[CANCER RESEARCH 41, 1263-1 270, April 1981 ]
0008-5472/81
/0041-OOOOS02.0O
Reduction of 1-/?-D-Arabinofuranosylcytosine and Adriamycin
Cytotoxicity following Cell Cycle Arrest by Anguidine1
Laura Teodori, Barthel Barlogie,2 Benjamin Drewinko, Douglas Swartzendruber,
and Francesco Mauro
Departments ol Developmental Therapeutics ¡L.T., B. B., D. S.] and Laboratory Medicine ¡B.D.], M. D. Anderson Hospital and Tumor Institute, Houston, Texas
77030, and Laboratorio dì
Dosimetria Biofisica, Comitato Nazionale Energia Nucleare, Rome, Italy {F. M.]
ABSTRACT
The protein synthesis inhibitor anguidine induced a frozen
cell cycle state in exponentially growing Chinese hamster ovary
cells, as demonstrated by serial DMA flow cytometric measure
ments in the absence and presence of Colcemid as a stathmokinetic agent. The minimally effective concentration of an
guidine for induction of cell cycle arrest was 0.1 jug/ml. As
demonstrated by tritiated thymidine labeling index and DMA
flow cytometric investigations in the presence of Colcemid, a
4-hr exposure of Chinese hamster ovary cells to >4 jug of
anguidine per ml effected a >12-hr cycle perturbation at no
cytotoxic expense. Preincubation of exponentially growing
Chinese hamster ovary cells for 4 hr with 5 fig of anguidine per
ml reduced the cytotoxicity from Adriamycin (1 hr; 0.1 to 10
jug/ml) and from 1-/J-D-arabinofuranosylcytosine
treatment (18
hr; 5 to 50 (ug/ml) by 1O- to 100-fold. Further investigation of
the concentration dependence and time course of this protec
tive effect of anguidine revealed a plateau at 1 jug of anguidine
per ml and lack of protection in case of anguidine exposure
subsequent to Adriamycin and 1-ß-D-arabinofuranosylcytosine
treatment. Prolongation of the treatment-free interval between
initial anguidine exposure and 1-hr Adriamycin treatment dem
onstrated partial recovery of DNA synthesis associated with
some loss in cytoprotection. Our results indicate that the largely
indiscriminate interference with cycle progression by anguidine
under noncytotoxic conditions affords significant protection
against 1-/8-D-arabinofuranosylcytosine
and Adriamycin-related cytotoxicity, the degree of which appears to be related to
the extent of reduction in cycle traverse rate. Thus, anguidine
may serve as a useful probe to study in detail drug-induced
lethal injury as a function of cycle traverse rate.
INTRODUCTION
Extensive investigations in experimental systems and, to a
lesser extent, in human neoplasms have revealed that the
cytokinetic determinants for tumor cell kill by antitumor agents
include the growth fraction, the cell cycle stage distribution,
and the cycle traverse rate (1 ). Available data in human neo
plasms suggest that malignant cells do not necessarily prolif
erate faster than their normal tissue counterparts, thus creating
a cytokinetic disadvantage for differential tumor versus normal
tissue damage by antitumor drugs (5). A number of treatment
strategies involving cytokinetic manipulations have been pro
posed to compensate for this disadvantage, but in human
disease, neither cell synchronization nor cell recruitment have
been documented or demonstrated to be superior to empirical
treatment designs (1).
More recently, attention has been drawn to the fact that, in
addition to the position in the cell cycle at the time of drug
administration, the rate of progression through this cycle stage
may also be an important factor determining the extent of cell
inactivation. In support of this concept, the in vitro lethal
efficacy to Sarcoma 180 cells of 2 S-phase-specific agents,
hydroxyurea and ara-C,3 was noted to be directly related to
the rate of DNA synthesis (15, 27).
Although not proven, it is conceivable that the observation of
amelioration of myelosuppression from nitrogen mustard, araC, and methotrexate by the protein synthesis inhibitors cycloheximide and L-asparaginase is at least in part related to delay
or block of cycle progression (6, 8, 9, 23, 30). A similar
mechanism may underlie the reduction of ara-C-induced inac
tivation of granulocytic colony-forming units in culture by infer
ieron (20).
The notion of cycle traverse rate-dependent tumor cell kill
together with the observation of normal tissue protection
against cytotoxic damage by protein synthesis inhibitors and
Interferon, possibly via reduction of cycle traverse rate, moti
vated our interest in the concept of normal tissue protection by
differential reduction in cycle traverse rate. Previous investi
gations on the lethal and cytokinetic effects of anguidine on
cultured human colon cancer (LoVo) cells revealed that this
protein synthesis inhibitor effectively induced a "frozen cell
cycle state" (i.e., complete block of cell progression through
all cycle phases) at virtually no cytotoxic expense (10). At the
same time, clinical Phase l-ll investigations had demonstrated
that anguidine could be safely administered to patients with
malignant disease but that there was no appreciable antitumor
effect at maximally tolerated dosages (24, 32).
Based on the above considerations, we initiated experiments
to test whether anguidine can reduce lethal toxicity from other
chemotherapeutic
drugs by delaying or blocking cell cycle
traverse. The long-term goal of this research aims at elucidation
of differential cell cycle arrest in normal versus tumor cells
and/or at differential protection of arrested normal versus
tumor cells against lethal toxicity from other agents. In this
paper, we demonstrate that anguidine does induce a concen
tration- and exposure time-dependent cell cycle arrest in ex
ponentially growing CHO cells, which is associated with pro
tection against lethal damage from ara-C and ADR.
MATERIALS
1 Supported
in part by Grants CA14528,
CA5831,
and CA23272
from the
National Cancer Institute. NIH. Bethesda. Md.
2 Junior Faculty Fellow of the American Cancer Society. To whom requests
for reprints should be addressed.
Received September 24, 1980; accepted December 30. 1980.
APRIL
AND METHODS
Cell Line. CHO cells were utilized throughout this investiga3 The abbreviations
used are: ara-C, 1-/S-D-arabinofuranosylcytosine;
CHO,
Chinese hamster ovary; ADR, Adriamycin.
1981
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1263
L. Teodori et al.
tion. Cultures were maintained in McCoy's 5A medium (Hsu's
modification; Grand Island Biological Co., Grand Island, N. Y.)
supplemented with 15% fetal calf serum and incubated at 37°
in a 5% CO2 atmosphere. Cell harvesting involved 5 min of
treatment with 0.125% of trypsin (Irvine Scientific, Santa Ana,
Calif.) and subsequent inactivation with medium.
Survival Studies. Cellular survival was measured by the
colony formation technique (25, 26). Cell suspension aliquots
were seeded into 60-mm Petri dishes (0.3 x 106 cells/dish).
Cultures were incubated at 37°in a 5% CO2 atmosphere in air
for 24 hr to reach exponential growth. The medium was then
discarded, and the cells were exposed to increasing concen
trations of drugs for a specified period of time at 37°.Following
drug treatment, the drug-containing medium was discarded,
and the cells were washed twice with Hanks' balanced salt
solution and counted using an electronic particle counter (Coul
ter Electronics Inc., Hialeah, Fla.). Known aliquots of cells were
dispensed into 60-mm Petri dishes, so that 50 to 100 colonies
would appear after incubation for 5 to 7 days. The surviving
colonies were stained with 2% crystal violet in 95% ethanol.
Cell survival for the different drug concentrations and exposure
times was normalized with respect to untreated controls, which
had a plating efficiency ranging from 70 to 80%. All experi
ments were repeated at least twice with triplicate samples for
each drug concentration and exposure time. For each experi
ment involving combined treatment with anguidine and ara-C
or ADR, control survival curves were obtained for each drug
alone.
Cytokinetic Studies. The effects of anguidine on cell cycle
progression were analyzed by serial measurements of DNA
content using DNA flow cytometry of fixed cells stained with
ethidium bromide and mithramycin as described previously (2).
CHO cells in exponential growth were exposed to increasing
concentrations of anguidine for various exposure times. Follow
ing treatment, cells were washed twice with 0.9% NaCI, har
vested with 0.125% trypsin, and fixed in 70% ethanol. Follow
ing fluorochromation, cells were measured in a Phywe ICP 11
pulse cytophotometer (Ortho Instruments, Westwood, Mass.)
as described previously (18). Routinely, 20,000 to 30,000 cells
were measured for each sample, and DNA histogram evaluation
was performed using an alogrithm developed by Johnston ef
al. (21).
In order to discern the effects of anguidine on protein syn
thesis, biparametric investigations of DNA and protein contents
were performed. Ethanol-fixed cells were stained with DAPI for
DNA (19) and sulforhodamine 101 for protein (16, 28) at final
concentrations of 0.3 and 5 ¿ig/mlat pH 7.5 (17). Biparametric
analysis of DNA and protein content was performed with the
use of a Phywe ICP 22 pulse cytophotometer (Ortho Instru
ments). Data analysis involved determination of mean relative
protein contents of Gì,mid S, and G2 anguidine-treated cells,
using both scattergram and 2-dimensional displays of DNA and
protein histograms. Instrument calibration was performed so
that the ratio of protein to DNA content of untreated d cells
would equal 1.0.
In order to further delineate the effect of anguidine on cycle
progression, cells in exponential growth were continuously
incubated with Colcemid at a concentration of 0.09 /ig/ml in
the presence and absence of anguidine (10, 11,31 ). Compar
ison between flow cytometrically determined G2 + M compart
ments and mitotic index (determined on 1000 nucleated cells)
1264
were made.
To assess the DNA-synthetic activity of anguidine-treated
cultures, treated and control cultures were incubated at spec
ified times with tritiated thymidine at a concentration of 5 /¿Ci/
ml (specific activity, 6.7 Ci/mM) for 60 min at 37°. Cells were
harvested in the routine fashion described above and trans
ferred onto slides by cytocentrifugation.
Autoradiographic
processing involved dipping of the slides into Kodak NTB2
emulsion, exposure for 7 days, and staining with May-Griinwald-Giemsa. The labeling index was determined as the pro
portion of cells with >5 grains overlying the nucleus among a
total of 500 nucleated cells.
Drugs. Anguidine, manufactured by Ben Venue Lab was
obtained from the Division of Cancer Treatment, National Can
cer Institute; ara-C was kindly provided as Cytosar by Upjohn
Co. (Kalamazoo, Mich.), and ADR was purchased as Adriblastin
from Farmitalia (Milan, Italy). Colcemid was available through
Grand Island Biological Co. All drug solutions were prepared
immediately before each experiment. Drugs were initially dis
solved in a small amount of 1 to 5 ml of 0.9% NaCI, and further
dilution was accomplished with McCoy's 5A growth medium
(Hsu's modification) enriched with 15% fetal calf serum. Ethid
ium bromide was purchased from Serva (Heidelberg, Ger
many), mithramycin was kindly provided by Pfizer, Inc. (New
York, N. Y.). Sulforhodamine 101 was obtained from Eastman
Kodak Co. (Rochester, N. Y.), and DAPI was purchased from
Serva.
RESULTS
Cellular Effects of Anguidine
Lethal Effects. Dose-dependent survival of exponentially
growing CHO cells is illustrated in Chart 1 for exposure times
ranging from 4 to 48 hr. One hundred % viability was observed
following 4-hr treatment with anguidine over the entire concen-
100
0.01
50 100
Concentration of Anguidine (/jg/ml)
Chart 1. Survival of exponentially growing CHO cells as a function of angui
dine concentration for different exposure times. Four-hr treatment is virtually
noncytotoxic, whereas exposure for >24 hr causes significant cell kill at concen
tration.«'
of >1 pg/ml.
CANCER
RESEARCH
VOL. 41
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Protection
tration range from 0.01 to 50 /tig/ml. Extension
ment to 12 hr reduced survival by approximately
of drug treat
60% in case
of treatment
with 50 fig/ml.
Cell kill of >50% occurred
only
after treatment
for >24 hr, where treatment
with a concentra
tion as low as 1 /¿gof anguidine
per ml reduced survival to 8%.
Cytokinetic
Effects. Chart 2 depicts the cycle stage distri
bution in Gì
/o, S, and G2 + M compartments
over time following
continuous
exposure
of exponentially
growing
CHO cells to
anguidine
at concentrations
of 0.1 to 50 /¿g/ml. Whereas
control cultures displayed
a significant
decrease in the propor
tion of cells in S phase from 53% at the start of the experiment
to 25% at 24 hr associated
with a corresponding
increase in
the percentage
of cells with a d DNA content from 28 to 50%,
there was no significant
change
in cell cycle compartment
distribution
for any of the treated cultures over the entire range
of anguidine
concentrations.
While accumulation
of untreated
cells in the G, ,, compartment
is well established
as a result of
aging with transition
into stationary
growth phase, the lack of
such reassortment
is consistent
with a frozen cell cycle state
(7).
In order to provide more direct evidence for the induction of
a frozen cell cycle state by anguidine,
exponentially
growing
CHO cells were jointly exposed
to anguidine
and Colcemid
(0.09 ¿ig/ml; Chart 3). Untreated
control cultures again dem
onstrated
a shift from S into the GVo compartment.
Following
treatment with Colcemid alone, a prompt G2 + M accumulation
of 95% was reached at 12 hr, associated
with evacuation
of
both S and G,/0 compartments.
Concurrent
exposure
of CHO
cells to Colcemid and anguidine
at the lowest concentration
of
0.01 /ig/ml
closely mimicked
the redistribution
pattern noted
previously
in the case of treatment
with Colcemid
alone. In-
against
100
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Lethal
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Cell Damage
by Cell Cycle Arrest
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Exposure Time (HR)
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Chart 3. Cell cycle distribution changes as measured by DNA flow cytometry
following continuous exposure to anguidine alone or in combination with Col
cemid (0.09 fig/ml). In the presence of Colcemid alone and in the case of joint
exposure to the lowest anguidine concentration (0.01 fig/ml), there is a prompt
accumulation of cells in the G2 + M compartment, reaching >90% at 12 hr. This
G? + M accumulation is associated with evacuation of G,- and S-phase com
partments. In contrast, concomitant exposure of CHO cells to Colcemid and
higher concentrations of anguidine produces largely constant cycle distributions
over the experimental time span from 3 to 24 hr. The 10 to 20% S-phase
increment during the first 3 hr is associated with a decrease in the G i/o and
G2 + M proportions by 5 to 10% each.
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0
0.1
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40
20
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12
24
Exposure Time (HR)
Chart 2. Cytokinetic response of exponentially growing CHO cells to contin
uous exposure of anguidine at increasing concentrations, as measured by serial
DNA flow cytometric analyses. Control cultures show a moderate reassortment
during 24 hr with an increase in the proportion of cells with a G, DNA content
associated with a decrease in the S-phase proportion. Following anguidine
treatment, there is virtually no change in the original cell cycle distribution, and
the final DNA compartment shift of control cultures at 24 hr is absent in the case
of treatment with 0.1 /ig of anguidine per ml.
APRIL
crease in anguidine concentration to 2:0.1 /tg/ml was associ
ated with a 10 to 20% increase in the proportion of cells in S
phase and a decrease in both Gi/0 and G2 + M compartments
by 5 to 10% within 3 hr of combined drug exposure. The cycle
stage distribution noted at 3 hr was then more or less main
tained throughout the remainder of the experiment (24 hr).
To further evaluate the G2 —»
M transition, mitotic determi
nation was performed both in the absence (Chart 4) and in the
presence of Colcemid (Chart 5). In the absence of Colcemid
and during continuous anguidine exposure, a concentrationdependent decrease of the mitotic index from a value of 3.5%
for untreated control cultures to 0.2% after treatment with 1
/ig of anguidine per ml was noted at 12 hr (Chart 4). At the end
of a 12-hr continuous joint exposure of CHO cells to anguidine
and Colcemid, the mitotic index dropped from 86% for cultures
treated with Colcemid alone to 12% for 0.1 ftg and to 8% for
50 /ig of anguidine per ml.
We next measured the relative protein content in relationship
to cell cycle distribution during a 12-hr continuous exposure of
CHO cells to anguidine concentrations ranging from 0.1 to 50
jig/ml (Chart 6). The data are expressed as protein:DNA con
tent ratios, and the flow cytometer was adjusted so that, at 0
hr, the protein:DNA content ratio was 1.0 for cells with a G,
DNA content. Untreated control cultures largely retained this
ratio throughout the experiment (Chart 6). There was a slight
decrease in protein:DNA content ratio from 1.0 to 0.8 at the
end of a 12-hr treatment regardless of cell cycle stage (Gì,mid
S, and G2 + M) and drug concentration.
1981
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1265
L. Teodorìet al.
Anguidine-induced Modification of Cell Kill by ara-C and ADR
The results illustrated in Chart 7 demonstrated that an effec
tive cell cycle arrest was maintained for at least 12 hr after a 4hr treatment with >4 /ig of anguidine per ml, conditions which
are nontoxic to exponentially growing CHO cells (see Chart 1).
Treatment with ara-C alone required prolonged drug exposure
1
3
6
Exposure
12
0.8
0.6
0.4
0.2
24
Time (HR)
Chart 4. Changes in the percentage of mitoses during continuous exposure
to increasing concentrations of anguidine. There is a time- and concentrationdependent decrease in mitotic index.
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Exposure
30
20 L
:Lllm
Time (HR)
Chart 6. Flow cytometric analysis of DNA and protein contents of CHO cells
stained with DAPI for DNA and sulforhodamine
101 for protein. The chart
illustrates the ratio of protein to DNA content of Gì,mid S, and G2 + M
compartments following continuous exposure to increasing concentrations of
anguidine. See text for further details.
0 0.1 1 10 50
Concentration of
Anguidine
Chart 5. Mitotic index following 12-hr joint exposure of exponentially growing
CHO cells to Colcemid (0.09 fig/ml) and increasing concentrations of anguidine.
Note the marked reduction in mitotic index in the presence of anguidine.
To assess the duration of anguidine-induced cell cycle arrest
as a function of drug concentration, experiments were de
signed to allow for a 12-hr cell recovery in the presence of
Colcemid. As detailed in Chart 7, 12-hr Colcemid treatment
following 4-hr treatment with drug-free medium promoted a
complete cycle distribution shift into the G2 + M compartment
with a small proportion of hyper-G2 + M cells, indicating some
degree of endoreduplication (Chart 76). If the Colcemid treat
ment was preceded by a 4-hr exposure to increasing concen
trations of anguidine, progressively larger Gì-and S-phase
proportions were retained. In the case of treatment with >4 jug
anguidine per ml (Charts 7, £and F), the cycle distribution of
treated cells mimicked very closely that of untreated cultures
(Chart 7A), except for a slightly more prominent S-phase com
partment. These data provided the necessary information for
drug combination experiments, investigating the impact of pre
treatment with anguidine as a cell cycle-freezing agent on
cytotoxicity from subsequently applied ara-C or ADR treatment.
1266
30
60
90
30
60
90
Channel Number (Relative DNA Content)
Chart 7. DNA compartment distribution of exponentially growing CHO cells
successively exposed to anguidine (4 hr) and Colcemid (12 hr). A, medium/
medium control; B, complete mitotic accumulation in the case of Colcemid
treatment without prior anguidine exposure; C to F, inhibitory effect on Colcemidinduced mitotic accumulation of 4-hr preincubation with anguidine at increasing
concentrations.
CANCER
RESEARCH
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VOL. 41
Protection against Lethal Cell Damage by Cell Cycle Arrest
intervals for effective cell killing (Chart 8). Pretreatment with 5
jug of anguidine per ml for 4 hr reduced the lethal damage from
18-hr treatment with ara-C by 30- to 100-fold over the entire
concentration range of 5 to 50 fig/ml (Chart 9). The antagonis
tic effect was most pronounced after >6 hr of ara-C treatment,
reaching a 10-fold difference in cell kill after 12 hr (25 and 50
jug/ml) and a 100-fold difference after 18 hr of ara-C treatment
by 50 jug/ml (Chart 8). In the case of ADR treatment, the same
condition of anguidine pretreatment effected a significant re
duction in cell kill (1'/2 log over a concentration range of ADR
1001
from 1 to 10 /ig/ml) (Chart 10).
100
o.o111
0.1
1
5
Concentration of ADR (^g/ml)
Chart 10. Survival of exponentially growing CHO cells treated with increasing
concentrations of ADR for 1 hr in the presence and absence of 4-hr preincubation
with anguidine at 5 /ig/ml. Note the marked reduction in cell kill in case of
pretreatment with anguidine.
100
10
0.01
46
12
ARA-C Exposure Time (HR)
Chart 8. Survival of exponentially growing CHO cells following increasing
durations of ara-C treatment (25 and 50 /ig/ml) with and without preincubation
in 5 (ig of anguidine per ml for 4 hr. Note the marked antagonistic effect in the
case of anguidine pretreatment, which is most obvious after 12 and 18 hr of araC exposure.
•Anguidine —ARA-C
o Anguidine—ADR
0.1
100
0.01
TT
0.1
10
2.5
Concentration
of Anguidine (^g/ml)
Chart 11. Evaluation of anguidine-induced reduction in cell kill from ADR (5
fig/ml; 1 hr) and ara-C (50/jg/ml; 18 hr). as a function of anguidine concentration
(preincubation for 4 hr). Note that a plateau of cytoprotection is reached at a
concentration of 1 /ig of anguidine per ml for both cytotoxic drugs.
0.01
5
10
25
Concentration of ARA-C (pg/ml)
Chart 9. Concentration-dependent
survival of exponentially growing CHO
cells following 18 hr of ara-C treatment in the presence and absence of 4-hr
preincubation with 5 jug anguidine per ml. There is a 30- to 100-fold reduction in
cell kill in the case of preincubation with anguidine over the entire range of araC concentrations.
We next examined the protection against ara-C and ADRinduced cell kill as a function of anguidine concentration. For
both cytotoxic agents, a plateau of protection was noted as the
anguidine concentration reached 1 /ug/ml (Chart 11).
The biological importance of the sequence of administration
of anguidine and cytotoxic treatment with ara-C and ADR was
investigated in experiments illustrated in Charts 12 and 13. It
is readily apparent that preincubation with anguidine was cru
cial to elicit an antagonistic effect against cell inactivation by
ADR, whereas concurrent or subsequent treatment with angui
dine did not afford any protection (Chart 12). In the case of
ara-C, requiring prolonged continuous exposure for significant
cytoreduction (see Chart 8), postincubation with anguidine
APRIL 1981
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1267
L. Teodori et al.
100
cytoprotection was accompanied by a gradual increase in the
labeling index following anguidine treatment from 0% at the
end of a 4-hr anguidine exposure to 13 and 51 %, 6 and 24 hr
after drug removal, respectively (Table 1). Thus, it appears that
the labeling index as an indicator of the proportion of cells
actively synthesizing DMA may sensitively reflect the degree
and duration of cell cycle arrest providing adequate protection
against subsequently administered cytotoxic chemotherapy.
10
DISCUSSION
0.1
o Anguldine Pré-Incubation
.-. Anguldine Simultaneous Incubation
e Anguidine Post-Incubation
A ADR Alone
0.01
1
5
10
Concentration of ADR (fjg/ml)
Chart 12. Evaluation of the schedule dependency of cytoprotection by anguidine (5 /ig/ml; 4 hr) against 1-hr treatment with increasing concentrations of
ADR. Amelioration of ADR cytotoxiciry was observed only in case of preincubation
with anguidine whereas concomitant initiation of treatment or postincubation with
anguidine closely followed the dose-response survival curve for ADR alone.
100
In this report, we have confirmed in an additional cell system
our previous finding that anguidine has the potential of effec
tively freezing cycle progression through all cell cycle stages
at virtually no cytotoxic expense (10, 29). Compared to our
previous report on human colon cancer (LoVo) cells, CHO cells
proved to be somewhat more sensitive to the toxic effects of
anguidine, but as in LoVo cells, the length of exposure was a
more important determinant for cell kill than drug concentra
tion. With regard to the cell cycle effects, almost identical
results were obtained in human colon cancer and CHO cells,
although the lower anguidine concentrations (0.01 and 0.1 /ig/
ml) used in this report were not examined in the previous paper.
Thus, in the current investigation, we demonstrated a threshold
concentration (0.1 jug/ml) below which anguidine does not
100
10
10
•Anguldln«—ADR
0-1
0.01
o Anguidine Pre Incubation
û Anguidine Simultaneous Incubation
•Anguidine Post-Incubation
•ARA-C Alone
5 10
Concentration
ADR Alón«
0.1
BO
of ARA-C (jjg/ml)
Chart 13. Schedule dependency of reduction in ara-C-induced cell kill (18 hr)
by exposure to anguidine (5 fig/ml; 4 hr). Approximately equal protection is
afforded in the case of either preincubation with anguidine or concurrent expo
sure to ara-C and anguidine, whereas survival in case of postincubation with
anguidine of CHO cells treated previously with ara-C was indistinguishable from
the survival pattern observed after treatment with ara-C alone.
mimicked the survival pattern for ara-C treatment alone (Chart
13). In contrast, simultaneous exposure to ara-C and anguidine
afforded protection of a similar extent as was observed after
pretreatment with anguidine.
We also investigated the biological importance of the time
interval between pretreatment with anguidine (5 /ig/ml; 4 hr)
and 1-hr pulse treatment with ADR (Chart 14). Increasing time
intervals between termination of anguidine exposure and treat
ment with ADR were associated with a decrease in cytoprotec
tion by almost 1 log within 12 hr, although still maintaining Vz
log protection from cell kill by ADR alone. This decrease in
1268
0.01
6
24
12
Drug-Free Interval (HR) Between
Anguidine and ADR Treatment
Chart 14. Examination of the reduction of ADR-induced cell kill (
hr) by preincubation with anguidine (5 fig/ml; 4 hr) as a function of the drug-free
time interval until ADR exposure. Note the moderate decrease in cytoprotection
with increasing time intervals after anguidine exposure. Compared to treatment
with ADR alone, a 12-hr exposure in drug-free medium still affords an approxi
mate '>i log protection. See Table 1 for comparison with labeling index following
termination of anguidine exposure.
Table 1
Labeling index following anguidine exposure (5 ng/ml; 4 hr)
Labeling index (",.)
hr6951
ControlAnguidineOhr6906hr621324
CANCER
RESEARCH
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Protection against Lethal Cell Damage by Cell Cycle Arrest
interfere with cycle progression. The relative increase in the
proportion of cells jn S phase during treatment with >0.1 jug of
anguidine per ml in the presence of Colcemid during the first
3 hr of exposure is reminiscent of results in the human colon
cancer system using continuous exposure to tritiated thymidine
(10). Thus, while the data on combined Colcemid and anguidine
exposure indicate an effective block of cycle progression
through all cell cycle stages, the early increase in the S-phase
compartment at the expense of cells in Gìand G2 + M
compartments is consistent with a delayed onset of the Gìand
G2 blocks compared to a prompt block of S-phase progression.
The persistence of a mitotic index of approximately 10% at 12
hr after joint exposure to Colcemid and anguidine at concen
trations ranging from 0.1 to 50 //g/ml is consistent with a
delayed establishment of an effective G2 block. The observation
of even a small percentage of mitotic cells following 3 and 6 hr
of continuous treatment with 0.1 and 1 /ug of anguidine per ml
does suggest an effective delay in the transition through mitosis
by anguidine in view of the lack of cell cycle compartment
distribution changes beyond 3 hr of combined exposure to
anguidine and Colcemid. In this investigation, we also approx
imated the duration of anguidine-induced cell cycle suspension
following 4-hr treatment as a function of drug concentration.
This determination was important in order to assure the per
sistence of the block throughout the cytotoxic exposure to araC. This block persisted for at least 12 hr. However, the labeling
index experiments indicated a partial recovery of DNA synthetic
activity by 24 hr, although the grain count distribution was still
markedly suppressed, suggesting that recovery was not yet
complete.
The availability of flow cytometric assessment of DNA and
protein content on a cell-by-cell basis allowed some insight
into the primary mechanism of action of anguidine, a purported
protein synthesis inhibitor (22). The virtually constant protein:
DNA content ratio for Gì,mid S, andG2 cells during a 12-hr
continuous exposure to a wide range of concentrations of
anguidine suggests that protein and DNA synthesis were
promptly suspended to an equal extent.
Experimental conditions associated with suspension of cell
cycle progression were associated with significant protection
against cell kill by ara-C and ADR. More detailed investigation
of this protective effect as a function of anguidine concentration
revealed that the protective effect was present at lower con
centrations than were required for prolonged suspension of
cycle progression. Interestingly, the critical anguidine concen
tration was quite similar (1 /¿g/ml)for both ADR and ara-C.
Protection against ara-C- and ADR-induced cytotoxicity by
pretreatment with anguidine does not necessarily indicate that
this antagonistic effect is related to the anguidine effect on
cycle progression. Yet, changes in schedule of administration
of anguidine and the cytotoxic agent demonstrated that angui
dine exposure following cytotoxic treatment was ineffective in
protecting against both ara-C and ADR. Lack of protection was
also noted in the case of concomitant exposure to anguidine
and ADR, but anguidine provided effective protection against
ara-C-induced cell kill. This difference between ara-C and ADR
is likely related to the requirement of prolonged ara-C exposure
to exert appreciable cytotoxicity (13).
The most convincing evidence that the anguidine protection
against cytotoxicity from ara-C and ADR was indeed related to
suspension of cycle progression was provided by the fact that
APRIL
the degree of protection decreased with the time interval be
tween anguidine exposure and ADR treatment. Yet, even after
a treatment-free interval of 12 hr, significant protection against
ADR-induced cell kill was observed. Such protection occurred
despite some degree of recovery of DNA synthesis and cycle
progression as documented in the experiment involving Col
cemid exposure following anguidine treatment.
The results of this investigation strongly support the conclu
sions of other investigators who indicated that, in addition to
cell cycle stage position, rate of cycle progression is an impor
tant determinant for extent of cell kill by antitumor agents (15,
27). Protein synthesis inhibitors in particular can provide pro
tection against cytotoxic damage from antimetabolites and nonphase-specific agents such as nitrogen mustard or in our case
ADR (6, 8, 9, 23, 30). We do not yet know the extent of cycle
traverse rate delay necessary for such protection, nor do we
understand the underlying biochemical mechanism. The lethal
efficacy of ADR is not restricted to cells undergoing DNA
synthesis (3, 12), although this agent is known to have de
creased cytotoxicity on cells in stationary phase of growth (4,
14). The observation of protection by pretreatment with angui
dine suggests that the rate of cycle traverse does determine
the extent of cell kill suffered during drug exposure in all stages
of the cycle.
Preliminary data4 on the modification of bleomycin- and
hyperthermia-induced
lethality by anguidine suggest that the
protection described for ADR and ara-C is a more general
phenomenon. If this approach is to be utilized clinically for the
design of more effective combination chemotherapy, differen
tial cell cycle arrest and/or protection of arrested cells against
cytotoxic damage in normal versus tumor cells needs to be
documented. In contrast to other cytokinetic strategies, such
as cell recruitment and synchronization, normal tissue protec
tion exploiting differential cycle traverse rate-dependent cell
kill is more readily applicable to the clinical setting, since
suspension of cycle progression is readily verified by combined
DNA flow cytometric and labeling index measurements.
ACKNOWLEDGMENTS
The authors wish to express their sincere appreciation
her excellent secretarial support.
to Mattie J. Scott for
REFERENCES
1. Barlogie, B , Drewinko, B . Dosik, G.. and Freireich, E. J. Cell kinetics and
the management of malignant disease in 1979. Acta Pathol. Microbio!.
Scand. Suppl., in press, 1981.
2. Barlogie, B., Spitzer, G., Hart, J. S., Johnston, D. A., Büchner,T., Schumann,
J., and Drewinko, B. DNA histogram analysis of human hemopoietic cells.
Blood, 48: 245-258, 1976.
3. Barranco, S. C. Review of survival and cell kinetics effects of Adriamycin
(NSC 123127) on mammalian cells. Cancer Chemother. Rep., 6: 147-152,
1975.
4. Barranco, S. C., and Novak, J. K. Survival response of dividing and nondividing mammalian cells after treatment with hydroxyurea, arabinosylcytosine, or Adriamycin. Cancer Res., 34: 1616-1618,
1974.
5. Baserga. R . Henegar, G. C . Kisieleski, W. E . and Lisco, H. Uptake of
tritiated thymidine by human tumors in vivo. Lab. Invest., //. 360-364,
1962.
6. Ben-lshay, Z., and Farber, E. Protective effects of an inhibitor of protein
synthesis, cycloheximide, on bone marrow damage induced by cytosine
* S. White. Lethal effects of bleomycin, cisplatin, and hyperthermia on anguidine-pretreated Chinese hamster ovary (CHO) cells in vitro. M. D. Anderson
Hospital Summer Student Research Symposium, August 1980.
1981
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1981 American Association for Cancer Research.
1269
L. Teodori et al.
arabinoside or nitrogen mustard. Lab. Invest., 33. 478-490, 1975.
7. Bhuyan. B. K., Fräser, T. J.. and Day, K. J. Cell proliferation kinetics and
drug sensitivity of exponential and stationary populations of cultured L1210
cells. Cancer Res.. 37. 1057-1063,
1977.
8. Capizzi, R. L. Biochemical interaction between asparaginase (A'ASE) and
methotrexate (MTX) in leukemia cells. Proc. Am. Assoc. Cancer Res., 75:
77, 1974.
9. Capizzi, R. L., Summers, W. P., and Benino. J. R. L-Asparaginase induced
alteration of amethopterin (methotrexate) activity in mouse leukemia L5178
Y. Ann. N.Y. Acad. Sci.. 786: 302-311, 1971.
10. Dosik. G.. Barlogie. B.. Johnston. D. A.. Murphy. W K., and Drewinko. B.
Lethal and cytokinetic effects of anguidine on a human colon cancer cell
line. Cancer Res.. 38 3304-3309,
1978.
11. Dosik, G., Barlogie. B., White. A.. Göhde. W., and Drewinko, B. A rapid
automated stathmokinetic method for determination of in vitro cell cycle
transit times. Cell Tissue Kinet., in press, 1981.
12. Drewinko, B., and Gottlieb, J. A. Survival kinetics of cultured human lymphoma cells exposed to Adriamycin. Cancer Res., 33: 1141 -1145, 1973.
13. Drewinko, B.. Ho, D. H., and Barranco, S. C. The effects of arabinosylcytosine on cultured human lymphoma cells. Cancer Res., 32: 2737-2742,
1972.
14. Drewinko, B., Patchen, M.. Yang. L. Y., and Barlogie. B. Differential killing
efficacy of twenty antitumor drugs on proliferating and non-proliferating
human tumor cells. Cancer Res., in press, 1981.
15. Ford. S. S., and Shackney, S. E. Lethal and sublethal effects of hydroxyurea
in relation to drug concentration and duration of drug exposure to Sarcoma
180 in vitro. Cancer Res.. 37: 2628-2637.
1977.
16. Freeman, P. A., and Crissman, H. A. Evaluation of six fluorescent protein
stains for use in flow microfluorometry. Stain Technol.. 50. 279-284, 1975.
17. Fu, C. T.. Johnston. D. A.. Thompson. D., and Barlogie, B. DNA/Protein
measurements by flow cytometry. Comparison of several techniques. Cell
Tissue Kinet. 13: 683, 1980.
18. Göhde, W.. Schumann, J., Büchner,T., Otto. F., and Barlogie, B. Pulse
cytophotometry: application in tumor cell biology and clinical oncology. In:
M. Melamed, P. Mullaney, L. Mortimer, and M. Mendelsohn (eds.). Flow
Cytometry and Sorting, pp. 599-620. New York: John Wiley & Sons, Inc.,
1979.
19. Göhde,W., Schumann, J., and Zante, J. The use of DAPI in pulse cytopho
tometry. In: D. Lutz (ed.), Third International Symposium on Pulse Cytopho
1270
tometry, pp. 229-232. Ghent: European Press Medikon, 1978.
20. Greenberg. P. L., and Mosny, S. A. Cytotoxic effects of Interferon in vitro on
granulocytic progenitor cells. Cancer Res., 37: 1794-1799,
1977.
21. Johnston, D. A., White, R. A., and Barlogie, B. Automatic processing and
interpretation of DMA distribution: comparison of several techniques. Cornput. Biomed. Res., 11: 393-404, 1978.
22. Liao. L. L.. Grollman. A. P., and Horwitz. S. B. Mechanism of action of the
12,13-epoxytrichothecene,
anguidine. an inhibitor of protein synthesis.
Biochim. Biophys. Acta. 454: 273-284, 1976.
23. Lieberman. M. W.. Verbin, R. S., Landay. M. M., Liang, H., Farber, E.. Lee,
T. N., and Starr, R. A probable role for protein synthesis in intestinal
epithelial cell damage induced in vivo by cytosine arabinoside. nitrogen
mustard, or X-irradiation. Cancer Res., 30: 942-951, 1970.
24. Murphy, W. K., Burgess, M. A., Valdivieso, M.. Livingston. R. B., Bodey, G.
P.. and Freireich, E. J. Phase I clinical evaluation of anguidine. Cancer
Treat. Rep.. 62. 1497-1502.
1978.
25. Puck, T. T., and Marcus, P. A rapid method for viable cell titration and clone
production with HeLa cells in tissue culture: the use of X-irradiated cells to
supply conditioning factors. Proc. Nati. Acad. Sei. U. S. A., 41: 432-438,
1955.
26. Roper, P., and Dewinko, B. Comparison of in vitro methods to determine
drug-induced cell lethality. Cancer Res., 36: 2182-2188,
1976.
27. Shackney, S. E., Erickson. B. W., and Lengel. C. E. Schedule optimization
of cytosine arabinoside (CA) and hydroxyurea (HU) in Sarcoma 180 in vitro.
Proc. Am. Assoc. Cancer Res.. 79. 225, 1978.
28. Stöhr, M., Eipel, H., Goerttler, K.. and Vogt-Schaden, M. Extended appli
cation of flow microfluorometry by means of dual laser excitation. Histochemistry, 51: 305-313, 1977.
29. Teodori. L., Fu, C. T., and Barlogie, B. In vitro cell cycle and lethal effects of
anguidine. Cell Tissue Kinet. 73: 683, 1980.
30. Weissberg. J. B.. Herion, J. C., Walker, R. I., and Palmer, J. G. Effect of
cycloheximide on the bone marrow toxicity of nitrogen mustard. Cancer
Res.. 38: 1523-1527.
1978.
31. White, R. A.. Barlogie, B., and Dosik, G. The biomédical sources of error in
determining cycle transit times by flow cytometry following Colcemid block.
Cell Tissue Kinet., in press, 1981.
32. Yap, M.-Y., Murphy. W. K., DiStefano. A., Blumenschein, G. R., and Bodey.
G. P. Phase II study of anguidine in advanced breast cancer. Cancer Treat.
Rep.. 63: 789-791, 1979.
CANCER
RESEARCH
VOL. 41
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Reduction of 1-β-d-Arabinofuranosylcytosine and Adriamycin
Cytotoxicity following Cell Cycle Arrest by Anguidine
Laura Teodori, Barthel Barlogie, Benjamin Drewinko, et al.
Cancer Res 1981;41:1263-1270.
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