Vol.
176,
No.
May
15,
1991
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
A RELATIONSHIP
BETWEEN MULTIDRUG RESISTANCE
DEPENDENT CYTOTOXICITY
1377-l
382
AND GROWTH-STATE
OF THE LYSOSOMOTROPIC DETERGENT
N-DODECYLIMIDAZOLE Patricia
D. Wilson, Department
University
David
of Physiology
of Medicine
Robert
March
and John
Wood Johnson
Lenard
and Biophysics
and Dentistry Medical
Piscataway, Received
Hreniuk
of New Jersey
School
New Jersey
(at
Rutgers)
08854
6, 1991
Multidrug resistance (MDR) in cultured cells and tumors is associated with overproduction of P-glycoprotein, a plasma membrane efflux pump normally present at very low levels. The cytotoxic action of N-dodecylimidazole (C,,Im), a lysosomotropic detergent, on cultured cells was previously shown to be with rapidly growing cells being most strongly dependent on growth state, We show here that this may be sensitive and confluent cells most resistant. due to a growth dependent increase in cellular P-glycoprotein activity. Both verapamil and nifedipine, structurally unrelated P-glycoprotein inhibitors, increased markedly the sensitivity of CHO fibroblasts to killing by C,z-Im; the increase was greater in confluent than in growing cells. Also, verapamil inhibitable 3H-daunomycin efflux was more efficient from confluent than from subconfluent cells. The MDR cell line CHRC, differed from all cell lines previously examined in that it did not show a growth-dependent decrease in C,,and sensitivity was not increased by verapamil or nifedipine. Im sensitivity, We suggest that a growth-dependent increase in MDR activity is a general property of cultured cells, except for those specifically overexpressing P0 1991 Academic Press, 1°C. glycoprotein.
Multidrug unrelated culture
resistance
drugs
after
exposure
MDR in vitro
(l-4).
accumulation mediated
(MDR), is
due to increased leading
the MDRl gene. normal
renal
colon
epithelia
and/or
drug
cytotoxic
to only
one,
proximal is
agents in
to MDR result
consistent
from
liver with
bile a role
to several
demonstrated
by decreased efflux. membrane
amplification
of P-glycoprotein
tubules,
in cells
cellular Drug
pump;
drug
efflux
is
elevated
levels
and overexpression in
canaliculi, in secretory
apical
cell
pancreatic transport
in
membranes ductules
of of of and
processes
(5). detergents
(6)
has been
a 170 kDa plasma
elimination
of resistance
characterized
The localization
Lysosomotropic accumulation
acquisition
energy-dependent
by P-glycoprotein,
p-glycoprotein
the
which
lysosomes,
such cause
cell
disruption
as N-dodecylimidazole death of the
by their lysosomal
(Ciz-Im)
are
acid-dependent membrane,
and release 0006.291X/91
1377
$1.50
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in arty form reserved.
Vol.
176,
No.
of cysteine
proteases
fibroblastic state
increasing levels
density
at several intracellular
The present association C,,-Im
between
AND
cytoplasm cell
during cell
BIOPHYSICAL
(7,8).
types
exponential densities (7).
were
carried
RESEARCH
Several
out cell
sensitivity
directly
with
to measured
whether
of cultured
lines,
of P-glycoprotein
in a growth
In BHK fibroblasts,
(7-9).
to determine state
in MDR and sensitive
to C,,-Im
decreasing
growth
COMMUNICATIONS
epithelial,
sensitive
corresponded
MDR and proliferative
and to the presence
are
a progressively
C,,-Im
studies
sensitivity
state
the showing
fashion,
cell of
into
and hematopoietic
dependent
cytotoxicity
BIOCHEMICAL
3, 1991
there cells
in relation
was an
by examining to growth
inhibitors.
METHODS Cell Culture. Chinese hamster ovary (CHO) fibroblast cell lines of the sensitive, parent strain (AuxB,) and the MDR strain (CHRC,), a generous gift of Dr. V. Ling (Ontario Cancer Institute, Canada), were routinely maintained in a-MEM Medium containing 10% fetal bovine serum (Gibco, Grand Island, NY). For viability and uptake studies, cells were plated at 3 x lo5 cells/ml in 12 well cluster plates (Corning) and allowed to reach various confluency states. All experiments were carried out in quadruplicate. Aliquots of cells were first extracted with 1% SDS for total cell protein determinations (11) or with 0.5% Triton X-100 (Mallinckrodt, Paris, KY) for determination of cysteine cathepsin B+L activity using carbobenzyloxphenylalanylarginyl-7-(4-methyl) coumarylamide (Peptide Institute, Osaka, Japan) as substrate (12,13). In some experiments, cells were preincubated in media containing verapamil (7j~M; Knoll, Hamburg, W. Germany) or nifedipine (5pM Sigma, St. Louis, MO). Toxicitv Assays. Cells were incubated for 2 hours at 37°C in 500~1 Hams/Hepes buffer, pH 7.6 containing 0,10,20,40,60,80,100 or 120pg/ml C,,Im. Viability was measured by release of lactate dehydrogenase (14); by the mitochondrial-dependent ability of cells to cleave 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT, Chemicon, El Segundo, CA; 15); and by Trypan Blue exclusion. 3H-Daunomvcin Uotake and Efflux Studies. Cells were grown to various confluency states and loaded with 3H-daunomycin (1 j&i/ml; 16), incubated for 1 h in Hepes (2OmM) buffered medium containing 3H-daunomycin, 1pM daunomycin and 1mM dinitrophenol, which was aspirated and replaced with glucosecontaining medium in the presence or absence of a non-toxic dose of verapamil To measure drug efflux, 100~1 media samples were collected at (7/1M). 0,1,2,3,4,5,10,20,30,40,50 and 60 minutes. Cellular content of 3H-daunomycin was determined by rapid rinsing with 3 changes of PBS, hypotonic lysis and scintillation counting of media samples and cell extracts. Cellular DNA content was analyzed in duplicate samples using mithramycin (Sanbio, Uden, Holland).
RESULTS Growth Resistant characteristic sigmoidal,
State Cells.
Denendence CHO cells
response and there
of C,,-Im of the
to CI,-Im:
was a dramatic
Cvtotoxicitv
in Sensitive
sensitive
parent
the dose
dependence
dependence 1378
cell
line of cell
of cytotoxicity
and Multidrug (AuxBr)
showed
killing
was
on growth
a
Vol.
176,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
100
100 $
f :
00
: 0 Eg
60
80
t
60
40 b L 20 0
1, 20
40
0
60
80
100
20
0
C,, -Imidazole
&ml
40
60
80
100
C,, -Imidazole
wdml
Figure 1. fibroblasts
Effect of degree of confluency (estimated as per cent) on sensitivity to Cu-Im-mediated cytotoxicity.
of AuxB,
Figure 2. fibroblasts
Effect of degree of confluency (estimated as per cent) on sensitivity to Cu-Im mediated cytotoxicity.
of CHsC,
state,
with
(Figure
greatest
cytotoxicity
occurring
at the
earliest
stages
of growth
1). By contrast,
confluent
confluent
AuxB,
pg/ml
(Figure
with
increasing
cells, 2).
Nifedinine.
for cells
48% of the cells
C,,-
channel
control with
increase
value.
Very
verapamil,
increase
in these
their
as 120 cells
sensitivity control
increase;
similar
effects
or
and nifedipine by the
fibroblasts
smaller
a chemically
of Veraoamil
mediated
AuxB,
to 37% of the
than
as high
verapamil
resistance in
not
sensitive
at doses
bv Addition
of confluent
a somewhat
much less
2).
antagonists drug
decreased
showed
(Figure
of C,,-Im
of multiple
Im
were cytotoxicity
to Clz- Im did
of confluency
in a substantial
LD,,
partial
resistance
Preincubation
resulted
only
of Cvtotoxicitv
inhibitors
(16,18-24).
of the
Also,
The calcium
powerful
growing
MDR CHRG5 cells
with
degree
Enhancement
the
COMMUNICATIONS
0 100% 0 50% A 25 %
p"
0'
RESEARCH
with
nifedipine
to C,,-Im value.
are
P-glycoprotein (Figure
the IDso decreased were
unrelated
seen after calcium
only
80
z 60 0 E8 40 f5 h 20 0
20 w/ml
Firure 3. Effect of nifedipine fibroblasts at two different
40
60
80
100
C,, -Imidazole
on C,,- Im-mediated cytotoxicity degrees of confluency (estimated 1379
to
preincubation
antagonist
100 $ l+i
3);
Exponentially
in AuxB, as per cent).
(not
Vol.
176,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
80 60 0 100
-
1002 100 X + Vorapamil
l
A 50 x v
20
40
pg/ml
0
80
60
100
findings
in determining
implicated
in the
C,,-Im
that
intracellular
By contrast,
presence
of 3H-daunamycin at two different
and suggested
P-glycoprotein,
the
20
Effect of nifedipine on C,,- Im-mediated at two different degrees of confluency
These
While
10
30 Time
Figure 5. Rate of efflux and absence of verapamil, per cent).
C,,-Im
.‘.‘.‘.‘.‘.’ 0
Clz-lmidazole
Figure 4. fibroblasts
shown).
50 X + Vompamil
sensitivity
showed
of nifedipine
(Figure
of subconfluent
of nifedipine to C,,- Im in
sensitivity
of CHRC, cells
for
in presence (estimated
as
the might
no increase
be important
to Cia-Im
confluent
the presence
in cytotoxicity
4) or verapamil
cells
verapamil,
or
sensitivity
from AuxBl fibroblasts degrees of confluency
activity
60
in CHRC5 as percent).
(estimated
as a substrate
50
levels.
the MDR CHRC, cells
presence
cytotoxicity
P-glycoprotein
Cl,-Im
40
(minutes)
(data
was unaffected
cells
showed
of nifedipine.
This
to C12-Im was mediated
of
not
shown).
by the
a decreased
suggested
by mechanisms
that
independent
of
P-glycoprotein. We had previously release the
of
that
altered
determined.
the
levels
confluent
and subconfluent
untreated
cells,
To provide growth
a more
states
exponentially lower growing growth inhibitor
differences
direct
growing
retention cells. states
were
between
of the
and stationary greater
ability
Efflux
was almost
(Figure
of the
in enzyme
levels
or verapamil
To exclude simply were between treated
and P-GlYcoDrotein
of normal
cells
efflux
cells
and
inhibited
Levels. at different
was measured
(Figure
5), consistent with its action P-glycoprotein pumping activity.
1380
might
was
cells.
in stationary
completely
above
activities
nifedipine
Efflux
(8).
cathepsin
and sensitive
AuxBr
efflux)
shown seen
3H-daunomycin
(i.e.,
cytoplasm
cysteine
on Daunomvcin drugs,
in C1a-Im toxicity
the
in toxicity
resistant
test
event
into
enzymes,
cells,
Effects
to pump out
lethal
differences
or between
Related
the
cathepsins
of these
No significant
Growth
that
cysteine
lysosomal
possibility
reflect
shown
5).
than
in
in
There was much exponentially
by verapamil as a competitive
in both
Vol.
176,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
DISCUSSION The experiments of cultured state,
cells
date,
in
very
this
has been
proceeded
or nifedipine, increase
which
is
cells
is growth
shown
for
a general on the
dependence
by Krishan
phenomenon
in cultured
surface
suggesting level
of AuxB,
that
cells
amounts
on the
of detectability
growth protein
is is
>48 hours constant studies
rate, have
induction
by which
not known. extremely (26),
it
of liver
is
possible
shown cell
carcinogen
administration
expression
and growth
heat
consensus
shock
association
between
established
(29).
(25),
cells.
that
shock
in
Verapamil both
for
confluent
than
to pump out (Figure cells
the
that
idea
3H-
5).
lymphoid
A
was this
is
of P-glycoprotein
in 100% confluent
of subconfluent
cells
activity
cells,
were
below
the
P-glycoprotein
is
levels
is
link
activation
and cell
to
or chemical between
implied
of
Previous
in response
hepatectomy
the MDRl gene promoter
gene
during
at a
proceeds.
A further
regulation
increase
synthesized
as proliferation
(3,27).
cycle
levels
et al. have shown that the with a half-time of turnover
by partial
to rats
heat
2).
cells
human
in MDR message
proliferation
in
used
observation).
an increase
elements
line
cells
supporting
P-glycoprotein
accumulates
or cell
greater
of AuxB,
possible
surface
lines,
a similar,
unpublished).
Immunolocalization
was only
cell
to C12-Im.
subconfluent
cultured
To
as growth
C12-Im
line
and is
However, since Gottesman stable in cultured cells,
and simply also
et al.
(unpublished
The mechanism(s)
in
idea.
showed
(Figure
cell
The ability from
growth
this
of P-glycoprotein,
AuxB,
than
of efflux
adriamycin
lines
P-glycoprotein
compounds, 3).
with
the CHRC, cell
inhibitors
in confluent
can vary
P. Wilson,
has been
parent
sensitivity
support
to
1; refs.7-9;
overexpresses
whether
25 different
cell
in sensitivity
so far
(Figure
greater
to over
of these
(Figure
both
Ci2-Im,
Our findings
all
decrease
for
to test
due to a growth-dependent
to be cytotoxic
of the
similar
sub-confluent
daunomycin
partly,
unrelated
sensitivity
Sensitization
particularly
origin;
structurally
the
drugs,
discovered
(lo),
designed
at least
confluency
exception
studies
similar
found
continuous
towards
The single
were
pump activity.
and tumorigenic
pronounced,
these
is,
P-glycoprotein
C,z-Im
of normal
here
to certain
and whether
increase
for
reported
by the (28) growth
since is
MDRl gene presence
of
the well
ACKNOWLEDGMENTS The authors are indebted to Dr. V. Ling, Ontario for of AuxB, and CHRC, cells and P-glycoprotein antibody. They appreciation to Drs. V. Ling and M. Gottesman for helpful E. Guerra for technical assistance and to Mrs. G. Trechock manuscript. This work was supported by NIH grant GM34050.
1381
the generous also offer discussion, for typing
gifts their to the
“-I VOI. 176,
No. 3,1991
BIOCHEMICAL
AND BlOPHYSlCAL
RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7.
a. 9. 10. 11. 12. 13. 14. 15. 16. 17.
18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
28. 29.
(1989) FASEB Journal, 3, Juranka, P.F., Zastawny, R.L., and Ling, V. 2583-2592. Endicott, J.A. and Ling, V. (1989) Ann. Rev. Biochem., 58, 137-171. Gottesman, M.M. and Pastan, I. (1988) J. Biol. Chem., 263, 12163-12166. Beck, W.T. (1987) Biochem. Pharmacol., 36, 2879-2887. Thiebaut, F., Tsuruo, T., Hamada, H., Gottesman, M.M., Pastan, I. and Willingham, M. (1987) Proc. Natl. Acad. Sci. USA 84, 7735-7738. Firestone, R.A., Pisano, J.M., Bailey, P.J. and Bonney, R.J. (1979) J. Med. Chem., 22, 1130-1133. Miller, D.K., Griffiths, E., Lenard, J. and Firestone, R.A. (1983) J. Cell Biol., 97, 1841-1851. Wilson, P.D., Firestone, R.A. and Lenard, J. (1987) J. Cell Biol., 104, 1223-1229. Wilson, P.D., Hreniuk, D. and Lenard, J. (1989) Cancer Research, 49, 507-510. Ling, V. and Thompson, L.H. (1974) J. Cell. Physiol., 83, 103-116. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem., 193, 265-275. Barrett, A.J., Kembhari, A.A., Brown, A., Kirschke, H., Knight, C.G., Tamai, M. and Hanada, K. (1982) Biochem. J., 201, 189-198. Barrett, A.J. and Kirschke, H. (1981) Methods Enzymol., 80, 540-541. Wroblewski, F. and LaDue, J.S. (1955) Proc. Sot. Exp. Biol. Med., 90, 210-213. (1983) J. Immunol. Methods, 65, 55-63. Mosmann, T. Cano-Gauci, D.J. and Riordan, J.R. (1987) Biochem. Pharmacol., 36, 21152123. Kartner, N., Evernden-Porelle, D., Bradley, G. and Ling, V. (1985) Nature, 316, 820-823. Fojo, A., Akiyama, S.-I., Gottesman, M. and Pastan, I. (1985) Cancer Res., 45, 3002-3007. Pradhan, S.G., Basrur, V.S., Chitnis, M.P. and Adwani, S.H. (1984) Oncology, 41, 406-408. Rogan, A.M., Hamilton, T.C., Young, R.C., Klecker, R.W., Jr. and Ozols, R.F. (1984) Science, 224, 994-996. Tsuruo, T., Iida, H., Tsukagoshi, S. and Sakurai, Y. (1983) Cancer Res., 43, 2267-2272. Tsuruo, T., Iida, H., Yamashiro, M., Tsukagoshi, S. and Sakurai, Y. (1982) Biochem. Pharmacol., 31, 3138-3140. Tsuruo, T., Iida, H., Nagamnuma, K., Tsukagoshi, S. and Sakurai, Y. (1983) Cancer Res., 43, 808-813. Yanovich, Y. and Preston, L. (1984) Cancer Res., 44, 1743-1746. Krishan, A., Dutt, K., Israel, M. and Ganapathi, R. (1981) Cancer Research, 41, 2745-2750. Richert, N.D., Aldwin, L,. Nitecki, D., Gottesman, M.M. and Pastan, I. (1988) Biochemistry, 27, 7607-7613. Thorgierson, S.S. Huber, B.E., Sorrell, S., Fojo, A, Pastan, I., Gottesman, M.M. (1987) Science, 236, 1120-1122. Chin, K., Tanaka, S., Darlington, G., Pastan, I. Gottesman, M. (1990) J. Biol. Chem., 265, 221-226. Bond, V., Schlessinger, M.J. (1987) Adv. in Genet. 24, l-29.
1382
176,
No.
May
15,
1991
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
A RELATIONSHIP
BETWEEN MULTIDRUG RESISTANCE
DEPENDENT CYTOTOXICITY
1377-l
382
AND GROWTH-STATE
OF THE LYSOSOMOTROPIC DETERGENT
N-DODECYLIMIDAZOLE Patricia
D. Wilson, Department
University
David
of Physiology
of Medicine
Robert
March
and John
Wood Johnson
Lenard
and Biophysics
and Dentistry Medical
Piscataway, Received
Hreniuk
of New Jersey
School
New Jersey
(at
Rutgers)
08854
6, 1991
Multidrug resistance (MDR) in cultured cells and tumors is associated with overproduction of P-glycoprotein, a plasma membrane efflux pump normally present at very low levels. The cytotoxic action of N-dodecylimidazole (C,,Im), a lysosomotropic detergent, on cultured cells was previously shown to be with rapidly growing cells being most strongly dependent on growth state, We show here that this may be sensitive and confluent cells most resistant. due to a growth dependent increase in cellular P-glycoprotein activity. Both verapamil and nifedipine, structurally unrelated P-glycoprotein inhibitors, increased markedly the sensitivity of CHO fibroblasts to killing by C,z-Im; the increase was greater in confluent than in growing cells. Also, verapamil inhibitable 3H-daunomycin efflux was more efficient from confluent than from subconfluent cells. The MDR cell line CHRC, differed from all cell lines previously examined in that it did not show a growth-dependent decrease in C,,and sensitivity was not increased by verapamil or nifedipine. Im sensitivity, We suggest that a growth-dependent increase in MDR activity is a general property of cultured cells, except for those specifically overexpressing P0 1991 Academic Press, 1°C. glycoprotein.
Multidrug unrelated culture
resistance
drugs
after
exposure
MDR in vitro
(l-4).
accumulation mediated
(MDR), is
due to increased leading
the MDRl gene. normal
renal
colon
epithelia
and/or
drug
cytotoxic
to only
one,
proximal is
agents in
to MDR result
consistent
from
liver with
bile a role
to several
demonstrated
by decreased efflux. membrane
amplification
of P-glycoprotein
tubules,
in cells
cellular Drug
pump;
drug
efflux
is
elevated
levels
and overexpression in
canaliculi, in secretory
apical
cell
pancreatic transport
in
membranes ductules
of of of and
processes
(5). detergents
(6)
has been
a 170 kDa plasma
elimination
of resistance
characterized
The localization
Lysosomotropic accumulation
acquisition
energy-dependent
by P-glycoprotein,
p-glycoprotein
the
which
lysosomes,
such cause
cell
disruption
as N-dodecylimidazole death of the
by their lysosomal
(Ciz-Im)
are
acid-dependent membrane,
and release 0006.291X/91
1377
$1.50
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in arty form reserved.
Vol.
176,
No.
of cysteine
proteases
fibroblastic state
increasing levels
density
at several intracellular
The present association C,,-Im
between
AND
cytoplasm cell
during cell
BIOPHYSICAL
(7,8).
types
exponential densities (7).
were
carried
RESEARCH
Several
out cell
sensitivity
directly
with
to measured
whether
of cultured
lines,
of P-glycoprotein
in a growth
In BHK fibroblasts,
(7-9).
to determine state
in MDR and sensitive
to C,,-Im
decreasing
growth
COMMUNICATIONS
epithelial,
sensitive
corresponded
MDR and proliferative
and to the presence
are
a progressively
C,,-Im
studies
sensitivity
state
the showing
fashion,
cell of
into
and hematopoietic
dependent
cytotoxicity
BIOCHEMICAL
3, 1991
there cells
in relation
was an
by examining to growth
inhibitors.
METHODS Cell Culture. Chinese hamster ovary (CHO) fibroblast cell lines of the sensitive, parent strain (AuxB,) and the MDR strain (CHRC,), a generous gift of Dr. V. Ling (Ontario Cancer Institute, Canada), were routinely maintained in a-MEM Medium containing 10% fetal bovine serum (Gibco, Grand Island, NY). For viability and uptake studies, cells were plated at 3 x lo5 cells/ml in 12 well cluster plates (Corning) and allowed to reach various confluency states. All experiments were carried out in quadruplicate. Aliquots of cells were first extracted with 1% SDS for total cell protein determinations (11) or with 0.5% Triton X-100 (Mallinckrodt, Paris, KY) for determination of cysteine cathepsin B+L activity using carbobenzyloxphenylalanylarginyl-7-(4-methyl) coumarylamide (Peptide Institute, Osaka, Japan) as substrate (12,13). In some experiments, cells were preincubated in media containing verapamil (7j~M; Knoll, Hamburg, W. Germany) or nifedipine (5pM Sigma, St. Louis, MO). Toxicitv Assays. Cells were incubated for 2 hours at 37°C in 500~1 Hams/Hepes buffer, pH 7.6 containing 0,10,20,40,60,80,100 or 120pg/ml C,,Im. Viability was measured by release of lactate dehydrogenase (14); by the mitochondrial-dependent ability of cells to cleave 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT, Chemicon, El Segundo, CA; 15); and by Trypan Blue exclusion. 3H-Daunomvcin Uotake and Efflux Studies. Cells were grown to various confluency states and loaded with 3H-daunomycin (1 j&i/ml; 16), incubated for 1 h in Hepes (2OmM) buffered medium containing 3H-daunomycin, 1pM daunomycin and 1mM dinitrophenol, which was aspirated and replaced with glucosecontaining medium in the presence or absence of a non-toxic dose of verapamil To measure drug efflux, 100~1 media samples were collected at (7/1M). 0,1,2,3,4,5,10,20,30,40,50 and 60 minutes. Cellular content of 3H-daunomycin was determined by rapid rinsing with 3 changes of PBS, hypotonic lysis and scintillation counting of media samples and cell extracts. Cellular DNA content was analyzed in duplicate samples using mithramycin (Sanbio, Uden, Holland).
RESULTS Growth Resistant characteristic sigmoidal,
State Cells.
Denendence CHO cells
response and there
of C,,-Im of the
to CI,-Im:
was a dramatic
Cvtotoxicitv
in Sensitive
sensitive
parent
the dose
dependence
dependence 1378
cell
line of cell
of cytotoxicity
and Multidrug (AuxBr)
showed
killing
was
on growth
a
Vol.
176,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
100
100 $
f :
00
: 0 Eg
60
80
t
60
40 b L 20 0
1, 20
40
0
60
80
100
20
0
C,, -Imidazole
&ml
40
60
80
100
C,, -Imidazole
wdml
Figure 1. fibroblasts
Effect of degree of confluency (estimated as per cent) on sensitivity to Cu-Im-mediated cytotoxicity.
of AuxB,
Figure 2. fibroblasts
Effect of degree of confluency (estimated as per cent) on sensitivity to Cu-Im mediated cytotoxicity.
of CHsC,
state,
with
(Figure
greatest
cytotoxicity
occurring
at the
earliest
stages
of growth
1). By contrast,
confluent
confluent
AuxB,
pg/ml
(Figure
with
increasing
cells, 2).
Nifedinine.
for cells
48% of the cells
C,,-
channel
control with
increase
value.
Very
verapamil,
increase
in these
their
as 120 cells
sensitivity control
increase;
similar
effects
or
and nifedipine by the
fibroblasts
smaller
a chemically
of Veraoamil
mediated
AuxB,
to 37% of the
than
as high
verapamil
resistance in
not
sensitive
at doses
bv Addition
of confluent
a somewhat
much less
2).
antagonists drug
decreased
showed
(Figure
of C,,-Im
of multiple
Im
were cytotoxicity
to Clz- Im did
of confluency
in a substantial
LD,,
partial
resistance
Preincubation
resulted
only
of Cvtotoxicitv
inhibitors
(16,18-24).
of the
Also,
The calcium
powerful
growing
MDR CHRG5 cells
with
degree
Enhancement
the
COMMUNICATIONS
0 100% 0 50% A 25 %
p"
0'
RESEARCH
with
nifedipine
to C,,-Im value.
are
P-glycoprotein (Figure
the IDso decreased were
unrelated
seen after calcium
only
80
z 60 0 E8 40 f5 h 20 0
20 w/ml
Firure 3. Effect of nifedipine fibroblasts at two different
40
60
80
100
C,, -Imidazole
on C,,- Im-mediated cytotoxicity degrees of confluency (estimated 1379
to
preincubation
antagonist
100 $ l+i
3);
Exponentially
in AuxB, as per cent).
(not
Vol.
176,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
80 60 0 100
-
1002 100 X + Vorapamil
l
A 50 x v
20
40
pg/ml
0
80
60
100
findings
in determining
implicated
in the
C,,-Im
that
intracellular
By contrast,
presence
of 3H-daunamycin at two different
and suggested
P-glycoprotein,
the
20
Effect of nifedipine on C,,- Im-mediated at two different degrees of confluency
These
While
10
30 Time
Figure 5. Rate of efflux and absence of verapamil, per cent).
C,,-Im
.‘.‘.‘.‘.‘.’ 0
Clz-lmidazole
Figure 4. fibroblasts
shown).
50 X + Vompamil
sensitivity
showed
of nifedipine
(Figure
of subconfluent
of nifedipine to C,,- Im in
sensitivity
of CHRC, cells
for
in presence (estimated
as
the might
no increase
be important
to Cia-Im
confluent
the presence
in cytotoxicity
4) or verapamil
cells
verapamil,
or
sensitivity
from AuxBl fibroblasts degrees of confluency
activity
60
in CHRC5 as percent).
(estimated
as a substrate
50
levels.
the MDR CHRC, cells
presence
cytotoxicity
P-glycoprotein
Cl,-Im
40
(minutes)
(data
was unaffected
cells
showed
of nifedipine.
This
to C12-Im was mediated
of
not
shown).
by the
a decreased
suggested
by mechanisms
that
independent
of
P-glycoprotein. We had previously release the
of
that
altered
determined.
the
levels
confluent
and subconfluent
untreated
cells,
To provide growth
a more
states
exponentially lower growing growth inhibitor
differences
direct
growing
retention cells. states
were
between
of the
and stationary greater
ability
Efflux
was almost
(Figure
of the
in enzyme
levels
or verapamil
To exclude simply were between treated
and P-GlYcoDrotein
of normal
cells
efflux
cells
and
inhibited
Levels. at different
was measured
(Figure
5), consistent with its action P-glycoprotein pumping activity.
1380
might
was
cells.
in stationary
completely
above
activities
nifedipine
Efflux
(8).
cathepsin
and sensitive
AuxBr
efflux)
shown seen
3H-daunomycin
(i.e.,
cytoplasm
cysteine
on Daunomvcin drugs,
in C1a-Im toxicity
the
in toxicity
resistant
test
event
into
enzymes,
cells,
Effects
to pump out
lethal
differences
or between
Related
the
cathepsins
of these
No significant
Growth
that
cysteine
lysosomal
possibility
reflect
shown
5).
than
in
in
There was much exponentially
by verapamil as a competitive
in both
Vol.
176,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
DISCUSSION The experiments of cultured state,
cells
date,
in
very
this
has been
proceeded
or nifedipine, increase
which
is
cells
is growth
shown
for
a general on the
dependence
by Krishan
phenomenon
in cultured
surface
suggesting level
of AuxB,
that
cells
amounts
on the
of detectability
growth protein
is is
>48 hours constant studies
rate, have
induction
by which
not known. extremely (26),
it
of liver
is
possible
shown cell
carcinogen
administration
expression
and growth
heat
consensus
shock
association
between
established
(29).
(25),
cells.
that
shock
in
Verapamil both
for
confluent
than
to pump out (Figure cells
the
that
idea
3H-
5).
lymphoid
A
was this
is
of P-glycoprotein
in 100% confluent
of subconfluent
cells
activity
cells,
were
below
the
P-glycoprotein
is
levels
is
link
activation
and cell
to
or chemical between
implied
of
Previous
in response
hepatectomy
the MDRl gene promoter
gene
during
at a
proceeds.
A further
regulation
increase
synthesized
as proliferation
(3,27).
cycle
levels
et al. have shown that the with a half-time of turnover
by partial
to rats
heat
2).
cells
human
in MDR message
proliferation
in
used
observation).
an increase
elements
line
cells
supporting
P-glycoprotein
accumulates
or cell
greater
of AuxB,
possible
surface
lines,
a similar,
unpublished).
Immunolocalization
was only
cell
to C12-Im.
subconfluent
cultured
To
as growth
C12-Im
line
and is
However, since Gottesman stable in cultured cells,
and simply also
et al.
(unpublished
The mechanism(s)
in
idea.
showed
(Figure
cell
The ability from
growth
this
of P-glycoprotein,
AuxB,
than
of efflux
adriamycin
lines
P-glycoprotein
compounds, 3).
with
the CHRC, cell
inhibitors
in confluent
can vary
P. Wilson,
has been
parent
sensitivity
support
to
1; refs.7-9;
overexpresses
whether
25 different
cell
in sensitivity
so far
(Figure
greater
to over
of these
(Figure
both
Ci2-Im,
Our findings
all
decrease
for
to test
due to a growth-dependent
to be cytotoxic
of the
similar
sub-confluent
daunomycin
partly,
unrelated
sensitivity
Sensitization
particularly
origin;
structurally
the
drugs,
discovered
(lo),
designed
at least
confluency
exception
studies
similar
found
continuous
towards
The single
were
pump activity.
and tumorigenic
pronounced,
these
is,
P-glycoprotein
C,z-Im
of normal
here
to certain
and whether
increase
for
reported
by the (28) growth
since is
MDRl gene presence
of
the well
ACKNOWLEDGMENTS The authors are indebted to Dr. V. Ling, Ontario for of AuxB, and CHRC, cells and P-glycoprotein antibody. They appreciation to Drs. V. Ling and M. Gottesman for helpful E. Guerra for technical assistance and to Mrs. G. Trechock manuscript. This work was supported by NIH grant GM34050.
1381
the generous also offer discussion, for typing
gifts their to the
“-I VOI. 176,
No. 3,1991
BIOCHEMICAL
AND BlOPHYSlCAL
RESEARCH COMMUNICATIONS
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