[CANCER RESEARCH 42, 3532-3537, September 1982]
0008-5472/82/0042-0000$02.00
CC-1065 (NSC 298223),
a Most Potent Antitumor Agent: Kinetics of
Inhibition of Growth, DMA Synthesis, and Cell Survival
Bijoy K. Bhuyan,1 Kenneth A. Newell, Sheri L. Crampton, and Daniel D. Von Hoff
The Upjohn Company, Ka/amazoo, Michigan
ID. D. V.¡
49001 [B. K. B., K. A. N., S. L. C.I, and University of Texas Health Science
ABSTRACT
CC-1065 (NSC 298223) is the most cytotoxic agent tested
against cells in culture in our laboratory. The 50% lethal doses
for exponentially growing B16 melanoma and Chinese hamster
ovary cells were 0.44 and 0.14 ng/ml, respectively, as com
pared to 35 and 500 ng/ml for Adriamycin. In the human
tumor-cloning assay, 1-hr exposure to CC-1065 (0.1 ng/ml)
caused ^50% lethality in a broad spectrum of tumors. The
dose-survival curves for B16 and Chinese hamster ovary cells
were characterized by an initial shoulder followed by an ex
ponential decline with increasing dose. CC-1065 was more
lethal to exponentially growing B16 cells (50% lethal dose =
0.44 ng/ml) than to plateau-phase cells (50% lethal dose =
1.2 ng/ml).
CC-1065 inhibited DNA synthesis much more than did RNA
or protein synthesis. After a 2-hr incubation with drug, inhibition
of DNA synthesis was low immediately (0 hr) after drug expo
sure and reached maximum inhibition about 20 hr later. The
doses for 50% inhibition of growth (0.18 ng/ml), survival (0.44
ng/ml), and DNA synthesis (0.15 ng/ml) were in the same
range, whereas RNA synthesis was inhibited 50% at a much
higher dose (5 ng/ml).
INTRODUCTION
CC-1065, a new antibiotic produced by Streptomyces zelensis, Dietz and Li nov. sp., was discovered and characterized at
The Upjohn Company, Kalamazoo, Mich. (4). It has significant
cytotoxicity in vitro and antitumor activity in vivo (4, 6). It is the
most potent agent tested in our laboratories against L1210
cells in culture. It caused 90% inhibition of the growth of L1210
cells at 0.05 ng/ml. This may be compared to the 90% inhibi
tion of the growth by some of the more cytotoxic antitumor
agents in this assay system: actinomycin D, 4 ng/ml; and
Adriamycin, 20 ng/ml (5). It was also active in vivo against the
murine tumors, P388 and L1210 leukemia, B16 melanoma,
CD8Fi mammary, and Colon 26 at doses ranging from 1 to 50
jug/kg (6, 8). The structure of CC-1065 was reported by Martin
et al. (7) and is shown in Chart 1.
Previous studies by Li et al. (5) showed that CC-1065 is
much more effective in inhibiting DNA synthesis than in inhibit
ing RNA or protein synthesis. The inhibition of DNA synthesis
is probably mediated through the strong binding of CC-1065
to DNA on the basis of studies of thermal melting temperature
of DNA, difference circular dichroism spectra, and Sephadex
chromatography (5). The drug binds only to double-stranded
DNA and not to heat-denatured DNA (5). The mechanism of
1To whom requests for reprints should be addressed.
Received January 6, 1982; accepted June 3, 1982.
3532
Center, San Antonio, Texas 78284
interaction of CC-1065 with DNA has been described by Swenson et al. (9).
Inasmuch as CC-1065 was so highly cytotoxic and interacted
strongly with DNA, it was assayed against human tumor cells
in the cloning assay (3) and against B16 melanoma and CHO
cells in culture. This paper compares the lethality of CC-1065
to that of several antitumor drugs and describes the kinetics of
growth inhibition, survival, and DNA synthesis in B16 cells. An
abstract of this study has appeared elsewhere (1 ).
MATERIALS
AND METHODS
CHO2 Cell Culture.
CHO cells were maintained in Ham's F-10
medium supplemented with 15% fetal calf serum. CHO cells grow
exponentially up to a cell density of about 5 x 106/75-sq-cm flask.
B16 Melanoma Cell Culture. The B16 (clone F-10) cell line was
obtained from Dr. I. J. Fidler (Frederick Cancer Research Center,
Frederick, Md.). The cells were grown as a monolayer in MEM (MEM
with Earle's salts) supplemented with 10% fetal calf serum, 1 HIM
sodium
pyruvate,
2 mw L-glutamine,
penicillin
G (60 ¡ig/m\), and
streptomycin (10 jig/ml). This medium was further supplemented with
MEM nonessential amino acids (10 ml of 100 x concentration per liter
of medium) and essential MEM vitamins (10 ml of 100 x concentration
per liter). Medium constituents were purchased from Grand Island
Biological Co., Grand Island, N. Y. The origin of the highly metastatic
F-10 line has been described (2). In this medium, the cells had a
doubling time of about 16 hr and grew exponentially
tration of about 107 cells/75-sq cm flask.
to a cell concen
Drug Exposure for Growth Inhibition and Cell Survival Determi
nation. B16 and CHO cells were planted (105 cells/25-sq cm flasks)
as a monolayer culture 24 hr before an experiment to assure exponen
tial growth during drug exposure. Plateau-phase B16 cells were ob
tained by planting 5 x 10s cells/25-sq
cm flask 3 days before an
experiment. On the second day, the old medium was replaced by fresh
medium, and on the third day the flasks contained 1.3 x 107 cells at
the time of exposure to drug. Flow cytometry showed that the majority
of these cells were in G0-Gi phase (10). Cells were exposed to drug at
37° in their respective growth medium. For each drug concentration
tested, 2 separate cultures were used.
To determine the percentage of survival after drug exposure, the
B16 or CHO cell monolayer was harvested with trypsin, and the cells
were centrifuged and washed to remove drug. The cells were diluted
in medium, and an aliquot was planted (to give about 20 to 100
colonies) in multiwell Linbro plates. Four wells were used per sample.
The plates were incubated in a humid 37°, 7% CO2 atmosphere for 8
days. Then, the colonies were fixed, stained with 0.2% méthylèneblue
in 70% ethanol, and counted. Cloning efficiency of exponentially grow
ing CHO and B16 cells and plateau-phase B16 cells ranged between
50 and 90%. In all cases, the cloning efficiency of the untreated
(control) cells was normalized to 100% and the cloning efficiency of
2 The abbreviations used are: CHO, Chinese hamster ovary; MEM, minimal
essential medium (Eagle's); LDso and LD9o, drug concentration to kill 50% and
90% of the cells, respectively; T-CFU, tumor colony-forming units.
CANCER
RESEARCH
VOL. 42
CC 1065-DNA Inhibition, Cell Kill Kinetics
the drug-treated samples as compared to the control. A decrease of
»70% in T-CFU was used because, in a prior retrospective study, it
was satisfactory for predicting which patient would respond to a partic
ular chemotherapeutic agent (11 ).
OH
OCH3
OCH3
RESULTS
Chart 1. Structure of CC-1065 (7).
Stability
the treated cells was expressed as a percentage of control survival.
The coefficient of variation (standard deviation expressed as a per
centage of the mean) in determining cell survival was about 15% within
each experiment. All experiments were repeated at least once.
For growth inhibition experiments, B16 cell monolayers (about 105
cells/75 sq cm) were exposed for different periods to the drug. The
cell monolayer was then washed to remove extracellular drug and was
incubated for 72 hr. At the end of 72 hr, the cells were harvested and
counted with a Coulter Counter. The percentage of growth inhibition
was calculated as:
100 x
f Cell no. in treated flask at 72 hr - cells inoculated\
\
Cell No. in control at 72 hr - cells inoculated
/
Determination
of Macromolecule
Synthesis by B16 Cells. DNA,
RNA, and protein syntheses were monitored by adding 1 ml of 10/iCi
[3H]thymidine (2 ftg/ml), or 20 /iCi [5-3H]uridine (10 /ig/ml), or [14C]leucine (5 /iCi/ml) to 9 ml medium covering the cell monolayer of about
106 cells/25-sq cm flask. To stop uptake of radioactivity, the radioac
tive medium was removed, and the cells were washed with 5 ml of cold
0.9% NaCI solution containing the appropriate unlabeled precursor
(thymidine, undine, or leucine) at 100 /ig/ml. The cells were then
harvested and resuspended in cold medium containing appropriate
unlabeled precursor (100 jug/ml).
One aliquot was counted in the Coulter Counter to give the cell
number, while 2 other aliquots of cells were filtered through glass
microfiber (Whatman GF/C) filters. The filters were washed 4 times
with 3 ml cold 10% trichloroacetic acid and once with 3 ml ethanol.
The filters were then placed in scintillation vials and heated with 0.5 ml
of 0.5 N perchloric acid at 70°for 1 hr. Diotol (15 ml) was added, and
the filters were counted in a scintillation counter.
Preparation of CC-1065 Solution. CC-1065 was dissolved
in di-
methylformamide at 10 to 100 /ig/ml and stored in a glass vial in the
freezer. CC-1065 is stable under these conditions. Since the drug
of CC-1065
CC-1065 was stable when stored as a solution in dimethylformamide in the freezer. The drug (at 0.2 ng/ml) was stable
in H2O in a glass vial for at least 24 hr. It was unstable when
stored in a plastic vial under the same condition, probably due
to binding to the plastic. In B16 medium containing serum,
about 15 to 20% of CC-1065 were lost after 2 hr and 50 to
60% were lost after 24 hr at 37°.
Inhibition of Growth of B16 Cells by CC-1065
Comparison of Glass and Plastic Culture Flasks. Although
CC-1065 binds to plastic, similar growth inhibition was ob
served when the drug was added to cell monolayers growing
in plastic or glass flasks. Therefore, we used plastic flasks in
all subsequent experiments. However, glass pipets and glass
vials were used to dilute and dispense CC-1065.
Time Course of Growth Inhibition. The effect of 72-hr
exposure to CC-1065 on the growth of B16 cells is shown in
Chart 2. Cells treated with 0.2 ng/ml grew exponentially at a
rate slower than the untreated control. Cells exposed to 0.4ng/ml of drug grew at a much slower rate, and cell growth
stopped after 2 cell divisions (i.e., 2 doublings of cell number)
subsequent to drug addition. At 0.8 ng/ml, cell growth stopped
after 1 cell division.
107_
CONTROL
5 x 106
binds rapidly to plastic, glass pipets and vials were used in preparing
dilute solutions. The CC-1065 solution was diluted in medium prior to
adding to the cells.
Human Tumor-cloning
Assay. The human tumor-cloning
assay was
done according to procedures described in detail previously (3, 12). In
brief, tumor cell suspensions were adjusted to a final concentration of
106 cells/ml and then incubated at 37°with either CC-1065 (0.1 ng/
ml) or the appropriate vehicle for 1 hr. The cells were then washed and
suspended in enriched Connaught Medical Research Laboratories
Medium 1066 supplemented with 15% horse serum and 0.3% agar to
yield a final concentration of 5 x 105 cells/ml. One ml of this mixture
was pipeted on top of 1 ml of enriched McCoy's Medium 5A solidified
106
5 x 10:
with 0.5% agar. Conditioned medium was not added. Cultures were
incubated at 37°in 7% CO2 in humidified air. All assays were set up in
triplicate.
Colonies (»50 cells) usually appeared in 10 to 15 days. The number
of colonies on control and drug-treated plates was counted on an
inverted-stage microscope at 30-fold magnification. A minimum of 30
colonies/plate
were required for an experiment to be considered
adequate for measurement of drug effect. Colony counts of the 3 plates
for a particular drug concentration were averaged to obtain one data
point. The standard error of the mean for individual data points aver
aged 14% of the mean.
The results are presented as a percentage of decrease in T-CFU in
SEPTEMBER
1982
10=
20
40
60
HOURS
8O
10O
Chart 2. Growth of B16 cells during 72 hr of continuous exposure to CC1065. The cells were planted in a 75-sq cm flask at 0 hr, and the drug was
added 20 hr later. Cells were exposed to the drug for 72 hr O.e., from 20 to 92
hr). At different times, cells were harvested from individual flasks, and cell number
per flask was determined by counting in the Coulter counter. This experiment
was repeated once and gave similar results. Bars, S.E.
3533
8. K. Bhuyan et al.
Effect of Contact Time on Growth Inhibition by CC-1065.
The growth inhibition obtained at different doses, with exposure
times ranging from 10 min to 72 hr, is shown in Chart 3. The
results are shown below.
CC-1065 acts very rapidly and irreversibly at high doses of
the drug. Thus, only 10 min exposure to CC-1065 (5 and 1
ng/ml) resulted in inhibition of growth during subsequent 72 hr
incubation. However, the action of the drug could be reversed
completely in cells exposed to 0.5 ng/ml for 10 min.
The extent of growth inhibition was dose and exposure time
dependent. Growth inhibition reached a plateau after a 2-hr
exposure. The dose for 50% growth inhibition (Table 1) ranged
from 1.8 ng/ml for 10 min exposure to 0.22 ng/ml for 72 hr
exposure. The concentration
x time value was similar for
exposures ranging from 10 min to 2 hr (see Table 1), which
indicates that up to 2-hr growth inhibition was proportional to
dose and exposure time. Since there was no further increase
in growth inhibition after 2 hr, there was a sharp increase in
the concentration x time value for 72 hr as compared to 2 hr.
Complete degradation of CC-1065 by the end of a 2-hr
incubation would account for the absence of any further in
crease in growth inhibition after 2 hr. Only 20% of CC-1065
was lost when the drug was incubated with medium alone for
24 hr at 37°. However when CC-1065 was incubated with a
monolayer culture of B16 cells, 90% of the CC-1065 was
removed from the supernatant medium (Table 2). This suggests
that CC-1065 was either bound to the cells or was degraded
by the cells during the 2-hr incubation. Since there was no CC1065 in the supernatant medium after 2 hr, there was no further
increase in growth inhibition after a 2-hr exposure.
Growth Inhibition in Medium with and without Fetal Calf
Serum. Because CC-1065 binds to a variety of substances, its
effect on cell growth was examined in serum-free medium. The
results (Table 3) are shown below.
CC-1065 was 10 to 20 times more cytotoxic in the absence
of serum than in its presence. Thus, after 10 min exposure, 5
ng/ml inhibited growth 85% in the serum-containing medium
compared to 91.7% inhibition by 0.5 ng/ml in the serum-free
medium.
In the absence of serum, even very low levels (0.25 ng/ml)
caused irreversible effects in 10 min.
I so
60
40
eg
Z
20
0.16
0.5
1
5
10
HOURS OF EXPOSURE
100
Chart 3. Inhibition of growth of B16 cells resulting from varying periods of
drug exposure. B16 cell monolayers were exposed to CC-1065 for periods
ranging from 10 min to 2 hr. After drug exposure, the cell monolayer was gently
washed, fed with fresh medium, and incubated for 70 hr. The growth of the drugtreated samples was expressed as a percentage of the growth in the untreated
(washed) control. For the 72-hr time point, cells were continuously exposed to
CC-1065 for 72 hr. This experiment was repeated once and gave similar results.
3534
(ng/ml)1.8
Exposure
(hr)0.166
time
i0.3
x
0.91
0.36
0.25
0.22C
0.5
1
2
72ID50a
0.45
0.36
0.5
15.8
IDso, concentration to inhibit growth by 50%. These values were calculated
from the data shown in Chart 3. C x t, ID5o x time of exposure.
Table 2
Growth inhibition by CC-1065 after incubation with medium alone or medium
plus 876 ce/te
growth3CC-1
% of control
065
(ng/ml)0.06
alone9
+90
0.125
0.25
0.5
0.6Medium
31Medium
90
60
16
9cells
Medium alone or medium plus (1.75 x 10 ) B16 cells was incubated with
different levels of CC-1065 for 2 hr at 37°. The supernatant medium was then
removed, and its ability to inhibit growth of a B16 culture was measured. CC1065 (0.6 ng/ml) incubated with medium plus cells gave the same growth
inhibition as CC-1065 (0.06 ng/ml) in medium alone. Therefore, 90% of the CC1065 was removed from the supernatant medium during a 2-hr incubation with
medium plus cells at 37°.
Table 3
Growth inhibition after drug exposure in medium with and without serum
B16 cell monolayers were washed twice with 0.9% NaCI solution and then
serum-free or serum-containing medium was added to the cells. CC-1065 was
diluted in the appropriate medium and was then added to the cells. After 10 or
30 min of contact, the cells were washed twice with 0.9% NaCI solution and then
incubated with fresh medium for 72 hr, at which time cell number was determined.
The experiment was repeated and gave similar results.
% of growth inhibition for exposure times of
10 min
i;t;- 1uba
(ng/ml)5
0.5
0.25Serum85
30 min
serum100
2.24.5No
91.7
74Serum99.3
serum100
50.5
9.3No
99.9
Cell Kill by CC-1065
Lethality for Human Tumor Cells. The lethality of CC-1065
(1-hr exposure to 0.1 ng/ml) for a number of human tumors
was determined by the human tumor-cloning assay; the results
are shown in Table 4. A decrease of ^70% in the number of T-
100
E
O
Table 1
ID50a values for different exposure times
CFU has been assumed to be necessary for this assay to
predict for results in the clinic (11). On this basis (3=70%
decrease in T-CFU, CC-1065 was active against tumors from
patients with cancer of lung (1 of 9 patients tested), pancreas
(1 of 2 patients tested), stomach (1 of 1 patient tested), small
bowel (1 of 1 patient tested), and adenocarcinoma of unknown
origin (1 of 2 patients tested). For many more tumors, the
percentage of decrease in T-CFU was 5=50%. On the basis of
3=50% decrease in T-CFU, CC-1065 was active against tumors
from patients with cancer of the breast (3 of 3 patients), lung
(3 of 9 patients), ovary (2 of 8 patients), pancreas (1 of 2
patients), multiple myeloma (2 of 2 patients), stomach (1 of 1
patient), testis (1 of 1 patient), small bowel (1 of 1 patient), and
adenocarcinoma of unknown origins (1 of 2 patients). CC-1065
was inactive (<50% decrease in T-CFU) against melanoma (3
CANCER
RESEARCH
VOL. 42
CC 1065-DNA Inhibit/on, Cell Kill Kinetics
samples tested), acute myelogenous leukemia (2 samples
tested), esophagus (1 sample tested), non-Hodgkin's
lym-
Inhibition of Macromolecule Synthesis in B16 Cells
phoma (1 sample tested), colon (3 samples tested), and sar
coma (2 samples tested).
Lethality for B16 and CHO Cells. The dose-survival curves
The effect of CC-1065 on DNA, RNA, and protein syntheses
was determined immediately following a 2-hr exposure to the
drug. In these experiments, labeled precursors were added
simultaneously with the drug and the cells were harvested 2 hr
later to determine radioactivity incorporation into DMA, RNA,
and protein. DMA synthesis was inhibited 19.5 ±5.3% (S.D.),
32.3 ±5%, and 65.7 ±4.7% at 0.5, 1, and 5 ng/ml, respec
tively. RNA and protein synthesis were not inhibited even at 5
ng/ml. Similar results have been reported by Li et al. (5) using
L1210 cells.
The dose needed for inhibition of DNA synthesis was much
greater than the doses needed for inhibition of cell growth and
survival. Thus, the doses required for 50% inhibition of growth,
for B16 and CHO cells are shown in Chart 4. With both cell
lines a sigmoid-survival curve was obtained which was char
acterized by an initial shoulder followed by an exponential (loglinear) decline in survival as the dose increased. These curves
were used to determine the D0 (1 /slope) of the exponential
portion of the curve and the LD50 and LD90 values (Table 5).
Plateau B16 cells were much less sensitive than were expo
nentially growing cells.
Table 5 compares the lethality of CC-1065 to several widely
used antitumor drugs. The results clearly show that CC-1065
is 50 to Stl000 times more cytotoxic than are most other drugs.
B16 cell-kill increased only slightly when exposure to CC1065 was increased from 2 to 24 hr. These results corroborate
the time course of growth inhibition and suggest that CC-1065
action was completed during a 2-hr exposure.
100
B16 Plateau
Table 4
Lethality of CC-1065 (0.1 ng/ml, 1-hr exposure) for human tumors
The lethality of CC-1065 was determined by a human tumor-cloning
assay.
No.
sensitiveNo.of
of tumors
tested2:70% tumors
de-Tumor
inT-CFU0/31 inT-CFU3/3
decrease
typeBreastLung
50)a3/9
(66, 53,
U
(adenocarcinoma)MelanomaOvaryPancreasAdenocarcinoma
(82)0/30/81/2(93)1/2(85)0/20/21/1
/9
54)0/32/8
(82, 67,
te
LU
57)1 (69,
(93)1
/2
origin)Acute
(unknown
leukemiaMultiple
myelogenous
myelomaStomachTestisEsophagusSmall
(85)0/21
/2
(68)1/1
/2
(70)0/10/11/1(70)1/1
(53)0/11/1
bowelLymphoma
(non-Hodgkin's)ColonSarcomacrease
1Numbers in parentheses,
0.01
(84)0/10/30/2^50%
(84)0/10/30/2
0.6
0.8
CC 1065 ng/ml
1.5 2.0
2.5
3.0
Chart 4. Dose-survival curves for B16 and CHO cells after 2 hr exposure to
CC-1065. Survival was determined by cloning. Results of several different
experiments in each group are shown by different symbols. Bars, S.E.
percentage of decrease in T-CFU.
Table 5
Lethality of CC-1065 and other antitumor agents for exponentially
growing B16 and CHO cells
Exponentially growing CHO and B16 cells were exposed to drug for 2 hr after which the percentage of
survival was determined by cloning assay.
(ng/ml)B16LDso3
LD500.44 LD90
CC-1065AdriamycinActinomycin
0.1435
50030
0.73
100
kill
Do
0.28
30
Do0.27
0.071
300
600
Dc/s-DiamminedichloroplatinumL-Phenylalanine5004
1033.6
X
10325
X
(NSC8806)1
mustard
x 103CHOLD90
-/8-D-Arabinofuranosylcytosine(NSC
63878)Cell
a LD50and LD»are the respective doses needed to kill 50 and 90% of the cells. Db is the reciprocal of the
slope of the straight exponential portion of the dose-survival curve. It is the dose which reduces survival by
a factor 1/e (=0.37) in the straight exponential portion of the curve.
SEPTEMBER 1982
3535
ß.K. Bhuyan et al.
survival, and DNA synthesis were about 0.25, 0.18, and 3.2
ng/ml, respectively. This discrepancy between the doses
needed for the different biological effects could be because
CC-1065 does not cause maximum inhibition of DMA synthesis
immediately after drug exposure but at some later time. The
inhibition of DMA synthesis at various times after a 2-hr drug
exposure is shown in Chart 5. The results show that the
inhibition of DMA synthesis increases with time after drug
exposure and reaches a maximum at about 18 to 21 hr. At 1
and 5 ng/ml, there was no change in the percentage of
inhibition after 20 hr, whereas at 0.25 and 0.5 ng/ml there was
a substantial decrease in inhibition after 20 hr.
Since the inhibition of DNA synthesis was maximum at about
18 to 21 hr after drug exposure, the inhibition of RNA and
protein synthesis was also measured at this time. The results
(Chart 6) show that, at 18 hr postexposure to 1 ng/ml, DNA
synthesis was inhibited 80% with no inhibition of RNA, or
protein synthesis. Significant inhibition (53%) of RNA synthesis
was seen only at 5 ng/ml. At 18-hr postdrug exposure, the
doses for 50% inhibition of growth (0.25 ng/ml), survival (0.44
100r
5 ng/ml A
10
60
20
30
4O
50
HOURS POST-DRUG EXPOSURE
70
Chart 5. Inhibition of DNA synthesis at various times after an initial 2-hr drug
exposure. B16 cells were exposed to CC-1065 (0.25 to 5 ng/ml) for 2 hr
following which the cell monolayer was washed gently with warm medium and
then the cells were incubated in fresh warm medium. At different times thereafter,
| H|thymidine (20 /iCi. 5 fig) was added for 1 hr. The cells were then harvested,
and radioactivity incorporation into DNA was measured. The specific activity of
DNA in the control sample for different times ranged between 3.7 x 10* to 5 x
10 ' cpm 1O6cells. The results of several experiments are shown A, A, 5 ng/ml;
D, •1 ng/ml; *. O, 0.5 ng/ml; and O, 0.25 ng/ml.
(0lULUZZ
DNA.Z"fX'^
—'"'
>
IO
«H
RNA0
Zm
u¡|2oc01OOso604020^—-—-•o
PROTEINo
,
0.2
0.5
CC-1065 (ng/ml)
•/Y//
o
1
yf —-c
5
Chart 6. Inhibition of DNA, RNA, and protein syntheses at 18 hr after an initial
2-hr drug exposure. Protocol was the same as in Chart 5 except that [5-3H]
uridine, ["CJIeucine. and |'H jthymidme were added at 18 hr post-drug exposure.
The results of several experiments
different symbols.
3536
to measure DNA inhibitions
are shown by
ng/ml), and DNA synthesis (0.15 ng/ml) were similar, whereas
about a 10-fold higher dose (5 ng/ml) was needed for inhibition
of RNA synthesis.
DISCUSSION
Comparative Cytotoxicity of CC-1065 and Adriamycin.
Several factors, including lethality to cells in culture and in the
human tumor-cloning assay, influence the selection of cancer
chemotherapeutic drugs for clinical trial. Adriamycin is being
compared to CC-1065, since it is a cytotoxic antitumor agent
that interacts with DNA and is active against a broad spectrum
of tumors in humans. CC-1065 (LDSO= 0.6 nM) was 100 times
more cytotoxic than was Adriamycin (LDSO= 64 nw) to B16
cells. Greater cytotoxicity of CC-1065 as compared to Adria
mycin was also seen with CHO and L1210 cells (1, 5).
CC-1065 can also be considered to be at least 100 times
more potent than Adriamycin in the human tumor-cloning as
say, in which CC-1065 was used at 0.1 ng/ml and Adriamycin
was used at 40 to 400 ng/ml. Many of the human tumors
(Table 4) were tested simultaneously for their sensitivity to CC1065 and Adriamycin. For 6 lung tumor samples, the percent
ages of survival after exposure to CC-1065 and Adriamycin,
respectively, were as follows: 33, 90; 18, 56; 46, 73; 61, 100;
97, 77; and 81, 59. These results show that 4 of the 6 tumor
samples were more sensitive to CC-1065 than the Adriamycin.
CC-1065 (34 and 50% survival) and Adriamycin (40 and 60%
survival) were equally lethal to 2 of the breast tumor samples.
For 4 ovarian tumor samples, the percentages of survival after
exposure to CC-1065 and Adriamycin, respectively, were as
follows: 31,100; 60,100; 63, 54; and 100, 75. Therefore, CC1065 was significantly more active than Adriamycin against at
least one ovarian tumor. These results also show that tumors
that were resistant to Adriamycin were sensitive to CC-1065;
i.e.. the drugs were not cross-resistant.
Kinetics of CC-1065 Cytotoxicity. Both growth inhibition
and cell kill studies showed that maximum effect was obtained
after a 2-hr exposure to B16 cells to CC-1065. There was no
further increase in growth inhibition or of cell kill for drug
exposure periods greater than 2 hr. This cessation of CC-1065
effect was not due to degradation of the drug, since only 15 to
20% of the cytotoxicity of CC-1065 was lost after 2 hr of
incubation in medium at 37°. The reason for this limitation of
CC-1065 activity is probably due to the removal of the drug
from the medium due to drug binding to the cells.
These results in vitro correlate well with in vivo experiments
in which the percentage of increase of life span of leukemic
(P388 or L1210) mice was the same irrespective of whether
the mice were given a single dose or several doses of the drug
(8).
Kinetics of Inhibition of DNA Synthesis. The primary site of
CC-1065 action is presumed to be its interaction with DNA
based on the following evidence, (a) There was marked inhi
bition of DNA synthesis with minimal inhibition of RNA and no
inhibition of protein synthesis (Chart 6; Ref. 5). (b) The dose
for 50% inhibition of DNA synthesis, growth, and cell survival
were similar and ranged between 0.15 and 0.45 ng/ml. The
50% inhibitory dose for RNA synthesis was about 10-fold
higher. Preferential inhibition of DNA synthesis as compared to
RNA or protein synthesis was also seen in L1210 cells (5).
CANCER
RESEARCH
VOL. 42
CC 1065-DNA Inhibition, Cell Kill Kinetics
Further studies by Swenson ei al. (9) have indicated that CC1065 does not intercalate with DNA but binds in the minor
groove of double-stranded DNA at A-T-rich sites. Therefore,
the inhibition of DNA synthesis probably occurs as a result of
the binding of CC-1065 to the DNA template.
The percentage of inhibition of DNA synthesis in cells ex
posed to 1 ng/ml was minimum at 4-hr postdrug exposure
(Chart 5). Preliminary experiments indicated that this decrease
was an artifact of the method of expressing DNA synthesis rate
as cpm/cell. Cell progression studies showed that CC-1065
caused cells to accumulate in S (1 ). Therefore, the percentage
of cells in S at 4 hr postdrug was higher than that in the control.
To take into account the varying percentages of S-phase cells,
the rate of DNA synthesis was recalculated as cpm/S-phase
cell. When this correction was made, the decrease in percent
age of inhibition of DNA synthesis at 4-hr postdrug exposure
was not observed, rather the percentage of inhibition gradually
increased to reach a maximum at 19 hr.
Maximum inhibition of DNA synthesis was seen at 18 to 20
hr after termination of drug exposure. Such an effect could be
caused by extensive cell death at this time, resulting in gener
alized loss of activity of enzymes involved in macromolecule
synthesis. However, RNA and protein synthesis continued at
this time, indicating that a generalized loss of metabolic activity
had not occurred. Therefore, maximum inhibition of DNA syn
thesis at 18 to 20 hr after drug exposure suggests delayed
expression of drug toxicity. Delayed expression of drug toxicity
was also seen in vivo as delayed deaths of drug-treated animals
(8).
SEPTEMBER
1982
REFERENCES
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