Download Protection from Adriamycin Cytotoxicity in LI210

[CANCER RESEARCH 5.1. 5237-5243. November 1. 1993]
Protection from Adriamycin Cytotoxicity in LI210 Cells by Brefeldin A1
Paul J. Vichi2 and Thomas R. Tritton
Vermont Cancer (.'enter ¡I*.
J. V., T. K. T\ and Department of Pharmacology (T. R. T.j, College of Medicine, University of Vermont, Burlington, Vermont 05405
ABSTRACT
We present studies which suggest that the cytotoxic action of Adriamy
cin (ADR) may involve intracellular pathways of vesicular transport. The
movement of proteins or lipids from the endoplasma- reticulum to the
plasma membrane via the Golgi organelle and associated compartments
exhibits several temperature-sensitive
steps between 15 < and 20°C.In
this same temperature range, ADR loses its cytotoxic capacity. Using the
inhibitor brefeldin A (BFA), we have investigated the possible role of
intravesicular trafficking in the loss of ADR activity and the induction of
protection from cytotoxicity at low temperature in I 121(1 cells. We show
here that cells pretreated at 37°Cfor 2 h with BFA could be protected from
a subsequent exposure to ADR. The concentration causing 50% inhibition,
determined by cloning in soft agar, was increased approximately 3.5 fold.
11210 cells could also be protected from the topoisomerase II inhibitors
etoposide and amsacrine, but to a lesser extent; the concentration causing
50% inhibition for the latter inhibitors were increased 2-fold. Spectrofluorometric analysis of intracellular ADR accumulation revealed that there
was no significant difference in the level of ADR in cells with or without
BFA pretreatment. In addition, examination of ADR-induced cleavable
Proteins present in the plasma membrane often require recycling
and/or modification to maintain an active function. If there are indeed
membrane proteins the interactions of which with distal sites are
important for the cytotoxic process initiated by ADR, then these
interactions should be susceptible to disruption by inhibiting intra
cellular protein modification or accessibility to specific sites. It has
already been demonstrated that the cytotoxicity of several topoisom
erase II inhibitors can be diminished by pretreatment of cells with
inhibitors of protein synthesis (3-5). However, no individual protein,
other than topoisomerase II, has been unambiguously identified as
being decisive in mediating the action of ADR. We summarize in this
report experimental studies describing the influence of disruption of
normal protein and lipid transport on the actions of ADR.
MATERIALS
AND METHODS
Cells. Murine leukemia L12IO cells were grown in suspension in McCoy's
complex formation by alkaline elution showed no significant difference in
the level of DNA strand breaks in cells which had been pretreated with
BFA even though there was a large difference in survival. Further exami
nation of the persistence of DNA damage after a period of up to 6 h of
repair revealed that cells which were pretreated with BFA removed DNA
strand breaks at rates equivalent to those of cells which had received ADR
directly. These results suggest that the protective effect induced by brefel
din A does not involve uptake, DNA damage, or repair but instead impli
cates protein or lipid interactions which may be independent of DNA
damage and which may influence critical events that take place after the
topoisomerase II-DNA complex has been formed.
INTRODUCTION
Previous work in our laboratory has investigated the reversible
property of the temperature-dependent cytotoxic action of ADR.3
Below 20°C,ADR loses its ability to kill cells even though potentially
toxic levels of the drug can still be accumulated within the cell (1).
Once cells are raised above 20°C,however, the cytotoxic process
5A medium (GIBCO. Grand Island. NY)) containing i.-glutamine at 2 IÃŽIM
and
10% heat-inactivated horse serum (GIBCO). The cells were maintained in a
humidified atmosphere at 37°Cand 10% CO2 and passaged twice each week.
The doubling time for L1210 cells was approximately 15 h. All experiments
were performed on log phase cells (2(K1,0(X)-5(K),(HK)cells/ml).
Chemicals. ADR at 20 mg/ml and m-AMSA at 9.8 /¿g/mlwere purchased
from Sigma, dissolved in 70% ethanol, and maintained at -20°C. Dilutions
used in cell treatments were made into 70% ethanol or McCoy's 5A medium.
Brefeldin A (Sigma) at 20 mg/ml was dissolved in 100% methanol and stored
at -2()°C.Taxol, supplied by the National Cancer Institute, was dissolved at 20
mg/ml in dimethyl sulfoxide, as was nocodazole (Sigma) at 1 mg/ml, and
maintained at -20°C. Etoposide at 59 fig/ml was dissolved in 70% ethanol and
maintained at -20°C.
Cloning Assay for Cell Survival. Survival of cells after a given treatment
was assessed by cloning in soft agar. The method has been published previ
ously (1). Briefly, following a given treatment cells were washed three times
in complete medium and resuspcnded at a concentration of lOO.(MK)cells/ml.
These cell suspensions were then serially diluted 1:20 (10 ml), followed by
1:10 (3 ml) into complete McCoy's 5A medium. The entire 1:10 dilution was
added to 27 ml of "cloning medium" containing McCoy's 5A medium with a
final concentration
of 15% horse serum, 2 ITIMi -glutamine and 0.42% Difco
ensues. Concomitant with the loss of activity is the inability to detect
DNA damage in cells treated with ADR at temperatures below 20°C Bacto-Agar. The cell suspension was aliquoted into five 60-mm tissue culture
(2), suggesting a requirement for DNA damage leading to drug-in
duced cytotoxicity. These studies also revealed that the protective
process is unique to whole cells and may involve proteins of extranuclear origin since isolated nuclei accumulated ADR-induced DNA
strand breaks in a dose-dependent fashion at low temperature. Fur
thermore, we have shown that the cytotoxic process can be inhibited
in whole cells by sequestration of extracellular drug with high mo
lecular weight DNA ( 1). The induction of complete cellular protection
in the presence of cytotoxic concentrations of intracellular drug sup
ports the role of cell surface interactions as an important component
in the initiation of cell death by ADR.
Received 5/20/93; accepted 8/26/93.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked iitlvt-ni^t-nicnt in accordance with
18 U.S.C. Section 1734 solely lo indicate this fact.
1Supported by National Cancer Institute Grants CA44729 and CA56Ü40.
: To whom requests for reprints should be addressed.
' The abbreviations used are: ADR. Adriamycin; m-AMSA, amsacrine; BFA. brefeldin
A; ER, endoplasmic reticulum; GRP. glucose-regulated protein; PBS. phosphate-buffered
saline; TON. /ran.v-Golgi network.
dishes/condition (Corning), 6 ml/dish, and allowed to gel at room temperature.
The 1:20 dilution for each condition was counted directly using a Coulter
Counter to determine the number of cells plated/condition. Culture dishes were
grown for 10-12 days in a humidified incubator at 37°Cand 10% CO2.
Colonies were counted, and the average cloning efficiencies were determined
for each condition. Survival was calculated by comparison to untreated con
trols.
Uptake of ADR. ADR was quantitated by fluorescence spectroscopy using
an SLM 4800 instrument. Approximately 2.5-3.5 X 10s log phase L1210 cells
were concentrated to 2-3 X 10'' cells/ml and treated with or without BFA (10
/J.M)for 2 h at 37°C.Initial treatments were then divided into equal aliquots (10
ml) and the cells were treated ±ADR for 2 h at 37°C.Cells were centrifugcd
at 500 x g for 5 min at 4°C,and the cell pellets were washed 3 times in
ice-cold PBS (pH 7.4). The cell pellet was then resuspended in 2 ml of Na2CO.ì
(pH 9.0), and the lysate was transferred to glass 18 mm x 150 mm test tubes.
ADR was extracted from the lysate by three successive additions (3 ml each)
of an organic buffer containing ethyl acetate and /i-propyl alcohol in a ratio
of 9:1, respectively. Tubes were vortexed vigorously and then centrifugcd at
1000 X g for 15 min at 4°Cto efficiently separate the organic layer from the
water layer. The organic layer was removed and pooled for each condition.
These were then dried under N2 and the residues were resuspended in 2 ml of
ethanol at 70%. ADR content for each condition was determined by fluores-
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PROTECTION
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BY BREFELDIN
A
cencc and comparison to known standards. Excitation was at 470 nm and the
emission was collected using a 550 nm long-pass filter.
reduced by disruption of vesicular movement. To test this hypothesis,
L1210 cells were pretreated for 2 h at 37°Cwith 10 /LIMBFA. The
Alkaline Elution. Total DNA strand breaks were assayed using alkaline
elution as described elsewhere (6). Forty ml of L1210 cells at 5 X IO4 cells/ml
in complete McCoy's 5A medium were labeled with either 0.1 ^iCi/ml of
[14C]thymidinc (ICN) or O.I ¿iCi/mlof pHlthymidine (ICN) for 24 h at 37°C
fungal metabolite BFA has been reported to reversibly disrupt anterograde vesicular trafficking between the ER and the Golgi apparatus (7)
by inducing the dissociation of a Mr 110,000 protein critical for
vesicular budding and fusion from the membrane of the Golgi organelle (8). After pretreatment with BFA, the cells were subsequently
exposed to various concentrations of ADR for 2 more h at 37°Cin the
and 10% COi. The cells were then centrifuged and resuspended in 50 ml of
complete McCoy's 5A medium without radionucleotide and chased for ap
proximately 18 h. The cells labeled with [l4C]thymidine were then counted
using a hemacytometer and treatments were performed. Following all treat
ments, the cells were washed with ice-cold PBS-6 rtiM EDTA (pH 7.4) and
resuspended at 1 X 10'' cells/ml in cold PBS-6 HIM EDTA. An aliquot of
untreated 14C'-labeled cells and a separate aliquot of BBS-washed, 3H-labeled
cells were exposed to 4(H) rads of ionizing radiation using a ^Co or 137Ce
source. The irradiated cells labeled with 14C served as a calibration curve for
each experiment, while the irradiated 3H-labeled cells served as an internal
standard for each condition. 5.0 X IO5 14C-labeled cells, with or without
treatment, and 5.0 X IO5 3H-labeled cells exposed to 400 rads were added onto
2.0 p.m polycarbonate filters. The cells were washed on the filters with 5 ml of
cold PBS-6 m.MEDTA and then lysed for l h at room temperature with lysis
buffer containing 2% sodium dodecyl sulfate, 30 ITIMdisodium EDTA, and 0.5
mg/ml proteinase K (pH 10. l ). Lysates were washed with 5 ml 20 HIMdisodium
EDTA (pH 10.1). The DNA was then eluted overnight with 20 ml of elution
buffer containing 0.1% sodium dodecyl sulfate, 2.0% tetrapropylammonium
hydroxide, and 20 ITIMEDTA (free acid; pH 12.2). Eight 2.5-ml fractions were
presence of BFA and cell survival was determined by cloning in soft
agar. As shown in Fig. IA, the percentage survival was significantly
increased if cells were pretreated with BFA prior to an exposure to
ADR. The concentration causing 50% inhibition was increased from
-0.03 /XMto 0.1 /AM.
The protection that cells acquired from ADR cytotoxicity was par
tially dependent on the concentration of BFA at which cells were
pretreated. Fig. IB indicates that the cell survival increased in cells
pretreated for 1 h with increasing concentrations of BFA prior to a 2-h
exposure to 0.1 JAMADR. Maximal protection was attained by pre
treatment with 100 JAMBFA, although the effect was not dramatically
increased over lower concentrations (10 /XM).BFA by itself was not
toxic at the concentrations and times used. It is important to note,
however, that although low temperature can provide complete protec-
collected/condition. Twelve ml of Ecolume (ICN) were added to each fraction
and the I4C and 'H content was quantitated on a Packard 2500TR liquid
scintillation counter. The polycarbonate filters from each condition were dis
solved in 750 ft\ of Protosol (NEN) and the isotope content was quantitated by
addition of 5 ml of Ecolume and liquid scintillation counting. The data were
graphed as the percentage of [3H]thymidine retained on the filter versus the
percentage of [l4C]thymidine retained on the filter. Calculations for singlestrand breaks (SSB) were quantitated using 14C values obtained for each
condition when 40% of the internal standard ('H + 400 rads) remained on the
Õ
3
M
filter.
SSB =
log(Ireated/untrcalctl)
log(X-ray/untreated)
The results were expressed in rad equivalents by multiplying the frequency
of single-strand breaks by the dose of radiation, in rads, of the calibration
1.00
0.10
curve. For all experiments, this was 400 rads.
Repair of DNA Damage. The extent of DNA breaks remaining after 3 or
6 h of repair was assayed using the alkaline elution technique described above
with the following modifications. After treatment of '4C-labeled cells with or
ADR
without 10 /AMBFA and 0.14 /AMADR, the cells were washed 3 times in chilled
PBS with or without 10 /AMBFA. The cells were then resuspended in complete
McCoy's 5A medium with 10% horse serum and allowed to repair at 37°Cfor
up to 6 h. The cells were then centrifuged and resuspended at 1 X IO6 cells/ml
in PBS-6 mm EDTA and layered on 2.0 /Ampolycarbonate filters (Nuclcopore).
The cells were washed and lysed, and the DNA was eluted as described in
"Alkaline Elution." The calibration line for repair experiments was created
using 14C- and 'H-labeled cells which had been exposed to 450 rads of ionizing
radiation from a '37Ce source.
tn
M
Measurement of DNA Synthesis. Log phase L1210 cells were centrifuged
and resuspended at 1 x K)1'cells/ml in complete McCoy's 5A medium with
L-glutamine and 10% horse serum. BFA (10 /AM)was added and the cells were
aliquoted into %-well plates at 100 /Al/well. The cells were incubated for 2 h
at 37°Cbefore addition of [3H]thymidine at 1 /ACi/well. The cells were incu
bated for an additional 2 h at 37°C,and the level of incorporation of [3H]-
5E-1
1
100
Brefeldin A ¿ÌM
thymidine was determined at various intervals. Cells were harvested onto glass
fiber filters and the content of ['Hjthymidine was determined using a Packard
Fig. 1. (A) Effect of brefeldin A pretreatment on Adriamycin-induced cytotoxicity.
L1210 cells at 2-4 X IO5 cells/ml were pretrealed with K) /J.MBFA for 2 h at 37°C.The
Matrix 96.
cells were then exposed to various concentrations of Adriamycin for an additional 2 h at
37°Cand washed, and the cell survival was measured by cloning in soft agar. Data
RESULTS
Effect of BFA on ADR-induced Cytotoxicity. If protein transport
processes contribute to ADR-induced cytotoxicity, then the degree of
cytotoxicity induced by a given concentration of ADR should be
represent the summation of 5 independent experiments with 5 replicates/data point for
each experiment. (B) Influence of increasing concentrations of brefeldin A during pre
treatment on the protection from ADR cytotoxicity. L1210 cells were pretreated with
various concentrations of BFA for 1 h at 37°C.The cells were then exposed to 0. l /J.MADR
for 2 h at 37°Cand washed; the percentage survival was determined by cloning in soft
agar. The average survival of nonpretreated cells was 8.3%. Data represent the mean of 3
independent experiments each with 5 replicates/data point. Bars, SD.
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PROTECTION
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tion from any concentration of ADR, the protection provided by BFA
was not absolute and could be completely overwhelmed at ADR
concentrations >0.25 JXM.
We also examined the effect of BFA on drug-induced cytotoxicity
of the additional topoisomerase II inhibitors etoposide and m-AMSA.
BY BREFELDIN
A
100
O - nocodazole
•¿
+ nocodazole
Fig. 2 indicates that cells could also be protected from etoposide and
m-AMSA by preincubation with 10 /J.MBFA for 2 h. The concentra
tion causing 50% inhibition for both agents was increased approxi
mately 2-fold. This protection was not as exaggerated as that observed
for ADR, however, and may be a reflection of the manner with which
each of these agents interacts with the cell.
Influence of Nocodazole and Taxol on ADR Cytotoxicity. In
order to test if microtubule-based transport is involved in the induction
of ADR cytotoxicity, L1210 cells were pretreated with 5 /J.Mnocodazole, an inhibitor of microtubule polymerization (9), for l h at 37°C
before exposure to ADR for an additional 2 h at 37°C.Nocodazole
0.000
0.025
0.050
0.075
ADR MM
was also present during exposure to ADR. Survival was assayed by
cloning in soft agar. The results illustrated in Fig. 3^4 show that
pretreated cells had an increased survival of approximately 2-fold
when subsequently exposed to ADR. Although the disruption of mi
crotubule formation can result in the inhibition of other cellular pro
cesses in addition to protein transport, most notably mitosis, there is
no evidence to suggest that such inhibition would lead to protection
120100
D
n
en
M
ADR MM
Fig. 3. (A) Effect of microtubule disruption at 37°Cby nocodazole on ADR- induced
cytotoxicity. L1210 cells were pretreated with 5 (ÃŒM
nocodazole for l h al 37°C.The cells
were subsequently exposed to various concentrations of Adriamycin for 2 h at 37°Cand
washed; survival was determined by cloning in soft agar. Data represent the mean of 3
independent experiments each with 5 replicates/data point. Bars. SD. (B) Stabilization of
microtubules at low temperature by taxol and the effect on ADR cytotoxicity. L1210 cells
were exposed to taxol (40 JUM)for 30 min at 37°C.The cells were transferred to ()°Cfor
2 h, exposed to ADR for an additional 2 h at ()°C,washed, and cloned in soft agar. Data
0.6
0.0
represent the mean of 3 independent experiments with 5 replicates/data point. Bars, SD.
120»
O—O
1000
0.000
from ADR-induced cell death. ADR has, by itself, been shown to
cause a blockade of cells in the G2-M phase of the cell cycle (10-13).
Stabilization of microtubules has been demonstrated to be an im
portant component of the activity of the antitumor agent taxol (1416). At low temperature, cytoplasmic microtubules have been shown
to be unstable, losing their filamentous organization (17, 18). This can
be prevented in whole cells by pretreatment with taxol (14, 16, 19).
These stabilized microtubules are resistant to depolymerization in
duced by low temperature as well as drugs. We hypothesized that if
destabilization of microtubules contributed toward the protection from
ADR which cells acquired below 20°C,then perhaps by maintaining
- BFA
•¿â€”•
+ BFA
0.025
0.050
0.075
0.100
m-AMSA MM
Fig. 2. (A) Effect of brefeldin A pretreatment on the cytotoxicity of eloposide (V7>/6).
L12IO cells were pretreated at 37°Cwith 10 ^LMBFA for 2 h. The cells were subsequently
exposed to different concentrations of etoposide for 2 additional h at 37°C,washed, and
cloned in soft agar. Data represent the mean of 3 independent experiments each with 5
replicates/data point. Bars. SD. (B) Effect of BFA pretreatment on the cytotoxicity
induced by m-AMSA. Cells were pretreated with IO U.MBFA for 2 h at 37°Cand then
exposed to various concentrations of m-AMSA for an additional 2 h at 37°C.The
percentage survival was determined by cloning in soft agar. Data represent the mean of
independent experiments each with 5 replicates/data point. Bars, SD.
microtubules at low temperature we might overcome the protective
process. We therefore attempted to stabilize microtubules by addition
of taxol prior to an exposure to ADR. Fig. 35 illustrates that pretreat
ment with 40 JUMtaxol did not significantly alter the protection cells
acquired from ADR at 0°C.L1210 cells were pretreated with taxol for
30 min at 37°Cto facilitate uptake and interactions before shifting to
0°Cfor an additional 2 h. At this point, the cells were exposed to ADR
for 2 h at 0°Cand their survival was determined. Virtually all cells
survived an exposure to 1 JU.M,2 /J.M,or 5 JIMADR with or without
taxol pretreatment. Although taxol has been shown to maintain mi
3
crotubules in platelets at low temperature, these structures differed
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PROTECTION
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A
with DNA. Using alkaline elution, we examined the amount of total
ADR-induced single-stranded DNA breaks in cells which had been
pretreated with 10 /J.MBFA for 1 h, prior to a 2-h exposure to various
concentrations of ADR. At all ADR concentrations examined, there
were no significant differences in the formation of strand breaks
in BFA-pretreated cells compared to cells receiving ADR directly
(Fig. 5).
Persistence of ADR-induced DNA Damage. The ability of BFApretreated cells to accumulate levels of DNA damage similar to those
of nonpretreated cells suggests that involvement of interactions which
may occur independent of DNA damage, or subsequent to the forma
tion of the cleavable complex, may have a critical role in whether the
cell dies. The persistence of DNA damage is one aspect which we
thought warranted investigation. It is possible, for example, that DNA
damage accumulated under BFA may be more readily removed or
repaired, thereby not contributing as significantly toward the cytotoxic
process. We therefore measured the levels of DNA strand breaks
which remained after a period of repair. The results presented in Fig.
6 show that after 3 or 6 h of repair, equivalent percentages of initial
DNA breaks remained in cells which had been pretreated with BFA
compared to cells treated directly with ADR.
Role of DNA Synthesis in Protection by BFA and Cytotoxicity
Induced by ADR. An additional area deserving investigation was the
effect that BFA may have on the ability of the cell to maintain DNA
synthesis. Other laboratories have shown that the cytotoxicity of the
topoisomerase agents m-AMSA and etoposide could be reduced if
cells were pretreated with agents which inhibited nucleotide synthesis
(20, 21). In order to test the possibility that BFA may be inducing
protection via an alteration of the rate of DNA synthesis, L1210 cells
were pretreated for 2 h at 37°Cwith 10 /MMBFA and then allowed to
incorporate [3H]thymidine for an additional 2 h at 37°Cin the pres
100
10
ADR O.tuM
BY BREFELD1N
ADR 1.0uM
Fig. 4. Effect of BFA prctrcatment on the uptake of ADR. Log phase cells were
concentrated and pretreated with 10 U,MBFA for 2 h at 37°C.The cells were subsequently
incubated with either 0.1 /¿M
or 1.0 ¡ÕM
ADR for 2 h at 37°Cand washed. Adriamycin was
ence of BFA. The results illustrated in Fig. 7 show that BFA did not
reduce the ability of L1210 cells to incorporate labeled nucleotide.
extracted and the content was quantitated by fluorescent analysis. Data are expressed in
pmol of Adriamycin accumulated/10'1 cells as a function of treatment. In each experiment
the conditions were assayed in duplicate. Data represent the mean of 3 independent
experiments. Bars, SE.
DISCUSSION
ADR is a topoisomerase II inhibitor which has enjoyed consider
able success in the clinic despite the lack of a full understanding of its
mechanism of action. While the drug has been shown to cause DNA
damage via stabilization of the topoisomerase II cleavage complex
(22), there is incomplete information describing various interactions
which may regulate the ability of ADR to stabilize the topoisomerase
significantly from platelets which were observed at 37°C(19). At low
temperature, microtubules were not organized into the circumferential
bands normally present in platelets but were seen more as masses or
bundles within the cell. Therefore, taxol may preserve or allow mi
crotubules to exist at low temperature but may not be sufficient to
maintain the preexisting architecture. Additionally, the stabilization of
microtubules by taxol may be insufficient to maintain normal trans
port between organelles if other processes such as fusion or budding
are inhibited by low temperature.
Effect of BFA on Drug Uptake. The protection from ADR ac
quired by cells pretreated with BFA could be explained if BFA altered
the level of ADR uptake. We therefore measured the steady-state
levels of ADR accumulated in cells with or without BFA pretreatment.
Fig. 4 shows that although there is a small change in the levels of ADR
accumulated at 0.1 /AM(92% ±13% of nonpretreated cells), this does
not appear to be sufficient to account for the greater than 3-fold
difference seen in survival. At higher doses of ADR such as 1 /XM,
there was a greater difference observed in total levels of intracellular
drug accumulated in pretreated cells; however, BFA does not protect
the cells against a drug insult of this magnitude. At this time we cannot
explain why BFA seems to effect ADR accumulation at higher con
centrations.
Effect of BFA on Cleavable Complex Formation. Although there
appear to be equivalent levels of ADR in BFA-pretreated and control
cells, it is possible that the effect of pretreatment is to cause a parti
tioning of the drug into specific cellular compartments, thereby re
ducing the amount of ADR which reaches the nucleus and can interact
300
250200-
SO
&
150-
m
100-
t
50-
-o
O—O
- BFA
•¿ •¿
+ BFA
-50
0.00
0.05
0.10
0.15
0.20
ADR /¿M
Fig. 5. Influence of BFA pretreatment on the level of topoisomerase-mediated
DNA
damage induced by ADR. LI210 cells, which had been labeled with [MC]thymidine, were
exposed to 10 ^M BFA for l h at 37°C.The cells were then exposed to various concen
trations of ADR for 2 h at 37°Cand washed; the level of total DNA strand breaks were
measured using alkaline elution. Data represent the mean of 3 independent experiments.
Bars, SE.
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PROTECTION
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100
m
•¿D
I
5
•¿s
M
Time of Repair (hours)
Fig. 6. Reversibility of ADR-induced DNA damage in cells with or without brefcldin
A prctrcatment. Log phase cells labeled with [14C]thymidine were pretreated for l h at
37°Cwith H) /IM BFA before exposure to 0.14 ¿IM
ADR for 2 h at 37°C.Cells were
washed 3 times in PBS with or without 10 /J.MBFA and resuspended at 1 X IO5 cells/ml
in complete McCoy's 5A medium at 37°C.The cells were allowed to repair for either 3
or 6 h and the levels of strand breaks remaining under each condition were assayed by
alkaline etution. Results represent the mean of 4 independent experiments. Bars, SE.
II-DNA complex as well as information illustrating events which take
place after the DNA has been compromised. When considering the
many different actions of ADR in addition to the ability to damage
DNA (23-25), it has been difficult to assert a clear relationship be
tween the level of drug-induced topoisomerase-mediated lesions and
cell death. It has been suggested that strand separation in the presence
of a stabilized, covalently bound topoisomerase II-drug complex
could lead to erroneous reassociation of topoisomerase II subunits and
nucleotide tails generating nonhomologous recombination events and
subsequent alterations in the DNA sequence (20). In accordance, it has
been demonstrated that the cytotoxicity of m-AMSA and etoposide
could be diminished in cells by pretreatment with inhibitors of nucleo
tide synthesis such as aphidicolin and cordycepin (20, 21). However,
the protective effect initiated by inhibitors of nucleotide synthesis has
not been demonstrated for ADR. Furthermore, the protection that cells
acquired by pretreatment with BFA was not the result of inhibition of
nucleotide incorporation into DNA. Treatment of L1210 cells with 10
/U.MBFA did not inhibit DNA synthesis. Other laboratories have shown
that inhibition of protein synthesis (3-5) or depletion of intracellular
calcium (26) prior to exposure to topoisomerase II inhibitors results in
similar events as we have described here. In each case, the reduction
in protein synthesis or calcium levels was not accompanied by a
diminution in the formation of drug-induced cleavable complexes.
Intravesicular trafficking of proteins to the plasma membrane, or
from the plasma membrane to the interior, involves several steps,
many of which display a tightly regulated temperature dependence
(27). At temperatures below 20°C,internalization and degradation
BY BREFELDIN
A
We used BFA and nocodazole in an attempt to disrupt cellular
transport mechanisms and measure the influence of such disturbances
on the ability of ADR to induce cell death. The drug BFA has been
shown to be extremely useful as a potent, reversible agent for disrup
tion of vesicular transport processes (7, 33, 34). BFA induces a rapid
redistribution of the coat protein ß-COPfrom the membranes of the
Golgi (8) resulting in the fusion of Golgi compartments, mixing of the
Golgi into the ER, and the subsequent inhibition of anterograde move
ment of proteins from the ER (33).
Our rationale for using nocodazole derives from past research
which has supported a role for microtubules as an important compo
nent in several membrane trafficking steps within the cell (35, 36). In
addition to helping maintain the organization of the Golgi apparatus,
microtubules have been shown to be involved in several stages of
protein movement, such as the transport of large endocytic vesicles
toward the nuclear region (37) as well as the movement of vesicles
between the ER and an intermediate or salvage compartment prior to
the Golgi (38, 39). The organization and kinetics of microtubules have
been shown to be dependent upon temperature. Observation of as
sembly kinetics for in vitro tubulin preparations revealed a switch
from biphasic to monophasic if the temperature was decreased below
30°C(18). Immunocytochemical examination of frog oocytes cooled
to 25°C,18°C,or 4°Cindicated a progressive disruption or fragmen
tation of the spindle and displacement of chromosomes (40). A 2-h
low-temperature treatment at 0°Cto 4°Cwas sufficient to disrupt all
but kinetochore microtubules in mitotic ptKl cells (41). We therefore
reasoned that disruption of microtubule assembly and/or Golgi mor
phology by low temperature may result in the partial loss of critical
architecture, microtubule-based vesicular transport, and Golgi func
tion in L1210 cells; these losses may contribute toward the protection
from ADR acquired at low temperature. The fact that BFA provided a
consistently higher degree of protection from ADR than nocodazole
may be related to the degree to which each of these agents disrupts
various transport processes. For example, microtubules do not appear
to be involved in transporting newly synthesized proteins between the
salvage compartment, Golgi cisternae or fra/w-Golgi (35, 37). Fur
thermore, Golgi units which had been disrupted with nocodazole were
shown to retain cis-trans polarity and functionality (42). Therefore,
although the addition of nocodazole to cells causes the depolymerization of microtubules, the resulting influence on transport may be
limited to specific interactions such as fusion between early and late
endosomes (37), disassembly of the Golgi stacks (36), formation of
rates of membrane proteins display a marked decline in activity (2729). The exit of proteins from the TON to the cell surface is also
significantly reduced below 20°C(30, 31). At still lower temperatures,
•¿such
as 15°C,vesicles have been shown to accumulate in an inter
mediate compartment between the ER and Golgi (27, 31). Stereological volume measurements of the Golgi compartment also indicate an
increase in the size of the TON at 2()°Cand a decrease in the surface
90
120
area and volume of preceding Golgi compartments (32). Although
Time (minutes)
ADR has no identified receptor and diffuses freely into the cell at both
Fig. 7. Effect of BFA on DNA synthesis. Log phase cells were concentrated to 1 X IO6
cells/ml and exposed to BFA (10 JIM) at 37°Cfor 2 h in 96-well plates (100 »¿I/well).
high and low temperature, albeit with reduced kinetics at low tem
[•'HjThymidincwas added at 1 /iCi/well and the cells were incubated for 2 h at 37°C.
perature, there may be important regulatory proteins or cofactors
Incorporation of ['H]thymidine was assayed at 30- or 60-min intervals. The results
which are important in mediating cytotoxicity, yet with modification
represent the mean of an individual experiment with 12 replicates/data point. Bars, SD.
or transport that is prevented by low temperature.
These results were repeated in 3 additional experiments.
5241
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PROTECTION
FROM ADRIAMYCIN
the TGN-early endosóme (43), movement of newly synthesized pro
teins from the ER to the Golgi or from the Golgi to the plasma
membrane, and recycling of receptors (34). As a result, microtubuledisrupting agents such as nocodazole may not universally inhibit the
flow of membrane traffic but have instead been shown to result in the
misdirection of vesicle movement (35). This lack of direction could
prevent a signaling protein from reaching its intended destination or
from being recycled to specific sites such as the plasma membrane or
ER. Such alterations in vesicle movement may be sufficient to account
for the limited protection from ADR observed in cells pretreated with
nocodazole.
Protection from cell death by pretreatment with BFA is most likely
related to the ability of BFA to inhibit some transport process rather
than an unidentified property of the drug, although this latter expla
nation cannot be ruled out. The fact that temperature disrupts some of
the same processes as BFA may also be coincidental; however, both
conditions lead to protection from ADR-induced cytotoxicity. Tem
peratures around 20°Cbegin to selectively inhibit various steps within
BY BREFELDIN
A
shown to protect cells against doxorubicin-induced cytotoxicity (53).
It is possible that the disruption of Golgi by BFA results in an eleva
tion of GRPs and subsequent protection from ADR similar to that
previously demonstrated (53). Additionally, part of the protective re
sponse induced by low temperature may be the ability to induce an
elevation of GRPs. This hypothesis will provide an active area for
future investigation.
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the transport pathways. Additional processes become inhibited as the
temperature is lowered to 15°C.Decreasing the temperature below
20°Cresults in complete protection from ADR cytotoxicity. The pro
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and may be related to the degree to which BFA can prevent the
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Protection from Adriamycin Cytotoxicity in L1210 Cells by
Brefeldin A
Paul J. Vichi and Thomas R. Tritton
Cancer Res 1993;53:5237-5243.
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