(CANCER RESEARCH 50, 2872-2876, May 15, 1990]
Activity of Quinone Alkylating Agents in Quinone-resistant Cells1
Asher Begleiter2 and Marsha K. Leith
Departments of Internal Medicine [A. B., M. K. LJ and Pharmacology and Therapeutics [A. B.J, University of Manitoba, and Manitoba Institute of Cell Biology [A. B.,
M. K. LJ, 100 Olivia Street, Winnipeg, Manitoba R3E OV9, Canada
ating activity of these agents. Studies have shown that the
alkylating activity of MMC is dependent on reduction of the
The role of the quinone group in the antitumor activity of quinone
quinone group (2, 5-9). Similarly, AZQ was shown to produce
alkylating agents, such as mitomycin C and 2,5-diaziridinyl-3,5DNA cross-links only after reduction (4, 10).
bis(carboethoxyamino)-l,4-benzoquinone, is still uncertain. The quinone
We have previously studied the activity of model quinone
group may contribute to antitumor activity by inducing DNA strand
alkylating
agents in LSI78Y lymphoblasts. BM and BDM
breaks through the formation of free radicals and/or by influencing the
showed significantly greater potency in L5178Y cells compared
alkylating activity of the quinone alkylators. The cytotoxic activity and
DNA damage produced by the model quinone alkylating agents, benzo- with the nonquinone alkylating agent, AM (11, 12). The qui
none agents induced both DNA strand breaks and DNA-DNA
quinone mustard and benzoquinone dimustard, were compared in LSI78Y
murine lymphoblasts sensitive and resistant to the model quinone anticross-links in L5178Y cells, and both their cytotoxic activity
tumor agent, hydrolyzed benzoquinone mustard. The resistant cell lines,
and strand break activity could be partially inhibited by the
L5178Y/HBM2 and L5178Y/HBM10, have increased concentrations of protective enzyme, catalase (11-13). Studies with these model
glutathione and elevated catalase, Superoxide dismutase, glutathione .Vcompounds in L5178Y cells resistant to alkylating agents also
transferase, and DT-diaphorase activity. L5178Y/HBM2 and L5178Y/
indicated a significant role for DNA-DNA cross-linking in the
HBM10 cells were 7.4- and 8.5-fold less sensitive to benzoquinone
cytotoxic
activity of BM and BDM (14).
mustard and 1.7- and 4.3-fold less sensitive to benzoquinone dimustard,
We have isolated and characterized LSI78Y cell lines resist
respectively, compared with sensitive cells, but showed no resistance to
ant to the nonalkylating quinone model compound, HBM (15).
the non-quinone alkylating agent, aniline mustard. The formation of DNA
double strand breaks by benzoquinone mustard was reduced by 2- and 8Resistance in these cell lines appeared to be multifactorial.
fold in L5178Y/HBM2 and L5178Y/HBM10 cells, respectively, while L5178Y/HBM2 cells, which were 2.5-fold resistant to HBM,
double strand break formation by benzoquinone dimustard was reduced
showed reduced drug uptake, had slightly elevated levels of
only in the L5178Y/HBM10 cells. The number of DNA-DNA cross
Superoxide dismutase and catalase activity, a 2-fold increase in
links produced by benzoquinone mustard was 3- and 6-fold lower, and
intracellular glutathione, and a 3-fold increase in GST and DTthe number produced by benzoquinone dimustard was 35% and 2-fold
diaphorase activity compared with L5178Y sensitive cells.
lower in L5178Y/HBM2 and L5178Y/HBM10 cells, respectively, com
L5178Y/HBM10 cells, which were 6-fold resistant to HBM
pared with L5178Y parental cells. In contrast, cross-linking by aniline
compared
with sensitive cells, showed reduced drug uptake and
mustard was unchanged in sensitive and resistant cells. Dicoumarol, an
similar increases in Superoxide dismutase activity and glutathi
inhibitor of DT-diaphorase, increased the cytotoxic activity of both
one levels. However, the L5178Y/HBM10 cells also had a 3benzoquinone mustard and benzoquinone dimustard in L5178Y/HBM10
fold increase in catalase activity, an 11-fold increase in GST
cells. This study provides evidence that elevated DT-diaphorase activity
activity, and a 24-fold increase in DT-diaphorase activity. HBM
in the resistant cells contributes to resistance to benzoquinone mustard
and benzoquinone dimustard, possibly by decreasing the formation of the
produced significantly reduced levels of DNA strand breaks in
semiquinone intermediates of these agents. The altered reduction of the
the resistant cells.
quinone groups in the resistant cells may be responsible for the decreased
In the present study we have examined the activity of the
DNA-DNA cross-linking and lowered induction of DNA strand breaks
model quinone alkylating agents, BM and BDM, in the quiby the quinone alkylating agents. These findings demonstrate that the
none-resistant cell lines in order to evaluate the role of the
quinone group can modulate the activity of quinone alkylating agents.
quinone group in the cytotoxic activity of these agents.
The study also suggests that the semiquinone intermediates of benzoqui
ABSTRACT
none mustard and benzoquinone dimustard may be the active alkylating
species of these two agents.
MATERIALS
INTRODUCTION
Antitumor agents such as MMC,1 AZQ, and triaziquone
contain both a quinone ring and alkylating groups in their
structure. It has been suggested that the quinone group may
play a role in the cytotoxic activity of these agents. Reductionoxidation reactions of the quinone group may lead to the
formation of oxygen free radicals which can induce DNA dam
age (1), and this has been observed with MMC (2, 3) and AZQ
(4). Additionally, the quinone group may influence the alkylReceived 4/26/88; revised 12/27/89.
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.
1Supported by the Medical Research Council of Canada.
2To whom requests for reprints should be addressed, at Manitoba Institute of
Cell Biology, 100 Olivia Street, Winnipeg. Manitoba R3E OV9. Canada.
3The abbreviations used are: MMC, mitomycin C; AZQ, 2.5-diaziridinyl-3,6bis(carboethoxyamino)-l,4-benzoquinone:
BM, benzoquinone mustard; BDM,
benzoquinone dimustard; AM. aniline mustard; HBM, hydrolyzed benzoquinone
mustard; GST. glutathione 5-transferase; DIO,concentration of drug reducing the
surviving cell fraction to 0.1.
AND METHODS
Materials. Benzoquinone mustard [di(2'-chloroethyl)amino-l,4-benzoquinone), benzoquinone dimustard |2,5-bis[di(2'-chloroethyl)amino]-l,4-benzoquinone|,
aniline mustard [A',A'-di(2'-chloroethyl)aniline], and hydrolyzed benzoquinone mustard (di(2'-hydroxymethyl)
amino-1,4-benzoquinone] were prepared as described previously (11,
13). Fischer's medium and horse serum were obtained from Grand
Island Biological Co., Grand Island, NY. ['4C)Thymidine (specific
activity, 50 mCi/mmol) and ['Hjthymidine (specific activity, 50 to 80
Ci/mmol) were obtained from Du Pont-New England Nuclear, Boston.
MA. Proteinase K was from E. Merck, Darmstadt, Federal Republic
of Germany; tetrapropylammonium hydroxide was from Eastman Ko
dak Co., Rochester, NY; polycarbonate filters (0.8 ^m and 2.0 Mâ„¢)
were from Nucleopore Corp., Pleasanton, CA; and dicoumarol was
from Sigma Chemical Co., St. Louis, MO.
Cell Lines. The L5178Y, L5178Y/HBM2, and L5178Y/HBM10 cell
lines used in this study have been described previously (15). L5178Y
cells were grown in Fischer's medium containing 12% horse serum,
L5178Y/HBM2 cells were grown in Fischer's medium containing 12%
horse serum and 0.2 mM HBM, and L5178Y/HBM10 cells were grown
in Fischer's medium containing 12% horse serum and 1.0 mM HBM.
Cytotoxicity Assays. L5178Y parental or resistant cells were incu-
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QUINONE ALKYLATORS IN QUINONE-RESISTANT
bated with different concentrations of the model antitumor agents for
l h at 37°Cin medium containing horse serum. Cytotoxic activity in
the parental and resistant cell lines was determined by a soft agar
cloning assay, as described previously (11, 16). Cloning efficiencies
ranged from 35 to 66% for L5178Y parental cells, from 51 to 90% for
L5178Y/HBM2 cells, and from 37 to 80% for L5178Y/HBM10 cells.
A linear regression analysis of each concentration-survival curve was
obtained, and the D,0 was derived from the negative reciprocal of the
regression slope as previously described (17). The degree of resistance
of the L5178Y/HBM2 and L5178Y/HBM10 cells was determined
from the ratio of the £>,o
value in the resistant cells to the D,a in the
parental cells. The cytotoxicity of each agent in the sensitive and
resistant cell lines was compared statistically by a i test comparing the
significance of the differences of the slopes of the concentration-survival
curves.
For inhibition studies with dicoumarol, L5178Y or L5178Y/HBM10
cells were pretreated with or without 100 MMdicoumarol for 15 min.
L5178Y cells were then treated with either 30 nM BM or 3 MMBDM,
while L5178Y/HBM10 cells were treated with either 0.5 MMBM or 10
MMBDM at 37°Cfor 1 h. Cytotoxicity was determined by clonogenic
assay. The results were analyzed statistically by a two-tailed / test
comparing the significance of the difference of the mean surviving cell
fraction in the absence or presence of dicoumarol. The concentration
of dicoumarol used in this study was not toxic to the cells.
Determination of DNA Double Strand Breaks. Cells labeled with
[14C]-thymidine were treated with various concentrations of HBM, BM,
or BDM for l h at 37°Cin medium containing horse serum. DNA
CELLS
treated with 30 nM BM or 3 nM BDM in the presence or
absence of 100 pM dicoumarol. Although there was a slight
increase in the cytotoxicity of both BM and BDM in the
presence of dicoumarol, this effect was not statistically signifi
cant. The concentration of BM and BDM used in these studies
produced similar levels of cell kill in LSI78Y and L5178Y/
HBM 10 cells in the absence of dicoumarol.
Induction of DNA Double Strand Breaks by Quinone Model
Compounds in L5178Y Sensitive and Resistant Cells. L5178Y
sensitive and resistant cells were incubated with various concen
trations of HBM, BM, or BDM at 37°Cfor 1 h. The number
of DNA double strand breaks induced in the L5178Y, L5178Y/
HBM2, and L5178Y/HBM10 cells was determined by elution
assay (Fig. 3). The induction of double strand breaks by HBM
was reduced by 2-fold in L5178Y/HBM2 cells and by 30-fold
in L5178Y/HBM10 cells compared with the level found in the
sensitive cells, and the latter effect was statistically significant
(P < 0.01). With BM, the level of DNA double strand breaks
was reduced by 2-fold and 8-fold in L5178Y/HBM2 and
L5178Y/HBM10 cells, respectively, compared with parental
cells, and again the latter result was statistically significant.
However, DNA double strand break formation by BDM was
not significantly different in L5178Y and L5178Y/HBM2 cells,
but was significantly reduced by only 40% to minimum detect
able levels in L5178Y/HBM10 cells (P < 0.01).
double strand breaks were measured using an elution assay as described
Production of DNA-DNA Cross-Links by Model Alkylating
previously (13, 18, 19). The level of DNA strand breaks was calculated
Agents
in LSI78Y Sensitive and Resistant Cells. LSI78Y sen
from the elution profiles and was expressed as rad equivalents (dose of
sitive and resistant cells were treated with various concentra
radiation inducing an equivalent number of breaks) as determined from
tions of BM, BDM, or AM at 37"C for 1 h, and the number of
calibration curves. A linear regression analysis of each concentrationDNA-DNA cross-links produced in the L5178Y, L5178Y/
activity curve was obtained, and the strand-breaking activity of each
agent in the sensitive and resistant cell lines was compared statistically
HBM2, and L5178Y/HBM10 cells was determined by elution
by a t test comparing the significance of the difference of the slopes of assay (Fig. 4). The production of cross-links by BM was reduced
the concentration-activity curves.
by 3-fold and 6-fold in L5178Y/HBM2 and L5178Y/HBM10
Determination of DNA-DNA Cross-Linking. Cells labeled with [14CJcells, respectively, compared with sensitive cells, while cross
thymidine were treated with various concentrations of BM, BDM, or
link formation by BDM was 35% and 2-fold lower in these
AM for l h at 37°Cin medium containing horse serum. DNA-DNA
cells,
respectively, compared with L5178Y cells. These effects
cross-links were measured using an elution assay as described previously
were statistically significant (P< 0.01). In contrast, the number
(13. 20). The level of cross-linking was calculated as described by Kohn
of cross-links produced by AM in sensitive and resistant cells
et al. (20). A linear regression analysis of each concentration-activity
cune was obtained, and the cross-linking activity of each agent in the
was not significantly different.
sensitive and resistant cell lines was compared statistically by a I test
comparing the significance of the difference of the slopes of the con
centration-activity curves.
RESULTS
Cytotoxicity of Model Compounds in LSI78Y Sensitive and
Resistant Cells. The cytotoxic activities of the model antitumor
agents, HBM, BM. BDM, and AM, in parental L5178Y cells
and in the resistant cells, L5178Y/HBM2
and L5178Y/
HBM 10, were determined by a soft agar cloning assay after
treatment of cells with drug for l h at 37°C(Fig. 1). A compar
ison of the D10values (Table 1) shows that the L5178Y/HBM2
and L5178Y/HBM10 cells were 2.5- and 6.2-fold resistant to
HBM, 7.4- and 8.5-fold resistant to BM, and 1.7- and 4.3-fold
resistant to BDM, respectively, compared with parental
L5178Y cells. In contrast, the resistant cells showed no crossresistance to the nonquinone model agent, AM.
Effect of Dicoumarol on the Cytotoxicity of Model Compounds
in L5178Y Sensitive and Resistant Cells. L5178Y/HBM10cells
were treated with 0.5 //M BM or 10 ^M BDM in the presence
or absence of 100 ^M dicoumarol. The presence of dicoumarol
significantly increased the cytotoxicity of both BM and BDM
in L5178Y/HBM10 cells (Fig. 2), but the level of resistance to
these agents was only partially reversed. L5178Y cells were
DISCUSSION
The quinone ring may play an important role in the cytotoxic
activity of a number of antitumor agents. Reduction-oxidation
reactions of the quinone group can lead to the formation of
oxygen free radicals and active oxygen species (1) which may
contribute to the antitumor activity (3, 21) and cardiotoxicity
of the anthracyclines (22-25) and to the antitumor action of
MMC (2, 3), AZQ (4), and streptonigrin (26). In addition, the
quinone group may influence the alkylating activity of alkylating quiñones,such as MMC (2, 5-9) and AZQ (4, 10), which
are activated by reduction of the quinone. In previous studies
with model compounds we have shown that the model quinone
alkylating agents, BM and BDM, show increased cytotoxicity
and cross-linking activity in L5178Y lymphoblasts compared
with a nonquinone alkylating agent, AM, and increased cyto
toxicity and DNA double strand break formation compared
with HBM (12). Additional work in alkylator-resistant cells
indicated an important role for cross-linking in the cytotoxic
activity of BM and BDM and suggested that the enhanced
DNA strand break formation by BM and BDM may be due to
the ability of these agents to generate free radicals close to their
target by binding to DNA (14).
We have isolated and characterized L5178Y cell lines resist-
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QUINONE ALKYLATORS IN QUINONE-RESISTANT
CELLS
Fig. 1. Concentration-survival
curves for
LSI78Y parental and resistant cells treated with
model antitumor agents. LSI78V parental cells
(•),L5178V/HBM2 cells (•).or L5178Y/
HBM10 cells (A) were incubated at 37°Cfor 1
h with HUM, BM, BDM, or AM at the concen
trations shown. The surviving cell fraction was
determined by a clonogenic assay as described
in the text and is plotted against the concentra
tion of drug used. Points, mean of 5 to 7 quad
ruplicate determinations: bars. SE. On occasion,
the confidence intervals are too small to be
shown.
io0
5
10 0
[HYDROLYZEDBENZOOUINONE
MUSTARD](mm)
0.25
0.5 0
[BENZOOUINONE
MUSTARD](¿IM)
20 0
10
BENZOOUINONE
15
ANILINEMUSTARD]
DIMUSTARD](,uM)
Table I Cytotoxic activity of model antitumor agents in 7.5778 Y sensitive and resistant cells
L5178Y, L5178Y/HBM2, or L5178Y/HBM10 cells were incubated with different concentrations of the model antitumor agents at 37'C for 1 h, and the surviving
cell fraction was determined by a clonogenic assay as described in the text. A linear regression analysis of each concentration-survival curve in Fig. 1 was obtained,
and the D\0 (mean ±SE) was derived from the negative reciprocal of the slope of the regression line. The cytotoxic activity of each model agent in the resistant cells
was compared with its cytotoxic activity in the sensitive cells by a 2-lailed t test, comparing the significance of the difference in the slopes of the linear regression
lines from the concentration-survival plots.
cells(0,0.
agentHydrolyzed
Model antitumor
benzoquinone mustard
Benzoquinone mustard
Benzoquinone dimustard
IO'5**P< Aniline mustardL5178Y
0.001.
* P = not significant.
M)0.56
±0.08 X
0.19 ±0.01 x
1.83±0.09x
1.20±0.08x
cells(0,0,
cells(0,o,10
M)1.37±0.06x
IO'3
IO'7
1.37 ±0.14 x
IO'6
3.14 ±0.12 x
IO'5L5178Y/HBM21.34±0.36x
M)3.48
10~w
IO"7"
10-M
10-'*L5178Y/HBM
±0.18 X 10-M
1.58 ±0.07 x IO-7"
7.86 ±1.31 x 10-"
1.22 ±0.18 x
HBM 10 cells are 6-fold resistant to this nonalkylating quinone
compared with parental cells. These cells may be resistant to
HBM because of reduced induction of DNA strand breaks
CONTROL
resulting from decreased formation and/or increased inactivaDICOUMAROL
tion of free radicals. Decreased formation of free radicals may
be due to reduced drug uptake or increased drug inactivation
-1
and extrusion resulting from elevated levels of glutathione and
10
GST activity (27). In addition, elevated levels of DT-diaphorase
may decrease the formation of the semiquinone radical of
HBM, and thus oxygen free radicals, by converting the quinone
agent directly to the hydroquinone (28). DNA strand breaks
may be further reduced in these cells by increased catalase and
Superoxide dismutase activity and elevated glutathione that can
-2
inactivate free radicals and hydrogen peroxide that are formed.
10
The present study provides evidence that the quinone group
plays an important role in the cytotoxic activity of quinone
alkylating agents. The quinone-resistant cell lines showed crossresistance to the quinone alkylating agents, BM and BDM, but
not to the nonquinone alkylating agent, AM. Since the
L5178Y/HBM2 and L5178Y/HBM10 cells did not demon
-3
strate any resistance to AM despite having elevated concentra
10
tions of glutathione and GST activity, it would appear that this
BENZOQUINONE
BENZOOUINONE
DIMUSTARD
MUSTARD
detoxification mechanism did not play a major role in resistance
Fig. 2. Effect of dicoumarol on cytotoxic activity of BM and BDM in L5I78Y'/
to alkylating agents in these cells.
HBM10 cells. L5178Y/HBM10 cells were preincubated with or without 100 Õ/M
Resistance mechanisms which can reduce the formation of
dicoumarol for 15 min at 37'C and then were incubated with either 0.5 ii\t BM
free radicals or inactivate these reactive species may contribute
or 10 iim BDM for 1 h. The surviving cell fractions were determined by a
clonogenic assay as described in the text and represent the mean of 4 to 11
to the resistance to BM and BDM. These include decreased
determinations. Bars, SE. For each quinone agent the results were evaluated
drug uptake; elevated GST, catalase, and Superoxide dismutase
statistically by 2-tailed r tests comparing the significance of the differences of the
means of cells treated with or without dicoumarol.
activity; and increased levels of glutathione. This suggestion
was supported by the finding that the formation of DNA double
ant to the model quinone antitumor agent, HBM (15). The strand breaks by BM and BDM was reduced in L5178Y/
HBM 10 cells. However, the induction of double strand breaks
cytotoxic activity of HBM appears to be due to the formation
of free radical-generated DNA strand breaks (11, 13). L5178Y/
by these agents is not significantly reduced in L5I78Y/HBM2
HBM2 cells are 2.5-fold resistant to HBM. while L5178Y/
cells, and the reduced formation of double strand breaks did
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QUINONE ALKYLATORS IN QUINONE-RESISTANT
CELLS
2000
Fig. 3. DNA double strand breaks induced
in L5178Y parental and resistant cells by
model quinone agents. L5178Y (•).LSI78V/
HBM2 (•).or L5178Y/HBM10 (A) cells were
incubated at 37°Cfor l h with the concentra
tions of HBM, BM, or BDM shown and were
assayed for DNA double strand breaks using
elution assays as described in the text. Double
strand breaks are presented as rad equivalents
(dose of radiation producing an equivalent
number of breaks) and represent the mean of
3 to 11 determinations. Bars, SE. For each
quinone agent, the results were evaluated sta
tistically by ( tests comparing the significance
of the differences of the slopes of the concen
tration-activity curves in the resistant cells to
the slope in the parental cells.
1500
1000
500
2
4 0
4
75
[HYDROLYZED BENZOQUINONE
[BENZOQUINONE
[BENZOQUINONE
MUSTARD] (mM)
MUSTARD] (¡M)
DIMUSTARD] (/4I)
150
600
Fig. 4. DNA-DNA cross-linking produced
by model alkylating agents in L5178Y parental
and resistant cells. L5178Y (•).L5178Y/
HBM2 (•).or L5178Y/HBM10 (A) cells were
incubated at 37°Cfor l h with the concentra
tions of BM, BDM. or AM shown and were
assayed for DNA-DNA cross-linking using
elution assays as described in the text. The
level of cross-linking is presented as rad equiv
alents and represents the mean of 3 to 7 deter
minations. Bars, SE. For each alkylating agent,
the results were evaluated statistically by t tests
comparing the significance of the differences
of the slopes of the concentration-activity
curves in the resistant cells to the slope in the
parental cells.
400
a:
o
ì
200
O
100
200
[ANILINE MUSTARD] (jM)
not correspond closely with the reduced sensitivity of the cells
to the antitumor agents. Thus, other mechanisms must also
contribute to resistance to BM and BDM in these cells.
There was a significant decrease in the production of DNADNA cross-links by BM and BDM in the L5178Y/HBM2 and
L5178Y/HBM10 cells, but cross-linking by AM was unchanged
in these cells compared with parental LSI78Y cells. This find
ing suggests that the decreased alkylating activity of the quinone
agents was not due to a direct effect on the nitrogen mustard
alkylating group. It has been shown previously that reduction
of the quinone group is required to activate the alkylating
activity of bioreductive alkylating agents, such as MMC and
AZQ (2, 8-10). In a similar manner, reduction of the quinone
group may enhance the cross-linking activity of BM and BDM.
Thus, mechanisms of resistance effecting reduction of the qui
none group may be responsible for the reduced cross-linking
observed in the L5178Y/HBM2 and L5178Y/HBM10 cells.
However, cytochrome P-450 reducÃ-ase activity, which is re
sponsible for the one electron reduction of quinone groups (29),
was unchanged in the resistant cells (15). In contrast, DTdiaphorase activity, which produces a two electron reduction of
the quinone directly to the hydroquinone (28), was 3- and 24-
O
0.1
0.2
[BENZOQUINONE
MUSTARD](pU)
0.3
O
20
40
[BENZOQUINONE
DIMUSTARD] (jM)
fold elevated in L5178Y/HBM2 and L5178Y/HBM10 cells
( 15), respectively, compared with parental cells. The importance
of DT-diaphorase in the mechanisms of resistance to the model
quinone agents in these cells was further supported by the
ability of dicoumarol, an inhibitor of DT-diaphorase activity,
to increase the cytotoxic activity of BM and BDM in the
resistant cells. The elevated DT-diaphorase activity in the re
sistant cells may result in increased reduction of BM and BDM
directly to their hydroquinone forms without formation of
semiquinone intermediates. These findings suggest that the
semiquinone intermediates, obtained by one electron reduction
of BM and BDM, may be responsible for the cytotoxic activity
of these antitumor agents. If semiquinones are the active alkyl
ating species of BM and BDM and are responsible for the
production of the DNA strand breaks by induction of oxygen
free radicals, then elevated levels of DT-diaphorase activity in
the resistant cell lines could decrease the formation of the
semiquinones and could, at least in part, account for the reduced
levels of cross-linking, double strand break formation, and
cytotoxicity of the quinone alkylating agents in these cells.
These findings contrast with results obtained with other
quinone alkylating agents. We have found that MMC produced
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QUINONE ALKYLATORS IN QUINONE-RESISTANT
increased cytotoxicity and DNA cross-linking in L5178Y/
HBM10 cells compared with parental L5178Y cells. Both of
these activities in L5178Y/HBM10 cells were decreased by the
addition of dicoumarol (30). Similarly, Siegel et al. (31) have
shown that AZQ was more cytotoxic to HT-29 human colon
carcinoma cells compared with BE human colon carcinoma
cells in vitro. HT-29 cells have approximately 140-fold greater
DT-diaphorase activity than do BE cells. Dicoumarol protected
against AZQ cytotoxicity in HT-29 cells but not in BE cells.
Thus, in contrast to BM and BDM, the hydroquinones of MMC
and AZQ, resulting from two electron reduction of the quinone
groups catalyzed by DT-diaphorase, may contribute to the
alkylating and cytotoxic activity of MMC and AZQ.
In summary, we have shown that L5178Y lymphoblasts
resistant to the model quinone antitumor agent, HBM, are
cross-resistant to the quinone alkylating agents, BM and BDM,
but not to the nonquinone alkylating agent, AM. Elevated levels
of DT-diaphorase activity in the resistant cells contribute to
resistance to BM and BDM, possibly by decreasing the forma
tion of the semiquinone intermediates of these agents. The
altered reduction of the quinone group may result in decreased
DNA-DNA cross-linking and lowered induction of DNA strand
breaks. Thus, the quinone group can modulate the activity of
quinone alkylating agents. These studies also suggest that the
semiquinone intermediates of BM and BDM may be the active
alkylating species of these two agents.
ACKNOWLEDGMENTS
The authors thank Agnes Warkentin for typing the manuscript.
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Activity of Quinone Alkylating Agents in Quinone-resistant Cells
Asher Begleiter and Marsha K. Leith
Cancer Res 1990;50:2872-2876.
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