[CANCER RESEARCH 30, 2636-2644,
November 1970]
Studies in Mouse L-cells on the Incorporation of 1-ß-DArabinofuranosylcytosine into DNA and on Inhibition
of DNA Polymerase by 1-0-o-Arabinofuranosylcytosine
5'-Triphosphate*
F. L. Graham2 and G. F. Whitmore
Department of Medical Biophysics, University of Toronto, and the Ontario Cancer Institute, Toronto, Ontario, Canada
SUMMARY
Studies have been carried out in mouse L-cells on the
incorporation of l-|3-D-arabinofuranosylcytosine
(ara-C) into
DNA and on the inhibition of DNA polymerase by the
5'-triphosphate of ara-C (ara-CTP) to determine whether either
of the two current models, incorporation into DNA or
inhibition of DNA polymerase, could account for ara-C action.
With a modification of the McGrath-Williams technique, it was
found that ara-C was initially incorporated into small
(Okazaki) pieces of DNA but shifted into longer DNA strands
when cells were washed and incubated in a medium free of
ara-C-3H. On degradation of DNA from ara-C-3 H-labeled cells
with micrococcal nuclease and spleen phosphodiesterase, it
was found that most of the ara-C appeared to be in
internucleotide rather than terminal linkages, suggesting that
chain elongation is not stopped by the addition of ara-C to a
growing strand. Studies on ara-C incorporation into nucleic
acids failed to show any correlation between the amount of
incorporation and the degree of lethality. With crude extracts
of L-cells, it was found that ara-CTP was a competitive
inhibitor of DNA polymerase, and values of 9.0 ±4.3 and 8.7
±5.2 X 10~6M were obtained for the Michaelis-Menten
constants of dCTP and ara-CTP, respectively. Calculations
based on these values and on measured values of the dCTP
and ara-CTP concentrations
in vivo indicated that the
predicted inhibition of DNA synthesis was significantly
smaller than that actually observed in whole cells. Never
theless, an evaluation of all of the available data suggests
that the most plausible model for the action of ara-C is that
DNA synthesis is inhibited by inhibition of DNA poly
merase.
INTRODUCTION
The previous paper (13) described studies on viability,
growth, and DNA synthesis of mouse L-cells exposed to
'Supported by the National Cancer Institute of Canada.
2Fellow of the National Cancer Institute of Canada.
Received February 19, 1970; accepted July 2, 1970.
2636
ara-C.3 Our results were consistent
with a model in which
inhibition of DNA synthesis is the result of inhibition of
DNA polymerase by ara-CTP and were inconsistent with
inhibition being the result of incorporation into DNA. The
present paper contains the results of further studies designed
to determine whether inhibition of DNA synthesis was the
result of inhibition of DNA polymerase or ara-C incorpora
tion into DNA. Momparler (23) has obtained evidence which
suggests that inhibition of DNA synthesis might be the result
of incorporation of ara-C into the 3'-hydroxyl terminal of
the newly synthesized strand. He found that the ara-C
incorporated by a partially purified calf thymus DNA poly
merase was confined almost exclusively to the 3'-hydroxyl
terminal of the DNA, suggesting that such incorporation
blocked further elongation of the chain.
We have undertaken a series of experiments to determine
whether a similar observation could be made in whole cells
and to determine whether any correlation could be made
between incorporation of ara-C into nucleic acids and loss of
viability.
Furth and Cohen (10) have shown that ara-CTP is a
competitive inhibitor of partially purified calf thymus DNA
polymerase and measured the Michaelis-Menten constants for
dCTP (Km) and ara-CTP (K¡).In order to determine whether
the inhibition observed in whole cells could be predicted
from the inhibition of DNA polymerase observed in vitro,
we felt that is was necessary to measure not only Km and K¡
but also the concentrations of dCTP and ara-CTP in araC-treated cells.
MATERIALS
AND METHODS
Materials
All experiments to be described in this paper were per
formed with mouse L-cells, strain L60T (36). Techniques for
3The abbreviations used are: ara-C, l-(3-D-arabinofuranosylcytosine;
ara-CTP, the 5'-triphosphate of ara-C; TCA, trichloroacetic acid; PBS,
phosphate-buffered saline.
CANCER
RESEARCH
VOL. 30
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ara-C: Incorporation-Inhibition
the maintenance and treatment of cell cultures have been
described (13). Deoxycytidine-3H, labeled in the 5-position
with specific activity 15.5 Ci/mmole and thymidine-3H
labeled in the methyl group with specific activity 17.4
Ci/mmole were purchased from the Radiochemical Centre,
Amersham, England. ara-C-3H, labeled generally with a
specific activity of 11 Ci/mmole, was purchased from New
England Nuclear Corp., Boston, Mass. Thymidine-2-14C, 54
mCi/mmole, and ara-C-3H, labeled nominally (more than
95%) in the 5-position with a specific activity of 26
Ci/mmole, were obtained from Amersham/Searle Corp., Des
Plaines, 111.ara-C-3H was always purified less than 1 week
prior to use by descending paper chromatography
with
1-butanol: water (86:14)
(20) (System
1). dCTP-5-3H
(specific activity, 27.3 Ci/mmole) and TTP-methyl-3H
(specific activity, 10.35 Ci/mmole) were purchased from
Schwarz BioResearch, Inc., Orangeburg, N.Y. 1-0-D-Arabinofuranosylcytosine
hydrochloride
was purchased from the
Sigma Chemical Co., St. Louis, Mo. All other nucleosides
were purchased from General Biochemicals, Inc., Chagrin
Falls, Ohio. The 5'-monophosphate of ara-C was synthesized
from ara-C and 2-cyanoethyl
phosphate by the Tener
method (35), and ara-CTP was synthesized from the 5'monophosphate of ara-C with the partially purified rat liver
kinase preparation described by Maley et al. (19). All other
nucleotides were obtained from P—L Biochemicals, Inc.,
Milwaukee,
Wis. Micrococcal nuclease (Staphylococcus
aureus), spleen phosphodiesterase (bovine spleen), alkaline
phosphatase (Escherichia coif), RNase-free DNase (DNase I,
bovine pancreas), and RNase (bovine pancreas) were pur
chased from Worthington Biochemical Corp., Freehold, N.J.
Bacteriophage X DNA was the generous gift of Dr. M. Gold
and Mr. S. McClure.
Velocity Sedimentation
of DNA
Method 1. One preliminary experiment (Chart 1) was
performed
using a variation of the McGrath-Williams
technique (22) described by Sambrook et al. (29). Approxi
mately IO6 labeled cells were layered onto alkaline sucrose
gradients, which were incubated overnight (14 to 16 hr) at
4°,then centrifuged for 6 hr at 76,000 X g in an SW 25.1
rotor. The gradients were then fractionated into 1.0-ml
fractions, which were precipitated with ice-cold 5% TCA,
filtered onto Whatman FG/C glass fiber filters, washed with
95% ethanol, dried, and counted in a toluene-based scintilla
tion fluid on a liquid scintillation counter.
Method 2. The size distribution of DNA labeled with
ara-C-3 H was studied with a variation of Method 1, details of
which will be published elsewhere (M. McBurney, F. L.
Graham, and G. F. Whitmore, in preparation). By allowing
very gentle lysis of cells after they have been layered on the
gradient, this technique permits the sedimentation of highmolecular-weight DNA (approximately 400 to 500 S, com
pared with 100 to 120 S obtained by Method 1). Labeled
cells (approximately
IO6 cells/gradient) were layered onto
alkaline sucrose gradients which were incubated overnight as
in Method 1 and centrifuged for 150 min at 95,000 X g in
NOVEMBER 1970
Studies
an SW 27 rotor. The gradients were then fractionated, filtered,
and counted as in Method 1.
The sedimentation coefficient of the rapidly sedimenting
DNA was obtained from the equation (2)
S20,w
ß-D
(rpm)2,
where D is the distance sedimented and t is time and where
the constant ßwas determined for our gradients by
centrifuging bacteriophage X DNA and using Studier's value
of 40.1 S for the sedimentation
in alkali.
coefficient of Xdg DNA (33)
Incorporation of Labeled Compounds
The determination of incorporation of labeled nucleosides
into acid-soluble intracellular pools and into acid-insoluble
material was made in the following way. Labeled cells were
centrifuged and washed twice with ice-cold PBS (9), and the
pellet was extracted 3 times with 0.5 ml of ice-cold 0.2 N
HC104. The supernatants from these 3 extractions were then
pooled and neutralized with 2.0 N KOH, and aliquots were
analyzed by descending chromatography on Whatman No.
3MM paper with 95% ethanol:!
M ammonium acetate
(75:30), pH 7.5 (36), (System 2). The radioactive com
pounds were then located and identified as previously
described (13). The acid-insoluble pellet was washed 2 more
times with 0.2 N HC1O4, dissolved in 1.0 ml of 0.5 N NaOH,
incubated for 36 hr at 37°to hydrolyze the RNA, cooled on
ice, and acidified by the addition of 75 ¿dof 12 N HC1O4,
and the resulting precipitate was removed by centrifugation.
This precipitate was sensitive to DNase and insensitive to
RNase. The supernatant (RNA) was decanted, and the
precipitate (DNA) was redissolved in 0.5 N NaOH, reprecipitated with HC1O4, filtered onto GF/C glass fiber filters,
washed with ice-cold 5% TCA, dried, and counted. Chroma
tography of acid-soluble extracts from ara-C-3H-labeled cells
was also carried out with 95% ethanol: l M ammonium
acetate saturated with sodium tetraborate (75:30) (System
3). In one experiment, the acid-soluble extract from cells
labeled for 4 hr with ara-C-3H was heated for 15 min at 80°
in 0.5 N HC104, neutralized and further hydrolyzed with
alkaline phosphatase, and chromatographed on System 1.
More than 96% of the radioactivity moved with the same Rp
as ara-C and was well separated from deoxycytidine, cytidine, cytosine, deoxyuridine, uracil, and thymidine. In one
experiment, the precipitates resulting from acidification of
the alkaline incubation mixture were digested with micrococcal nuclease and spleen phosphodiesterase by the method
of Josse et al. (14). Aliquots of the resulting digest were
then chromatographed
on System 2 to determine the
amount of radioactivity released in the nucleoside form, and
the remainder was further hydrolyzed by alkaline phos
phatase and chromatographed for 72 hr with 1-butanol:5%
sodium tetraborate in water (86:14) (18) (System 4) to
identify the incorporated radioactivity with ara-C.
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F. L. Graham and G. F. Whitmore
Determination
of the dCTP Pool
Measurements on total endogenous dCTP pools in L-cells
were made as follows. L-cells were centrifuged, washed twice
with ice-cold PBS, and extracted 3 times with 1.0 ml of
ice-cold 0.2 N HC104. Along with the first 1.0 ml of HC1O4
was added 1.0 juCi of dCTP-3H (specific activity, 27.3
Ci/mmole) to serve as a chromatography marker and as a
recovery standard. The acid-soluble extract was neutralized
with 2.0 N KOH immediately after decanting from the
acid-insoluble pellet, and, after removal of the insoluble
KC104, it was chromatographed on a Dowex 1-X8 (C03)
(200 to 400 mesh) column, eluting with triethylammonium
bicarbonate (19). The dCTP fraction was then evaporated to
dryness, heated in 0.5 N HC1O4 at 80° for 15 min,
neutralized with KOH, and treated with alkaline phosphatase
to hydrolyze it further to the deoxynucleoside. A final
chromatography was performed on System 1 to separate
deoxycytidine from any other deoxynucleosides which may
not have been eliminated by the column chromatography.
Finally, the deoxycytidine was assayed for total deoxy
cytidine content with the microbiological assay described
below and assayed for radioactivity by counting a small
aliquot on a scintillation counter. Thus, from the final
specific activity and the specific activity and amount of
dCTP-3H added initially, the total amount of dCTP original
of ara-C into nucleic acids. Recently, Momparler (23) has
presented evidence that, in vitro, ara-C incorporation
appeared to be limited to the 3'-hydroxyl position of the
DNA strand, possibly implying that the further addition of
nucleotides to ara-C-terminated
strands is blocked. Con
siderable evidence now exists that DNA is initially
synthesized in the form of short (Okazaki) pieces (26, 28,
30, 34) which are joined together at a later stage. If, in vivo,
ara-C incorporation prevented further addition of deoxynucleotides to newly synthesized strands, then it might be
expected to block the elongation of Okazaki pieces, and the
incorporated ara-C would never be found in large DNA. One
way to determine whether this was true was to determine
the size distribution of DNA containing ara-C-3H.
The results shown in Chart 1 indicate that in L-cells a short
pulse of thy nudine-3 H results in incorporation primarily into
small DNA (20 to 30 S), which is later converted into large
material (120 S). To measure the effect of ara-C, cells were
labeled with ara-C-3 H for 2 hr, centrifuged, either washed
with ice-cold PBS and stored at 0°or resuspended in fresh
medium free of ara-C and containing deoxycytidine, and
incubated for various times to allow DNA synthesis to
resume and Okazaki pieces to elongate, if possible. Finally,
all cell samples were layered onto alkaline sucrose gradients,
centrifuged, and fractionated as described in "Materials and
ly present in the cells could be calculated by isotope
dilution.
The microbiological assay for deoxycytidine was carried
out with Lactobacillus acidophilus R26 (ATCC 11506) as
described by Siedler et al. (31), except that the assay volume
was 0.5 ml and growth was measured by counting on a
Model A Coulter Counter with a 30-/n aperture (Coulter
Electronics, Hialeah, Fla.). With this modification, the assay
was capable of measuring 2 X 10~12 mole of deoxycytidine
and was linear from 0 to 100 X lo"12 mole/assay tube.
a)
400
300
200
100
DNA Polymerase Assay
DNA polymerase activity was assayed in cell-free lysates of
L-cells by the methods described by Gold and Helleiner (12).
The reaction mixture contained 20 Amóles of phosphate
buffer, pH 7.5; 2 Amólesof 2-mercaptoethanol; 2 /nmoles of
MgCl2; 120 Mg of heat-denatured calf thymus DNA; 60
mpmoles
each of dATP, dGTP, and TTP-3H (IO4
cpm/m/itmole); varying amounts of dCTP and ara-CTP; and
0.05 to 0.2 mg of extract protein in a total volume of 0.3
ml. The reaction mixture was incubated at 37°for 30 min,
then the reaction was stopped by the addition of 10 ¿/moles
of Na4P207 and ice-cold 5% TCA, and the mixture was
filtered onto glass fiber filters, which were dried and counted
on an Ansitron scintillation counter.
0
1
10
15
20
25
30
b)
12,000
9,000
6,000
3,000
RESULTS
Chart 1. The sedimentation properties of (a) L-cell DNA labeled by a
1-min pulse with thymidine- H, 1.0 nCi/ml (specific activity, 17.4
Ci/mmole) and (o) Inceli DNA from cells labeled 1 min with
thymidine-3 H, then centrifuged and incubated for 2 hr in fresh medium
Incorporation
of ara-C into DNA. In spite of evidence
presented in the previous paper (13) that ara-C was probably
not irreversibly inhibiting DNA synthesis by incorporation, it
was of interest to examine in more detail the incorporation
containing deoxycytidine and thymidine at 3 mM. Labeled cells were
layered onto alkaline sucrose gradients according to Method 1, then
centrifuged for 5 hr at 23,000 rpm in an SW 25.1 rotor. Fractions were
collected from the top, precipitated with ice-cold 5% TCA, filtered, and
counted.
2638
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ara-C: Incorporation-Inhibition
Methods" (Method 2). Two ara-C concentrations were used
in these experiments: 2.5 X 10~6 M, which is nonlethal
1200
during a 2-hr treatment, although DNA synthesis is severely
inhibited (see Charts 3 and 7 of Ref. 13), and 2.5 X IGT5 M,
eoo
which induces loss of viability of S phase cells within 2 hr
(Chart 6 of Ref. 13). The results of these experiments are
shown on Charts 2 and 3. For purposes of comparison,
Chart 2a shows the sedimentation profile of DNA from
control cells labeled for 16 hr with thymidine-2-14C in the
absence of ara-C. Chart 26 shows the sedimentation profile
of DNA labeled by a 2-hr treatment with ara-C-3H at 2.5 X
IO"6 M (specific activity, 1.4 Ci/mmole). Almost all of the
Studies
o)
400
o
b)
300
200
counts are found at the top of the gradient, as expected if
ara-C is initially incorporated into small DNA strands and if
the continued presence of ara-C blocks further DNA
synthesis. Chart 2c shows the result of washing the cells free
of unincorporated
ara-C after a 2-hr treatment
and
incubating them for 4 hr at 37° in medium containing
100
deoxycytidine (pulse-chase) before layering the cells onto the
alkaline sucrose gradient. Within 4 hr most of the in
corporated radioactivity has shifted into material with a
sedimentation coefficient similar to the control. Chart 3
shows a similar experiment carried out at 2.5 X 105 M
no
o
c)
ISO
50
••••^^•^^^
o
Top
10
20
30
Bottom
Fraction number
Chart 3. Alkaline sucrose sedimentation of (a) DNA from cells labeled
for 2 hr with 2.5 X 10~s M, ara-C-3H (specific activity, 1.4 Ci/mmole),
(ft) DNA from cells labeled with ara-C-3H as in a, then centrifuged,
4.000
resuspended, and incubated for 2 hr at 37 in fresh medium containing
deoxycytidine at 10 mM, and (c) DNA from cells labeled with
ara-C- H, then incubated for 6 hr in fresh medium containing 10 mM
deoxycytidine.
Labeled cells were layered onto gradients and
centrifuged, and the fractions were collected, filtered, and counted as in
Chart 2.
2.000
ara-C, a lethal treatment, which gave qualitatively similar
results.
Thus both at a lethal and a nonlethal concentration
incorporation of ara-C into Okazaki pieces did not prevent
the subsequent elongation of a large fraction of these pieces
once the cells were removed from the presence of ara-C and
DNA synthesis was allowed to resume. The only apparent
difference between results obtained at 2.5 X 1(T6 and at 2.5
X 10~s M is that the initial distribution of counts is more
I
sharply distributed towards the top of the gradient for 2.5 X
10"s M and may take slightly longer to shift to faster
sedimenting material. Some of the radioactivity remaining at
the top of the gradients may be due to ara-C-3 H in
20
Fraction number
Bottom
Chart 2. The sedimentation properties of (a) L-cell DNA from cells
labeled for 16 hr with thymidine-2-' 4C, (ft) DNA from cells labeled for
2 hr with ara-C-3H at 2.5 X IO"6 M (specific activity, 1.4 Ci/mmole),
and (c) DNA from cells labeled 2 hr with ara-C as above, then
centrifuged and resuspended in fresh medium containing deoxycytidine
at 10 mM and incubated for an additional 4 hr. Labeled cells were
layered onto alkaline sucrose gradients by Method 2 and centrifuged at
23,000 rpm in an SW 27 rotor. Fractions were collected from the top,
precipitated with ice-cold 5% TCA, filtered, and counted.
corporated into RNA which has been only partially degraded
by the alkali.
The above results strongly suggested that DNA strands
terminating in ara-C could be further elongated by the
addition of nucleotides to the 3' end, but there is at least one
other possible interpretation.
For instance, the shift of
radioactivity from very short pieces into high molecular
weight DNA could be the result of the joining together of
Okazaki pieces not terminating in ara-C with the 5' end of a
strand containing ara-C at its 3' terminus, as illustrated in
Chart 4.
NOVEMBER 1970
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1970 American Association for Cancer Research.
2639
F. L. Graham and G. F. Whitmore
S 5'
Õ51
oro-C
3'5'
1200
.CR,
COR
UOR
TdR
.UR.
1000
polynucleotide
ligase
800
S1
3'
ora-C
Chart 4. A possible mechanism by which ara-C- H incorporated into
Okazaki pieces could be chased into high-molecular-weight DNA in the
absence of addition of nucleotides to ara-C-terminated strands.
To determine more directly whether nucleotides could be
added to ara-C-terminated DNA, the following experiment
was performed. A culture of cells was exposed to ara-C-3H
at 1.0 X IO6 M (specific activity, 1.4 Ci/mmole), and after
4 hr the culture
washed in ice-cold
The other part was
containing 1CT4 M
was split, and 1 part was centrifuged,
PBS, and set aside for subsequent assay.
centrifuged, resuspended in fresh medium
deoxycyUdine, and incubated at 37°.At
10 hr, 6 hr after washing the cells, the remaining cells were
centrifuged and washed with PBS and, along with the cells
removed at 4 hr, extracted with HC1O4 and treated with
NaOH as described in "Materials and Methods." The DNA
which precipitated on addition of HC104 to the alkaline
solution was finally digested with micrococcal nuclease and
spleen phosphodiesterase according to the method of Josse
et al. (14). If the incorporated ara-C was confined to the
3'-hydroxyl position of the DNA, then it would be expected
that all the radioactivity would be released in the nucleoside
form. Instead, when aliquots of the digests were chromatographed on System 3, it was found that only 4% of the
radioactivity from the cells harvested at 4 hr was released as
ara-C and only 2% from cells harvested at 10 hr. To ensure
that the radioactivity
was ara-C-3 H and to determine
whether the nuclease digestion had gone to completion, the
remainder of the hydrolysate resulting from the micrococcal
nuclease and spleen phosphodiesterase digestion was further
degraded with alkaline phosphatase and chromatographed on
System 4. The data from one of the resulting chromatograms
are illustrated in Chart 5, and a comparison of the radio
activity in the nucleoside peak with the total radioactivity
indicates
that the digestion was approximately
73%
complete. The radioactivity in the nucleoside form can be
identified as ara-C. Thus, at least 70% of the incorporated
ara-C was not located at the 3'-hydroxyl terminus of the
DNA but rather was incorporated in internucleotide linkage.
The results of the enzymatic digestion described above are
summarized in Table 1. The observed incorporation of ara-C
into internucleotide linkage was expected from the experi
ments on Okazaki pieces, but is not in agreement with
observations made in vitro by Momparler (23).
One important difference between the conditions in whole
cells and those of Momparler's experiments in vitro is that
whole cells contain dCTP at significant concentrations (3 to
4 m¿imoles/108 cells), as will be shown later, while in vitro,
2640
600
400
200
10
15
2O
25
Distance from origin! inches)
35
30
Chart 5. Chromatography of products resulting from the successive
digestion of ara-C- H-labeled DNA by micrococcal nuclease, spleen
phosphodiesterase, and alkaline phosphatase. An aliquot of the final
digest was chromatographed on Whatman No. 3MM paper for 72 hr,
with l-butanol:5% sodium tetraborate in water (86:14), and the
chromatogram was cut into strips and counted as previously described
(13). Horizontal bars, ara-C, cytosine (CR), deoxycytidine (CdR),
deoxyuridine (UdR), thymidine (TdR), uridine (UR), cytosine (C),
and uracil (U). Radioactivity remaining at the origin presumably
represents oligonucleotides resulting from incomplete digestion by
micrococcal nuclease and spleen phosphodiesterase.
Table 1
Enzymatic digestion of ara-C-3H-labeled DNA
l^cells were treated for 4 hr with 1.0 tiM ara-C-3H (specific activity,
1.4 Ci/mmole) or treated for 4 hr followed by a 6-hr chase with lö~4M
deoxycytidine, then centrifuged, washed with ice-cold PBS, and
extracted with 0.2 N HC1O4.The acid-insoluble fraction was incubated
for 36 hr at 37 in 0.5 N NaOH. Finally, the DNA was digested with
micrococcal nuclease and spleen phosphodiesterase, according to the
method of Josse et al. ( 14). Aliquots were chromatographed on System
2 to determine the proportion of the radioactivity in the nucleoside
form, and some of the digest was further hydrolyzed with alkaline
phosphatase and chromatographed on System 4 to identify the radio
activity as ara-C and to determine the efficiency of degradation by
micrococcal nuclease and spleen phosphodiesterase.
1.1 X IO8 cells
harvested at 4 hr
cpm
1.3 X IO8 cells
harvested at lOhr
cpm
%
Nucleoside3'-NucleotideOligonucleotideTotal1,00016,7006,90024,600468281001,30046,40
in the reaction mixture which resulted in ara-C being
incorporated
almost exclusively into the 3'-hydroxyl
position, dCTP was completely lacking (23). It may be that
in vitro as well ara-C can be incorporated into inter-
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ara-C: Incorporation-Inhibition
nucleotide linkage within the polydeoxynucleotide chain if a
small amount of dCTP is provided during the reaction or
shortly after.
On the basis of the results shown in Charts 2 and 3 and the
fact that ara-C is not confined to the 3'-hydroxyl terminal of
DNA, it seems unlikely that inhibition of DNA synthesis is
due to incorporation. It could still be responsible for the
lethal effect of ara-C by altering the DNA in some detri
mental way. For instance, it has been shown that the 2'
position of the nucleoside sugar may be an important factor
in the determination of the conformation of nucleic acids (1,
21), and therefore it is possible that the 2'-hydroxyl of the
arabinose sugar could distort the DNA molecule sufficiently
to be lethal. Since it was known that exposure to con
centrations below 4 X IO"6 M ara-C over a 4-hr period was
nonlethal for L-cells, while concentrations above 7 X 10~6 M
were toxic for S phase cells within 2 hr (13), an attempt was
made to observe a correlation between incorporation and
lethality.
When cells were exposed to ara-C-3 H at various con
centrations for 4 hr, the incorporation into DNA and RNA,
as illustrated in Chart 6, was already quite appreciable at
concentrations of only 0.1 and 0.2 X IGT6M relative to the
incorporation observed at lethal concentrations. Yet in the
previous paper (Ref. 13, Chart 1), it was shown that, at 0.3
X IO"6 M ara-C, L-cells were able to multiply for many
weeks, with no sign of any cumulative
their doubling time was increased. At
concentrations shown in Chart 6 of the
incorporation
into DNA levels off and
decrease at 2.0 X 10~s M, a concentration
Studies
Inhibition of DNA Polymerase. Since it appeared that
incorporation of ara-C could not account for the inhibition
of DNA synthesis, the possibility that it was acting by
inhibiting DNA polymerase was investigated. In this study, 2
sets of parameters were determined: the Michaelis-Menten
constants (Km and K¡)of DNA polymerase for dCTP and
ara-CTP (necessarily measured in vitro) and the concentra
tions of dCTP and ara-CTP in whole cells.
The measurements on inhibition of DNA polymerase were
carried out with the procedures described by Gold and
Helleiner (12). The reaction mixture contained dGTP, dATP,
and TTP-3H at saturating concentrations; the dCTP con
centration was varied in the absence of ara-CTP to determine
Km and varied with ara-CTP present to determine K¡.In the
absence of any dCTP the rate of incorporation of TTP-3H
was approximately 25% of the rate found in the presence of
all 4 nucleotides and was unaffected by the presence of
ara-CTP. This was presumably due to the terminal addition
of nucleotides and was subtracted before attempting to
determine Km and K¡.The results of a typical experiment
are plotted as a double reciprocal plot in Chart 7. Since
straight lines can be drawn passing through the same point
on the ordinate, the inhibition is apparently competitive, as
has been shown previously for partially purified calf thymus
damage, although
the higher ara-C
present paper, the
even appears to
which is rapidly
lethal for S phase cells. Thus the amount
corporation into nucleic acids is apparently
correlated with lethality.
of ara-C in
not directly
O.24
- 0.20
«
- 0.16
- 0.12
-OX)8
- 0.06
- 0.04
22
Chart 6. Incorporation of ara-C- H (specific activity, 11 Ci/mmole)
into ara-CTP, RNA, and DNA during a 4-hr treatment at various
concentrations of ara-C.
-0.04
O
O04
l/dCTP(IOSM)
0.08
0.12
Chart 7. Inhibition by ara-CTP of DNA polymerase activity in
crude lysates of I^cells. The reaction mixture (0.3 ml) contained 20
Mmoles of phosphate buffer, pH 7.5; 2 /¿molesof 2-mercaptoethanol;
2 junóles of MgCl2; 120 ng of heat-denatured calf thymus DNA; 60
mamóles each of dATP, dGTP, and TTP-3H (IO4 cpm/mfimole); 0.15
mg of extract protein; and varying amounts of dCTP. After 30 min
at 37 , the reaction was terminated, and incorporation into acidinsoluble material was determined as described in "Materials and
Methods." The incorporation in the absence of dCTP has been sub
tracted from these data. »,no ara-CTP; A, 63 nM ara-CTP; A, 126 MM
ara-CTP.
NOVEMBER 1970
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2641
F. L. Graham and G. F. Whitmore
DNA polymerase (10). From 7 determinations of Km and
6 determinations of K¡values of 9.0 ±4.3 and 8.7 ±5.2
X IO"6 M (mean ±95% confidence limits), respectively, were
obtained. Furth and Cohen (10) obtained values of 3 and 1
X IO"6 M, respectively, for Km and Kj with calf thymus
DNA polymerase. These values seem significantly lower than
our values obtained with L-cell DNA polymerase.
The intracellular ara-CTP concentration was determined by
treating cells with ara-C-3H and chromatographing aliquots
of the acid-soluble fraction. The data of Chart 6 show that
at low concentrations of ara-C after a 4-hr treatment the
intracellular ara-CTP was proportional to the ara-C con
centration in the medium, but it deviated from linearity
above 10~s M. Usually 80 to 90% of the acid-soluble pool of
ara-C-containing compounds in cells washed with PBS con
sisted of ara-CTP. Measurement of the intracellular pool of
dCTP was carried out as described in "Materials and
Methods" with approximately IO8 untreated cells or 10s
cells which had been treated with 2.0 X lo"5 M ara-C for 4
hr. The values obtained were 3.2 mamóles of dCTP/108
control cells and 4.1 m/nnoles/lO8 ara-C-treated cells, values
which are similar to the pool si/e of total deoxycytidine
nucleotides in L5178Y cells (24). Treatment with ara-C, and
the resulting inhibition of DNA synthesis, did not appre
ciably alter the intracellular dCTP pool, although these
amounts of dCTP are sufficient to provide for only 3 to 4
min of DNA synthesis.
If Michaelis-Menten kinetics hold within cells, then from a
knowledge of the constants Km and K¡and the concentra
tions of dCTP and ara-CTP it should be possible to predict
what inhibition of DNA synthesis would be expected in cells
treated with ara-C. In carrying out the calculation, it was
assumed that ara-CTP and dCTP were uniformly distributed
over the total cell volume, which from the volume of a
packed cell pellet and from pulse height analysis of L-cell
suspensions was found to be 0.15 ml/108 cells. Since the
data described in the preceding paragraph indicated only a
slight variation of dCTP concentration with changes in ara-C
concentrations, it was also assumed that the concentration of
dCTP was independent of the concentration of ara-CTP.
The inhibition actually observed in .cells treated for 4 hr
with ara-C was determined from experiments on thymidine-3H incorporation
of the type described in the
previous paper (See Ref. 13, Chart 7). Chart 8 of this paper
illustrates the predicted and observed inhibitions plotted as a
function of the ara-C concentration in the medium. Aside
from the log-log scale, which was chosen to permit the use of
data obtained over a wide range of ara-C concentrations, the
values are plotted according to the method of Dixon (7). If
the inhibition follows Michaelis-Menten kinetics, then:
where v and v¡are the control and inhibited rates of DNA
synthesis, respectively, and S and / are the concentrations of
substrate (dCTP) and inhibitor (ara-CTP). The curve for the
observed inhibition of DNA synthesis is approximately
parallel to the predicted inhibition suggesting that in whole
cells inhibition of DNA synthesis, expressed as (v/i>/) - 1, is
2642
Chart 8. A comparison between the observed inhibition of DNA
synthesis in l^cells exposed to ara-C and the inhibition predicted
from in vitro measurements of inhibition of DNA polymerase by
ara-CTP. The observed inhibition (X) was calculated from the rate of
incorporation of thymidine- H into acid-insoluble material in cells
exposed for 4 hr to various concentrations of ara-C. Thymidine- H
incorporation was measured as described in Chart 9 of Ref. 13. The
predicted inhibition of DNA synthesis (*) was calculated with the
values determined in vitro for Km and Kj and the values determined
in vivo for dCTP and ara-CTP, assuming Michaelis and Menten
kinetics is valid in whole cells. - - -, maximum inhibition which would
be predicted within the experimental errors of Km and K¡,i.e., Km =
9.0 ±4.3 nM and K¡
= 8.7 - 5.2 jiM.
proportional to the ara-CTP concentration; however, the
observed inhibition is 60 times greater than that predicted
from inhibition of DNA polymerase in vitro. The dashed
curve of Chart 8 indicates that even the maximum inhibition
which could be expected within the experimental values of
Km and Ki; i.e.. Km = (9.0 + 4.3) X 1(T6 M, Kj = (8.75.2) X 10~6 M, could not completely account for the
observed inhibition. Possible reasons for this discrepancy will
be considered in the discussion to follow.
DISCUSSION
Incorporation of ara-C into DNA. The results of our studies
on incorporation of ara-C into the DNA of mouse L-cells
and the studies reported in the previous paper (13) suggest
that incorporation of ara-C into DNA is not the cause of
inhibition of DNA synthesis. As evidence we cite the
following facts.
ara-C can severely inhibit DNA synthesis without affecting
viability. Thus inhibition must be reversible, since synthesis
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ara-C: Incorporation-Inhibition
must resume in order for the cells to divide and form
colonies.
Incorporation of ara-C into newly synthesized strands of
DNA (Okazaki pieces) does not prevent the subsequent
elongation of these pieces when the cells are incubated in
the absence of ara-C.
When DNA labeled with ara-C-3 H was digested with micrococcal nuclease and spleen phosphodiesterase, it was found
that more than 70% of the radioactivity was released as a
3'-phosphate and was therefore incorporated into internucleotide linkage. It is not obvious how ara-C incorporation
would affect DNA synthesis if it does not block the addition
of nucleotides to ara-C-terminated strands.
Although ara-C incorporation does not appear to inhibit
DNA synthesis, it is still conceivable that incorporation
might be lethal through the introduction of some deleterious
alteration in the DNA structure. However, our results
indicate that incorporation of ara-C is not correlated with
lethality. It has also been suggested that the lethal effect of
ara-C might be due to its incorporation into RNA (5). Since
our results indicate that the incorporation into RNA at lethal
concentrations is not strikingly different from that at nonlethal concentrations, and since ara-C is specifically toxic for
S phase cells, while RNA synthesis occurs in all parts of the
cell cycle except mitosis (8, 27), it appears that incorpora
tion of ara-C into RNA is not the lethal event.
Inhibition of DNA Polymerase. Of the three current models
proposed to explain the inhibition of DNA synthesis by
ara-C, inhibition of CDP reducÃ-ase (3), inhibition of DNA
synthesis by incorporation (23, 32), and inhibition of DNA
polymerase (10), the model that ara-C inhibits DNA
synthesis via inhibition of DNA polymerase appears to be
most consistent with the following facts. First, inhibition of
DNA synthesis is apparently reversible, since cells survive and
form colonies after an inhibition of DNA synthesis as severe
as 97%. Second, ara-C does not appear to interfere with the
synthesis of either dCTP or TIP (at least not from exogenously
supplied deoxynucleosides),
yet inhibits their
incorporation into DNA. Third, ara-CTP does inhibit DNA
polymerase in vitro. Finally, the concentration of ara-CTP
within ara-C-treated cells is appreciable relative to the dCTP
concentration.
One serious difficulty,
however, is the
discrepancy between the observed and predicted inhibition
of DNA synthesis illustrated in Chart 8. The observed
inhibition in whole cells is approximately 60 times more
severe than that predicted from our in vitro studies on
inhibition of DNA polymerase by ara-CTP. There are a
number of possible explanations for this difference.
ara-C may be inhibiting DNA synthesis by some mechanism
other than inhibition of DNA polymerase. As we have
mentioned previously, ara-C does not appear to block DNA
synthesis by interfering with the production of either dCTP
or TTP. The possibility that ara-C affects the synthesis of
deoxypurines has not been investigated and cannot be
completely disregarded, although we know of no evidence
suggesting that this occurs. The work of Moore and Cohen
(25) on the effect of arabinonucleotides on ribonucleotide
reduction would suggest that at least ara-C does not interfere
with the reduction step in purine biosynthesis.
Studies
The conditions of the in vitro assay for DNA polymerase
activity may be so unlike the conditions in vivo that no
comparison can legitimately be made. For instance, in the in
vitro experiments the concentrations of the 3 deoxynucleotides, dGTP, dATP, and TTP, and the concentration of DNA
primer were at saturating levels, while in vivo this condition
may not hold. (The dCTP concentration has been found to
be approximately 3 X IO"5 M in I^cells. If the concentra
tions of dGTP, dATP, and TTP are comparable or lower,
then they would be far below the 2 X 10" M concentration
used in vitro.) In view of this, it may be naive to expect
quantitative agreement between in vivo and in vitro observa
tions.
The properties of the DNA polymerase enzyme in vitro
may be quite different from its properties in vivo. It has
been suggested that in vivo DNA replication may require an
enzyme complex containing nuclease, polymerase, and ligase
activities (11), and it is possible that DNA polymerase might
be altered by dissocation from this complex. Indeed, recent
studies (6, 16) on E. coli could be interpreted as suggesting
that E. coli DNA polymerase is not responsible for the major
synthesis of DNA, but may only act as a repair enzyme.
Because of the discrepancy between the observed and
predicted inhibition of DNA synthesis, the model in which
ara-C acts by inhibiting DNA polymerase cannot be con
sidered proven. At least part of the observed inhibition of
DNA synthesis, however, can be accounted for on the basis
of inhibition of DNA polymerase.
At this point, it seems appropriate to summarize the
possible action of ara-C in terms of a model in which
inhibition of DNA synthesis is the result of inhibition of
DNA polymerase.
ara-C must be converted to ara-CTP in order to inhibit
DNA synthesis. The ability of deoxycytidine to protect cells
against the effects of ara-C is presumably due, at least in
part, to the prevention of formation of ara-CTP from ara-C
by competition at the kinase level (15, 17). Thus resistance
to ara-C could result from a decrease in activity of deoxy
cytidine kinase, as has often been observed (4).
ara-CTP inhibits DNA polymerase and, consequently, DNA
synthesis. Since this inhibition is competitive with dCTP, the
ability of deoxycytidine to protect against ara-C-induced
inhibition of DNA synthesis may be in part due to competi
tion at the polymerase-binding site.
Inhibition of DNA synthesis can cause some type of
damage (as yet unknown) to cells synthesizing DNA at the
time of induction of the block which permanently prevents
cell proliferation after removal of the block. The induction
of this damage evidently depends on the severity of the
inhibition.
Therefore, it appears that inhibition of DNA polymerase
could account for all of the observed actions of ara-C, and,
while additional work is required to prove the model, this
seems the most satisfactory hypothesis at the present time.
ACKNOWLEDGMENTS
We thank Dr. M. Gold for his interest in this work and his helpful
advice and Dr. J. E. Till and Dr. W. R. Bruce for their comments on
the manuscript.
NOVEMBER 1970
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2643
F. L. Graham and G. F. Whitmore
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CANCER RESEARCH VOL. 30
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Studies in Mouse L-cells on the Incorporation of 1-β
-d-Arabinofuranosylcytosine into DNA and on Inhibition of DNA
Polymerase by 1- β-d-Arabinofuranosylcytosine 5′-Triphosphate
F. L. Graham and G. F. Whitmore
Cancer Res 1970;30:2636-2644.
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