a variable
portion of the coding region. Superoxide dismulase was 0.6 kilobases;
calalase, 2.1 kilobases; glulathione peroxidase, 0.6 and 0.3 kilobases;
DT-diaphorase, 1.2 kilobases; and carbonyl reducÃ-ase,0.9 kilobases.
Nucleic Acid Isolation and Restriction Digest
High molecular weight genomic DNA was isolated by automated
extraction on a ABI model 340 A nucleic acid extractor, by the method
suggested by the manufacturer (Applied Biosystems, Foster City, CA).
Restriction digests were performed on aliquots of 30 to 50 ^g of DNA
in 1200-^1 solutions, containing 10 units of enzyme/Mg of DNA, for 25 h at 37'C.
Total cellular RNA was isolated by the guanidinium isothiocyanate/
cesium chloride centrifugation method of Maniatis et al. (33), with
minor modifications.
Gel Electrophoresis and Nucleic Acid Transfer
Denaturing RNA Gels. Denaturing glyoxal/formaldehyde gels were
prepared and run according to the method of Maniatis et al. (33).
Twenty /¿gof total RNA were loaded per lane. After electrophoresis,
3 Forrest et al.. Induction of a human carbonyl reducÃ-asegene located on
chromosome 21. submitted for publication.
IN DOXORUBICIN
RESISTANCE
gels were soaked for 5 min in distilled water, followed by l h in two
changes of 10 x SSC.4 The RNA was then transferred to Genetrans
nylon filter paper (Plasco, Inc. Woburn, MA), by capillary transfer with
10 x SSC buffer. Upon completion of the transfer, filters were baked
under vacuum for 2 h at 80°C.
DNA Gels. Five- to 10-j/g aliquots of enzyme-restricted genomic
DNA were electrophoresed on 1.0-1.2% agarose gels in 40 niM TrisHCI, pH 7.4, 20 mM sodium acetate, 1 m.\t EDTA buffer at 50 V, at
room temperature for 2 h, stained with 0.5 Mg/ml ethidium bromide,
and photographed. DNA was depurinated vvilh 0.25 M HC1 for 10 min
and then transferred to Genetrans nylon filter paper by capillary transfer
in 0.4 M sodium hydroxide. After transfer, filters were rinsed in 2 x
SSC and baked under vacuum at 80°Cfor 2 h.
Hybridizations. RNA blots were prehybridized in 5 x Denhardt's
solution, 2 x SSPE, 1% SDS, 0.5 mg/ml denatured herring sperm
DNA, 20 Mg/ml yeast RNA, 50% formamide, at 42°Cfor 4 h. Blots
were then hybridized for 15 h at 42°Cin the above solution containing
10% dextran sulfate plus 1-2 x IO6cpm/ml [12P]-dCTP-labeled probe.
Probes were labeled with the Amersham random priming kit (Amersham. Inc., Clearbrook, IL). Specific activities of labeled probes were
routinely 0.8-1.0 x IO9 cpm/Mg. DNA blots were hybridized in the
same fashion, except that the salt concentration was increased to 3 x
SSPE.
After hybridization, filters were washed in 2 x SSC, 0.1 % SDS, at
room temperature for 15 min; 0.5 x SSC, 0.1% SDS, at room temper
ature for 15 min; 0.1 x SSC, 0.1% SDS, at room temperature for 15
min; followed by 0.1 x SSC, 0.5% SDS, at 50°Cfor 1 h. Autoradiograms of the filters were made on Kodak XAR-5 film (Kodak, Roch
ester, NY) with two Dupont Cronex Lightning-Plus intensifying screens
(DuPont, Wilmington, DE), at —¿70°C.
Signal intensities of the autoradiograms were quantified by densitometry on a Bio-Rad computerized
video densitometer (Bio-Rad, Richmond, CA). Although it is common
practice to use the level of fi-actin mRNA to normalize specific gene
expression, this was not possible in these experiments because the 2.0kilobase (f-actin mRNA was increased by 1.8-fold in MCF-7/Dox as
compared to MCF-7 (Fig. 2, lanes 7 and 8). Hence, signal intensities
were normalized to the intensity of the 28S ribosomal RNA band, to
account for variability in gel loading and transfer efficiency.
Enzyme Assays
Cytosol Preparation. Cell pellets containing 1-5 x IO7 cells were
resuspended in 0.25 M sucrose, 5 mM jV-2-hydroxyethylpiperazine-Ar/2-ethanesulfonic acid, pH 7.4, 1 m,M EDTA, and sonicated for 2 min
on ice (Branson Sonifier Cell Disruptor 200; Branson Sonic Power,
Danbury, CT). Sonicates were centrifuged at 16,500 x g for 10 min at
4°C;the supernatants were then centrifuged at 105.000 x # at 4°Cfor
16 min and the cytosolic supernatants were assayed immediately for
enzymatic activities.
Assays. Catalase and glutathione peroxidase were assayed by the
method of Beutler (34, 35), Superoxide dismutase by the method of
McCord and Fridovich (36), DT-diaphorase by the method of Ernster
(37), and carbonyl reducÃ-aseby the method of Ahmed et al. (15), with
the following modification. Rather than quantifying daunorubicinol
production by thin layer chromatography, we utilized the high perform
ance liquid chromatography method described by Cusack et al. (38).
Proteins were assayed by the method of Lowry et al. (39).
RESULTS
Drug Sensitivity. The IC50 for MCF-7 cells using a 1-h
exposure to doxorubicin was 1.0 fiM; the IC50 for MCF-7/Dox
was approximately 500 /ÃŒM
(Fig. 1). Therefore, the MCF-7/
Dox cells used in this study were at least 500-fold resistant to
doxorubicin.
Antioxidant Enzyme Gene Expression. A 0.9-kilobase band
4 The abbreviations used are: SSC, standard saline citrate buffer, 20 x SSC is
3 M sodium chloride/0.3 M sodium citrate; SSPE, standard saline phosphate
EDTA buffer, 20 X SSPE is 3 M NaCI, 0.23 M NaH2PO4. 20 mM EDTA; SDS,
sodium dodecyl sulfate. cDNA. complemenatry DNA.
1398
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1990 American Association for Cancer Research.
ANTIOXIDANT ENZYME GENE EXPRESSION IN DOXORUBICIN RESISTANCE
12
90 \
34
56
78
_\
\
70 _\
O
E h
§50
.
tt
LU
g
\
I
40
30
\
20
I
10
i
0.751.52.25
l
l
I
U
5.0 0 100200300400500
[DOXORUBICIN],
Fig. 1. Clonogenic survival of MCF-7 (left) and MCF-7/Dox (right) cells after
a 1-h exposure to doxorubicin at the concentrations shown, as described in
"Materials and Methods." After drug exposure, cells were allowed to grow for 8
days; colonies of >40 cells were scored. Each data point represents the mean of
triplicate determinations of the colony counts of drug-exposed cells divided by
the colony counts of the control (no drug exposure) cells x 100. SD were routinely
less than 10% of the mean value.
Fig. 2. Autoradiogram of a Northern blot of MCF-7 RNA (eren-numhered
lanes) and MCF-7/Dox RNA (odd-numbered lanes). The blot was probed,
stripped, and reprobed in succession with glutathione peroxidase (lanes I and 2),
catatase (lanes 3 and 4). Cu-Zn Superoxide dismutase (lanes 5 and 6), and tf-actin
(lanes 7 and 8) cDNA probes. The blot shown is representative of three replicate
blots. Twenty ^g/lane of total RNA were electrophoresed, blotted, probed, and
autoradiographed as described in "Materials and Methods." Autoradiographic
signal intensities were estimated by video densitometry. The upper band observed
in lanes I and 2 is 28S ribosomal RNA: the lower hand in lane /only is glutathione
peroxidase mRNA. The 28S ribosomal RNA band is visible due to cross-hybrid
ization with the glutathione peroxidase cDNA probed under the hybridization
and washing conditions used in these studies.
Southern blot suggests that glutathione peroxidase is part of a
gene family. MCF-7 cells also express both 0.7- and 0.9kilobase Cu-Zn Superoxide dismutase mRNA species, similar
to other human cells (28). When normalized to the 28S RNA
band, no difference in steady state Cu-Zn Superoxide dismutase
mRNA level was observed between the parent MCF-7 line and
MCF-7/Dox (Fig. 2, lanes 5 and 6). This correlated well with
enzyme specific activities, where little difference was observed
between the parent and doxorubicin-resistant cells (Table 1). A
2.4-kilobase band of catalase mRNA was observed in both lines
(Fig. 2, lanes 3 and 4). As with Superoxide dismutase, there was
no significant difference in steady state catalase mRNA levels
between the parent line and MCF-7/Dox. Catalase enzyme
activity, however, was diminished approximately 2-fold in
MCF-7/Dox compared to MCF-7 cells (Table 1; P < 0.025).
DT-Diaphorase and Carbonyl ReducÃ-ase Gene Expression.
The cDNA sequence for human DT-diaphorase has recently
been published (31). The 3' region contains alternative poly-
corresponding to glutathione peroxidase mRNA was detected
in MCF-7/Dox cells (Fig. 2. lane 1). No corresponding band
was visualized, even after prolonged autoradiography, in the
parent line (Fig. 2, lane 2). Slot blot analysis suggested that the
enhancement of glutathione peroxidase mRNA expression in
MCF-7/Dox was greater than 40-fold (data not shown). Glutathione-dependent H2O2 peroxidase activity was detectable at
a very low level in the parent cells but was increased by more
than 25-fold in MCF-7/Dox (Table 1); f-butyl hydroperoxidase
activity was increased 50-fold in MCF-7/Dox. The glutathione
peroxidase gene from MCF-7 cells was examined by Southern
analysis. Digestion of high molecular weight DNA with Pstl
produced a single major band hybridizing with the glutathione
peroxidase probe at 15.2 kilobases and several minor bands
adenylation signals: three mRNA species of 1.2, 1.7, and 2.7
(Fig. 3, lanes 1 and 2). Pstl digest bands were identical in kilobases may be produced. The 1.2- and 2.7-kilobase species
mobility and intensity in the parent MCF-7 and MCF-7/Dox
were detected in MCF-7 cells; both bands were diminished
approximately 3-fold in MCF-7 Dox, as compared to the sen
lanes, demonstrating that there was no major gene amplifica
sitive cells (Fig. 4). Correspondingly, DT-diaphorase specific
tion to account for the increased glutathione peroxidase mRNA
in MCF-7/Dox. This was confirmed by digestion with Mspl,
activity was also reduced 4.2-fold in MCF-7/Dox (Table 1; P
which revealed a 1.1-kilobase band, a 0.35-kilobase band, and < 0.005).
multiple bands of <0.25 kilobases in both the parent line and
Daunorubicin reducÃ-asehas been considered to play an im
MCF-7/Dox (Fig. 3, lanes 3 and 4). The sequence for the portant part in anthracycline metabolism, because of its role in
human glutathione peroxidase gene has been elucidated (40). It reducing the C-9 side chain carbonyl to an alcohol. Daunorub
contains at least 16 Mspl recognition sites, cutting the gene icin reducÃ-aseactivily measured at pH 6.0 was present in equal,
into a 0.35-kilobase band and multiple bands of <0.15 kilo- albeil low, amounls in Ihe 12,000 x gsupernatants from MCFbases. The consistent presence of a 1.1-kilobase band in our
7 and MCF-7/Dox (Table 1). This was somewhal surprising.
1399
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1990 American Association for Cancer Research.
ANTIOXIDANT
ENZYME GENE EXPRESSION
IN DOXORIIBICIN
RESISTANCE
Table 1 Antioxidant and xenobiotic-metaholi:ing en:yme activities
Assays of enzyme activities were performed on 105,000 x g cell supcrnatants as described in "Materials and Methods." The values shown represent the mean ±
SD of 2-9 determinations. Statistical validity of the null hypothesis (no difference between mean enzyme activities of MCF-7 and MCF-7/Dox) was evaluated by the
r statistic.
peroxidase
(nmol NADPH/min/mg)r-Butyl
lineMCF-7
Cell
dismutasc
Gig/mg)0.42
u.niulH2O2/min/mg)4.8
hydroperoxide0.4
H2O2
±0.04
±1.40
±0.24
0.46 ±0.04"Catalase
2.0 ±0.40*Glutathione 10.4±2.8f
MCF-7/DoxSuperoxide
* Not significantly different than control (P > 0.15).
* Significantly different than control (P < 0.025).
' Significantly different than control (P < 0.005).
12
reducÃ-ase
(nmol
(nmol
NADPH/min/mg)509daunorubicinol/
min/mg)0.8
0.2 ±0.15
±117
±0.1
10.2±0.7fDT-diaphorase
117 ±4'Daunorubicin 0.9 ±0.1°
34
-1.4
1.1
0.9
0.6
0.3
0.2
Fig. 3. Autoradiograms of Southern blots of />s/I-restricted (lanes I and 2)
and A/i/>l-restricted (lanes 3 and 4) genomic DNA isolated from MCF-7 (oddnumbered lanes) and MCF-7/Dox (even-numberedlanes). Blots were probed with
a full-length glutathione pcroxidase cDNA probe as described in "Materials and
Methods."
Fig. 4. Autoradiograms of Northern blot of MCF-7 (lane I) and MCF-7/Dox
(lane 2) RNA probed with a 1.2-kilobase DT-diaphorase cDNA probe, as de
scribed in "Materials and Methods." Bands were observed at 1.2 and 2.7 kilobases.
toxicity of doxorubicin in human (MCF-7) and murine tumor
cells by addition of exogenous Superoxide dismutase, catalase,
and the synthetic low molecular weight glutathione peroxidase
congener PZ 51 has been reported by our laboratory (26, 41).
We have also found that selective augmentation of intracellular
glutathione peroxidase activity using mechanical methods to
introduce the purified enzyme into MCF-7 cells provided sig
nificant protection from doxorubicin toxicity (42). Sinha et al.
(10) have observed diminished hydroxyl radical production by
DISCUSSION
doxorubicin in a multidrug-resistant MCF-7 breast cancer cell
line related to the one used for our current experiments; they
Recent evidence suggests that antioxidant enzymes may exert
attributed this finding to augmented glutathione peroxidase
a protective effect against the cytotoxic activity of anticancer
quiñones in certain tumor cell lines. Protection against the activity, with a consequent presumed decrease in the level of
1400
since a 1.2-kilobase mRNA homologous to a cDNA whose
sequence is identical to that assigned by Wermuth et al. (32) to
human carbonyl reducÃ-asewas abundant in both MCF-7 and
MCF-7/Dox (Fig. 5). Similar to DT-diaphorase, expression of
this mRNA species was diminished 2.5-fold in MCF-7/Dox, as
compared to MCF-7.
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1990 American Association for Cancer Research.
ANTIOXIDANT
ENZYME GENE EXPRESSION
1 2
Fig. 5. Autoradiograms of Northern blot of MCF-7 (lane I) and MCF-7/Dox
(lane 2) RNA probed with a 0.9-kilobase carbonyl reducÃ-asecDNA probes, as
described in "Materials and Methods."
doxorubicin-related Fenton chemistry. Recently, Mimnaugh et
al. (11) reported that doxorubicin-resistant MCF-7 cells are
also resistant to oxyradical stress induced by xanthine/xanthine
oxidase or hyperoxia. They found a 12-fold increase in glutathione peroxidase activity associated with a 2-fold increase in
Superoxide dismutase activity in their MCF-7 cells that were
resistant to doxorubicin, which were descended from the origi
nal MCF-7/Dox line described by Batist and colleagues (8).
Our doxorubicin-resistant MCF-7 variant also exhibited a >25fold increase in glutathione peroxidase activity, slightly dimin
ished catatase activity, and a decreased non-protein reduced
thiol pool (data not shown), similar to that reported by Mim
naugh et al.; however, in contrast to the findings of Mimnaugh
et al., but similar to those of Sinha et al. (10), there was no upregulation of Superoxide dismutase activity. Possibly, the in
creased Superoxide dismutase activity observed by Mimnaugh
and associates represents a further evolution in antioxidant
enzyme gene expression not yet undergone by our cells.
Our data strongly suggest that the increased glutathione
peroxidase activity of MCF-7/Dox cells is due to overrepresentation of selenium-dependent glutathione peroxidase mRNA.
We did not detect glutathione peroxidase gene amplification,
however, suggesting that the augmented mRNA level was due
to either enhanced transcription, altered processing, or en
hanced stability of the mRNA. Our current data do not permit
us to distinguish among these possibilities. Studies in murine
IN DOXORUBICIN
RESISTANCE
erythrocyte-nonerythrocyte
hybrid cells suggest that glutathi
one peroxidase mRNA expression could be regulated by trans
acting elements, which regulate the appearance of DNase I
hypersensitive sites (43). It is also possible that multidrugresistant MCF-7 cells contain an alteration in transregulation.
Altered glutathione peroxidase gene expression does not ap
pear, however, to be a general phenomenon associated with the
multidrug-resistant phenotype. Several multidrug-resistant cell
lines do not exhibit increased glutathione peroxidase activity
(44).
Not only is antioxidant enzyme gene expression altered in
MCF-7/Dox, expression of DT-diaphorase is altered as well.
Our data are consistent with those of Ivy et al. (9), i.e., DTdiaphorase activity is diminished approximately 3-fold in MCF7/Dox. Furthermore, our results suggest that basal (uninduced)
transcription of the DT-diaphorase gene is down-regulated in
MCF-7/Dox. It is difficult to relate repressed DT-diaphorase
gene expression directly to doxorubicin resistance; however, it
is possible that DT-diaphorase down-regulation may be part of
the response of a group of genes which are coordinately regu
lated by xenobiotic insult. For example, DT-diaphorase expres
sion is inducible by activation of the Ah receptor; Ivy et al. (9,
45) have observed that Ah receptor activity is repressed in
MCF-7/Dox.
Daunorubicin reducÃ-ase converts the C9 carbonyl of both
daunorubicin and doxorubicin to their corresponding alcohols
(13-16). It has been proposed that diminished daunorubicin
reducÃ-aseactivily may be the cause of the relative insensilivily
of some human leukemic cells lo these anlhracyclines (17, 18).
However, daunorubicin reducÃ-aseaclivities at pH 6.0 in MCF7 and MCF-7/Dox were comparable, suggesling lhat altered
cellular metabolism is not an importanl componenl of doxo
rubicin insensitivity in MCF-7/Dox. The finding of low level
daunorubicin reducÃ-ase activity in MCF-7 and MCF-7/Dox
was somewhat surprising, since mRNA homologous lo carbonyl
reducÃ-asecDNA was quite abundant in both lines. It has been
proposed that carbonyl reducÃ-ase, a quinone reducÃ-ase, and
daunorubicin reducÃ-ase, an aldo-keto reducÃ-ase, are ideniical
enzymes (46). Our dala suggest thai ihe assignment of dauno
rubicin reducÃ-aseactivily lo carbonyl reducÃ-asemusi be consid
ered tentative. It is interesting that carbonyl reducÃ-aseexpres
sion is repressed in MCF-7/Dox, similar to DT-diaphorase.
Carbonyl reducÃ-asehas been assigned a quinone detoxification
role, similar to DT-diaphorase (46), and receñÃdala suggesl
coordinate regulation of carbonyl reducÃ-aseand DT-diaphorase
gene expression.5
In summary, MCF-7/Dox conlains alterations in the mRNA
levels of a variety of enzymes which may affect Ihe abilily of
doxorubicin lo damage cells. These include ihe imporlant per
oxide-disposal enzyme glutathione peroxidase, as well as xenobiotic-melabolizing enzymes including DT-diaphorase and
carbonyl reducÃ-ase.These data suggest thai doxorubicin resislance is complex and multifactorial and may originate, al leasl
in pari, at the RNA level.
ACKNOWLEDGMENTS
We would like to acknowledge the excellent technical assistance of
Florinda Matsumoto and Judith Stetzler. We also thank Sunny Aure
for her excellent secretarial assistance.
REFERENCES
1. Dimarco. A., and Arcamene, F. DNA-complexing antibodies: daunomycin.
Adriamycin and their derivatives. Arzneimittel-Forsch., 25: 368-375. 1975.
' Forrest et al., manuscript in preparation.
1401
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1990 American Association for Cancer Research.
ANTIOXIDANT
ENZYME GENE EXPRESSION
2. Bachur, N. R., Gordon, S. L., and Gee, M. V. A general mechanism for
microsomal activation of quinone anticancer agents to free radicals. Cancer
Res., 38: 1745-1750, 1978.
3. Tritton, T. R.. and Yee, G. The anticancer drug Adriamycin can be actively
cytotoxic without entering cells. Science (Wash. DC), 217: 248-250, 1982.
4. Muindi, J. R. F., Sinha, B. K.. Gianni, Z., and Myers, C. T. Hydroxyl radical
production and DNA damage induced by anthracycline-iron complex. FEBS
Lett., / 72: 226-230, 1984.
5. Tewey, K. M., Rowe, T. C., Young, L., Halligar, B. D.. and Liu, L. F.
Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II. Science (Wash. DC). 226: 466-468, 1984.
6. Cowan. K. H.. Batist, G., Tulpule, A., Sinha. B., and Myers, C. E. Similar
biochemical changes associated with multidrug resistance in human breast
cancer cells and carcinogen-induced resistance to xenobiotics in rats. Proc.
Nati. Acad. Sci. USA, 83: 9328-9332, 1986.
7. Fairchild, C. R., Ivy, S. P., Chien-Song, K. S.. Whang-Peng. J.. Rosen, N.,
Israel, M. A., Melera, P. W., Cowan, K. H., and Goldsmith, M. E. Isolation
of amplified and overexpressed DNA sequences from Adriamycin-resistant
human breast cancer cells. Cancer Res., 47: 5141-5148, 1987.
8. Batist, G.. Tulpule. A., Sinha. B. K., Katki, A.. Myers, C. E., and Cowan, K.
H. Overexpression of a novel anionic glutathione transferase in multidrugresistant human breast cancer cells. J. Biol. Chem., 261:15544-15549,1986.
9. Ivy, S. P.. Tulpule. A.. Fairchild. C. R., Auerbuch, S. D., Myers, C. E.,
Nebest, D. W., Baird, W. M., and Cowan, K. H. Altered regulation of P-450
IA1 expression in a multidrug-resistant MCF-7 human breast cancer cell
line. J. Biol. Chem., 263: 19119-19125, 1988.
10. Sinha, B. K.. Katki. A. G.. Batist, G., Cowan, K. H.. and Myers, C. E.
Differential formation of hydroxyl radicals by Adriamycin in sensitive and
resistant MCF-7 human breast tumor cells: implications for the mechanism
of action. Biochemistry, 26: 3776-3781, 1987.
11. Mimnaugh, E. G., Dusne, L., Atwell, J., and Myers, C. E. Differential oxygen
radical susceptibility of Adriamycin-sensitive and -resistant MCF-7 human
breast tumor cells. Cancer Res.. 49: 8-15. 1989.
12. Kramer. R. A., Zakher. J., and Kim. G. Role of the glutathione redox cycle
in acquired and de novo multidrug resistance. Science (Wash. DC), 241:694697, 1988.
13. Felsted, R. L., Gee, M., and Bachur. N. R. Rat liver daunorubicin reducÃ-ase:
an aldo-ketoreductase. J. Biol. Chem., 249: 3672-3679. 1974.
14. Bachur. N. R. Cytoplasmic aldo-ketoreductases: a class of drug metabolizing
enzymes. Science (Wash. DC). 193: 595-597. 1976.
15. Ahmed, N. K., Felsted, R. L., and Bachur. N. R. Heterogeneity of anthracycline antibiotic carbonyl reductases in mammalian livers. Biochem. Pharmacol.. 27: 2713-2719. 1978.
16. Ahmed, N. K., Felsted, R. L., and Bachur, N. R. Daunorubicin reduction
mediated by aldehyde and ketone reductases. Xenobiotica. //: 131-136,
1981.
17. Anbel-Sadion. G.. and Londos Gagliardi. D. Daunorubicin and doxorubicin.
anthracycline antibiotics, a physiochemical and biological review. Biochimie
(Paris). 66: 333-352. 1984.
18. Huffman. D. H., and Bachur, N. R. Daunorubicin metabolism in acute
myelocylic leukemia. Blood, 39: 637-643. 1972.
19. Greene, W., Huffmann, D., Wiernik, P. H.. Schimpft, S., Benjamin. R.. and
Bachur, N. R. High dose daunorubicin therapy for acute nonlymphoblastic
leukemia: correlation of response and toxicity with pharmacokinetics and
intracellular daunorubicin reducÃ-aseactivity. Cancer (Phila.). 30:1419-1427,
1972.
20. Gola, A. Daunorubicin reducÃ-aseactivity in leukemia leukocyle homogenales.
Arch. Immunol. Ther. Exp., 27: 815-818, 1979.
21. Vasanlhakumar. G.. and Ahmed. N. K. Uptake and metabolism of dauno
rubicin by human myelocytic cells. Cancer Chemolher. Pharmacol.. 15: 3539, 1985.
22. Goren, M. P., Ahmed, N. K.. and Tereba, A. Use of Somalie cell hybrids lo
analyze role of specific enzymes in daunorubicin cytoloxicily. Cancer Res.,
47: 1924-1929. 1987.
23. Lind. C., Hochstein. P., and Ernster, L. DT-diaphorase as a quinone reduc
Ã-ase:a cellular control device against semiquinone and Superoxide radical
formation. Arch. Biochem. Biophys., 216: 178-185, 1982.
IN DOXORUBICIN
RESISTANCE
24. Galaris, D., and Rydstrom. J. Enzyme induction by daunorubicin in neonatal
heart cells in culture. Biochem. Biophys. Res. Commun., 110: 364-370,
1983.
25. Wallin. R. Adriamycin and DT-diaphorase. Cancer Lett., 30: 92-101, 1986.
26. Doroshow, J. H. Prevention of doxorubicin-induced killing of MCF-7 human
breasl cancer cells by oxygen radical scavengers and iron chelating agents.
Biochem. Biophys. Res. Commun., 135: 330-335, 1986.
27. Sanger, F., Nicklus, S., and Coulsen, R. DNA sequencing with chainterminaling inhibilors. Proc. Nati. Acad. Sci. USA. 74: 5463-5467, 1977.
28. Groner, Y., Lieman-Hurwilz, J., Dafni, N., Bernstein, Y., Sherman, L.,
Levanon. D., Danciger. E.. and Elroy-Stein, O. The human Cu-Zn Superoxide
dismutase gene family: architecture and expression of the chromosome 21encoded functional gene and ils relaled processed pseudo genes. In: G. Rotulo
(ed.), Superoxide and Superoxide Dismutase in Chemistry, Biology and
Medicine, pp. 257-265. Amsterdam: Elsevier Publishers, 1986.
29. Quan, F., Korneluk, R. G., Tropak, M. B., and Gravel, R. A. Isolation and
characterization of the human catalase gene. Nucleic Acid Res., 14: 53255334, 1986.
30. Mullenbach. G. T., Tabrizi, A., Irvine, B. D., Bell, G. I., and Hallewell, R.
A. Sequence of a cDNA coding for human glutalhione peroxidase confirms
TGA encodes active site selenocysteine. Nucleic Acids Res., 15: 5484, 1987.
31. Jaiswal, A. K., McBride, O. W.. Adesnik, M., and Neben, D. W. Human
dioxin-inducible cylosolic NAD(P)H: menadione oxidoreduclase. J. Biol.
Chem., 263: 13572-13578, 1988.
32. Wermuth, B., Bohren, K. M., Heinemann, G., von Westburg, J. P., and
Gabbay, K. Human carbonyl reducÃ-ase.J. Biol. Chem., 263: 16185-16188,
1988.
33. Maniatis, J., Fritsch, E. F., and Sambrook, J. Molecular Cloning, A Laboralory Manual, p. 196. Cold Spring Harbor, NY': Cold Spring Harbor
Laboralory, 1982.
34. Beuiler, E. Caialase. In: E. Beutler (ed.). Red Cell Metabolism, A Manual of
Biochemical Melhods, pp. 89-90. New York: Academic Press, 1975.
35. Beuller, E. Glulathione peroxidase (GSH-Px). In: E. Beutler (ed.). Red Cell
Metabolism, A Manual of Biochemical Methods, pp. 71-73. New York:
Academic Press, 1975.
36. McCord, J. M., and Fridovich, I. Superoxide dismutase: an enzymatic
function for erylhrocupein. J. Biol. Chem., 244: 6049-6055, 1969.
37. Ernsler, L. DT-diaphorase. Melhods Enzymol., 10: 309-317, 1967.
38. Cusack, B. J.. Tesnohlidek, D. A., Loseke, V. L., Vestal, R. E., Brenner, D.
E.. and Olson, R. D. Effect of phenyloin on ihe pharmacokinelics of doxo
rubicin and doxorubicinol in ihe rabbil. Cancer Chemolher. Pharmacol., 22:
294-298, 1988.
39. Lowry, O. H., Rosebrough. N. J., Farr, A. L., and Randall, R. J. Prolein
measuremenl wilh ihe Folin Phenol reagenl. J. Biol. Chem., 193: 265-275,
1951.
40. Ishida. K., Morino, T., Takagi. K.. and Sukenaga. Y. Nucleolide sequence of
a human gene for glulalhione peroxidase. Nucleic Acids Res., 15: 10051,
1987.
41. Doroshow, J. H. Role of hydrogen peroxide and hydroxyl radical formalion
in the killing of Ehrlich lumor cells by anlicancer quiñones.Proc. Nail. Acad.
Sci. USA, W: 4514-4518, 1986.
42. Doroshow, J., Esworthy, S., Burke, T., Chu, F.-F., and Akman, S. Doxorub
icin resislance conferred by seleclive enhancemeni of intracellular glutalhione
peroxidase or Superoxide dismutase content in human MCF-7 breast cancer
cells. Proc. Am. Assoc. Cancer Res., 30: 496, 1989.
43. Affara, N., Fleming. J., Goldfarb, P. S.. Black, E.. Thiele, B., and Harrison.
P. R. Analysis of chromatin changes associated with ihe expression of globin
and non-globin genes in cell hybrids belween erylhroid and other cells.
Nucleic Acids Res.. 13: 5629-5644, 1985.
44. Ross, K. L., Akman, S. A.. Sikic, B. L., and Doroshow, J. H. Free radical
detoxifying enzymes in Adriamycin-resistanl cell lines. Proc. Am. Assoc.
Cancer Res., 29: 503, 1988.
45. Ivy, S. P., Baird, W. M., Myers, C. E., and Cowan, K. H. Inhibilion of TCDD
inducible cylochrome P1-450 expression in mulidrug resislanl MCF-7 cells
by a Ã-ranÃ--acling
repressor. Proc. Am. Assoc. Cancer Res., 29: 314, 1988.
46. Wermulh, B., Plans, K. L., Seidel. A., and Oesch, F. Carbonyl reducÃ-ase
provides the enzymalic basis of quinone deloxification in man. Biochem.
Pharmacol., 35: 1272-1282, 1986.
1402
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1990 American Association for Cancer Research.
Antioxidant and Xenobiotic-metabolizing Enzyme Gene
Expression in Doxorubicin-resistant MCF-7 Breast Cancer Cells
Steven A. Akman, Gerald Forrest, Fong-Fong Chu, et al.
Cancer Res 1990;50:1397-1402.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/50/5/1397
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1990 American Association for Cancer Research.