Download DNA Strand Breaks Produced by Etoposide

(CANCER RESEARCH 48, 5096-5100, September 15, 1988]
DNA Strand Breaks Produced by Etoposide (VP-16,213) in Sensitive and Resistant
Human Breast Tumor Cells: Implications for the Mechanism of Action
Biraiulra K. Sinlia,1 Nissiin Haim,2 Lata Dusre, Donna Kerrigan,
and Yves Pommier
Clinical Pharmacology Branch, Clinical Oncology Program fB. K. S., N. H., L. D.J, and Laboratory of Molecular Pharmacology ¡D.K., Y. P.], National Cancer Institute
N IH. Bethesda, Maryland 20892
ABSTRACT
Pleotropic resistant human breast cancer cells (MCF-7), selected for
resistance to Adriamycin, were used to study the production of DNA
strand breaks by etoposide (VP-16) and its relationship to drug cytotoxicity. It was shown that the resistant MCF-7 cell line was cross-resistant
to VP-16, and the degree of resistance was found to be 125-200-fold.
Alkaline elution studies indicated that the parental cell line was very
sensitive to VP-16 which caused extensive DNA strand breakage. In
contrast, little DNA strand breakage was detected in the resistant MCF7 cells, even at very high drug concentrations, indicating a good agreement
between strand breaks and cytotoxicity. Further studies indicated that
the nuclei isolated from the parental cell line were more resistant to VI'
16-induced DNA strand breaks than the intact cells, while the opposite
was found in the resistant cell line. In addition, the alkaline elution
studies in isolated nuclei showed only a 2-fold reduction of VP-16-induced
DNA breaks in nuclei from the resistant cells. In agreement with this
result, it was found that nuclear extract from the resistant cells produced
2-3-fold less VP-16-induced DNA breaks than that from the sensitive
cells in 32P-end-labeled SV40 DNA. VP-16 uptake and efflux studies
indicated that there was a 2-3-fold decrease in net cellular accumulation
of VP-16 in the resistant cells. Although the reduced uptake of VP-16
and decreased drug sensitivity of topoisomerase II appear to contribute
to the mechanism of action and the development of resistance to VP-16,
they do not completely explain the degree of resistance to VP-16 in this
multidrug-resistant MCF-7 cell line indicating that other biochemical
factors, such as activation of VP-16, are also involved in drug resistance
and suggesting that the resistance is multifactorial.
INTRODUCTION
The epipodophyllotoxin derivative, etoposide, produces both
single- and double-strand DNA breaks in mammalian cells and
isolated cell nuclei (1-7). Earlier work of Loike and Honvitz
(8) has shown that the presence of cellular components and a
free hydroxyl group at C-4' in the E-ring was essential for the
DNA damage to occur. Subsequently, it was shown in an in
vitro system that VP-163-induced DNA cleavage is caused by
the interaction of the drug with topoisomerase II, resulting in
a covalent enzyme-DNA complex (9, 10). Therefore, it is be
lieved that the intracellular target of VP-16 and the related
compound VM-26 is nuclear DNA topo II and that the topo
II-mediated DNA strand breaks are responsible for VP-16dependent cell killing of human tumors (2, 3, 7).
Studies reported from our laboratory have shown that VP16 is metabolized either by cytochrome P-450 or by peroxidases
(prostaglandin or horseradish) to dihydroxy- and o-quinone
derivatives of VP-16 (11-15). Furthermore, such enzymatic
activation of the drug also forms reactive intermediates that
covalently bind to purified DNA and proteins (12, 13, 15). We
have therefore postulated that the metabolism of VP-16 and
formation of reactive species from VP-16 and subsequent bind
ing of these species to cellular macromolecules may be impor
tant in VP-16-induced cell killing.
Studies in drug-resistant (or multidrug-resistant) cells with
VP-16 have shown a reduced formation of protein-associated
DNA strand breaks (16-19). This decrease in drug-induced
DNA damage has been reported to arise from the modification
of topo II activity in the resistant cells. We have recently
reported that human breast tumor (MCF-7) cells made resistant
to Adriamycin (ADRR) also became resistant to VP-16 and had
the multidrug-resistant phenotype (20). We have also shown
that the development of resistance in the MCF-7 cells also
induces changes in activation and detoxification enzymes, i.e.,
glutathione peroxidase and glutathione transferase, resulting in
enhanced elimination of peroxides and free radicals (21-23).
In the present study, we have used both MCF-7-sensitive
(WT) and -resistant cell (ADRR) lines to study VP-16 uptake,
VP-16-induced DNA strand breaks, and their relationship to
cytotoxicity. Our findings show that VP-16 resistance was
accompanied by reduced uptake of the drug and decreased VP16-induced DNA breaking activity due to alterations in nuclear
topo II activity. However, the decrease in the drug accumulation
(2-3-fold), and alteration in topo II activity (2-3-fold) in ADRR
cells do not completely explain the degree of resistance to VP16 (125-200), indicating that other factors may also be involved
in resistance to VP-16 in this multidrug-resistant MCF-7 cell
line.
MATERIALS
AND METHODS
VP-16,213 (NSC 1415140) was a gift from Bristol-Myers Co. (Syr
acuse, NY) and was dissolved in dimethyl sulfoxide immediately before
experiments. Control cells were treated with equivalent concentrations
of dimethyl sulfoxide. [3H]VP-16 labeled in the aromatic rings only
(400 mCi/mmol; >99% pure) was obtained from Moravek Biochemicals, Inc., Brea, CA. [14C]Thymidine (56 mCi/mmol), and [3H]thymidine (80.9 mCi/mol) were obtained from New England Nuclear, Bos
ton, MA. SV40 and \Hindlll DNAs; Banl, EcoRl, Hpall restriction
endonucleases; T4 polynucleotide kinase; and agarose were purchased
from Bethesda Research Laboratories (Gaithersburg, MD). Phosphatase was purchased from New England Biolabs (Beverly, MA) and [732P]ATP from New England Nuclear Research Products (Boston, MA).
XAR-5 films (Eastman Kodak Co., Rochester, NY) were used for
autoradiography.
The human breast tumor (MCF-7) WT and ADR" cells (kindly
provided by Dr. Ken Cowan) were grown in monolayer in exponential
growth phase in IMEM supplemented with 2 mM glutamine, gentatnicin (100 units/ml; 10 ml/liter) and 5% fetal bovine serum (Grand Island
Biological Co., Grand Island, NY) under an atmosphere containing 5%
CÛ2.The selection of the resistant cells was achieved by treating WT
cells with stepwise increasing concentrations of Adriamycin (Drug
Development Branch, National Cancer Institute, NIH, Bethesda, MD)
(20,21).
VP-16-induced cytotoxicity was determined by either colony forma
tion (drug treatment for 1 and 2 h) or growth inhibition assay (contin
uous exposure). The cells were trypsinized, plated (15,000 cells/3 ml
5096
Received 10/21/87; revised 3/1/88, 6/7/88; accepted 6/14/88.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' To whom requests for reprints should be addressed, at Bldg. 10, Room 6N119, National Cancer Institute, NIH, Bethesda, MD 20892.
' Present address: Department of Radiation and Clinical Oncology, Hadassah
University Hospital, Jerusalem, Israel 91120.
3 The abbreviations used are: VP-16, etoposide (VP-16, 213); topo II, topoi
somerase II; ADR2, Adriamycin resistant; NB, nucleus buffer (150 mM NaCl-1
mM KHjPO.,-5 min MgCI¡-l min ethyleneglycol bis(/3-aminoethyl ether)/WVyV'JV'-tetraacetic acid-0.1 mM dithiothreitol, pH 6.4).
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VP-16-INDUCED DNA DAMAGE IN HUMAN TUMOR CELLS
for WT and 30,000 cells/3 ml for ADR" for continuous exposure and
450 cells/2 ml for WT and 500 cells/2 ml for ADR" for the clonogenic
assay in triplicate) in six-well Linbro dishes, and allowed to attach for
18 h at 37°C.The drug was removed following exposure (1 or 2 h), and
cells were allowed to grow for 12-14 days. Colonies were stained with
0.5% méthylène
blue in 50% methanol and counted. The plating
efficiencies of the untreated cells were 20-25% for both WT and ADR"
cells and were used to normalize for VP-16-induced cytotoxicity.
For uptake studies, WT and ADRR cells were grown to 50-70%
confluency and treated with 10 ^M VP-16 containing 0.2 nC\/m\ [3H]VP-16. The cells were incubated at 37°C.All uptake studies were
carried out similarly to those described by Schilsky et al. (24). The
uptake studies with different concentrations of VP-16 were similarly
carried out and that the incubation time was 60 min. Efflux experiments
were carried out following 60-min incubations with VP-16. The drug
was removed by washing the cells three times with ice-cold buffer and
incubating the cells in fresh drug-free medium at 37°Cfor the indicated
times.
For the determination of the drug-induced DNA breaks in the WT
and ADRR MCF-7 cells, the alkaline elution technique was used as
described by Kohn et al. (25). Briefly, MCF-7 cells were seeded (2.5 x
10" cells/150 cm2 for WT and 3 x 106/150 cm2 for ADRR) and labeled
with [14C]thymidine (0.02 ¿iCi/ml)for 24 h. The medium was then
changed, and the cells were grown in fresh medium for an additional
24 h to allow the incorporation of the label into high molecular weight
DNA. The cells were then exposed to different concentrations of VP16 for 1 h. The drug was removed by washing the cells three times with
excess Hanks' balanced salt solution at 0°C.Cells were scraped in
Hanks' balanced salt solution. Aliquots were mixed with [3H]thymidinelabeled internal standard LI210 cells that had been irradiated with
1500 rails and served as internal standards as described previously (25).
Nuclei from the [14C]thymidine-labeled MCF-7 cells were isolated
from 10s cells as described previously (26). Pelleted cells were sus
pended in NB at 4°Cand the cells were washed with NB and resus-
gels were autoradiographed with Kodak XAR-5 films (28).
Autoradiography films were scanned with a DU-8B Beckman spectrophotometer interfaced with a computer for data collection and
analysis. The intensity of topo H-mediated DNA cleavage at any given
site was computed by calculating the area (absorbance x distance) under
selected peaks. Comparison of topo 11mediated cleavage produced by
nuclear extracts from LI210, MCF-7, and MCF-7/ADR" cells was
done by plotting the corresponding
extract concentrations.
DNA cleavage areas at various
RESULTS
The relative cytotoxic effects of VP-16 to WT and ADRR
cells under different exposure conditions, e.g., l h and during
continuous exposures, are shown in Fig. 1, and the relative
x50% inhibitory concentration and the resistance index ob
tained under various conditions are summarized in Table 1.
These data clearly show that the ADRR cell line is highly
resistant to VP-16 and, that the VP-16-induced cytotoxicity
increases with the time of drug exposure.
Since multidrug-resistant cells in general have transport de
fects (29, 30) which may explain the differential cytotoxic
effects of the drug, we examined the cellular accumulation of
VP-16 in these two cell lines. As shown in Fig. 2A, the steady
state concentration of VP-16 was significantly higher (2-3-fold)
in the WT cells than in the ADRR cells. The uptake of VP-16
in both cell lines was rapid and linear up to 5 min.
The efflux of VP-16 from WT and ADRR cells with time was
100
pended in the buffer. Nuclei were obtained by incubating the cells in
0.27% Triton X-100 for 10 min at 4°C.The nuclear pellets were then
isolated by centrifugation. Drug treatments of isolated nuclei were for
30 min at 37"C. VP-16 was removed by diluting the nuclei suspensions
(20-fold) in NB at O'C. [3H]Thymidine internal standard LI210 cells
in NB at 0°Cwere then mixed with the l4C-labeled nuclei and alkaline
elution was performed as described above.
Nuclear extracts from the MCF-7 cells were prepared as described
previously (27). All extractions were performed at 4. The nuclei pellets
were suspended in NB containing 0.35 M NaCl (final concentration).
The salt extraction was carried out by gentle rotation for 30 min. The
nuclei were then spun down at 1800 rpm for 20 min. The supernatants
were collected and recentrifuged for 10 min to remove any nuclei or
insoluble materials.
DNA topo II was purified from leukemia (L1210) cell nuclei, as
described previously (16). End-labeling of SV40 DNA was carried out
as follows: (a) native supercoiled SV40 DNA was first linearized by
cutting at the Ban\ restriction site; (b) the 5'-DNA termini were then
dephosphorylated with calf alkaline phosphatase and labeled with [732P]ATP in the presence of polynucleotide kinase; and (c) finally, a
ADRR
10
1
g
IO-8
10-'
10-6
10-5
10-4
10-3
VP-16, (M)
second cut was introduced with Hpall. This second cut being at ap
Fig. 1. Relative survival of human breast cancer (MCF-7) WT and ADR" cells
proximately 50 base pairs from the labeling site, any fragment bigger
following exposures to various concentrations of VP-16. The cells were either
than 50 base pairs could arise only from the longer fragment.
exposed to VP-16 for 1 h for the clonogenic assay (•.A) or continuously for the
DNA cleavage reactions were carried out by reacting 32P-end labeled
growth inhibition assay (•,D).
DNA with purified topo II or nuclear extracts in 0.01 M Tris-HCl (pH
7.5)-0.05 M KC1-5 mM MgCI2-0.1 mM EDTA containing 15 /ig/ml
Table 1 Relative cytotoxicity of VP-16 in sensitive (WT) and resistant (ADR")
bovine serum albumin for 30 min at 37°C.Reactions were performed
MCF-7 cells
in 40-fil volumes and stopped by adding sodium dodecyl sulfate (1%
OIM)ADR"600
Exposure
final concentration). Proteinase K (0.5 mg/ml) was then added and the
index'120
reaction mixtures were incubated for an additional 30 min at 50°C.
time(h)12
The DNA was extracted with phenohchloroform (1:1). Loading buffer
was added to each sample, which was then heated at 65°Cfor 2 min
before being loaded into 1% agarose gels in Tris-borate-EDTA buffer.
32P-end labeled fragments of XHindlll EcoRl DNA were used as DNA
markers and were run in the same gels. Gels were run at 2-3 v/cm
overnight and then dried on a No. 3MM paper with a lyophilizer. Dried
170
600
ContinuousWT53.5 0.08IC,o°
20Resistance
250
" ICso, VP-16 concentration required for 50% cell kill as determined
by
clonogenic assay (1 and 2 hr treatment) or 50% growth inhibition (continuous
exposure).
* IC50 ADR" cells/IC» WT cells.
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VP-16-INDUCED
DNA DAMAGE IN HUMAN TUMOR CELLS
2000
2 5
10
Fig. 2. (A) Time course of VP-16 accumulation and retention in WT and ADR" MCF-7 cells. The cells were incubated at 37'C with 10 >iMVP-16 containing 0.2
»iCi/ml[3H)VP-16. At the end of the incubation times, cells were washed three times with ice-cold PBS and dissolved in l N NaOH. The protein content and the
radioactivity were determined. Arrow, beginning of the time course of the drug efflux. The cells were incubated with the drug for 60 min as in the uptake experiments,
washed and placed in the drug-free medium. Bars, SE. (B) Steady state VP-16 intracellular concentration as a function of extracellular drug concentrations in the
MCF-7 WT and ADR" cells. The cells were incubated with VP-16 for 60 min. All points represent the mean of at least 3 different experiments.
rapid and most of the drug was released within 10-15 min.
However, a small fraction (5-10%) of VP-16 remained associ
ated with the cells even after 60 min and may represent an
irreversibly bound fraction of the drug and/or metabolites.
Cellular accumulation of VP-16 was found to be linear as a
function of VP-16 concentrations, up to 100 MM,and at steady
state, a 2-3-fold difference in drug accumulation was observed
between the two cell lines at all drug concentrations examined
(Fig. 2B).
Small differences (2-3-fold) in the net cellular accumulation
of VP-16 in these two cell lines do not appear to account for
the large difference (200-fold) in the cytotoxicity and resistance
to VP-16. Since it is possible that cytotoxicity of VP-16 results
from topo II-mediated DNA cleavage, we compared the pro
duction of DNA breaks by VP-16 in the WT and ADRR cells.
As shown in Fig. 3, very few DNA strand breaks were produced
in the ADRR cells, even at VP-16 concentrations up to 500 MM.
In contrast, a significant amount of DNA breaks were produced
by low concentrations of VP-16 in WT cells. DNA singlestrand breaking frequency in rad equivalents as a function of
drug concentration in the two cell lines is summarized in Fig.
3A.
Experiments were then carried out in isolated nuclei to in
vestigate further the possibility that any drug transport or
activation defect could be responsible for differential VP-164000
induced DNA single-strand breaking activity in the intact cells.
VP-16 treatment were performed for only 30 min because
longer incubation resulted in the appearance of endogenous
DNA breaks in the untreated nuclei which rendered the analysis
of VP-induced breaks less clear. The data are presented in Fig.
3B. The difference between isolated nuclei from WT and ADRR
cells was much smaller than that observed in the intact cells. It
is noteworthy that VP-16 caused significantly less DNA breaks
in isolated nuclei from WT cells than in the intact cells while
more DNA breaks were detected in isolated nuclei from the
ADRR cells than from the intact ADRR cells. A comparison of
the data in intact cells and nuclei is presented in Table 2, which
shows that VP-16 is 2-3-fold less active in isolated nuclei from
ADRR cells than in isolated nuclei from WT cells. These results
suggest that nuclear modifications in the resistant MCF-7 cells
are partially responsible for VP-16 resistance.
In order to examine further whether the reduced formation
of VP-16-induced DNA breaks in isolated nuclei from ADRR
cells was due to altered topo II activity, nuclear extracts from
both cell lines were compared. Increasing concentrations of
nuclear extracts were then reacted with "P-end-labeled SV40
DNA in the presence of 50 MM VP-16 (Fig. 4). The DNAbreaking activity of these nuclear extracts was compared to
those of purified LI210 topo II and LI210 nuclear extracts.
The DNA cleavage patterns induced by VP-16 in the presence
of nuclear extracts from LI210, WT, and ADR" cells were
similar and were comparable to that induced in the presence of
purified LI210 topo U. L1210 nuclear extracts, however, were
more potent than the WT nuclear extracts (Fig. 4). The ADRR
nuclear extracts were less sensitive to VP-16 than WT nuclear
extracts suggesting that ADRR cells had lower VP-16-sensitive
topo II activity.
Autoradiography films corresponding to Fig. 4 and additional
experiments performed with 0.5-Mg nuclear extracts (12.5 Mg/
Table 2 VP-16 concentration (fiM) required to produce 1000 rad equivalent single
strand breaks in the intact MCF-7 cells and isolated nuclei
500
O
100
200
The drug exposure times were 1 and 0.5 h for the intact cells and the nuclei,
respectively. The data are extrapolated from Fig. 4.
600
VP-16 CONCENTRATION IMMI
Fig. 3. DNA single strand breaks produced in WT and ADR" cells (A) and in
nuclei isolated from these cells (B). Cells were incubated with the indicated drug
concentrations for l h and the isolated nuclei were incubated for 30 min.
WT cells
ADR" cells
Cells
Nuclei
3.5
>500
215
425
5098
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VP-16-INDUCED
C?
*
/
y.
///
DNA DAMAGE IN HUMAN TUMOR CELLS
y.
5.0
10.0
NUCLEAR EXTRACT (ng/ml)
Fig. 5. Topoisomerase II-mediated double strand breaks produced by nuclear
extracts from LI210 (A), WT (•),and ADR" (D) cells in the presence of 50 /IM
VP-16 in "P-end labeled SV40 DNA. Autoradiography films (as described in
Fig. 4) were scanned and DNA cleavage was determined at selected sites by
computing areas [absorbance x distance].
Fig. 4. Double strand breaks induced by VP-16 in the presence of WT and
ADR* MCF-7 cell nuclear extracts in SV40 DNA. "P-end-labeled SV40 DNA
(control SV40 DNA) was reacted with purified LI210 topo II in the absence or
presence of VP-16 (50 <JM)([topo II] and [topo II + VP-16] lanes, respectively).
The indicated amounts of nuclear extract were reacted with DNA in the presence
or absence of VP-16. Reactions were stopped after 30 min incubation at 37'C by
adding 1% sodium dodecyl sulfate and 0.5 mg/ml proteinase K (tinnì
concentra
tion). After an additional 30-min incubation at 50'C, the DNA was extracted
with phenol-chloroform and loaded into a 1% agarose gel in Tris-borate EDTA
buffer. The gel was dried and autoradiography was performed. "P-end-labeled
Hindlll EcoRI restriction fragments of A were used as DNA markers. The size of
these markers in base pairs is: 564, 831, 983, 1584, 1904, 2027, 3530, 4277, and
4973 (bottom to top).
ml) were used to quantify topo II mediated DNA cleavage
produced in the presence of 50 ^M VP-16 at several DNA
cleavage sites and is summarized in Fig. 5. Topo II-mediated
DNA cleavage was greater with L1210 nuclear extract than
with nuclear extract from WT and ADRR cells. WT nuclear
extract was approximately twice more potent than the ADRR
extract. These results are in good agreement with those obtained
with isolated nuclei (Fig. 4B) and indicate a 2-fold resistance
of topo II from ADRR cells to VP-16.
DISCUSSION
Several investigators have reported that topo II-catalyzed
DNA strand breaks are responsible for the antitumor activity
of VP-16 (2,3,5-7). Cellular resistance to VP-16 and antitumor
DNA intercalators, is accompanied by decreased drug-induced
topo II-mediated DNA-cleaving activity in both intact cells and
isolated nuclei (17-19). The results presented here show that
MCF-7 cells selected for resistance to Adriamycin are also
significantly resistant to VP-16. In the resistant cell, there is a
marked decrease in VP-16-induced DNA single strand breaks
and this decrease (in single strand breaks) (100-200-fold) is in
good agreement with the degree of resistance observed for VP16 in this cell line. Furthermore, there is a good correlation
with the topo II-mediated DNA cleavage and VP-16-induced
cytotoxicity. The decrease in DNA strand breaks was also
observed in isolated nuclei of the ADRR cells compared to WT
cells. The decreased formation of VP-16-induced DNA single
strand breaks observed in the ADRR cells appears to be related
partially (2-3-fold) to a decrease in the nuclear activity of topo
II in the ADRR cells. Topo II modifications have also been
documented with topo H-active drugs in other resistant cell
lines (17-19, 28, 31). However, our observation in MCF-7/
ADRR cells is unique because this cell line has been character
ized as typical pleotropic cells with overexpression of the M,
170,000 protein.
Resistance to VP-16 in MCF-7 cells was also accompanied
by a 2-3-fold decrease in the net cellular accumulation of VP16 in the ADRR cells. Thus, the development of VP-16 resist
ance in the multidrug-resistant MCF-7 cells is, in part, due to
defects in the transport of VP-16 in the resistant cells and
alteration in the topo II activity resulting in decreased formation
of DNA strand breaks. These two observations, i.e., a 2-3-fold
decrease in cellular accumulation for VP-16 and 2-3-fold de
crease in VP-16-induced topo II-mediated DNA cleavage activ
ity, taken together, however, are not sufficient to explain 125200-fold resistance in MCF-7 cells. Thus, it appears that other
biochemical mechanisms contribute to the development of re
sistance to VP-16 ADRR cells. Furthermore, the findings that
the nuclei prepared from WT cells formed significantly less
VP-16-induced DNA breaks than the intact cells would suggest
that topo II activity (including its activation) was lost selectively
during the isolation of nuclei from WT cells. Similar observa
tions have been described previously in other cell lines (7, 26).
It is also possible that the intracellular distribution of VP-16 is
different in the resistance cells or that some other factors in the
cytosol of the WT cells were necessary for the VP-16-induced
DNA strand-breaking activity.
The biochemical nature of the "necessary factors" is under
study at the present time. It is possible to postulate that VP-16
is enzymatically activated by cytoplasmic enzymes to a reactive
o-quinone derivative or a quinone methide which binds irre
versibly to proteins and DNA (12-13, 15, 32). This activation
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VP-16-INDUCED
DNA DAMAGE IN HUMAN TUMOR CELLS
of the drug has been shown to be catalyzed by cytochrome P450. Of interest is our previous finding that the ADRR have
very low levels of inducible P-450-dependent drug-metabolizing
enzyme activity compared to the WT cell line (20). This would
suggest that in the WT MCF-7 cell line, but not in the resistant
cell line, the P-450-dependent metabolism of xenobiotics and
VP-16 is likely. Thus, it is possible that VP-16 is activated to a
reactive species that may then alkylate DNA and proteins,
including topoisomerase II which may contain functional
sulfhydryl groups at the active site(s). Therefore, it is likely that
during the isolation of the nuclear fraction the cytosolic or
endoplasmic reticulum activating factors are lost from the WT
cells, thereby decreasing the DNA strand-breaking activity in
the nuclei or nuclear extracts compared to the intact cells.
The decreased DNA strand breaks and therefore the resist
ance to VP-16 observed in the ADRR cells may, in part, be due
to the inability of the ADRR cells to activate the drug to reactive
intermediates. It is also likely that reactive VP-16-derived spe
cies are rapidly detoxified in the ADRR cells since ADRR cells
show a significant increase in both glutathione peroxidase and
glutathione transferase activities (20, 21). The function of these
two enzymes is to detoxify organic free radicals and reactive
alkylating species. The fact that ADRR cells are also resistant
to benzo(fl)pyrene (20) is consistent with the activation/detox
ification hypothesis.
In conclusion, the association of decreased drug uptake,
decreased formation of topo Il-mediated drug-induced DNA
strand breaks as a result of alteration of topo II, and lack of
drug activation and or increased detoxification of the reactive
intermediates in the resistant cell line may have broad impli
cations in the phenomenon of multidrug resistance.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
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Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1988 American Association for Cancer Research.
DNA Strand Breaks Produced by Etoposide (VP-16,213) in
Sensitive and Resistant Human Breast Tumor Cells:
Implications for the Mechanism of Action
Birandra K. Sinha, Nissim Haim, Lata Dusre, et al.
Cancer Res 1988;48:5096-5100.
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