(CANCER
RESEARCH
36, 3789-3797,
October,
1976]
The Effect of [email protected] Cell
Viability, DNA Synthesis, and Chromatid Breakage in
Synchronized Hamster Fibrosarcoma Cells1
Peter A. Jones,2Mary S. Baker, and William F. Benedict@
Division of Hematology-Oncology, Department of Medicine, Childrens Hospital, Los Angeles, California 90027
SUMMARY
Hamster fibrosarcoma cells were synchronized by mitotic
selection and exposed to varying concentrations of i-fJ-D
arabinofuranosylcytosmne (ama-C)for 2 hr in mid-S phase.
There was a direct relationship between DNA synthesis inhi
bition and cytotoxicity produced by ama-Conce DNA synthe
(12). Furthermore, ama-Ccan produce oncogenic transfor
mation in hamster and rat cells (ii , 16) and morphological
transformation in mouse cells (1).
This study was undertaken in an attempt to determine
what parameterswere relevant
toama-Ccytotoxicity,
as well
as to gain some insight into the mechanisms of ama-Cproduced transformation.
The cell line used in this study
sis was decreased by over 85%. The noncytotoxic concen was the hamster fibmosancomaline, A(11)Cl-3,developed in
tration of 10@M ama-Cproduced little chnomatid breakage; our laboratory. This cell line was chosen for the following
reasons: it can be easily synchronized by mitotic selection
(19); it has a diploid chromosomal number (4); and it grows
rapidly, forming discrete colonies which can be counted
servations that chromatid breakage is highly correlated with electronically (3).
cytotoxicity.
The experimental design was to expose cells to ama-Cfor
Predominantly small DNA was synthesized when cells a 2-hr period in the middle of S phase. The effects of various
were treated with both 10@ and 10@ M ama-C,and this DNA concentrations of ama-Con the rate of [3H]thymidine incor
pomation and on cytotoxicity were then studied under these
could be completely chased into high-molecular-weight
DNA after addition of deoxycytidine. Both concentrations of defined conditions. Preliminary results of the experiments
ama-C also inhibited,
to different
degrees,the joiningof showed that i0@ M ama-Cmarkedly inhibited DNA synthesis
but was not cytotoxic, in contrast to i0@ M ara-C which
intermediate-size DNA fragments into larger DNA; thus nei
ther parameter appeared directly related to the ama-C-pro inhibited DNA synthesis to an even greaten extent and was
highly cytotoxic.
duced cytotoxicity.
Further studies were then carried out at these 2 ama-C
concentrations in an attempt to elucidate the mechanism of
INTRODUCTION
ama-C cytotoxicity. The parameters chosen were: (a) the
recoveries of DNA synthesis and cell mitotic ability; (b) the
ama-C4inhibits DNA synthesis in various mammalian cells appearance of chromatid breaks and chromosomal rear
(9, 13, 15, 23). It is believed that this inhibition results from
rangements; (c) the size of DNA synthesized during the
the conversion of the nucleoside to the tniphosphate, ama treatment period; and (d) the effect of ama-Con DNA chain
CIP, which in turn inhibits DNA replication at the level of elongation.
the polymemase enzyme (8, 10, 18). This hypothesis is sup
ported by studies on isolated Escherichia co!i enzymes,
which show that replicative synthesis is more sensitive than
MATERIALSAND METHODS
but extensive chromatid breakage and chromosomal rear
rangement were seen in cells treated with the cytotoxic
concentration of 10@M ama-C,thus supporting earlier ob
is repair synthesis(7, 24) and that DNApolymeraseI is more
resistant than polymemaseII on Ill to inhibition by ara-CIP
(22). ama-Cis also highly cytotoxic to cells in the S phase of
the cell cycle (5, 14, 26), causing chromatid damage, which
has been highly correlated with the degree of cytotoxicity
I This
work
was
supported
by
Grant
CA-i
4226
from
the
National
Cancer
Institute, NIH.
2 Present
address:
Department
of
Medical
Biochemistry,
Medical
School,
of
Career
Development
Award
CA-70996
from
the
National
Cancer Institute. To whom requests for reprints should be addressed.
4 The
abbreviations
used
are:
are-C,
1-@-D-arablnofuranosylcytosine;
stemfibmoblastsA(I1)Cl-3 (4). They were propagated in sus
pension culture in McCoy's spinner medium (Grand Island
Biological
Co.,Grand Island,
N.Y)containing
10% fetal
calf
serum (Flow Laboratories, Rockville, Md.) and seeded into
75- or 150-sq cm flasks (Corning Glass Works, Corning, N.
V.)at6 or12x i0@cells/flask
thedaybeforemitoticharvest
Box 63, Tygerberg 7505, Republic of South Africa.
S Recipient
Culture Conditions and Synchronization Technique. The
cells used were the cloned line of ama-C-transformed ham
ara
CTP, i-ft-D-arabinofuranosylcytosine 5'-triphosphate; CdA, 2'-deoxycyti
dine; PBS. Dulbecco's phosphate-buffered saline [NaCI (8 g/liter), KCI (0.2 g/
liter), Na@HPO4
(1.15 g/liter), and KH,P04 (0.2 g/liter), pH 7.2].
Received December 15, 1975; accepted July 8, 1976
ing. The medium was then changed to McCoy's medium 5A
containing 10% fetal calf serum after the cells had attached
(2 hr at 37°),and this medium was used in all subsequent
experiments. Synchronized cells were obtained from these
cultures, using the technique of mitotic detachment as de
OCTOBER 1976
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1976 American Association for Cancer Research.
3789
p. A. Jones
et a!.
scnibed by Momparler et a!. (19), and seeded into Petni
dishes (Falcon Plastics, Oxnand, Calif.).
Exposure to ara-C. ama-Cand CdR hydrochloride were
purchased from Sigma Chemical Co. , St. Louis, Mo. , and
were dissolved at 100-fold final concentration in PBS and
filter sterilized immediately before use. ama-Cwas added
directly to the cell culture medium 5 hr after mitotic selec
tion (i.e. , in mid-S phase). The treatment time was always 2
hm, after which either the cells were scraped off 60-mm
dishes with a rubber policeman or the medium was changed
to fresh medium containing 10@ IACdR. In all of the expeni
ments reported in this paper CdR was added to all cultures
at 7 hr after plating. Cell killing was determined by seeding
200 mitotic cells into 60-mm dishes (4/treatment) and ex
posing them to ama-Cexactly as indicated above. Medium
was changed to 10@ M CdR after the 2-hr exposure period,
and the dishes were incubated at 37°for 5 days. On the 5th
day the colonies were fixed in methanol, stained with
Giemsa, and counted electronically with an automatic col
ony counter (New Brunswick Scientific, New Brunswick, N.
J.).
Measurement of DNA Synthesis. Mitotic cells were
seeded at approximately 105/35-mm dish and treated 5 hr
material was then dissolved in 0.4 ml of 0.05 M sodium
citrate, pH 5.0; the small amount of potassium chlorate was
removed by centnifugation; and 0.1-mI aliquots were ana
lyzed by high voltage paper electrophonesis on Whatman
No. 3MM paper. The voltage gradient was 50 V/cm, and the
run time was 1 hr in 0.05 M sodium citrate, pH 5.0. The
paper was cut into 1-cm strips after drying, and these were
counted in the scintillation counter.
Studies on Chromatid Breakage and Metaphase Index.
Approximately 1 x 1O@cells were cultured in 60-mm tissue
culture dishes in a similar manner as described above and
exposed for 2 hr to 10@ and 10@ PAama-C5 hr after plating.
At various times after treatment, Colcemid (Grand Island
Biological Co.) was added for 30 mm at a final concentra
tion of 1 @g/ml.The cells were then trypsinized, and chro
mosomal preparations were made (2). Chromatid breakage
was scoredas previously
described(2).
One hundred metaphases were analyzed for each time
period for chromatid breaks on gaps. A gap was considered
to be a lesion at least the width of a chromatid, and a break
was a gap with a different angle than the adjacent intact
chromatid arm. All these lesions were reported as “breaks―
for simplicity. Metaphases were also scored for the pres
after plating with varying concentrations of ama-Cfor 2 hr.
ence of abnormal chromosomal configurations, particularly
The dishes were also exposed to [methy!-3H]thymidmne, 1
@Ci/ml (20 Ci/mmole; New England Nuclear, Boston,
Mass.), throughout the treatment period. The radioactive
medium was then removed; the cells were washed carefully
with 1 ml of PBS and then lysed in the dish with 1 ml of a
solution of 1% sodium dodecyl sulfate containing bovine
serum albumin , 500 @g/ml.The lysates were treated with
0.3 ml of 50% tnichlomoacetic acid and filtered through
Whatman GF/C filters that had been thoroughly washed
first with 5% tnichloroacetic acid and then 96% ethanol,
dried, and counted in scintillation fluid in a Packard In
Carb liquid scintillation spectmophotometem(Packard Instru
ment Co. , Downers Grove, III.).
The inhibition of DNA synthesis by ama-Cwas also studied
by autonadiography.
Cells treated with ama-C and
[3H]thymidine, 5 pCi/mI, as above were chased for 2 hr with
CdR-containing medium, scraped off the dish; and fixed
with methanol:acetic acid (3:1). Slides were made from the
cell suspension, stained with acetooncein and dipped in
Kodak NIB-2 nuclear track emulsion. After standing for 6
days in the dark, the slides were developed and scored for
tninadial and quadmiradial formations.
The same slides used for chromosomal analysis were also
scored for the percentage of metaphases present. One
thousand cells were analyzed for each time period and the
labeling.
Measurement of Thymidine Phosphates. Mitotic cells
were seeded into 100-mmdishes (1 x 10@/dish)and treated
with ama-Cplus [methy!-3H]thymidine, 2 pCi/mI, 5 hr after
plating for 2 hr. The radioactive medium was then removed,
the culture were washed once with 10 ml PBS, and the
dishes were placed on ice before the addition of 2 ml of ice
cold 0.2 N perchlonic acid. The cells were scraped off the
dishes and transferred to centrifuge tubes; after standing
for 20 mm in ice, the tubes were centrifuged for 5 mm at
2000 x g. The precipitate
was reextracted
with0.5 ml of
penchlonic acid and the combined supemnatants were neu
tralized to pH 7 with 2 N potassium hydroxide. The precipi
tated potassium perchlorate was removed by centnifuga
tion, and the supemnatantwas lyophilized. This freeze-dried
3790
percentage of mitotic cells was defined as the metaphase
index.
Alkaline Sucrose GradIents. Cells to be analyzed on alka
line sucrose gradients were exposed simultaneously to
[methy!-3H]thymidine, 5 @Ci/ml,and ama-C as indicated
above. At the end of the 2-hr treatment time, either they
were directly analyzed on gradients on the radioactive me
dium was removed and replaced with fresh medium con
taming 10-s M CdR for various chase periods. In some
experiments,
cultures received a 5-mm pulse with
[3H]thymidine, 10 @Ci/ml,immediately before exposure to
ama-C.The radioactive medium was removed after 5 mm; the
dishes were washed with 5 ml of medium and then treated
with ama-C.The controls were either scraped into cold PBS
on chased with freshmedium for2 hr.
Cells were harvested by scraping with a rubber policeman
in 1 ml of PBS. They were then counted on a hemacytome
ten, the cell concentration was adjusted to 25 x 10@cells/mI
and made 0.01 M with respect to EDIA, and 0.2 ml of this
solution was carefully layered over the gradients. The gma
dient procedure was essentially that described by Peterson
et a!. (21) with the exception that a cushion of 2.2 M sucrose
was used.A 30-mIlinear
5 to25% alkaline
sucrosegradient
was formed over a 4-mI cushion of 2.2 M sucrose in a 2.5- x
8.6-cm cellulose nitrate tube. All of the gradient solutions
contained 1 IA NaCI, 1 mM EDTA, and 0.06 M sodium p
aminosalicylate at a final pH of 12.5. The gradients were
overlaid with 0.2 ml of 1 N NaOH immediately before the
cells (0.2 ml; 5 x 10@cells) were added. The cells were
allowedtolysefor30 mm atroom temperatureon topofthe
gradient and then centrifuged for 3 hr at 24,000 rpm in the
Beckman SW-27 motorwithout the brake. Deceleration time
CANCER RESEARCH VOL. 36
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1976 American Association for Cancer Research.
Effect of ara-C on Hamster Fibrosarcoma Ce!!s
was 30 mm. A 20-gauge needle was inserted to the cushion
interface, and 30 sequential fractions of 17 drops (about 1
ml) were collected. Carrier DNA (200 pg/fraction) was then
added, and the radioactivity precipitable by tnichlonoacetic
acid was determined as outlined above.
The S values given are approximate and were obtained
from the data of Parkhunst et a!. (20). They were checked
using 32P-labeled bacteriophage 17 DNA (37 5) which was a
gift from
Dr. John
Leavitt,
Johns
Hopkins
University,
I00
50
Balti
more, Md.
-J
0
I—
RESULTS
Cell Killing and Inhibition of DNA Synthesis. The killing
of synchronized hamster fibrosancoma cells by ama-Cin mid
S phase is shown as a function of dose in Chart 1. For the 2hr exposure period used, no cell kill was observed until a
concentration of 3.3 x 10@ M ama-Cwas reached. Ihereaf
ten, the cell kill obtained was concentration dependent,
reaching a maximum of 75% kill at 10@ M ama-C.Chart 1
also shows the inhibition of [3Hjthymidine incorporation by
ama-Cas a function of concentration in similarly synchmo
nized cells. Clearly, the cells are able to survive considera
ble inhibition of thymidine incorporation without death. A
concentration of 10@ M ama-Ccaused an 88% inhibition of
thymidine incorporation, but it was ineffective in causing
cell death. A concentration of 10@ M ama-C,however, killed
75% of the cells and inhibited thymidine incorporation by
97%. It seems, therefore, that the cells can withstand an
88% inhibition of thymidine incorporation for 2 hr without
death but that, above this level of inhibition, the cells die at
a matethat may be correlated with the extent of inhibition.
Although the difference between an 88 and a 97% inhibition
of DNAsynthesis may not appear large, cells incorporated 3
z
010
0
\
5.
I
lo@6
@@
5 y-4
l0@3
C0N@ENTRATI0N
ARA-C
(M)
Chart 1. Cell killing and inhibition of DNA synthesis by are-C. Synchro
nized cells were treated in mid-S phase for 2 hr with the indicated concentra
tions of are-C (5 to 7 hr after plating). The effect of this treatment on
[3H]thymidine incorporation into DNA per culture during the exposure period
was measured ( 0). Cytotoxicity (•)
was measured by a colony-forming assay
in similarly
treated cultures
that were subsequently
exposed
to i0@ M CdA.
Bars,rangeof resultsfoundin 3 separateexperiments.
to 5 times more thymidine in the presence of 10@ M ama-C
than with 10@ M ama-C.This large difference may therefore
Table 1
be significant in terms of survival. The differences in incom Theconversionof (3HJthymidineto thymidine nucleotides in ara-C
treated cells
ponation were not due to drug-induced cell detachment,
since the cell numbers found in treated cultures were identi
cal to those in untreated cultures (not shown).
The experiments shown in Table 1 were carried out to
investigate whether these 2 concentrations of ama-C in
hibited the conversion of the 3H-pmecursomto thymidine
nucleotides. Cleanly, this was not the case, since a slight
stimulation of the level of radioactivity in TIP was found.
The patterns of acid-soluble radioactivity in cells treated
with 2 concentnations of ama-Cwere very similar. Also, since
the acid-soluble levels of radioactivity in ama-C-treated cells
Synchronized
cells were treated for 2 hr in mid-S phase with the
indicated concentrations of ara-C and [3H]thymidine, 2 pCi/mI.
Acid-soluble
radioactivity
in these cells was then analyzed
midine nucleotides by high-voltage paper electrophoresis.
for thy
Results
given are the averagevalues ofnuns.Incorporation
2 electrophometic
cells)Treatment
[3H]TTPNone
ara-C(10@M)
ama-C(10@M)
(cpm/lOe
[3H]TMP
[3H]TDP
660
868
820
604
1 224
764
16,188
21,360
22,744
were similar to those in untreated cultures, the inhibition of
thymidine incorporation observed was not due to an inhibi
tion of precursor uptake. In other studies (not shown), it
was found that the degree of inhibition of thymidine incor
poration into DNA was independent of the amount of radio
activity added over a 25-fold mangein concentration (0.2 to 5
@Ci/ml),arguing against large differences in endogenous
poolsizes.
The experiments shown in Fig. 1 and Table 2 were under
taken to confirm that inhibition of DNA synthesis occurred
in all treated cells, since it might be argued that the de
crease in thymidine incorporation was due to selective inhi
OCTOBER
1976
bition in some cells with other cells being more resistant to
ama-C. Autoradiognaphy showed that the majority of un
treated cells were heavily labeled (Fig. 1A), while those
exposed to 10@and 10@ M ama-Cwere moderately on lightly
labeled, respectively (Fig. 1, B and C). Both concentrations
of ama-Cinhibit DNA synthesis in all treated cells in a uni
form manner. Following exposure to 10@ M ama-C,no heav
ily labeled
cells were
seen (Table
2). Table
2 also indicates
the high degree of synchrony achieved with at least 90% of
the cells being labeled during the 2-hr pulse.
The recovery of DNA synthesis and mitotic activity in cells
3791
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1976 American Association for Cancer Research.
P. A. Jones
et a!.
slowly and showed little thymidine incorporation 9 hr after
Table 2
Autoradiographyof cells exposedsimultaneouslyto (3H)thymidine plating (2 hr after removal of ama-C). Subsequently, the
Synchronized
indicated doses
CdR-containing
labeling scored
and ara-C
cells were treated for 2 hr in mid-S phase with the
of ama-C and [3H]thymidine, 5 pCi/mI. After 2 hr in
medium, autoradiography
was performed and the
as heavy (Fig. 1A), moderate (Fig. 18), light (Fig.
incorporation of thymidine increased substantially, but the
curve was broader than that found either for the control on
for 10@M ama-C-treatedcultures. Maximum mitotic activity
was delayed by 9 hr compared with the control cells and by
1C),or none(Fig.1C).Resultsgivenwereobtainedbyexamination 6 hr compared with the 10@ M ama-C-treated cells.
of 500 cells in each case.
Table 3 contains a summary of data comparing chromo
somal damage in cells treated with the 2 concentrations of
totalcellsHeavyModerateLightNolabellabellabellabel76143777878013729
% of
ama-C.No metaphases were found with 4 or more chromatid
breaks pen metaphase when the A(I,)Cl-3 cells were treated
Treatment
in mid-S phase with 10@ M ama-C.Also no chnomosomal
None
ara-C (10'
abnormalities
M)
such as tniradial or quadminadial configura
tions were seen at this dose. In contrast, numerous meta
ara-C (10@M)
phases were found with greater than 4 chromatid breaks pen
metaphase at the cytotoxic level of 10@ M ara-C. Of particu
lamsignificance were the number of metaphases seen within
the 1st mitotic period following 10@ M ama-Cwhich had 5 or.
more chromatid breaks together with several abnormal tn
radial and quadnimadial chromosomal configurations (Fig.
2). After at least 1 additional cell cycle following treatment
with 10@ M ama-C(more than 25 hr after plating the synchro
nized cells), there was a gradual decrease in the number of
metaphases with greater than 4 chromatid breaks per meta
phase, along with the number of abnormal tnimadial and
quadnmnadialconfigurations. However, a few cells with these
chromosomal aberrations still persisted in the population.
0
Sizeof DNASynthesized
duringara-CTreatment.In all
experiments we found that the sedimentation profiles ob
(1)
4
0
2
4
6
8
HR AFTER
K@ 12
14
IS
@20
PLATING
Chart 2. Effect of era-C on the recovery of [‘H)thymidineincorporation
and mitotic activity of treated cells. Cells were synchronized and treated with
no ara-C (•),10@ M ara-C ( 0), or 10' M are-C (U) for 2 hr 5 to 7 hr after
plating. Medium was then changed to 10@ M CdR. a. At the indicated times
after plating, cultures were pulsed with [‘H]thymidine,1 @Ci/ml,for 30 mm
and the acid-insoluble radioactivity was determined . b . Cells were harvested
and chromosome preparations made. The percentage of metaphases in
these preparations was determined on a sample of 1000 nuclei.
treated with ama-Care shown in Chart 2. Cultures treated
with 10@ M ama-Crapidly regained their DNA-synthetic abil
ity. Significant thymidine incorporation occurred by 9 hr
after plating (2 hr after removal of ama-C),and a sharp peak
of DNA synthesis was observed at 11 hr. Maximal mitotic
activity was seen 13 hr after plating. The cultures treated
with 10@ M ama-Crecovered their DNA-synthetic ability more
3792
tamed for material sedimenting at less than 160 5 were
reproducible from experiment to experiment. However, me
suIts obtained for the profiles of DNA sedimenting wittj an S
value higher than 160 5 were inconsistent, presumably me
flecting problems in aggregation of high-molecular-weight
DNA. The results of the gradient experiments are shown in
both chart and table form for clarity.
The sedimentation pattern in alkaline sucrose gradients
of DNA labeled in mid-S phase (5 to 7 hr after plating) is
shown in Chart 3a and Table 4. Much of the radioactivity
(56%) is found in DNA sedimenting at greater than 160 S.
Lighten material found in the upper regions of the gradient,
which presumably represents replication intermediates, can
be largely chased into high-molecular-weight
material
(>160 5) when the [3H]thymidine-containing medium is me
placed with fresh medium (Chart 3b; Table 4).
Chart 4a and Table 4 show the size of DNA synthesized in
mid-S phase in the presence of 10@ M ama-C.Although 24%
of the DNAsynthesized is >160 5, a large proportion of the
labelisincorporated
intoa broadheterogeneousband with
a peak at approximately 64 S. This material was chased into
the medium- and high-molecular-weight regions of the gra
dient after 2 hr in CdR-containing medium (Chart 4b; Table
4). At this time DNA synthesis in cultures treated with 10@ M
ama-Chad begun to recover (Chart 2a). A further 2 hr in
CdR-containing medium caused another shift in the sedi
mentation profile (Chart 4c; Table 4), making it similar to
that seen in untreated controls.
The DNA synthesized during exposure to 10@ M ara-C
differed in size distribution from that in both the control and
105 M ama-C-treatedcultures (Chart 5; Table 4). Only 14% of
CANCER RESEARCH VOL. 36
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Effect of ara-C on Hamster Fibrosarcoma Cells
Table3
Comparatiyechromosomal changes following treatment of synchronized A(T,)Cl-3
withMeta
noncytotoxic (1O@M) and cytotoxic (10@ M) concentrations of ara-Ccells
phasesNo.
of metaphaseswith indicated breaks/cell
with trira
dial or
—
Concen
Time aftertration ofdial
plating>tions9None
(hr)ama-C
(M)
quadmima
fomma
0
1
3
4
5-7
01110@
97
3
0
0
0
0
001384
001590
94
5
11
0
2
1
3
0
0
0
0
10
0
0
0
0
17
20
4
14
12
4
3
8
0
5
4
0
21
14
2
4
8
0
2
12
0
2
4
1
0
0
0
22
4
0
0014.510@
1001534
871764
24111942
32
28172570
135096
2
8 or
11
cause of our reservations on high-molecular-weight DNA
(see above), we cannot attach any significance to this.
Since the results outlined above could be explained by
degradation of the low-molecular-weight DNA rather than
by its subsequent appearance in high-molecular-weight ma
tenial, experiments were conducted to determine the con
servation of the radioactivity incorporated during pulsing.
Cells were pulsed with [3H]thymidmne, 1 @Ci/ml,during the
2-hr ama-Cexposure period, and the acid-insoluble madioac
tivity was determined in duplicate samples at 0, 2, 4, and 6
hr after exposure. These experiments (not shown) failed to
demonstrate the loss of any [3H]thymidine in either the
control on treated cultures. We therefore concluded that all
of the DNA made during ama-Cexposure is retained.
Effect of ara-C on Chain Elongation. Chart 6a shows the
U)
I-
z
:3
0
C)
.J
30
TOP
size distribution of DNA synthesized during a 5-mm pulse
with [3H]thymidine. Most of the label was incorporated into
a broad band of material with a peak sedimentation coeffi
cient of approximately 56 5. This material was chased into
more rapidly sedimenting DNA after 2 hr in fresh medium
(Chart 6b). Chart 6c shows that 10@ M ama-Ctreatment for 2
hr following the 5-mm pulse strongly inhibited the extension
of these DNA chains into large DNA, although the profile did
the labeled DNA was of high molecular weight (>1 60 5) with shift toward a broad peak of 80 to 112 5. ama-C, 10@ M,
most of the DNAsynthesizedbeing found as low-molecular caused more marked inhibition of this process with the
weight DNA at the top of the gradient in a peak of approxi
peak moving to the 72 to 80 S position (Chart 6d). Both
mately40 5 (Chart5a).Iwo hrafter
exposure,the40 S peak concentrations of ama-Ctherefore inhibit the conversion of
began to decrease, and the profile shifted to more rapidly intermediate size DNA chains into high-molecular-weight
sedimenting material (Chart Sb; Table 4). A further 2 hr in cellular DNA.
CdR-containing medium resulted in a marked shift in the
FRACTION NUMBER
Chart 3. Alkaline sucrose gradient sedimentation profiles of DNA synthe
sized during a 2-hr (3H]thymidmnepulse in mid-S phase in untreated A(T,)Cl-3
cells. Synchronized cells were pulsed 5 to 7 hr after plating with 5
[3Hjthymidine, MCi/mI, and the DNA was analyzed (a) at the end of the pulse
period; or (b) following a 2-hr chase in CdA-containing medium (i.e. , 9 hr
after plating).
size of the DNAthat had been synthesized, and 43% of the
radioactivity
was
now
found
in the
midnegion
of the
DISCUSSION
gra
dients (Chart 5c; Table 4). At this time, DNA synthesis in
treated cultures had increased (Chart 2a). Chart 5d and
Table 4 show that, 6 hr after the ama-Cis removed, most of
the intermediate-size DNA has disappeared and the distni
bution of radioactivity is very similar to that of control cul
tunes.Thus, the small DNAmadeduring exposure to 10@M
ama-C later becomes associated with large cellular DNA.
Although the profile shown in Chart 5d appears different
from the control (Chart 3b) or 10@ M ama-C(Chart 4c), be
The dose-response curves (Chart 1) that are obtained for
cytotoxicity and DNA synthesis inhibition produced by ama-C
suggest that ama-Ckill may be related to the inhibition of
DNA synthesis within certain limits. Theme is a direct nela
tionship between ama-C-produced cytotoxicity and DNA syn
thesis inhibition at high ama-Cconcentrations, i.e. , cell kill
ing and DNA inhibition are parallel once the cell-killing
response begins, although the cells can withstand a signifi
cant threshold level of inhibition without damage. This con
OCTOBER 1976
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1976 American Association for Cancer Research.
3793
P. A. Jones et a!.
Table 4
Size distribution of DNA synthesizedduring exposure to ara-C
SynchronizedA(T,)CI-3cells wereexposedto the indicated concentrationsof ama-C5 to 7 hr after
plating
and pulsed
simultaneously
with
[3H]thymidine,
5 pCi/mI.
Then either
cellular
DNA was
directly analyzed on alkaline sucrose gradients or the medium was changed to CdR-containing
medium
for varying
chase
periods
before
analysis.
Thirty
fractions
were collected
from
each
gradient, and the distribution of radioactivity in 3 broad areasof the gradientswasdetermined (i.e.,
<80 S; >80 S, <160 S; >160 S). Results are the mean values obtained
of separate experiments.Time
from the indicated
numbers
inTubes1-1011-20
% recovenedcpm
(>8021-30Treatmentplating
afterChase
5)None7
(hr)nod
3ara-C
pe
No. of ex
5)5,
(hm)periments(>160
<160 5)(<80
90
25
256
7435
249
9
2
3
34
57
110
43
124
7042
2734
9
2
2
26
43
11
130
4
64
2
214
27
6828
67
3058
(10' M)7
3ara-C(103M)7
9
31
7
3
C')
z:@
I-
0
0
-J
4
U)
g
8
z
I'—
I.
0
10
BOTTOM FRACTION
20
30
NUMBER
Chart 4. Alkaline sucrose gradient sedimentation profiles of DNA synthe
sized during a 2-hr (3H]thymidine pulse in S phase in A(T,)Cl-3 cells simulta
neously treated with 10' M are-C. Conditions were as in Chart 3 and the DNA
was analyzed (a) at the end of the pulse period; (b) following a 2-hr chase in
CdR-containing medium (i.e. , 9 hr after plating); or (C) following a 4-hr chase
(i.e. , 11 hr after plating).
0
clusion is supported by Chou et a!. (6) in an in vivo study
proposing that the inhibition of the last few percentages of
DNA synthesis might be critical for cell death.
We were not able rigorously to exclude the possibility that
the different inhibitions of thymidine incorporation that we
observed were due to changes in endogenous thymidine
pool sizes in response to ama-Ctreatment. However, we feel
3794
BOTTOM
10
20
30
TOP
FRACTION M..NBER
Chart 5. Alkaline sucrose gradient sedimentation profiles of DNA synthe
sized during a 2-hr (3Hjthymidine pulse in S phase in A(T)Cl-3 cells simulta
neously treated with 1O@N ama-C.Conditions were as in Chart 3 and the DNA
was analyzed (a) at the end of the pulse period; (b) following a 2-hr chase in
CdA medium (i.e. , 9 hr after plating); (C) following a 4-hr chase (i.e. , 11 hr
after plating) and; (d) following a 6-hr chase (i.e. , 13 hr after plating).
CANCER RESEARCH VOL. 36
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1976 American Association for Cancer Research.
Effect of ara-C on Hamster Fibrosarcoma Cells
that this would be an unlikely explanation of our results,
because the large differences required would probably have
caused perturbations in the levels of radioactivity in thymi
dine nucleotides. Thus, if the differences in incorporation of
thyrnidine into the DNA of cells treated with 10@ and 10@ M
ama-Cwere due to changes in pool size, it would require a 3to 5-fold increase in endogenous TIP. We therefore feel
that our results, taken together, argue more strongly that
the inhibitions of radioactive incorporation into acid-insolu
ble material represent differing levels of DNA synthesis.
The curves shown in Chart 2 illustnate that cells treated
with the noncytotoxic concentration of ama-Cresume DNA
synthesis and mitotic activity shortly after removal of the
ama-C.In contrast,lethally
treatedcellsshowed a more
heterogeneous recovery, which presumably reflects the
damage inflicted by ama-C.Since it is possible that residual
intracellular ara-CTP following treatment with 1O@M ama-C
may still significantly inhibit DNA synthesis, the increased
z
:3
0
0
-J
4
I—
0
I—
recoverytime may also be partially due to a longer effective
exposure to the drug. We are currently examining these 2
possibilities in more detail.
The results obtained on the size of DNA synthesized dur
ing ama-Cexposure (Charts 3 to 5; Table 4) showed that the
molecular weight of DNA synthesized at the cytotoxic dose
of 10@ M ama-Cwas lower than that at the noncytotoxic 10@
IA ama-C dose.
This
finding
may
be a reflection
of our
0
obser
BOTTOM
vation that 10@M ama-Cis a more potent inhibitor of the
joining of preformed DNA pieces than is 10@ M ama-C
(Chart 6). However, since significant inhibition of the join
ing of preformed DNA pieces occurs at both 10@and 10@ M
ama-C,this effect of ama-Con DNA synthesis does not appear
to be related to cytotoxicity. Ihe inhibition by ama-Cof the
joining of preformed DNA pieces is especially interesting in
10
20
30
TOP
FRACTIONNUMBER
Chart 6. Inhibition of DNA chain joining by are-C. Five hr after seeding,
synchronized A(T,)CI-3 cells were pulsed for 5 mm with (3H]thymidine, 10
@Ci/ml,immediately before treatment. The DNA was analyzed on alkaline
sucrose gradients. a, profile of DNA synthesized during the pulse period
without a chase; b , as in a but following a 2-hr chase in fresh medium ; C, as in
b, but chase medium contained 1Ø-aPAare-C; d, as in b but chase medium
contained 1O@M ara-C.
view of the observation of Leeet a!. (17) that 3 x 10@IAama
C has no effect on the rejoining of radiation-induced single
strand breaks in L1210 cells. Repair of DNA breaks theme
fore probably progresssesby a different pathway than join
ing of newly synthesized single-stnand pieces. The in
creased time required to chase the small DNA pieces made
in the presence of 1O@IA ama-Cinto larger DNA may, how
ever, still be significant.
Studies on chromosomal abnormalities following ama-C
treatment (Table 2) showed that the noncytotoxic dose of
10_s IA produced no metaphases with more than 3 chroma
tid breaks, although DNA synthesis was inhibited by 86%.
Following the highly cytotoxic dose of 10@ IA ama-C,how
ever, 50% of the metaphasesexamined were found to have
greatenthan 4 breaks per metaphase 19 hr after plating of
the cells. Numerous tniradial and quadmimadial configura
tions were also found. Previous work in our laboratory with
Don-C cells has shown 4 on more chromatid breaks per
metaphase to be highly correlated with cytotoxicity (12). In
addition, a relationship has been found between transfom
mation in hamstercells and the production of chromosomal
imbalances (4, 25). The chromosomal abnormalities ob
served in the present study in cells treated with 10@ IAama-C
may thus be involved both in the cytotoxicity (chmomatid
breakage) and the transformation produced by ama-C(chro
mosomal rearrangement). Thus, we believe that knowledge
regarding the molecular events responsible for ama-C-pro
duced chromosomal breaks and rearrangements is critical
to our understanding of the mechanism(s) of ama-C-pro
duced cytotoxicity and transformation , respectively.
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Fig. 1. Autoradiography of cells exposed simultaneously to [3H]thymidine and ara-C. Synchronized A(T,)Cl-3 cells were treated for 2 hr in S phase (5 to 7 hr
after plating) with [3H]thymidine, 5 @Ci/ml,and iO@ or 10' PAare-C. Slides were prepared following a 2-hr chase in CdA-containing medium, dipped in
emulsion, and developed after 6 days. A, untreated cells heavily labeled; B, cells exposed to 1O@M ara-C, moderately labeled; C, cells exposed to 1O@M are
C, lightly labeled with 2 unlabeled cells visible.
Fig. 2. A, metaphase after 2 hr treatment with 1O@M are-C showing numerous chromatid breaks and rearrangements; arrows, chromatid breaks. B,
metaphase after ara-C treatment showing triradial configuration (arrow). C, metaphase after treatment with iO@ M ara-C showing extensive chromatid breaks
and fragments.
3796
CANCER
RESEARCH
VOL. 36
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1976 American Association for Cancer Research.
@
@.
Effect of ara-C on Hamster Fibrosarcoma Cells
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1976
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1976 American Association for Cancer Research.
3797
The Effect of 1-β-d-Arabinofuranosylcytosine on Cell Viability,
DNA Synthesis, and Chromatid Breakage in Synchronized
Hamster Fibrosarcoma Cells
Peter A. Jones, Mary S. Baker and William F. Benedict
Cancer Res 1976;36:3789-3797.
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