Taylor CW, Dalton WS, Parrish PR, Gleason MC, Bellamy WT, Thompson FH, Roe DJ, Trent JMDifferent mechanisms of decreased drug accumulation in doxorubicin and mitoxantrone resistant variants of the MCF7 human breast cancer cell line. Br J Cancer 63: 923-929

Abstract

We selected two drug resistant variants of the MCF7 human breast cancer cell line by chronic in vitro exposure to doxorubicin (MCF7/D40 cell line) and mitoxantrone (MCF7/Mitox cell line), respectively. The cell lines are similar in growth characteristics including doubling time, DNA synthetic phase and cell size. Resistance to mitoxantrone conferred only partial resistance to doxorubicin; whereas resistance selected for doxorubicin appeared to confer complete resistance to mitoxantrone. Both agents selected for cross resistance to the Vinca alkaloids. MCF7/D40 cells display a classic-multi-drug resistance phenotype with expression of P-glycoprotein, decreased drug accumulation relative to the parental line and reversal of drug accumulation and drug resistance by verapamil. MCF7/Mitox cells likewise display resistance to multiple drugs, but in contrast to MCF7/D40 cells do not express P-glycoprotein by immunoblot or RNA blot analysis. Net drug accumulation in MCF7/Mitox cells was decreased relative to the parental cells but there was no selective modulation of drug accumulation or in vitro drug resistance by the addition of verapamil. Efflux of mitoxantrone was enhanced in both the MCF7/D40 and MCF7/Mitox cell lines relative to the MCF7/S cell line. We conclude that the two drug resistant cell lines have different mechanisms of decreased drug accumulation.

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Figure 4

Figure 5

Full text

Br.
J.
Cancer
(1991),
63,
923
929
©
Macmillan
Press
Different
mechanisms
of
decreased
drug
accumulation
in
doxorubicin
and
mitoxantrone
resistant
variants
of
the
MCF7
human
breast
cancer
cell
line
C.W.
Taylor,
W.S.
Dalton,
P.R.
Parrish,
M.C.
Gleason,
W.T.
Bellamy,
F.H.
Thompson,
D.J.
Roe
&
J.M.
Trent
Section
of
Hematology/Oncology,
Department
of
Internal
Medicine,
Arizona
Cancer
Center,
University
of
Arizona,
Tucson,
Arizona
85724,
USA.
Summary
We
selected
two
drug
resistant
variants
of
the
MCF7
human
breast
cancer
cell
line
by
chronic
in
vitro
exposure
to
doxorubicin
(MCF7/D40
cell
line)
and
mitoxantrone
(MCF7/Mitox
cell line),
respectively.
The
cell
lines
are
similar
in
growth
characteristics
including
doubling
time,
DNA
synthetic
phase
and
cell
size.
Resistance
to
mitoxantrone
conferred
only
partial
resistance
to
doxorubicin;
whereas
resistance
selected
for
doxorubicin
appeared
to
confer
complete
resistance
to
mitoxantrone.
Both
agents
selected
for
cross
resistance
to
the
Vinca
alkaloids.
MCF7/D40
cells
display
a
classic-multi-drug
resistance
phenotype
with
expression
of
P-glycoprotein,
decreased
drug
accumulation
relative
to
the
parental
line
and
reversal
of
drug
accumulation
and
drug
resistance
by
verapamil.
MCF7/Mitox
cells
likewise
display
resistance
to
multiple
drugs,
but
in
contrast
to
MCF7/D40
cells
do
not
express
P-glycoprotein
by
immunoblot
or
RNA
blot
analysis.
Net
drug
accumulation
in
MCF7/Mitox
cells
was
decreased
relative
to
the
parental
cells
but
there
was
no
selective
modulation
of
drug
accumulation
or
in
vitro
drug
resistance
by
the
addition
of
verapamil.
Efflux
of
mitoxantrone
was
enhanced
in
both
the
MCF7/D40
and
MCF7/Mitox
cell
lines
relative
to
the
MCF7/S
cell
line.
We
conclude
that
the
two
drug
resistant
cell
lines
have
different
mechanisms
of
decreased
drug
accumulation.
Breast
cancer
is
responsive
to
a
wide
variety
of
single
and
combination
chemotherapy
regimens.
Unfortunately,
after
an
initial
response
to
chemotherapy
most
patients
with
metas-
tatic
breast
cancer
ultimately
develop
recurrences
(Dalton,
1990).
In
such
patients,
clinical
drug
resistance
(failure
to
respond
to
drugs
which
were
initially
effective)
is
a
common
phenomenon.
Clinical
drug
resistance
is
likely
due
to
a
number
of
factors
such
as
tumour
growth
kinetics,
develop-
ment
of
pharmacologic
sanctuaries
due
to
loss
of
vascular
supply,
development
of
hypoxia
in
large
tumours,
and
devel-
opment
of
structural
and
metabolic
changes
in
individual
tumour
cells.
Doxorubicin
(DOX)
is
the
most
active
single
agent
cur-
rently
available
for
the
treatment
of
breast
cancer
(Tormey,
1975).
For
previously
treated
and
untreated
patients
the
res-
ponse
to
DOX
as
a
single
agent
varies
between
28
and
43%
respectively.
In
addition,
DOX
is
an
integral
component
of
many
combination
chemotherapy
regimens
for
breast
cancer.
Mitoxantrone
(Mitox)
belongs
to
a
new
class
of
synthetic
chemotherapeutic
agents,
the
anthracenediones
(Alberts
et
al.,
1980).
Numerous
comparisons
have been
made
between
DOX
and
Mitox
in
that
both
compounds
possess
dihydroxy-
quinones
and
are
believed
to
intercalate
DNA
(Shenkenberg
&
Von
Hoff,
1986).
Mitox
is
active
as
a
single
agent
in
patients
with
breast
cancer
(Smyth
et
al.,
1984;
Yap
et
al.,
1981).
Individual
tumour
cells
may
develop
resistance
to
a
broad
range
of
structurally
unrelated
drugs
and
thus
display
the
multidrug
resistance
(MDR)
phenotype
(Gerlach
et
al.,
1986).
A
170,000
dalton
cell
membrane
protein,
termed
P-
glycoprotein
is
over-expressed
in
some
MDR
cells
and
func-
tions
as
a
drug
efflux-pump
(Gerlach
et
al.,
1986;
Pastan
&
Gottesman,
1987).
A
DOX-resistant
MCF7
breast
cancer
cell
line
(DOXR
MCF7)
was
previously
reported
and
shown
to
have
a
decreased
intracellular
drug
accumulation,
over-
expression
of
P-glycoprotein
and
over-expression
of
an
ani-
onic
form
of
the
enzyme
glutathione
transferase
(Batist
et
al.,
1986;
Cowan
et
al.,
1986).
Another
DOX-resistant
breast
cancer
cell
line
(MDA-ADR)
was
found
to
express
P-glyco-
protein
gene
sequences
(Fuqua
et
al.,
1987).
In
comparison
to
DOX
resistance,
little
has
been
reported
about
the
devel-
opment
and
mechanisms
of
Mitox
resistance
in
human
cancer
cell
lines.
Recent
reports,
however,
indicate
that
resis-
tance
to
Mitox
in
colon
cancer
(WiDr)
and
leukaemia
(HL-
60)
cell
lines
confers
a
unique
MDR
phenotype
unrelated
to
the
over-expression
of
P-glycoprotein
(Dalton
et
al.,
1988;
Harker
et
al.,
1989).
We
have
selected
DOX
and
Mitox
resistant
variants
of
the
MCF7
human
breast
cancer
cell
line
and
provide
evidence
that
these
two
drug
resistant
cell
lines
share
some
similarities
in
MDR
phenotype
and
both
have
decreased
drug
accumu-
lation
and
enhanced
drug
efflux.
The
decrease
in
drug
accumulation
appears
to
be
partially
responsible
for
the
MDR;
however,
the
mechanisms
of
decreased
drug
accumu-
lation
in
the
two
cell
lines
are
different.
Material
and
methods
Cell
culture
The
MCF7
parent
cell
line
was
obtained
from
the
American
Type
Culture
Collection
(ATCC,
Rockville,
MD).
Authen-
ticity
was
confirmed
by
cytogenetic
analysis.
The
MCF7
cells
were
adherent
to
plastic
and
grown
in
RPMI
1640
medium
supplemented
with
10%
(v/v)
foetal
bovine
serum,
1%
(v/v)
penicillin
(100
units
ml
1),
1%
(v/v)
streptomycin
(100
jig
ml-
'),
and
1%
(v/v)
L-glutamine
(GIBCO,
Grand
Island,
NY).
Cells
were
maintained
at
37°C
in
a
5%
C02-95%
air
atmosphere.
Drugs
DOX
was
obtained
from
Adria
Laboratories
(Columbus,
OH);
Vincristine
(VCR)
from
Eli
Lilly
Laboratories
(Indiana-
polis,
IN);
Verapamil
(VER)
from
Knoll
Pharmaceuticals
(Whippany,
NJ);
Mitox
from
Lederle/Cyanamid
(Pearl
River,
NY);
amsacrine
(m-AMSA)
from
Ben
Venue
Laboratories,
Inc.
(Bedford,
OH).
Vinblastine
(VBL)
and
5-fluoracil
(5-FU)
were
obtained
from
LyphoMed,
Inc.
(Rosemont,
IL).
Etopo-
Correspondence:
W.S.
Dalton,
University
of
Arizona,
Arizona
Can-
cer
Center,
Tucson,
Arizona
85724,
USA.
Received
1
August
1990;
and
in
revised
form
4
February
1991.
Br.
J.
Cancer
(1991),
63,
923-929
'."
Macmillan
Press
Ltd.,
1991

924
C.W.
TAYLOR
et
al.
side
(VP-16)
and
mitomycin-C
(MitoC)
were
obtained
from
Bristol
Laboratories
(Evansville,
IN).
Cisplatin
(CDDP),
mel-
phalan
(L-PAM)
and
gramacidin-D
(Gram-D)
were
obtained
from
Sigma
Chemicals
(St.
Louis,
MO).
3H
VCR
(specific
activity
of
6.2
Ci
mmole
',
6.72
mCi
mg-')
was
obtained
from
the
Amersham
Corp.
(Arlington
Heights,
IL);
"4C
DOX
(specific
activity
23.3
mCi
mmole
1,
40.2
yCi
mg-')
obtained
from
SRI
International
(Menlo
Park,
CA);
"4C
Mitox
(specific
activity
8.1
mCi
mmole
',
18.2
giCi
mg-')
obtained
from
Research
Triangle
Institute
(Research
Triangle
Park,
NC).
Selection
of
drug
resistant
cells
MCF7
parental
cells
were
exposed
to
DOX
or
Mitox,
each
at
an
initial
concentration
of
1
x
10-8M.
Fresh
drug
was
added
when
the
medium
was
changed
(approximately
three
times
weekly).
As
allowed
by
cell
growth,
the
concentration
of
each
drug
was
slowly
increased
in
a
multiple
step
procedure.
Over
a
period
of
19
months
the
concentration
of
DOX
was
in-
creased
from
1
x
10-8M
to 7
x
10-8M.
An
additional
12
months
were
required
to
reach
the
final
DOX
concentration
(4
x
10-7M,
representing
a
40-fold
increase)
and
full
develop-
ment
of
the
DOX
resistant
variant
(MCF7/D40)
for
a
total
selection
time
of
31
months.
The
Mitox
resistant
cell
line
(MCF7/Mitox)
was
selected
in
a
similar
fashion
by
increasing
the
Mitox
concentration
from
1
x
10-8M
to
8
x
10-8M
over
a
6
month
period.and
maintaining
this
concentration
over
the
next
18
months.
Prior
to
any
experiments,
cells
were
main-
tained
in
drug-free
medium
for
I
week.
In
vitro
drug
assays
Cytotoxicity
was
determined
using
a
modified
MTT
(3-(4,5-
dimethylthlazol-2-yl)-2,5-diphenyl
tetrazolium)
dye
assay
(Carmichael
et
al.,
1987;
Denizot
&
Lang,
1986).
This
is
a
colorimetric
assay
based
on
the
ability
of
viable
cells
to
reduce
MTT
to
a
blue
formazan
product.
In
dose
response
curves
the
data
are
presented
as
the
per
cent
of
an
optical
density
value
obtained
for
untreated
cells.
Cells
were
plated
into
96-well
microtiter
plates
(Falcon,
Becton
Dickinson
and
Company,
Oxnard,
CA)
at
1
x
104
cells/well
in
0.2
ml
of
media
containing
appropriate
concentration
of
drug
with
replicates
of
six
(standard
deviations
were
within
±
10%
of
the
mean
values)
VER
(6
,g
ml-',
13.2
pM)
was
added
15
min
prior
to
drug
and
was
present
continuously
during
the
period
of
incubation
with
drug.
After
incubation
for
4
days
at
37°C,
50
1l
of
MTT
dye
(2
mg
ml-')
was
added
to
each
well
and
incubated
for
4
h.
Plates
were
then
centrifuged
at
500
g
for
5
min,
media
aspirated,
dimethyl
sulfoxide
(DMSO)
added
to
each
well
(100
tlI),
plates
mechanically
agitated
for
5
min
and
optical
density
at
570
nm
determined
on
a
microplate
reader
(Dynatech
Labs,
Alexandria,
VA).
Each
experiment
was
repeated
a
minimum
of
three
times
and
data
from
a
representative
experiment
are
shown.
A
clonogenic
assay
(Malinin
&
Perry,
1967)
was
used
to
confirm
results
obtained
with
the
MTT
assay
for
Mitox
with
and
without
VER.
The
concentration
of
drug
which
pro-
duced
a
50%
inhibition
of
cloning
efficiency
(ICs)
was
cal-
culated
by
linear
regression
analysis
of
the
linear
portion
of
the
dose
response
curves.
Cell
growth
characteristics
For
the
determination
of
doubling
time,
cell
growth
curves
were
established
for
each
cell
line
by
plotting
cell
number
versus
growth
time.
Doubling
time
was
determined
directly
from
the
linear
portion
of
the
plot.
The
fraction
of
cells
in
S-phase
and
relative
cell
size
were
determined
using
flow
cytometry
(FACStar
Flow
Cytometer,
Becton
Dickinson)
after
propidium
iodide
staining
(Krishan,
1975;
Deitch
et
al.,
1982).
Actual
tumour
cell
diameters
were
determined
by
direct
measurements
of
100
tumour
cells
per
cell
line
using
an
inverted
microscope
equipped
with
a
reticle
and
an
internal
standard.
The
results
are
expressed
as
the
mean
cell
diameter
and
the
standard
deviation.
Chromosome
analysis
Cultures
were
harvested
for
karyotypic
analysis,
slides
pre-
pared
and
G-banding
performed
as
previously
described
(Trent
&
Thompson,
1987).
A
minimum
of
30
cells
per
cell
line
were
analysed,
with
results
expressed
according
to
ISCN
recommendations
(Harden
&
Klinger,
1985).
Karyotypic
in-
formation
on
the
parental
MCF7
cell
line
has
been
presented
previously
(Osborn
et
al.,
1987).
Drug
accumulation
Cellular
accumulation
of
drug
was
determined
during
a
1
h
exposure
of
an
aliquot
of
cells
(1
x
106
cells
plated
2
days
previously
into
a
35
mm2
petri
dish)
to
"'C
DOX-5.0
tM,
"1C
Mitox-10.0
1M,
and
3H
VCR-1.G
gM
at
37°C.
These
drug
concentrations
were
chosen
based
on
the
relative
specific
activities
of
the
three
radiolabelled
agents
in
an
attempt
to
achieve
similar
counts
per
minute
(c.p.m.)
in
MCF7/S
cells.
One
hour
after
the
addition
of
radiolabelled
drug,
the
cells
were
washed
twice
with
ice
cold
phosphate
buffered
saline
(PBS)
trypsinised,
incubated
with
0.2
N
NaOH
for
2
h
and
neutralised
with
0.2
N
HCI
(equivalent
volumes).
The
amount
of
radiolabel
(c.p.m.)
was
determined
using
liquid
scintilla-
tion
counting.
Each
experiment
was
repeated
a
minimum
of
three
times
and
data
from
a
representative
experiment
are
shown.
For
all
experiments
involving
VER,
cells
were
incubated
in
media
containing
6
gg
ml-'
(13.2
jLM)
VER
for
15
min
prior
to
drug
exposure
and
during
the
1
h
drug
exposure.
Mitoxantrone
efflux
studies
Cells
were
incubated
with
"'C
Mitox
for
1
h
as
described
in
the
Drug
Accumulation
section.
At
the
end
of
1
h,
cells
were
placed
on
ice
and
washed
once
with
ice
cold
PBS;
drug-free
RPMI
complete
media
(2%
foetal
bovine
serum)
was
added,
and
cells
were
incubated
at
37°C
for
up
to
60
min.
At
the
end
of
each
time
period,
cells
were
washed,
trypsinised,
digested,
and
counted
as
described
previously.
Efflux
at
each
time
point
was
determined
by:
c.p.m.
"'C
Mitox
c.p.m.
"'C
Mitox
at
time
0
Each
experiment
was
repeated
a
minimum
of
three
times
and
data
from
a
representative
experiment
are
shown.
Non-protein
sulJhydryl
(NPSH)
measurements
The
NPSH
content
of
the
MCF7
cell
lines
was
measured
using
the
method
of
Sedlak
and
Lindsay
(1968).
A
total
of
5
x
10'
cells
was
washed
twice
with
iced
PBS
(pH
7.4)
and
transfered
to
a
microcentrifuge
tube
where
they
were
lysed
by
sonication
(model
250
Branson
sonifier,
Danbury,
CT).
Cel-
lular
protein
was
precipitated
by
addition
of
5%
sulfosal-
icylic
acid.
The
cell
lysate
was
then
centrifuged
at
12,000g
for
5
min
at
room
temperature
and
a
1
ml
aliquot
of
the
supernatant
was
transferred
to
a
tube
containing
0.2
M
Tris
buffer
(pH
8.9).
To
each
tube,
100
ftl
of
0.01
N
5,5'-dith-
iobis(2-nitrobenzoic
acid)
in
absolute
methanol
was
added.
The
contents
were
mixed
and
the
absorbance
of
each
sample
was
measured
at
412
nm.
The
concentration
of
NPSH
in
the
sample
was
determined
by
comparing
the
optical
density
reading
of
the
sample
to
a
standard
curve
constructed
using
reduced
glutathione.
NPSH
levels
were
analysed
4
days
post
cell
passage
in
all
lines.
Protein
measurements
were
deter-
mined
according
to
the
method
of
Lowry
et
al.
(1951).
Immunoblot
analysis
for
P-glycoprotein
Plasma
membranes
were
purified
from
2
x
108
cells
(Riordan
&
Ling,
1979).
Polyacrylamide
gel
electrophoresis
(50
fig
pro-
tein
per
lane)
was
performed
according
to
the
method
of
Laemmli
(1970).
Protein
was
transferred
from
sodium
dode-

DRUG
ACCUMULATION
IN
RESISTANT
MCF7
BREAST
CANCER
CELLS
925
cyl
sulphate-polyacrylamide
gels
onto
nitrocellulose
filter
paper
according
to
the
method
of
Towbin
et
al.
(1979).
The
nitrocellulose
filters
were
then
incubated
overnight
at
room
temperature
in
tris-PBS
(TPBS)
containing
the
C219
mono-
clonal
antibody
(IgG)
(kindly
provided
by
Dr
Victor
Ling,
Ontario
Cancer
Institute,
Toronto,
Ontario,
Canada)
at
a
concentration
of
10
ng
ml-'
(Kartner
et
al.,
1985).
Following
wash
steps
to
remove
unbound
antibody,
the
filters
were
incubated
overnight
at
room
temperature
in
50
ml
of
TPBS
containing
25
fig
of
1231-labelled
rabbit
anti-mouse
IgG
(spec-
ific
activity
600
ACi
ml-',
New
England
Nuclear,
Boston,
MA).
The
filters
were
again
washed
to
remove
the
unbound
secondary
antibody,
dried,
and
exposed
to
X-Omat
AR
film
(Kodak).
A
membrane
preparation
from
8226/DOX40
multi-
ple
myeloma
cells
was
added
as
a
positive
control
for
P-
glycoprotein
(Dalton
et
al.,
1986).
RNA
analysis
RNA
was
isolated
from
MCF7
cells
using
guanidium
isothio-
cynate
and
cesium
chloride
centrifugation
(Maniatis
et
al.,
1982).
Slot
blot
analysis
of
total
cellular
RNA
was
carried
out
as
described
(Davis
et
al.,
1986)
using
a
640
bp
cDNA
fragment
of
mdr-1,
p-CHP-1,
isolated
from
a
colchicine-
resistant
CHO
cDNA
library
(Riordan
et
al.,
1985).
The
probe
was
oligolabelled
according
to
the
method
of
Feinberg
and
Vogelstein
(1983).
Human
multiple
myeloma
cells
dis-
playing
the
multidrug
resistant
phenotype
(8226/DOX40)
were
included
as
positive
controls
and
the
drug
sensitive
parent
cells
(8226/S)
as
negative
controls.
The
blots
were
also
probed
with
32P-labelled
human
tubulin
cDNA
(American
Type
Culture
Collection,
Rockville,
MD)
to
confirm
the
amount
of
RNA
contained
in
each
sample.
Statistical
methods
The
P-values
for
the
comparison
of
drug
accumulation
with
and
without
VER
were
calculated
using
a
two-sample
inde-
pendent
t-test.
The
P-values
for
the
difference
in
drug
accumulation
between
cell
lines
were
calculated
using
Tukey's
Studentised
Range
Test
and
the
Bonferroni
multiple
com-
parisons
procedure
(Miller,
1981).
The
95%
confidence
limits
of
the
doubling
times
were
determined
from
the
slope
of
the
linear
portion
of
the
growth
curves
(Mood
et
al.,
1974).
The
slopes
of
the
Mitox
efflux
curves
were
evaluated
by
Analysis
of
Covariance
(Snedecor
&
Cochran,
1980).
Results
Growth
characteristics
The
growth
characteristics
of
the
sensitive,
parental
line
(MCF7/S),
DOX
resistant
variant
(MCF7/D40)
and
Mitox
resistant
variant
(MCF7/Mitox)
are
listed
in
Table
I.
The
doubling
times
for
MCF7/S,
MCF7/D40
and
MCF7/Mitox
cells
were
similar.
No
differences
between
the
fraction
of
cells
in
S-phase
were
demonstrated
between
the
three
cell
lines.
The
mean
cell
diameter
varied
slightly
among
the
three
cell
lines,
but
the
confidence
intervals
(standard
deviation)
were
overlapping.
Dose
response
curves
Dose
response
curves
for
MCF7/S
and
MCF7/D40
cells
after
exposure
to
DOX
(with
and
without
VER)
as
determined
by
the
MTT
assay
are
depicted
in
Figure
la.
Similar
curves
for
MCF7/Mitox
cells
after
exposure
to
Mitox
(with
and
without
VER)
are
depicted
in
Figure
lb.
Significant
resistance
to
DOX
and
Mitox
was
observed
for
both
MCF7/D40
and
MCF7/Mitox
cells,
respectively.
However,
the
addition
of
6
C5
4-a
cJ
0
L-
100
90
80
70
60
50
40
30
20
10
a
).001
0.01
0.1
1.0
10
100
Micromolar
concentration
of
doxorubicin
b
100
90
80
70-
60
50-
40-
30
20-
10
0
0.001
0.01
0.1
1.0
10
100
Micromolar
concentration
of
mitoxantrone
Figure
1
Dose
response
of
MCF7/S
(0)
and
MCF7/D40
(-)
cells
to
DOX
alone
(close
symbols)
and
in
combination
with
VER
(open
symbols)
(VER-61igml',
13.2iLM,
MTT
assay)
(a).
Dose
response
of
MCF7/S
(0)
and
MCF7/Mitox
(U)
cells
to
Mitox
alone
(closed
symbols)
and
in
combination
with
VER
(open
symbols)
(VER-6jLgml-',
13.2I1M,
MTT
assay)
(b).
The
data
are
presented
as
the
per
cent
of
control
optical
density
(o.d.).
Table
I
Growth
characteristics
Cell
line
Characteristic
MCF7/S
MCF7/D40
MCF7/Mitox
Doubling
time,
hours
41.6
35.2
40.3
(95%
confidence
(32.1-59.0)
(25.5-56.8)
(35.0-47.6)
limits)
aFraction
of
cells
in
38%
38%
31%
S-phase
bRelative
cell
1.00
1.09
1.00
size
Mean
cell
diameter
in
18.5
21.0
17.5
microns
(SD)
(3.5)
(6.1)
(3.0)
aDetermined
using
propidium
iodide
and
flow
cytometry.
bDetermined
using
flow
cytometry,
MCF7/S
normalised
to
1.00.

926
C.W.
TAYLOR
et
al.
VER
affected
resistance
only
for
the
MCF7/D40
cell
line.
VER
alone
was
minimally
toxic
to
the
cells
(data
not
shown).
The
50%
inhibitory
concentration
(IC50)
of
DOX
(with
and
without
VER)
against
MCF7/D40
cells
and
the
IC50
of
Mitox
(with
and
without
VER)
against
MCF7/Mitox
cells
are
shown
in
Table
II.
With
the
addition
of
VER
the
IC50
of
DOX
against
MCF7/D40
cells
decreased
10-fold
(30
uM
vs
3.1
SAM).
In
contrast,
the
IC50
of
Mitox
against
MCF7/Mitox
cells
was
essentially
unchanged
with
VER
exposure
(64
tLM
vs
91
pM).
The
experiments
performed
with
the
MTT
assay
were
repeated
using
a
clonogenic
assay
(Malinin
&
Perry,
1967)
for
Mitox
with
and
without
VER
in
all
three
cell
lines
(Table
II).
The
IC50s
from
the
clonogenic
assays
were
gen-
erally
lower
than
those
obtained
from
the
MTT
assays.
However,
the
relative
degree
of
resistance
between
the
cell
lines
was
similar.
In
addition,
the
IC50
from
the
clonogenic
assay
for
Mitox
against
MCF7/Mitox
cells
remains
un-
changed
with
VER
exposure
(7.7
jiM
vs
7.4
jiM).
Cross
resistance
patterns
Table
II
demonstrates
the
sensitivities
of
the
3
MCF7
cell
lines
to
a
number
of
chemotherapeutic
agents.
The
data
are
expressed
as
the
IC50
and
as
the
relative
amount
of
resistance
of
MCF7/D40
and
MCF7/Mitox
cells
to
MCF7/S
cells
(IC50
ratio).
A
high
degree
(75
fold)
of
resistance
to
DOX
was
observed
in
MCF7/D40
cells.
In
addition,
MCF7/D40
cells
were
cross
resistant
to
a
number
of
other
drugs:
VCR
(190
fold),
Mitox
(153
fold),
VLB
(93
fold),
m-AMSA
(41
fold).
The
addition
of
VER
partially
reversed
the
resistance
of
MCF7/D40
cells
to
DOX,
VCR
and
Mitox.
MCF7/Mitox
cells
displayed
a
very
high
degree
of
resis-
Table
II
Cross
resistance
patterns
IC50a,
AM
(IC50ratiob)
Drug
MCF7/S
MCF7/D40
MCF7/Mitox
DOXC
0.4
30
3.3
(75)
(8.3)
DOX
+
VER
0.37
3.1
3.5
(8.4)
(9.5)
VCR
0.0039
0.74
0.086
(190)
(22)
VCR
+
VER
0.0031
0.065
0.16
(21)
(52)
Mitox
0.053
8.1
64
(153)
(1208)
[O.01
1
]d
[0.14]
[7.7]
Mitox
+
VER
0.053
1.2
91
(23)
(1717)
[0.0071]
[0.034]
[7.4]
VLB
0.0070
0.65
0.30
(93)
(43)
CDDP
1.3
628
(4.6)
(21.5)
VP-16
2.9
33
76
(11)
(26)
m-AMSA
0.86
35
36
(41)
(42)
L-PAM
16
40
91.4
(2.5)
(5.7)
MitoC
0.72
5.3
6.3
(7.4)
(8.8)
Gram
D
0.15
gml-'
1.2
jgml-'
0.98
1gml-'
(8)
(6.5)
5FU
30
103 117
(3.4)
(3.9)
'50%
Inhibitory
Concentration,
as
determined
by
MTT
assay;
bRatio
of
IC50
for
the
drug
resistant
cell
line
to
MCF7/S;
cAbbreviations:
DOX
=
Doxorubicin
VP-16
=
Etoposide
VCR
=
Vincristine
m-AMSA
=
Amsacrine
VER
=
Verapamil
L-PAM
=
Melphalan
Mitox
=
Mitoxantrone
MitoC
=
Mictomycin
C
VLB
=
Vinblastine
Gram
D
=
Gramacidin
D
CDDP
=
Cisplatinum
SFU
=
5-Fluorouracil;
d[]
=
IC50
as
determined
by
clonogenic
assay.
tance
to
Mitox
(1208
fold)
which
was
not
reversed
by
VER.
A
lesser
degree
of
cross
resistance
to
DOX
(8.3
fold)
and
VCR
(22
fold)
was
observed.
VER
was
ineffective
in
revers-
ing
resistance
to
both
DOX
and
VCR
in
MCF7/Mitox
cells.
The
MCF7/Mitox
cell
line
was
cross
resistant
to
VLB
(43
fold),
m-AMSA
(42
fold)
VP-16
(26
fold)
and
CDDP
(21.5
fold).
Only
low
levels
of
resistance
to
L-PAM,
MitoC,
Gram
D
or
5FU
were
seen
in
either
MCF7/D40
or
MCF7/Mitox
cells.
Drug
accumulation
studies
The
1
h
net
intracellular
accumlations
of
14C
DOX,
3H
VCR
and
'4C
Mitox
are
displayed
in
Figure
2.
In
both
the
MCF7/
D40
and
MCF7/Mitox
cell
line,
accumulation
of
14C
DOX
(Figure
2a),
3H
VCR
(Figure
2b)
and
"4C
Mitox
(Figure
2c)
was
decreased
relative
to
the
MCF7/S
cell
line.
The
MCF7/
Mitox
cell
line
had
less
net
intracellular
drug
accumulation
for
DOX
and
Mitox
compared
to
the
MCF7/D40
cell
line
(P<0.05
for
DOX
and
Mitox)
and
the
MCF7/S
parent
cell
line
(P<0.05
for
DOX
and
Mitox).
Both
resistant
cell
lines
had
decreased
VCR
net
intracellular
accumulation
relative
to
MCF7/S
(P<0.05
for
MCF7/D40
and
MCF7/Mitox)
but
were
not
different
from
each
other
(P>0.05).
The
effects
of
VER
in
increasing
DOX
and
VCR
intracel-
lular
drug
accumulation
were
limited
to
the
MCF7/D40
resis-
tant
cell
line
(41%
and
298%
increased,
respectively)
with
no
effect
observed
in
the
MCF7/S
or
MCF7/Mitox
cell
lines.
A
slight
but
significant
increase
in
Mitox
accumulation
was
seen
in
both
the
MCF7/S
(25%)
and
MCF7/Mitox
(24%)
cell
lines
with
a
greater
increase
seen
in
the
MCF7/D40
cell
line
(63%).
Curves
of
Mitox
efflux
versus
time
in
minutes
for
MCF7/S,
MCF7/D40
and
MCF7/Mitox
cells
were
depicted
in
Figure
3.
At
each
time
point
tested,
relatively
less
drug
persisted
in
MCF7/Mitox
and
MCF7/D40
cells
as
compared
with
MCF7/S
cells.
At
the
5min
time
point
MCF7/D40
and
MCF7/Mitox
cells
contained
34%
(2149
c.p.m.)
and
24%
a
p
<
0.0001
nl
DOX
U
DOX
+
VER
p
=
0.10
U1)
C.)
tD
0
x
a)
Q.
05
UL
b
5000
-
4000
-
3000
-
2000
-
1000
L
0
p
=
0.12
p
<
O.OOC
VCR
*
VCR
+
VER
p
=
0.31
-n
I
I
<
0.0001
p
<
0.0001
MCF7/S
MCF7/D40
[:
Mitox
*
Mitox
+
VER
p
=
0.0045
MCF7/Mitox
Figure
2
One
hour
net
accumulation
of
"1C
DOX
(5.0
pM),
3H
VCR
(1.0
IM)
and
1'C
Mitox
(10.0
JM)
in
MCF7/S,
MCF7/D40
and
MCF7/Mitox
cells
with
(closed
bars)
and
without
(open
bars)
VER
(6
jig/ml-
'),
13.2
1AM).
Error
bars
indicate
the
standard
error
of
the
mean
with
replicates
of
three.
P-values
for
the
effect
of
VER
are
indicated
above
the
respective
open
and
closed
bars.
The
decreased
accumulation
of
each
of
the
three
drugs
alone
in
MCF7/D40
and
MCF7/Mitox
cells
relative
to
MCF7/S
cells
were
statistically
significant
(P<0.05
in
each
instance).

DRUG
ACCUMULATION
IN
RESISTANT
MCF7
BREAST
CANCER
CELLS
927
"
MCF7/S
*-v
MCF7/MITOX
A--RArP7/IrAn
kD
0
x
0
0
O
CD
gzs
Q
co
200-
0
10
20
30
Time
40
s0
60
Figure
3
Efflux
of
Mitox
in
MCF7/S
(0),
MCF7/D40
(A) and
MCF7/Mitox
(v)
cells
versus
time
(minutes).
Data
points
are
expressed
as
per
cent
of
14C
Mitox
present
at
time
0.
Error
bars
indicate
the
standard
error
of
the
mean
with
replicates
of
three.
(1526
c.p.m.)
respectively,
of
the
amount
of
drug
contained
in
MCF7/S
cells
(6299
c.p.m.).
These
values
were
statistically
significant
at
P
<0.05
for
MCF7/S
cells
compared
to
both
MCF7/D40
and
MCF7/Mitox
cells.
Furthermore,
in
MCF7/
Mitox
and
MCF7/D40
cells
drug
efflux
was
more
rapide
during
the
initial
5
min
as
compared
to
MCF7/S
cells.
Statis-
tical
comparison
of
the
slopes
of
the
efflux
curves
during
this
time
period
revealed
P
=
0.055
and
0.0063
for
MCF7/S
cells
compared
to
MCF7/D40
and
MCF7/Mitox
cells,
respec-
tively.
At
time
points
beyond
10
min
the
slopes
of
the
efflux
curves
were
not
significantly
different.
P-glycoprotein
detection
An
immunoblot
probed
for
the
presence
of
P-glycoprotein
using
the
C219
antibody
is
seen
in
Figure
4.
The
8226/
DOX40
cell
line
was
included
as
a
positive
control
for
P-
glycoprotein
(Dalton
et
al.,
1986).
P-glycoprotein
was
detec-
ted
in
the
MCF7/D40
cell
line
but
was
not
seen
in
the
MCF7/S
and
MCF7/Mitox
cell
lines.
RNA
analysis
Slot
blot
analysis
revealed
no
evidence
of
mdr-1
message
in
the
MCF7/S
or
MCF7/Mitox
cell
lines
but
was
found
to
be
expressed
in
the
MCF7/D40
line
thus
confirming
our
findings
using
western
blotting
(Figure
5).
Thus,
selection
with
the
anthracycline
DOX
resulted
in
an
expression
of
the
P-
glycoprotein
message
while
selection
with
the
antracenedione
mitoxantrone
did
not.
97.4-
68-
Figure
4
Immunoblot
for
the
presence
of
P-glycoprotein
(C219
antibody)
in
MCF7/S,
MCF7/Mitox
and
MCF7/D40
cells
(8226/
DOX40
cell
line
-
positive
control).
Cytogenetic
studies
Chromosomal
analysis
of
the
MCF7
parent
cell
line
and
its
DOX
and
Mitox
resistant
sublines
were
performed
using
G-banding
analysis.
Detailed
cytogenetic
analysis
of
the
parental
line
has
been
published
previously
(Osborne
et
al.,
1987).
There
were
obvious
similarities
between
all
three
cell
lines
clearly
indicating
their
common
origin.
All
three
cell
lines
demonstrated
a
near-triploid
chromosome
number
(72-80)
with
numerous
structural
and
numeric
alterations
(Table
III).
The
MCF7/D40
cell
line
deviated
the
most
significantly
from
the
parental
cell
line
(primarily
by
the
addition
of
new
marker
chromosomes).
The
MCF7/Mitox
cell
line
more
closely
resembled
the
parental
line
with
the
only
differences
being
a
loss
of
the
marker
chromosomes
lp-,
6q-,
7pHSR,
13q+,
16q+
and
the
addition
of
one
marker
unique
to
this
cell
line
(3p+).
The
parental
cell
line
(which
is
known
to
be
amplified
for
N-ras)
(Graham
et
al.,
1985)
displayed
cytologic
evidence
of
gene
amplification
in
the
form
of
a
homogeneous
staining
region.
This
marker
was
lost
in
both
resistant
sublines
and
neither
displayed
double
minutes
or
other
HSRs.
8226/S
8226/DOX40
MCF7/S
MCF7/D40
MCF7/MITOX
Figure
5
Slot
blot
analysis
of
MCF7
RNA.
Serial
two-fold
dilutions
of
total
cellular
RNA
(10,
5,
2.5,
1.25,
0.6,
0.3
fig,
left
to
right)
were
applied
to
the
filter.
After
hybridisation
with
the
p-CHP-l
cDNA
probe,
the
filter
was
exposed
for
24
h
with
an
intensifying
screen
at
-
80C.
180
-
160
-
140
-
8
100~
a,
80-
0
cJ
a60-
40
-
20
-
)
P-gly
fir-'&
IVILIr//L)4u

928
C.W.
TAYLOR
et
al.
Table
III
Cytogenetic
analysis
of
MCF7
parental
and
resistant
sublines
Marker
MCF7/S
MCF7/D40
MCF7/Mitox
lp-
+
2q+
+
+
der(2)
+
2q-
+
3p-
+
+
+
3p
+
+
5p
+
+
+
+
6p+
+
6q+
++
+
6q-
+
+
7p+
+
+
7pHSR
+
inv(7)
+
+
+
9p+
+
12q-
+
+
+
13q+
+
+
16q+
+
17q+
+
t(l2;19)
+
+
+
t(22;22)
+
Xq-
+
+
Xq+
+
t(X;?)
+
Non-protein
sulfhydryl
(NPSH)
measurements
The
MCF7/S
cell
line
was
found
to
have
significantly
ele-
vated
amounts
of
NPSH
compared
to
the
other
two
cell
lines
(25.43
±
0.34
nmol
10-6
cells;
P<0.001;
Student's
t-test).
NPSH
levels
in
MCF7/D40
and
MCF7/Mitox
cells
were
decreased
at
16.23
±
0.28
and
15.83
±
0.21
nmol
10-6
cells
respectively
(not
significant,
Student's
t-test).
Total
protein
measurements
revealed
no
differences
among
the
three
cell
lines
(data
not
shown).
Discussion
In
this
report
we
describe
the
development
of
two
multidrug-
resistant
cell
lines
established
from
a
common
parental
cell
line
using
DOX
and Mitox
as
selecting
agents.
Of
note
is
the
fact
that
Mitox
resistance
developed
much
quicker
(6
months)
than
DOX
resistance
(greater
than
2
years).
In
establishing
the
drug
resistant
variants
the
concentrations
of
the
selecting
agents
were
increased
as
rapidly
as
allowed
by
cell
growth.
Indeed,
the
concentration
of
DOX
could
be
increased
only
very
slowly
in
the
selection
of
the
MCF7/
DOX
cell
line.
However,
in
spite
of
a
different
degree
of
selection
pressure
for
the
MCF7/Mitox
cell
line
(lower
final
Mitox
concentration,
shorter
selection
time)
relative
to
the
MCF7/DOX
cell
line,
a
greater
degree
of
Mitox
resistance
(1208
fold
vs
75
fold)
developed.
Formal
fluctuation
analysis
tests
were
not
performed
and
the
rate
of
mutation
to
resis-
tance
for
the
individual
drugs
cannot
be
stated.
The
DOX
resistant
(MCF7/D40)
and
Mitox
resistant
(MCF7/Mitox)
cell
lines
have
both
similarities
and
differ-
ences
in
cross
resistance
to
other
agents.
A
broad
range
of
drugs
with
varied
mechanisms
of
action
were
studied:
DNA
binding/intercalating
agents
(DOX,
Mitox),
tubulin
binding
agents
(VCR,
VLB),
alkylating
agents
(L-PAM,
CDDP,
Mito-C)
topoisomerase
II
inhibitors
(DOX,
Mitox,
VP-16,
m-AMSA),
an
antimetabolite
(5
FU)
and
a
cell
membrane
ionophore
(Gram-D).
In
addition
to
being
resistant
to
DOX,
MCF7/D40
cells
were
highly
resistant
to
the
tubulin
binding
agents,
moderately
resistant
to
the
topoisomerase
agents
and
displayed
lower
levels
of
resistance
to
the alkylating
agents
and
the
antimetabolite
5
FU.
Cross-resistance
to
Mitox
was
also
high
in
the
MCF7/D40
cell
line.
The
MCF7/Mitox
cell
line
displayed
a
number
of
unique
characteristics.
It
devel-
oped
a
very
high
degree
of
resistance
(1208
fold)
to
the
primary
selecting
agent
(Mitox)
with
relatively
minor
cross
resistance
to
DOX
(8.3
fold)
and
VCR
(22
fold).
In
addition,
MCF7/Mitox
cells
were
partially
cross
resistant
to
CDDP,
an
agent
not
commonly
associated
with
the
multidrug-resistance
phenotype
(21.5
fold
resistant).
Other
Mitox
resistant
cell
lines
have
been
reported
and
are
similar
to
the
Mitox
resistant
MCF7
cell
line
in
that
P-
glycoprotein
is
not
over-expressed.
Wallace
et
al.
first
repor-
ted
a
human
colon
carcinoma
cell
line
selected
for
resistance
to
Mitox
(WiDr)
(Wallace
et
al.,
1987).
This
line
was
further
characterised
by
Dalton
et
al.
demonstrating
decreased
in-
tracellular
accumulation
of
both
Mitox
and
DOX
with
no
increased
expression
of
P-glycoprotein
(Dalton
et
al.,
1988).
Harker
et
al.
recently
reported
an
HL-60
leukaemic
cell
line
selected
for
resistance
to
Mitox
which
also
lacked
P-glyco-
protein
overexpression
(Harker
et
al.,
1989).
Marsh
et
al.
reported
a
multi-drug
resistant
HL-60
cell
line
which
over-expressed
a
150,000
dalton
membrane
protein
distinct
from
P-glycoprotein
(Marsh
&
Center,
1988).
These
investigators
felt
that
the
P150
protein
was
involved
in
the
resistance
mechanism
and
contributed
to
the
decreased
in-
tracellular
drug
accumulation.
Whether
the
Mitox
resistant
cell
lines
over-express
a
novel
drug-resistance
related
mem-
brane
protein
remains
to
be
determined.
Studies
are
currently
in
progress
to
further
describe
the
mechanism
of
enhanced
drug
efflux
in
the
MCF7/Mitox
cell
line.
Other
drug
resistance
mechanisms
unrelated
to
drug
trans-
port
may
be
important
in
conferring
drug
resistance.
A
number
of
'atypical'
MDR
cell
lines
have
recently
been
reported.
Danks
et
al.
reported
a
leukaemic
cell
line
(CEM/
VM-1)
selected
for
resistance
to
the
epipodophyllotoxin,
VM-
26
(Danks
et
al.,
1987).
This
cell
line
was
felt
to
display
'atypical'
MDR
because
it
was
not
cross
resistant
to
Vinca
alkaloids
and
did
not
display
a
decreased
cellular
accumula-
tion
of
drug.
Slovak
et
al.
reported
two
DOX
resistant
cell
lines
which
had
different
mechanisms
of
resistance;
one
associated
with
P-glycoprotein,
the
other
more
atypical
in
its
mechanism
of
resistance
(Slovak
et
al.,
1988).
Mirski
et
al.
reported
a
DOX
resistant
human
small
cell
lung
cancer
cell
line
(Mirski
et
al.,
1987)
which
displayed
a
typical
cross
resistance
pattern
to
VP-16,
Vinca
alkaloids
and
colchicine
but
did
not
over-express
P-glycoprotein.
In
summary,
this
study
demonstrates
that
two
compounds
(DOX
and
Mitox)
with
structural
similarities
result in
diff-
erent
mechanisms
of
resistance
when
used
as
selecting
agents
in
the
same
human
breast
cancer
cell
line.
Reduced
drug
accumulation
secondary
to
enhanced
drug
efflux
accounts,
at
least
in part,
for
the
multidrug
resistant
phenotype
in
both
resistant
cell
lines.
However,
only
the
DOX
resistant
cell
line
over-expresses
P-glycoprotein
compared
to
the
drug
sensitive
parent
cell
line.
It
remains
to
be
determined
why
DOX,
a
natural
product,
induces
P-glycoprotein
mediated
MDR;
whereas,
Mitox,
a
synthetic
compound
induces
a
different
mechanism
of
MDR.
The
authors
would
like
to
thank
Judith
Gooley
for
her
technical
assistance
and
B.
Kathryn
Monroe
for
typing
the
manuscript.
This
work
was
supported
in
part
by
National
Cancer
Institute
Grants
CA17094,
CA43043
and
CA41183.
C.W.T.
was
a
recipient
of
a
Clinical
Oncology
Career
Development
Award
from
the
American
Cancer
Society.
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