Download Modulation of the Cell Cycle-dependent

[CANCER RESEARCH 52. 3515-3520. July I. I992|
Modulation of the Cell Cycle-dependent Cytotoxicity of Adriamycin and 4Hydroperoxycyclophosphamide by Novobiocin, an Inhibitor of Mammalian
Topoisomerase II1
Francis Y. F. Lee,2 Deborah J. Flannery, and Dietmar W. Siemann
Tumor Biology Division, university of Rochester Cancer Center, Rochester, New York 14642
ABSTRACT
MATERIALS AND METHODS
Centrifugal elutriation was used to obtain synchronized cell populations
in various cell cycle phases without prior growth-perturbing manipulation.
Treatment of these subpopulations with novobiocin (NOVO), a putative
inhibitor of the mammalian topoisomerase II enzyme, revealed a unique
cell cycle phase-dependent cytotoxicity for this agent. At a concentration
of 0.3 m\i, NOVO was cytotoxic only to a specific cell subpopulation in
the ( ; i-S phase boundary. Cells in other cell cycle phases were completely
unaffected. Additionally, S and (.M phase cells progressed through the
cell cycle relatively unaffected by NOVO but were blocked at the d-S
boundary.
NOVO treatment protected tumor cells from Adriamycin (ADR)induced lethality but sensitized them to the toxic action of 4-hydroperoxycyclophosphamide, an alkylating agent. These opposing effects of
NOVO were demonstrated in all of the four tumor cell lines investigated:
A431 and HEp3 (derived from human squamous cell carcinomas); MLS,
a human ovarian cancer cell line; and a Chinese hamster ovary cell line.
The degree of protection against ADR was the greatest for S-phase cells,
intermediate for cells in early (;, and M phases, and the least for late d
cells. This cell cycle-dependent protection by NOVO, which is identical
to the cell cycle-dependent cytotoxicity of ADR, was consistent with the
idea that NOVO interfered directly with the cell-killing mechanism of
ADR. In contrast, even though the cytotoxic activity of 4-hydroperoxycyclophosphamide exhibited significant cell cycle dependency, NOVO
enhanced 4-hydroperoxycyclophosphamide lethality equally for all cell
cycle phases.
Cell Culturing. All cell lines were grown as monolayer cell cultures.
Chinese hamster ovary cells were maintained in Ham's F-IO medium,
HEp3 in Ham's F-12 medium, A431 and MLS in »-minimumessential
INTRODUCTION
There is considerable current interest in the role of Topo II'
in determining the sensitivity of tumor cells to chemotherapeutic agents, notably the epipodophyllotoxins (1, 2), anthracyclines (3), and alkylating agents (4-6). It has been demonstrated
in several studies that tumor cell lines that were resistant to
DNA intercalators, whether acquired or innate, had decreased
Topo II activity (7-9), while those resistant to alkylating agents
had increased Topo II activity (1, 6). Clearly, these directly
opposing relationships between Topo II activity and the antitumor efficacy of two of the most frequently used classes of
anticancer agents have important implications for the optimal
use of these agents in polychemotherapy. However, the precise
role of Topo H-mediated processes in determining antitumor
efficacy is still not well understood. Here we describe experi
ments, using the putative Topo II inhibitor novobiocin, aiming
at defining the possible role of Topo II in the cell cycledependent cytotoxicity of ADR and 4-OOH-CP.
medium. All media were obtained from Gibco and were supplemented
with 10% fetal calf serum, 5 ITIMglutamine, and 10 ITIM4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acid. Three-day-old exponentially
growing cells were used in all experiments.
Drugs and Drug Treatment. ADR was obtained from Adria Labora
tory (Columbus, OH); 4-OOH-CP was kindly provided by Dr. R. Borch
(University of Rochester, Rochester, NY) and Dr. P. Hilgard (AstaWerke AG, Bielefeld, Germany). NOVO was purchased from Sigma
Chemical Co. Cells were generally treated with cytotoxic agents while
still attached to Petri dishes. In some experiments, cells were treated
in suspensions, at a concentration of 2-3 x IO5 cells/ml, following
standard trypsinization procedures to obtain single cells. Cells were
suspended in complete »-minimumessential medium in a type 1vial at
37°Cas described by Whillans and Rauth (10) and were continuously
gassed with 95% air-5% CO;. For both treatment procedures, a 10-iil
aliquot of a stock solution of the appropriate drug was given per ml of
medium.
Clonogenic Cell Survival Assay. At the end of drug exposure, cells
were washed and single cell suspensions were obtained. The cell number
was counted and a variable number of cells were plated to determine
survival by clonogenic assay. At an appropriate interval following
plating, the exact length of time being cell line dependent, dishes were
stained with crystal violet. Colonies containing more than 50 cells were
counted to determine cell survival.
Centrifugal Elutriation. Cells in various cell cycle phases were isolated
by centrifugal elutriation. which separates cells according to size and
density (11). Briefly, single cell suspensions of tumor cells were elu
triated in ice-cold complete media containing 10% fetal calf serum.
During the elutriation, the reservoir, rotor, and collection flasks were
kept at 4°C.After 10*cells were loaded into a Beckman JE-6 elutriator
rotor, subfractions were collected at decreasing rotor speeds. Cell
volume and number were determined with a Coulter Channelyzer. Since
most tumor cells are aneuploid. flow cytometry was used as a second
measure of tumor cell purity. Cells were fixed with 70% methanol and
stained with mithramycin or propidium iodide, and the fluorescence
intensity was quantilated by a Coulter EPICS V flow cytometer.
Glutathione Assay by High Performance Liquid Chromatograph).
Cellular GSH content was analyzed by the high performance liquid
chromatography technique described previously (11). Briefly, tumor
cells (2-5 x IO5) were washed with phosphate-buffered saline, and
centrifuged at 400 x g for 10 min at 4°C,and the cell pellet was stored
at -70°C before analysis. Thawed cell pellets and frozen-solid tumor
cubes (1 mm') were homogenized with 200 n\ or 20 volumes (w/v),
respectively, of a 20 ITIM5-sulfosalicylate solution and centrifuged in
an
Eppendorf microcentrifuge for 40 s. The supernatant was derivatized
Received 1/2/91 ; accepted 4/21/92.
using the fluorescent reagent monobromobimane. The fluorescent GSH
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
conjugate was eluted in a Waters Radial-PAK reversed-phase bonded
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
octadecylsilane (CIK)cartridge column (8 mm inside diameter) using
1This work is supported by NIH Grant CA-36858 and BRSG Sub
isocratic conditions consisting of a mobile phase of 23% acetonitrile in
S7RR05403-27.
2To whom requests for reprints should be addressed, at Experimental Thera
40 ITIMammonium phosphate buffer, pH 7.2, containing 5 mM tetrapeutics Department, Pharmaceutical Research Institute. Bristol-Myers Squibb
butylammonium hydroxide, and a flow rate of 3 ml/min. Fluorescence
Co., 5 Research Parkway, P. O. Box 5100, Wallingford. CT 06492-7660.
of the effluent was detected at 410 nm with excitation at 340 nm. GSH
"The abbreviations used are: Topo II, topoisomerase 11; NOVO, novobiocin;
concentration was determined from peak height with reference to a
ADR, Adriamycin; GSH. glutathione; 4-OOH-CP, 4-hydroperoxycyclophos
phamide.
linear calibration curve constructed using synthetic GSH standards.
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NOVOBIOCIN MODULATION
OF ANTICANCER DRUG ACTION
Typical performance characteristics of this assay are: lower limit of
detections, 8.0 pmol on column; coefficient of variation, 4-8%; accu
racy, 92-109%.
RESULTS
Cell Cycle Selective Cytotoxicky of NOVO
NOVO exhibits pronounced cell cycle phase selectivity in its
toxic action. Following a 4-h treatment with 0.3 HIM NOVO,
lethality was observed only for a small population of cells in
the G]-S boundary of the cell cycle (Fig. 1). For this particular
subpopulation, clbnogenic cell survival was reduced to ~30%
of control. Cells in other parts of the cell cycle were completely
unaffected.
4 hr
4 hr
C
O
•¿
<—t
*j
1 ü
11
CO
24
5-,
i-i
hr
24
hr
24
hr
00
C
CO
G2M
0.1
2500
2000
3000
Cell volume
3500
(/urn )
Fig. 1. Cell cycle-specific cytotoxicity of NOVO. Monolayer cultures of MLS
cells in exponential growth were treated with 0.3 IHM NOVO or phosphatebuffered saline for 4 h. Single cell suspensions were then prepared for centrifugal
elutriation. Cell cycle phase distribution was determined by flow cytometry
following DNA labeling with propidium iodide.
Fig. 3. Effects of two different concentrations of NOVO (0.3 and 1.8 m\i) on
the cell cycle transit of S and ( ;. M cells. A mixture of S and G2M cells obtained
by centrifugal elutriation were exposed to NOVO at time 0. DNA content was
determined by flow cytometry analysis of propidium iodide-labeled cells at the
indicated time.
Effects of NOVO on Cell Cycle Progression
c
'S
IDO
E
80
CJ
O
ü
8X10
7X105
M
S
0)
O
6X10
5
5X10
O
0)
O
tt
3X10
10
Time
20
30
(hr)
Fluorescence
Intensity
Fig. 2. Effects of NOVO (0.3 m.\t) on the cell cycle progression and growth of
the human ovarian tumor cell line MLS. Purified G, cells were plated at time 0
in complete medium with or without NOVO. At various times after, triplicate
plates of cells were trypsinized, cell number was counted, and DNA content was
determined by flow cytometry. A, percentage of G, cells remaining at various
times after plating (initial percentage of d cells, 89%). The major effect of
NOVO (•)was the blockage of G, to S phase transition as compared with
controls (O). As a result, cell division was inhibited (B). Cani D, DNA histograms
of initially purified G, cells at various times following plating with or without
NOVO, respectively.
NOVO at a concentration of 0.3 HIM inhibited the cell cycle
progression from G, to S phase (Fig. 2). Fig. 2, A and C, shows
that under normal conditions the number of G, cells in a Gì
phase-enriched cell population declined progressively with time
as individual cells transited the cell cycle. At 16 h, the number
of G, cells reached a minimum (31%), while 44 and 25% of the
cells were in S and G^M phase, respectively. With Novo, the
G, to S transition was blocked (Fig. 2, A and D). At 16 h, 73%
of the cells remained in the late G, phase of the cell cycle while
17 and 10% were in S and G:M phases, respectively. The effects
of NOVO on cell cycle progression are confirmed by cell growth
measurement (Fig. 2Ä).Under control conditions the number
of cells roughly doubled 30 h following the plating of G, cells.
With NOVO in the growth medium, however, no increase in
cell number was apparent.
The effects of NOVO (0.3 HIM) on cell cycle progression
appeared to be specific for cells in the G,-S transition phase.
Cells in S and G:M phases progressed normally through the
cell cycle at similar rates as cells under control conditions (Fig.
3). However, at very high NOVO concentration (1.8 HIM),cell
cycle progression was blocked at the G:M phase (Fig. 3).
Cell Cycle-dependent Cytotoxicity of ADR and 4-OOH-CP
ADR. The four different cell lines investigated in this study
all exhibited similar patterns of cell cycle phase-dependent
responses to ADR (Figs. 4A and 5Ä).Cells in the G, phase of
the cell cycle were always the most resistant, whereas S and
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NOVOBIOriN
MODULATION
OF ANTICANCER DRUG ACTION
NOVO after 4-OOH-CP was removed produced no sensitization effect (Fig. 6Ä).
«,•<<sç£>
•¿'" >-...
C
0u
Effects of NOVO on Glutathione Metabolism
-
À S"AÀ
Gi
oCi_Dr/l0.01
Because GSH is known to be a critical determinant of the
cytotoxicity of 4-OOH-CP (12-14), we have carried out studies
on the effects of NOVO on the status of GSH metabolism
using the MLS tumor cell line. As depicted in Fig. 8/Õ,NOVO
treatment alone did not affect the GSH level of cells in all
phases of the cell cycle. These results show that the enhance
ment of 4-OOH-CP cytotoxicity by NOVO was not simply due
to a direct effect on the level of GSH per se. However, the
sensitizing action of NOVO may nevertheless involve GSH.
This possibility was suggested by the observation that NOVO
enhanced the GSH depletion induced by 4-OOH-CP. Previous
dose-response studies where tumor cells were incubated with
various concentrations of 4-OOH-CP have shown that deple
tion of GSH occurred only with cytotoxic concentration of 4OOH-CP. Significant depletion invariably indicates a break
down of the cellular defense mechanism which protects cells
from damage by the toxic metabolites of 4-OOH-CP (14).
Additional data suggested that in treated cells GSH was lost
through conjugation reaction with the GSH-reactive toxic me
tabolites of 4-OOH-CP, acrolein and phosphoramide mustard.
The present finding, shown in Fig. 8Ä,that cells treated with
s
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A
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1800
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4800
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500
1500
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2500
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I,
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Fig. 4. /4, cell cycle-dependent cytotoxicity of ADR on the survival of clonogenic cells of three cell lines. O, CHO; A, A43I; •¿,
HEp3. Cells were treated
with 0.5 ¿ig/mlADR for 3 h. B, cell cycle-dependent cytotoxicity of 4-OOH-CP
on the survival of clonogenic cells of three cell lines. O, CHO; A, A431;T, MLS.
Cells were treated with 10, 50, and 25 MM4-OOH-CP for 3 h, respectively.
\Q/Y°\à '°\3
•¿Â¿
;^0
'
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0.010-D(0.002-0A
10
G^M phase cells were invariably more sensitive.
4-OOH-CP. A radically different pattern of cell cycle-de
pendent responses was observed for 4-OOH-CP. For this cytotoxic agent, cells in the early G, phase of the cell cycle were
always the most sensitive to the cytotoxicity of 4-OOH-CP. As
cells traversed the cell cycle from G, through S to G2M their
sensitivities to 4-OOH-CP became progressively reduced (Fig.
45).
Effects of NOVO on the Cell Cycle-dependentCytotoxicities of
ADR and 4-OOH-CP
ADR. NOVO protected exponentially growing cells (both
Chinese hamster ovary and A431) against the cytotoxicity of
ADR (Fig. 5A). The protective efficacy of NOVO was concen
tration dependent; significant protection was observed at a
concentration of 0.01 HIM.In addition, the protective efficacy
of NOVO was cell cycle dependent; cells in S phase were
protected to a greater degree than cells in G, phase (Fig. 5B).
4-OOH-CP. NOVO enhanced the cytotoxicity of 4-OOHCP (Fig. 6A). The sensitizing activity of NOVO was dose
dependent; its effectiveness increased with increasing NOVO
concentration, from 0.01 to 0.3 IHM(Fig. dB). However, in
contrast to its effects on ADR, the enhancement by NOVO of
cellular 4-OOH-CP chemosensitivity was not cell cycle depend
ent (Fig. 7). The timing of NOVO treatment also appeared to
be critical for its activity. Simultaneous presence of NOVO and
4-OOH-CP was essential for interaction; treatment of cells with
0.2
1.[Novobiocin]
0.4
0.6
0.8
(mM)
C
0.1 -
o
4_>
o
IS
t-,
OD
C
0.01 -
2000
4000
3000
Cell volume
(/um
Fig. 5. . I. dose-dependent protection of two cancer cell lines against ADR
cytotoxicity by NOVO. O, A431; »,HEp3. Cells were incubated with NOVO
(0.3 HIM) for 1 h initially followed by the coincubation with ADR (0.5 ng/ml) and
NOVO (0.3 mM) for an additional 3 h. Bars, SD. B, cell cycle-dependent
protection by NOVO (0.3 m.M)against the cytotoxic action of ADR in the MLS
cancer cell line. Cells were treated for 3 h with 0.25 fig/ml ADR. Single cell
suspensions were then prepared for centrifugal elutriation. O, ADR alone; •¿,
ADR plus NOVO. Bars. SD.
3517
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NOVOBIOCIN MODULATION
OF ANTICANCER DRUG ACTION
both NOVO and 4-OOH-CP exhibited greater degrees of GSH
depletion than cells treated with 4-OOH-CP alone suggests that
NOVO may act through a mechanism which resulted in an
increase in the level of toxic 4-OOH-CP metabolites. The
precise mechanism by which this may occur is not yet known.
DISCUSSION
The problem of acquired drug resistance to antineoplastic
agents by cancer cells is currently attracting a good deal of
research interest. For the DNA intercalator class of antineo
plastic agents, a number of biochemical mechanisms of acquired
resistance have been identified (for a review see Ref. 13).
However, the existence of innately resistant cell subpopulations
within the solid tumor mass may also be an important cause of
treatment failure in patients receiving first-line chemotherapy.
A good case in point is the innate resistance of nonproliferating
cells to Topo II-interactive antineoplastic agents (e.g., Adria-
10
0
10
20
30
40
50
[4-OOH-CP]
1E-1
1
15-
|E-2J
GO
t)
"o
e
I
10 -
1e-40.1
Novobiocm
0.2
conc.
0.3
c
o
o
(mM)
Fig. 6. A, effects of NOVO on the cytotoxicity of 4-OOH-CP in the MLS
ovarian cancer cell line. Cells were incubated with NOVO (0.3 mM) for l h
followed by coincubation with various concentrations of 4-OOH-CP for an
additional 3 h. NOVO enhanced the cytotoxicity of 4-OOH-CP by a factor of 2.1
at the 10% survival level. Bars. SD. B, effects of incubation with NOVO (0.3
mM), simultaneously with or following 4-OOH-CP exposure, on the cytotoxicity
of the alkylating agent. The MLS human ovarian cancer cells were treated either
simultaneously with 4-OOH-CP and NOVO for 4 h (•),or in two stages: first
with 4-OOH-CP for 4 h, washed and then incubated with NOVO for an additional
4 h (A). Bars, SD.
{--Õ
1.0 -.
5 -
1
00
U
1500
2000
2500
Cell volume
B
(/um
3
3500
)
100 -
DC
co
Ü
ï-NoYOblocm (lona
3000
80 -
13
13
•¿
(•H
O
4-OOH-CP llana
ï
0
•¿l-<
1-1
O
v>
0.01 -
0
10
20
30
40
50
[4-OOH-CP]
0.001
1800
2400
3000
3600
Cell Volume (pm3)
Fig. 1. Effects of NOVO on the cell cycle-dependent cytotoxicity of 4-OOHCP in the MLS ovarian cancer cell line. Cells were treated with NOVO (0.3 mM)
for 1 h followed by coincubation with 25 JIM4-OOH-CP for another 3 h. Single
cell suspensions were then prepared for centrifugal elutriation. Bars, SD.
Fig. 8. A, effects of NOVO on the GSH content of MLS human ovarian cancer
cells in different phases of the cell cycle. Exponentially growing cells were treated
with NOVO (0.3 mM) for 4 h prior to separation into the various cell cycle phases
by centrifugal elutriation. Bars, SD. B, NOVO enhanced the GSH contentdepleting effects of 4-OOH-CP. MLS cells were incubated with NOVO (0.3 m\i)
for 1 h followed by coincubation with various concentrations of 4-OOH-CP for
another 3 h. Bars, SD.
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NOVOBIOCIN MODULATION
OF ANTICANCER DRl'Ci ACTION
mycin, etoposide). Several recent studies have associated the
"resistance" of nonproliferating cells to Topo II-interactive
antineoplastic agents with reduced Topo II activity in these
cells (7, 9,15). Similarly, the greater sensitivity of cells in active
DNA synthesis (S phase) has been correlated with increased
Topo II activity in this cell cycle phase (9, 16). The present
results obtained using a variety of different tumor models clearly
support this hypothesis.
An important feature of the present investigation is the use
of countercurrent centrifugal elutriation to obtain cell popula
tions in different cell cycle phases. This technique has two
advantages: (a) cells in different cell cycle phases can be ob
tained simultaneously from the same exponentially growing cell
populations; (/»)
prior manipulation of growth conditions is not
required, thus avoiding complications resulting from growth
perturbation (11). The characteristic cell cycle-dependent cytotoxicity of ADR demonstrated for the four different cell lines
in this study is in agreement with previously published data
(17, 18). Cells in S phase as a rule showed the greatest and G,
cells the least sensitivity to ADR (Figs. 4A and 5B). The findings
that NOVO abolished the cell cycle-dependent cytotoxicity of
ADR (Fig. 5B) are consistent with the hypothesis that the
cytotoxicity of ADR is partly caused by DNA strand breakages
resulting from the stabilization of DNA "cleavable complexes"
(19, 20). According to this hypothesis the greater sensitivity of
proliferating cells to ADR, as compared to stationary phase
cells, is a consequence of the higher Topo II activities in actively
dividing cells (9, 15). The present findings suggest that NOVO
may abrogate the Topo Il-mediated cell cycle-dependent cyto
toxicity of ADR. This interpretation is in agreement with other
studies using DNA intercalators other than ADR (9, 15, 21). It
should be noted, however, that the work of Smith and Bell (22)
suggested that at least part of the effects of NOVO may be
attributed to a reduction in cellular accumulation of ADR.
The effects of NOVO on 4-OOH-CP cytotoxicity have not
been previously reported, although a number of studies have
investigated its effects on other alkylating agents (4, 5). In this
study we showed that NOVO augmented the cytotoxicity of 4OOH-CP in exponentially growing MLS human ovarian cancer
cells by a dose enhancement factor of 2.1 at the isoeffect of 1
log cell kill (Fig. 6/1). Unlike its effects on ADR, however, the
enhancement of 4-OOH-CP cytotoxicity appeared not to be
cell cycle dependent (Fig. 7). In addition, 4-OOH-CP treatment
alone did not exhibit the same cell cycle-dependent cytotoxicity
as that described above for ADR (cf. Fig. 4, A and B). Further
more, for the two cell lines investigated thus far, namely A431
and MLS, significant differences in sensitivity to 4-OOH-CP
were not observed for exponential versus stationary cells (results
not shown). These findings argue against the direct involvement
of Topo II perse in the mechanism of alkylating agent toxicity
enhancement by NOVO. Other known effects of NOVO may
play important roles in this respect. NOVO has been shown to
inhibit replicative and repair DNA syntheses (23), affect mitochondrial structure and ATP metabolism (24), and impair DNA
and RNA polymerases activity (25). The observation in this
study, that NOVO treatment immediately following a 3-h ex
posure to 4-OOH-CP did not enhance the activity of the cytotoxic agent (Fig. 6Ä),suggests that NOVO probably effected a
process(es) which occurs concurrently with 4-OOH-CP expo
sure. We have obtained preliminary results showing that com
bined 4-OOH-CP and NOVO caused a greater degree of glutathione depletion than 4-OOH-CP alone. In previous studies
we have established that glutathione depletion is an effective
indicator of cytotoxicity because it accurately reflects the
amount of alkylating toxic species formed from the parent 4OOH-CP (14, 26). Therefore, the greater depletion of GSH by
combined NOVO and 4-OOH-CP may indicate that the for
mation of the toxic phosphoramide mustard and acrolein from
the parent 4-OOH-CP was enhanced by NOVO. However, the
mechanism by which this occurs is not known at present.
There is strong evidence that NOVO impairs Topo II activity
by inhibiting the ATPase portion of this enzyme (27, 28). The
present study shows that treatment of tumor cells with NOVO
at concentrations that can inhibit cell cycle progression (from
G, to S phase), protect against ADR cytotoxicity, and produce
synergistic cell killing with 4-OOH-CP did not in fact cause
significant cytotoxicity. It appears therefore that NOVO is
comparatively nontoxic for mammalian cells. It should be noted
that at extremely high concentrations (>500 n\\) NOVO has
been reported to be toxic to mammalian cells (5, 22) and to
cause the accumulation of cells in G?M phase of the cell cycle
(22, 29). However, as shown in this study, NOVO was effective
in protecting against ADR cytotoxicity at the concentration of
50 UMor less (Fig. 5Ä);additionally, Utsumi et al. (21) found
that 100 ¿¿M
NOVO can abrogate the cytotoxicity of the DNA
intercalator 4'-(9-accidinylamino)methanesulfon-w-anisidide.
Thus, assuming that the above protective effects of NOVO were
the result of its inhibition of Topo II, it is likely that the toxic
action of NOVO at high concentrations is not a direct conse
quence of Topo II inhibition. The only toxicity that we observed
with low NOVO exposure (0.3 m\i for 4 h), as revealed in the
cell fractionation experiments, was uniquely cell cycle phase
specific and therefore would not otherwise be detected in an
unfractionated cell population. Only cells in the Gi-S boundary
(which usually constitute less than 5% of the whole population
of cells) appeared to be killed by NOVO (Fig. 1). These results
suggest that the NOVO treatment may be deleterious to survival
only for cells the DNA of which was in the process of being
unwound in preparation for DNA synthesis. In addition,
NOVO prevented the cell cycle transition from G, to S phase
(Fig. 2). Presumably, cells were blocked in the G,-S boundary
at a point immediately before the unwinding of the supercoiled
DNA. Additional studies concerning the exact position in the
G]-S boundary at which cells were blocked, relative to those
produced by hydroxyurea and nutrient deprivation, are in prog
ress in this laboratory.
In conclusion, we have demonstrated that NOVO can abolish
the cell cycle-dependent cytotoxicity of ADR and enhance the
toxicity of 4-OOH-CP in a variety of tumor cell lines. Prelim
inary evidence suggests that these two effects of NOVO may be
mediated by different mechanisms. Further investigations into
the effects of NOVO on glutathione metabolism, drug uptake
and accumulation, and DNA repair may provide important
information into the mode of action of this clinically safe agent.
This may ultimately aid in the design of combined regimens of
NOVO and cytotoxic alkylating agents for the treatment of
cancer.
ACKNOWLEDGMENTS
The authors wish to thank the Cell Separation Facility of the Uni
versity of Rochester Cancer Center for technical support; Dr. R. Borch
and Dr. P. Hilgard for the supply of 4-hydroperoxycyclophosphamide;
and B. King for excellent technical assistance.
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NOVOBIOCIN
MODULATION
OF ANTICANCER DRUG ACTION
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3520
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1992 American Association for Cancer Research.
Modulation of the Cell Cycle-dependent Cytotoxicity of
Adriamycin and 4-Hydroperoxycyclophosphamide by
Novobiocin, an Inhibitor of Mammalian Topoisomerase II
Francis Y. F. Lee, Deborah J. Flannery and Dietmar W. Siemann
Cancer Res 1992;52:3515-3520.
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Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1992 American Association for Cancer Research.