(CANCER RESEARCH 51. 1829-1835. April 1. 1991]
Inhibition of Primer RNA Formation in CCRF-CEM Leukemia Cells by
Fludarabine Triphosphate1
Carlo V. Catapano,2 Kimberley B. Chandler, and Daniel J. Fernandes'
Department of Biochemistry, Bowman Cray School of Medicine of H ake Forest University, Winston-Salem, Sorth Carolina 2 7103
ABSTRACT
The effects of fludarabine triphosphate (Fara-ATP), l-/8-r>-arabinofuranosylcytosine 5'-triphosphate (ara-CTP), and aphidicolin on primer
RNA and DNA synthesis in human CCRF-CEM leukemia cells were
investigated. RNA-primed Okazaki fragment synthesis was monitored
by first incubating whole cell lysates for 10 min in the presence or absence
of the compound and then following the incorporation of [a-32P|ATP and
|'H|dTTP into the primer RNA and DNA portions, respectively, of the
Okazaki fragments. In whole cell lysates the degree of DNA synthesis
inhibition induced by Fara-ATP was directly related to the extent of
primer RNA synthesis inhibition over the entire range of Fara-ATP
concentrations tested (10-50 MM).In contrast, primer RNA formation
was stimulated by concentrations of ara-CTP (25-200 MM)and aphidi
colin (0.5-5 Mg/ml) that inhibited DNA synthesis. The primer RNA
recovered from cell lysates incubated with either Fara-ATP, ara-CTP, or
aphidicolin was of normal length, predominately 11 nucleotides. FaraATP was a more potent inhibitor of the polydeoxythymidylate primase
activity than of the DNA polymerase a/a activities present in the 100,000
x g supernatants of CCRF-CEM cells. Fara-ATP was a noncompetitive
inhibitor of DNA primase with respect to ATP [50% inhibitory concen
tration, 2.3 ±0.3 (SD) MM,K, = 6.1 ±0.3 (SE) MM|and the tfm(ATP)/
K¡(Fara-ATP) was 25. The 50% inhibitory concentration values of FaraATP for DNA nul) nicrascs a/6 activities on calf thymus DNA were 43
±1.6 (SD) MMand >100 MMwith respect to dATP and dTTP. The
effects of ara-CTP and aphidicolin on these enzymes were opposite those
seen with Fara-ATP, since 50% inhibitory concentrations of either araCTP or aphidicolin for DNA polymerases a/i did not inhibit polydeoxy
thymidylate primase activity. The results provide evidence that fludara
bine phosphate blocks DNA synthesis in CCRF-CEM cells through
inhibition of primer RNA formation. In contrast, the accumulation of
primer RNA and RNA-primed Okazaki fragments that is induced by
ara-CTP and aphidicolin could lead to the rereplication and amplification
of chromosomal DNA segments.
INTRODUCTION
Many of the clinically important anticancer agents interfere
with tumor cell growth by inhibiting DNA replication. One of
the key reactions involved in DNA replication is the synthesis
of primer RNA that takes place on the lagging strand of the
replication fork (1-4). Primer RNA formation is catalyzed by
DNA primase, which unlike other RNA polymerases, is insen
sitive to high concentrations of either actinomycin D or aamanitin (5-8). This primer RNA, which is about 10 nucleo
tides in length (9, 10), provides the free 3'-hydroxy terminus
required by the DNA polymerase «-primase complex for the
discontinuous synthesis of an Okazaki fragment of 100-200
nucleotides (11, 12). Following the elongation of the primer
RNA into a mature Okazaki fragment, the primer RNA is
Received 8/20/90; accepted 1/22/91.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Grant CH-226 from the American Cancer
Society and by USPHS Grant CA-44597 awarded by the National Cancer
Institute.
3 Supported in part by a Berti Fellowship from Champion Industries, WinstonSalem. NC.
3 Scholar of the Leukemia Society of America. Inc.; to whom requests for
reprints should be addressed.
removed, the gap filled, and the fragment ligated to higher
molecular weight DNA by DNA ligase (13).
In addition to DNA primase, both DNA polymerases a and
ôare involved in DNA replication (14-16). Specifically, DNA
polymerase o is thought to be the leading strand replicase,
whereas DNA primase and polymerase «are likely involved in
Okazaki fragment synthesis on the lagging strand of the repli
cation fork (15-17). A drug that selectively inhibits DNA
primase without blocking DNA polymerases a or 6 could be of
significant therapeutic value. DNA primase activity is required
only in DNA replication, whereas DNA polymerase «and
possibly óare involved in DNA repair as well as DNA replica
tion (18, 19). Thus, a primase-specific drug might be less toxic
and mutagenic to normal tissues than an inhibitor of polymer
ase «or ¿.The nucleoside triphosphate metabolites of various
anticancer agents, including ara-CTP4 (20, 21) and Fara-ATP
(22), inhibit DNA primase activity in cell-free systems. One
approach for evaluating the relevance of primase inhibition
measured in vitro to cytotoxicity would be to determine the
effects of these metabolites on both primer RNA and DNA
synthesis in a whole cell system. Under these conditions, drug
effects would be monitored in the presence of the four rNTPs
and dNTPs, the natural DNA template, and true DNA repli
cation forks.
However, it has previously been impractical to determine the
effect of an anticancer agent on primer RNA formation in a
whole cell system because of the difficulties involved in the
isolation of the primer RNA. Even in rapidly proliferating cells,
primer RNA comprises less than 1% of the total cellular RNA
(3, 10). This laboratory has recently shown that in human
CCRF-CEM leukemia cells essentially all of the primer RNA
is tightly bound to the insoluble nuclear matrix (10). Thus, by
incubating whole cell lysates with the anticancer agent and
subsequently isolating nuclear matrices, it was possible to mon
itor drug effects on primer RNA formation in the presence of
the natural DNA template and all of the rNTPs and dNTPs.
In this system Fara-ATP, the active metabolite of the new
anticancer agent, Fludara, was a potent and specific inhibitor
of DNA primase activity and primer RNA formation.
MATERIALS
AND METHODS
Materials. The human CCRF-CEM leukemia cell line was propa
gated at 37°Cunder 95% air-5% CO2 in Fischer's medium supple
mented with 10% heat-inactivated horse serum, penicillin (20,000
units/liter), and streptomycin (20 mg/liter). Tissue culture medium,
serum, and antibiotics were obtained from GIBCO/BRL, Grand Island,
NY. Cells were checked periodically for Mycoplasma contamination
with the Gen-probe Mycoplasma ribosomal RNA hybridization kit
4 The abbreviations used are; ara-CTP, 1-tf-D-arabinofuranosylcytosine 5'triphosphate; Fara-AMP. l-rf-D-arabinofuranosyl-2-fiuoroadenine 5'-monophosphate. Fludara; Fara-ATP, l-#-D-arabinofuranosyl-2-fluoroadenine
5'-triphosphate; BuPd-GTP. A'2-(p-«-butylphenyl)-9-(2-deoxy-/i-D-ribofuranosyl)guanine;
dNTP, rNTP, a deoxyribo- and ribonucleoside triphosphate, respectively, having
an unspecified base; PMSF. phenylmethylsulfonyl fluoride; DTT, dithiothreitol;
IC50, concentration of a compound required to inhibit enzyme activity by 50%
compared to the untreated control; poly(dT), polydeoxythymidylate; ara-C, 1-0D-arabinofuranosylc) tosine.
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EFFECTS OF Fara-ATP AND ara-CTP ON PRIMER RNA
obtained from Fisher Scientific Co., Raleigh, NC. Fara-ATP and aphidicolin were generous gifts of Dr. William Parker of the Southern
Research Institute, Birmingham, AL and Dr. Maurizio D'Incaici of the
Mario Negri Institute for Pharmacological Research, Milan. Italy,
respectively. The purities of the various Fara-ATP preparations varied
between 86 and 93% as determined by reversed-phase high-performance
liquid chromatography analysis. The concentrations of Fara-ATP in
the stock solutions were determined spectrophotometrically (261 nm,
f = 14, 800. pH 7). Poly(dT), proteinase K, and all of the rNTPs and
dNTPs were purchased from Sigma Chemical Co., St. Louis, MO.
RNase-free pancreatic DNase I with a specific activity of 2200 Kunitz
units/mg protein was obtained from Worthington Biochemical Corp.,
Freehold, NJ. Endonuclease-free DNA polymerase I (Klenow frag
ment), RNasin (ribonuclease inhibitor), and polynucleotide kinase were
purchased from Promega, Madison, WI. Oligoribonucleotide and oligodeoxyribonucleotide size markers were obtained from Pharmacia
LKB Biotechnology, Piscataway, NJ. All oligonucleotide size markers
were end labeled with [7-32P]ATP and 10 units of polynucleotide kinase
as described previously (23). [«-"PJATP and [7-"P]ATP with specific
radioactivities of approximately 3000 Ci/mmol. and [2-uC)thymidine,
[»H?/Aj'/-3H]tnyinidine,[methyl-*H]dTTP, [8-'H]dATP, and [5-'H]
dCTP with specific radioactivities of 0.056, 64, 66, 12, and 24 Ci/
mmol, respectively, were purchased from ICN Radiochemicals, Irvine,
CA. Low salt buffer consisted of 10 mM Tris-HCl (pH 7), 1 miviMgCl2,
10 mM NaCl, and 1 mM PMSF. High salt buffer consisted of 10 mM
Tris-HCl (pH 7), 0.6 mM MgCl2, 1.5 M NaCl. and 1 mM PMSF. TE
buffer consisted of 10 mM Tris-HCl (pH 7.8) and 2 mM EDTA.
Extraction of Nuclear Matrix-bound DNA and RNA. In order to
uniformly label the total nuclear matrix DNA, exponentially growing
CCRF-CEM cells (2 x IO7 cells/group) were incubated for 72 h (ap
proximately 3 doubling times) with 0.05 ^Ci/ml of ('Hjthymidine. The
tumor cells were then resuspended in fresh medium at a density of 2 x
10' cells/ml. After 24 h the cells were harvested by centrifugation at
4°C,and the pellets were washed with a solution of 140 mM NaCl, 2
mM KCI, 8 mM Na2HPO4, and 1.5 HIM KH2PO4 at pH 7. The cells
were then resuspended at a density of 2 x IO7cells/ml in 10 mM TrisHCl (pH 7) buffer
cells were allowed
with 25 strokes in
lysate were each
incubated for 10
that contained 2 mM MgCl2 and 1 mM PMSF. The
to swell on ice for 15 min and then were disrupted
a Dounce homogenizer. One-mi aliquots of the cell
mixed with 0.25 ml of incorporation buffer and
min at 25°Cin the presence or absence of the
anticancer compound. The final concentrations in the reaction mixtures
were 40 mM NaCl, 5 mM MgCl2, 50 mM sucrose, 30 mM 4-(2hydroxyethyl)-l-piperazineethanesulfonic
acid, 0.4 mM CaCl2, 5 mM
phosphoenolpyruvate, 0.8 mM DTT, 2 MMdTTP, 100 MMconcentra
tions of the remaining dNTPs, 1 MM[a-32P]ATP, and 1 mM concentra
tions of the remaining rNTPs at a final pH of 7.8. After the 10-min
incubation period, the lysates were immediately layered over a solution
of 45% (w/v) sucrose at 4°Cand the nuclei were isolated by centrifu
gation (1900 x g for 30 min at 4°C).Newly synthesized RNA and
DNA were extracted from the salt-insoluble nuclear matrices with
phenol/chloroform as previously described (10). The recovery of newly
replicated DNA was greater than 95% following this procedure.
Isolation of Primer RNA and RNA-primed Nascent DNA. These
intermediates were isolated from the nuclear matrices according to a
method that was previously developed in this laboratory (10). The
radiolabeled nucleic acids were purified as described above, heated at
90°Cfor 5 min to separate any RNA/DNA hybrids, immediately cooled
on ice, and then centrifuged to equilibrium at 101,000 x g in a cesium
chloride gradient. Since primer RNA is an oligonucleotide that is
covalently attached to the nascent DNA, the RNA-primed DNA bands
at a lower density in the cesium chloride gradient (DNA density region
of 1.72-1.75 g/ml) than the higher density bulk RNA (10). Gradient
fractions of 0.45 ml were collected from the bottom of the tube and the
amount of radioactivity in a 50-^1 aliquot of each fraction was deter
mined by liquid scintillation counting. The fractions collected from the
DNA density region of the cesium chloride gradient were pooled and
diluted 4-fold with TE buffer that contained 13 mM MgCl2 and 5.6 ¿ig/
ml of yeast tRNA. The DNA- and RNA-primed DNA were precipitated
overnight with 3 volumes of ethanol at 4°C,and were then recovered
by centrifugation in a swinging bucket rotor (40,000 x g, 60 min, 4°C).
The pellets were washed twice with 70% (v/v) ethanol (40,000 x g, 15
min, 4°C)and then resuspended in 45 M!of 10 mM Tris-HCl (pH 7.8)
buffer containing 1 mM DTT and 1 unit/Ml of RNasin. Aliquots of the
samples were removed for liquid scintillation counting, and the remain
ders of the samples were divided into two fractions such that each
fraction contained an equal amount of ['HjDNA. To one fraction
MgCl2, Cad;, and DNase I were added to yield final concentrations of
1 mM, 0.1 mM, and 12 units/Ml, respectively. To the second aliquot
only EDTA was added to a final concentration of 2 mM to inhibit any
endogenous DNase activity. The samples were incubated for 22 h at
37°C.Following this, the samples were heat denatured at 90'C in 7 M
urea for 5 min and then cooled on ice for 5 min. The samples were
then electrophoresed in a 20% polyacrylamide-7 M urea gel and the
primer RNA and RNA-primed nascent DNA were visualized by autoradiography as previously described (10). Oligo- and polynucleotide
size markers consisted of I5'-"P](A)3, [5'-'2P](U)6, 15'-"P](A)8, and a
ladder of repeating [5'-"P]d(GACT)8_32. The effects of drug treatment
on the relative amounts of primer RNA and RNA-primed nascent DNA
in cell lysates were determined by scanning the autoradiographic neg
atives with a laser scanning densitometer (Biomed Instruments, Inc.,
Fullerton, CA). As a check on the accuracy of this densitometric
technique for quantitating primer RNA, electrophoresis was carried
out following the loading of known amounts of [5'-"Pl(A)8. The area
beneath the density curve was a linear function (r = 0.99) of the amount
of 32Ploaded on the electrophoresis gel lane.
Measurement of Nuclear Matrix DNA Synthesis. The DNA of
CCRF-CEM leukemia cells was uniformly prelabeled for 72 h with 0.5
MCi/ml of [uC)thymidine. The cells were than resuspended in fresh
medium at a density of 2 x IO5cells/ml. After 24 h, whole cell lysates
were prepared and pulse labeled for 10 min with 2 MM[3H]dTTP (final
specific radioactivity of 6.4 Ci/mmol) in the presence or absence of the
anticancer compound as described above. Nuclear matrices were pre
pared and washed as described above. The nucleic acids were then
precipitated overnight with ice-cold 10% (w/v) trichloroacetic acid. The
samples were then heated at 90°Cfor 30 min in 5% trichloroacetic acid
to extract the DNA as previously described (24). The incorporation of
['H]dTTP into DNA was linear for at least 15 min under these condi
tions. Inhibition of DNA synthesis was revealed as a decrease in the
specific activity of DNA ([1H]/['4C]) in cells incubated with anticancer
agent compared to the specific activity of DNA ([3H]/[I4C]) in untreated
control cells.
DNA Primase and Polymerase Activities. The activities of these
enzymes were measured in extracts of 2 x IO7 logarithmically growing
CCRF-CEM cells. The cells were harvested by centrifugation at 4°C,
washed with Fischer's medium at 4°C,and then disrupted in a volume
of 1 ml by Dounce homogenization as previously described (24). The
lysate was centrifuged at 1,900 x g for IO min at 4°Cto pellet the
nuclei and cellular debris. Tris-HCl (pH 8), DTT, and glycerol were
added to the supernatant to yield final concentrations of 20 mM, 1 mM,
and 10% (v/v), respectively. The supernatant was then centrifuged for
l h at 100,000 x g, and 4°Cand dialyzed overnight against 500 volumes
of the above buffer at 4°C.DNA primase activity in the supernatant
was measured using a poly(dT) template in a coupled assay with the
Klenow fragment of Escherichia coli DNA polymerase I. The standard
reaction was carried out in the presence and absence of inhibitor at
37°Cfor 45 min with cell extract (approximately 9 Mgof protein), 60
mM Tris-HCl (pH 8), 1 mM DTT, 8 mM MgCl2, 750 Mg/ml heatinactivated bovine serum albumin, 800 MMATP, 300 MM poly(dT)
(dTMP concentration), 0.66 unit of the Klenow fragment, 75 MM[3H]dATP (final specific radioactivity of 0.07 Ci/mmol) in a final volume
of 50 M'.The following preliminary experiments were done in order to
ensure that the incorporation of ['HjdATP into acid-insoluble product
was directly dependent on the priming of the poly(dT) by DNA primase
present in the 100,000 x g supernatants. Primase activity was depend
ent on the concentration of ATP and the amount of sample protein
present in the assay. The products synthesized in these reactions were
predominately alkaline-labile oligomers of 5-10 nucleotides in length.
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EFFECTS OF Fara-ATP AND ara-CTP ON PRIMER RNA
No significant activity was detected in the absence of either ATP,
poly(dT), or cell extract. Moreover, the priming activity appeared
distinct from other RNA polymerases. The combination of 50 Mg/ml
each of actinomycin D and a-amanitin, which decreased total RNA
synthesis by greater than 50%, had no effect on DNA primase activity.
In the primase assay inhibition of [3H]dATP incorporation by a com
pound was the result of a blockade of DNA priming and not the
extension of the primer RNA by the Klenow fragment of DNA polymerase I. At the highest concentrations used in this study, neither FaraATP, ara-CTP, nor aphidicolin inhibited DNA poiymerase I activity
on an activated calf thymus DNA template.
DNA poiymerase activities were assayed in the same 100,000 x g
supernatants as DNA primase. Reactions were carried out in the
presence and absence of inhibitor at 37°Cfor 30 min with an activated
calf thymus DNA template and either 50 ßM
[3H]dCTP, ['HjdATP, or
[3H]dTTP (final specific radioactivity of 0.2 Ci/mmol) and 100 MM
concentrations of the remaining dNTPs in a final volume of 50 n\. In
the reactions which contained the SJK 132-20 antibody to DNA
poiymerase a, the antibody was incubated with the enzyme preparation
for l h at 4°Cprior to the start of the poiymerase reaction by the
addition of substrate. Following completion of the reactions, the sam
ples from the primase and poiymerase assays were spotted on Whatman
3MM filters and the acid-insoluble radioactivity remaining on the
washed filters was determined as previously described (25). The DNA
primase and poiymerase activities of the purified DNA poiymerase aprimase complex, which was ¡mmunoaffinity purified from human Tleukemia cells obtained by leukapheresis (26), were assayed as described
above.
RESULTS
A recent report from this laboratory has described the syn
thesis and distribution of primer RNA and RNA-primed DNA
in lysates of CCRF-CEM leukemia cells (10). Essentially all of
the primer RNA and RNA-primed DNA (Okazaki fragments)
were located within the insoluble matrix fraction of the nucleus.
The predominant primer RNAs isolated from the nuclear ma
trix were 8-10 nucleotides in length and showed a random
distribution of ribonucleotides at the 3' end. The primer RNAs
were covalently linked to newly replicated DNA as evidenced
by their buoyant density in cesium chloride, sensitivity to DNase
I, and phosphate transfer analysis.
Effects of Fara-ATP, ara-CTP, and Aphidicolin on Primer
RNA and DNA Synthesis in Whole Cell Lysates. Whole cell
lysates of exponentially growing CCRF-CEM cells were incu
bated for 10 min with either [12P]ATP or [3H]dTTP in the
presence or absence of these compounds. The whole cell lysates
contained intact nuclei. Since primer RNA is present in very
low concentrations in cells, the lysate system was used to permit
the incorporation of high specific activity [32P]ATP into the
M 50 •)
0~"~
50
100
150
AraCTP (fiU)200
150
5100
u
50
012345
Aphidicolin (/¿g/ml)
Fig. I. Effects of Fara-ATP (A), ara-CTP (B), and aphidicolin (C) on primer
RNA and DNA synthesis in whole cell lysates. Primer RNA synthesis was
measured by preincubating CCRF-CEM cells for 72 h with [3H]thymidine to
uniformly label the DNA. Whole cell lysates prepared from these cells were
incubated with and without drug for 10 min at 25°Cin the presence of [32P]ATP.
RNA-primed DNA was isolated following cesium chloride gradient centrifuga
tion. Control samples contained an average of 1,721 32P and 77.010 3H dpm,
respectively. Inhibition of primer RNA synthesis was revealed as a decrease in
the "P-primer RNA/['H]DNA ratio obtained from lysates incubated with the
drug compared to the specific activity of primer RNA from untreated controls.
DNA synthesis was measured in a manner similar to that of primer RNA synthesis
with the exception that the cells were prelabeled with ['4C]thymidine and then
pulse labeled with [3H]dTTP. Inhibition of DNA synthesis was revealed as a
decrease in the [3H]/['4C] ratio compared to control. Control samples for DNA
synthesis contained an average of 4883 ±1268 (SD) 3H and 3782 ±1009 (SD)
MCdpm, respectively. Points, mean of either two (primer RNA synthesis) or three
(DNA synthesis) separate experiments. O. primer RNA synthesis: •.DNA
synthesis.
tested both ara-CTP and aphidicolin stimulated primer RNA
synthesis but inhibited DNA synthesis.
The effects of these compounds on primer RNA formation
were further evaluated by gel electrophoresis. Fractions were
pooled from the DNA density regions of the above cesium
chloride gradients and the RNA-primed DNA was concentrated
by ethanol precipitation and then resuspended in buffer. In
order to control for any differences in the recoveries of the
nucleic acids, aliquots that contained equal amounts of [3H]-
primer RNA. Thus, this technique also allowed one to monitor
drug effects on both primer RNA formation and the extension
of the primer RNA with [3H]DNA. Following the preparation
of the nuclear matrices from the cell lysates, the RNA-primed
DNA were removed from each sample. Each aliquot was then
nascent DNA was isolated by cesium chloride density gradient
exhaustively digested with pancreatic DNase I to remove the
centrifugation. Previous results from this laboratory indicated
nascent DNA attached to the primer RNA and then electrothat at least 94% of the cellular RNA-primed DNA is recovered
phoresed on a strand-separating 20% polyacrylamide-urea gel.
under these conditions. Fig. IA shows that Fara-ATP, the active
metabolite of Fludara, inhibited the incorporation of [32P]ATP It can be seen (Fig. 2, Lanes 1-9) that the radiolabel from all
into primer RNA and [3H]dTTP into DNA to a similar extent
the control and drug-treated lysates was concentrated in a
over the entire range of Fara-ATP concentrations (10-50 UM). product of about 11 nucleotides, although a smaller amount of
ara-CTP (27, 28) and aphidicolin (29, 30), known inhibitors of an oligomer of about 4 nucleotides was also present in each
lane. This laboratory has previously demonstrated that the 11DNA poiymerase a, were also evaluated. In contrast to that
nucleotide product is full-length primer RNA, whereas the less
observed with Fara-ATP, no relationship was seen between
inhibition of primer RNA and DNA synthesis with either ara- intense band that migrated at 4 nucleotides primarily represents
partially degraded primer RNA (10). A 10-min incubation of
CTP or aphidicolin (Fig. l, B and C). At all concentrations
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EFFECTS OF Fara-ATP AND ara-CTP ON PRIMER RNA
1
2
3
32-
24 16-
10 63A
100 55 36
100 192 213
100 150 123
B
100 56
100
100
44
FaraATP
58
40
AraCTP
79
63
Aphidicolin
Fig. 2. Eleclrophoretic analysis of the effects of Fara-ATP, ara-CTP, and
aphidicolin on primer RNA formation in whole cell lysates. Whole cell lysates
were incubated with and without the anticancer agent for 10 min. and then the
RN A-primed DN A was isolated following cesium chloride gradient centrifugation
as described in Fig. 1. Fractions from the gradients that contained the RNAprimed DNA (density of 1.72-1.75 g/ml) were pooled, subjected to ethanol
precipitation, and then exhaustively digested with RNase-free DNase I. Aliquots
of the samples that contained equal amounts of DNA were electrophoresed on a
strand-separating 20% polyacrylamide-urea gel. Lanes 1-3. primer RNA obtained
from cell lysates incubated with either 0, 25, or 50 UM Fara-ATP. respectively:
Lanes 4-6. primer RNA from lysates incubated with either 0, 50, or 100 /JM araCTP, respectively; Lanes 7-9. primer RNA from lysates incubated with either 0,
0.5, or 1 jig/ml of aphidicolin. respectively. Nucleotide size markers are shown
on the ordinate. Below each lane is given: Row A. percentage of control primer
RNA content as determined by scanning densitometry; Row B. percentage of
control DNA synthesis as determined by [3H]dTTP incorporation into DNA.
cell lysates with either 25 MM Fara-ATP (Lane 2) or 50 ¿IM
Fara-ATP (Lane 3) reduced total primer RNA synthesis to 55
and 36% of control, as determined by densitometric scanning
of the autoradiographic negative. At each Fara-ATP concentra
tion, the extent of primer RNA synthesis inhibition was similar
to the degree of DNA synthesis inhibition. Conversely, with
ara-CTP (Lanes 4-6) and aphidicolin (Lanes 7-9), inhibition
of DNA synthesis was accompanied by an accumulation of
primer RNA in the cell lysates. These results are in agreement
with those shown in Fig. 1 and suggest that Fara-ATP inhibits
DNA synthesis by blocking DNA primase and primer RNA
formation. The effects observed with ara-CTP and aphidicolin
were consistent with their known mechanism of action, i.e.,
inhibition of DNA polymerization.
Effects of Fara-ATP, ara-CTP, and Aphidicolin on DNA Pri
mase and DNA Polymerase Activities. Further studies were
carried out to determine if the inhibition of primer RNA
formation in the whole cell system by Fara-ATP (Fig. 2) was
the result of a selective blockade of DNA primase, rather than
of other DNA polymerases active at the replication fork. Polym
erase activities were assayed in the 100,000 x g supernatants
of exponentially growing CCRF-CEM cells. The polymerase
activity measured in the high speed supernatants was a combi
nation of DNA polymerases «and ó.DNA polymerases a and
5 activities are extensively inhibited by 150 mivi KC1, whereas
polymerase ßactivity is stimulated at this salt concentration
(18). We observed that 150 HIM KC1 inhibited 98% of the
polymerase activity (data not shown), which implied that our
standard polymerase assay detects little polymerase ßactivity
in the high speed supernatants. Fig. 3 shows that low concen
trations of the specific inhibitors of DNA polymerase a, the
SJK 132-20 monoclonal antibody to polymerase a and BuPdGTP (26, 31), completely inhibited the activity of immunoaffinity-purified DNA polymerase a. In contrast, a maxi
mum of about 50% inhibition of the polymerase activity present
in the 100,000 x g supernatants was observed with excess
concentrations of either the SJK 132-20 antibody or BuPdGTP. Taken together, these results indicate that on calf
thymus DNA the ratio of DNA polymerase a activity to polym
erase ôactivity in the 100,000 x g supernatant is about 1:1,
which is in agreement with the results reported by others using
extracts from various types of mammalian cells (14, 15). Ac
cordingly, DNA polymerase activity measured in the high speed
supernatant will be referred to as polymerase a/5.
Fig. 4A shows that Fara-ATP is a much more potent inhibitor
of DNA primase than DNA polymerases «/¿.
The lC5o of FaraATP for poly(dT) primase activity determined in the presence
of saturating substrate (800 /ZMATP) was 2.3 ±0.3 (SD) /IM.
Incubation of the enzyme preparation with Fara-ATP and
poly(dT) for 5 min prior to the initiation of the reaction with
substrate did not change the IC50value. This suggested that the
formation of the Fara-ATP-primase complex was rapid and not
time dependent under our assay conditions. Evidently the 2-F
substitution of Fara-ATP is important for interaction with
DNA primase, since ara-ATP is a less potent inhibitor of the
primase [IC50 = 10.6 ± 1 (SD) MM]. It was of interest to
K
20
SJK 132-20
40
(/¿g/ml)
60
150
200
•
B
75
50
25'
50
100
BuPdGTP
(/¿M)
Fig. 3. Effects of the SJK 132-20 monoclonal antibody (A) and BuPdGTP
(A) on purified DNA polymerase a activity and the DNA polymerase i»/¿
activities
in cells extracts. DNA polymerase activities »eremeasured on an activated calf
thymus DNA template with 50 JIM[3H] dTTP and 100 J*Mconcentrations of the
remaining dNTPs. Points, mean of three determinations. The SD of each mean
was smaller than the size of the symbol. For purified DNA polymerase «and the
DNA polymerase a/èactivities present in the 100,000 x g supernatants, 100%
of control activity represents the incorporation of 14,629 ±139 and 9,978 ±102
(SD) 'H dpm, respectively, into acid-insoluble product over 30 min. •,purified
DNA polymerase «activity; O, DNA polymerase n/¿activities in the 100.000 x
i: supernatants.
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EFFECTS OF Fara-ATP AND ara-CTP ON PRIMER RNA
100»
75
50
MëC
O.0
0)
0
0\
.
25 100FaraATP
50
75
(¿iM)10017550250B\«AX^
~*~"
*
0uoX25
•3
A—
50
100
AraCTP
150
200
2
4
6
8
Aphidicolin 0¿g/ml)
10
100*.
0
0'SM5025QA^«^==t==
sè0
Fig. 4. Effects of Fara-ATP (A), ara-CTP (B), and aphidicolin (C) on DNA
primase and DNA polymerase activities. DNA primase activity was measured by
using poly(dT) as a template, 800 MMATP, 75 MM[3H]dATP, and DNA polym
erase I. DNA polymerase activities were measured on an activated calf thymus
DNA template with either 50 MM[3H)dTTP, [3H]dATP, or [3H]dCTP, and 100
MMconcentrations of the remaining dNTPs. Points, mean of 4 determinations.
The SD of each mean was smaller than the size of the symbol. For DNA primase,
100% of control activity represents the incorporation of 52,264 ±928 (SD) 3H
dpm into acid-insoluble product over 45 min. For DNA polymerase a/6, 100%
of control activity represents the incorporation of 13,919 ±300, 10,966 ±707,
and 9,873 ±618 (SD) 3H dpm into acid-insoluble product over 30 min with [3H]
dTTP, [3H]dATP, and [3H]dCTP as substrate, respectively. O, DNA primase
activity; •,DNA polymerase a/6 with 50 MM[3H]dTTP; •DNA polymerase «/
6 with 50 MM[3H]dATP; A, DNA polymerase a/6 with 50 MM[3H]dCTP.
determine if Fara-ATP could serve as a partial agonist in the
reaction catalyzed by DNA primase. However, Fara-ATP
showed no substrate activity in the poly(dT) primase assay,
since Fara-ATP at various concentrations between 1 and 300
MMdid not promote any detectable product formation when
incubated with the enzyme preparation and poly(dT) in the
absence of ATP. Taken together, the above data suggest that
Fara-ATP inhibits DNA primase and is incapable of supporting
the synthesis of functional primers that can be utilized by DNA
polymerase I. Fara-ATP is a much weaker inhibitor of DNA
polymerase a/o than the primase, since the ICso values of FaraATP for polymerase a/b were 43 ±1.6 (SD) MMand >100 pM
with respect to the corresponding purine substrate (50 ¿tM
dATP) and a pyrimidine substrate (50 /¿M
dTTP), respectively
(Fig. 44). DNA polymerase o activity in the high speed supernatants was determined as the activity remaining following
complete inhibition of DNA polymerase a with either 50 Mg/
ml of the SJK 132-20 antibody or 5 MMBuPdGTP. Preliminary
studies suggest that the polymerase 5 activity in the 100,000 x
g supernatant was about equally sensitive to Fara-ATP as the
immunoaffinity-purified DNA polymerase a (data not shown).
The effects of ara-CTP and aphidicolin on these enzyme
activities (Fig. 4, B and C) were opposite those seen with Fara-
ATP. Concentrations of either ara-CTP or aphidicolin that
produced 50% inhibition of DNA polymerases a/a did not
significantly interfere with primase activity. These results are
in agreement with those of others who reported that neither
ara-CTP nor aphidicolin inhibited poly(dT) primase activity
(21,32).
A kinetic analysis of the inhibition of DNA primase by FaraATP is given in Fig. 5. Initial velocity measurements, carried
out with varying concentrations of ATP in the absence of
inhibitor, revealed positive cooperativity. These data are best
described by a Hill equation with a coefficient (nH) of 1.8 ±0.2
(SE). A similar degree of positive cooperativity was observed
by Parker and Cheng (22) when DNA primase activity was
likewise measured on a poly(dT) template. The Hill coefficients
determined in the presence of 0.9 and 3.3 MMFara-ATP are 1.7
±0.2 and 1.9 ±0.3 (SE) /¿M,
respectively. Since Fara-ATP did
not significantly change the Hill coefficient, this suggests that
Fara-ATP did not alter the cooperative interaction of the sub
strate ATP with its binding sites on the primase.
The linear Lineweaver-Burk plot shown in Fig. 5 was ob
tained by plotting 1/v versus 1/[ATP]18 as described by Segel
(33). Fara-ATP is a noncompetitive inhibitor of the primase
with respect to ATP, and replots of the slopes and intercepts
yield a K, of 6.1 ±0.3 (SE) MM. The significance of this
noncompetitive inhibition to the mechanism of Fara-ATP in
teraction with DNA primase is difficult to determine because
of the complex kinetics of this processive enzyme. However,
other analyses of the kinetic data were informative. The Km of
ATP is 151 ±4 (SE) MM(Fig. 5) and, therefore, the Km/K¡is
25. In contrast, the K, of Fara-ATP for either DNA polymerase
a or e is about equal to the Km for dATP (34, 35). These
observations further suggest that Fara-AMP is more active
intracellularly as a DNA primase inhibitor than as an inhibitor
of DNA polymerase a or t.
DISCUSSION
It seems reasonable that DNA primase could be an important
target for certain anticancer agents. Many of the clinically useful
anticancer agents inhibit DNA replication, and primer RNA is
a key intermediate involved in this process. However, little
information is available concerning the effects of any anticancer
agent on primer RNA formation in a whole cell system. The
enrichment of primer RNA in the nuclear matrix of CCRFCEM cells has facilitated our isolation of this intermediate and
-60
[ATP]""
(mM)
Fig. 5. Kinetic analysis of DNA primase inhibition by Fara-ATP. The concen
tration of ATP varied between 0.125 and 1.0 mM. The concentration of [3H]dATP was 75 MM(0.06 Ci/mmol). v is expressed as: (3H dpm incorporated into
acid-insoluble product over 45 min) x 10'. nH is the Hill coefficient, which was
determined experimentally to be 1.8. The inset shows the replots of the slopes
and intercepts of the double reciprocal plot. O, no Fara-ATP; D, 0.9 MMFaraATP; A, 3.3 MMFara-ATP.
1833
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EFFECTS OF Fara-ATP AND ara-CTP ON PRIMER RNA
the subsequent studies of drug effects on primer RNA formation
in lysates of these cells.
Fara-AMP (Fludara) is a purine antimetabolite that is active
as a single agent against chronic lymphocytic leukemia (36) and
non-Hodgkin's lymphoma (37). A number of studies were
aimed at defining the intracellular locus of Fara-AMP action.
Early investigations revealed that Fara-ATP inhibited partially
purified ribonucleotide reducÃ-aseat low micromolar concentra
tions (38). However, concentrations of Fara-AMP that pro
duced maximal inhibition of DNA synthesis in human CCRFCEM leukemia cells did not induce significant changes in any
of the dNTP pools (39). This suggested that ribonucleotide
reducÃ-aseis not a primary targel for Fara-AMP in Ihese cells.
Il has been reported lhal Fara-ATP also inhibils DNA polymerase a and DNA primase aclivilies in vitro (22, 35). One
cannot be certain thai Ihese resulls reflecl ihe effecls of FaraAtTP in whole cells because of Ihe complexily of cellular DNA
replicalion compared lo DNA synlhesis in purified syslems.
For ihis reason, ihe effecls of Fara-ATP were examined in a
whole cell syslem which conlained ihe nalural DNA tempiale,
aclual DNA replication forks, and ihe four rNTPs and dNTPs.
We found Fara-ATP lo be a potent and specific inhibitor of
primer RNA formation in CCRF-CEM leukemia cells. The
extenl of inhibition of primer RNA formation was direclly
relaled lo Ihe degree of DNA synthesis inhibition over a wide
range of Fara-ATP concentrations. This strongly suggested lhat
Fara-ATP blocked DNA synthesis by inhibiting DNA primase.
Parker et al. (22, 35) obtained no evidence that Fara-ATP
induced primer RNA chain terminalion. The resulls described
herein are consislenl wilh Ihose of Parker et al., bui do not
completely rule oui this potentially importani effecl of FaraATP. The primer RNA synthesized in the presence of FaraATP was of full length (Fig. 2), and Fara-ATP showed no
subslrale aclivily for DNA primase on a poly(dT) template.
However, it was still possible that a single Fara-AMP was
incorporated at the 3' end of the full length primer. The primer
RNAs which contained a lerminal 3'-Fara-AMP may noi have
been utili/ed by eilher DNA polymerase a in Ihe whole cell
syslem or by ihe Klenow fragmenl of DNA polymerase I presenl
in Ihe poly(dT) primase assay.
In centrasi lo the inhibilory effecl of Fara-ATP, ara-CTP
and aphidicolin induced an accumulalion of primer RNA in
whole cell lysales. The primer RNA synthesized in the presence
and absence of these agents was about 11 nucleolides. This
indicated thai the enhanced amounts of primer RNA recovered
from lysates incubated with eilher ara-CTP or aphidicolin could
noi be attributed to the synthesis of primers of increased length.
An alternalive hypolhesis is lhal ara-CTP and aphidicolin in
duce an inlracellular accumulation of primer RNA by inhibiting
its enzymatic removal. The degradation of the primer RNA is
thought to occur after extension of the primer into full length
Okazaki fragmenls (13). Therefore, agenls which inhibil DNA
polymerase «or ô(e.g., ara-CTP, aphidicolin) could increase
Ihe inlracellular concentrations of primer RNA by inhibiting
primer RNA removal. An indication lhal ara-CTP mighl inlerfere wilh primer RNA removal is provided by the work of Tseng
and Goulian (12), who showed by phosphale Iransfer analysis
lhal ara-CTP slowed the rate of disappearance of the 3' termi
nal rNMP presenl al Ihe primer RNA/DNA junclion.
Anolher inleresting possibility is thai ara-CTP and aphidi
colin induced primer RNA accumulation by indirectly promot
ing increased binding of the primase lo DNA. This mechanism
may be relaled lo ihe previous mechanism, since primer RNA
synthesis is closely coupled to primer RNA removal (13).
Inhibition of DNA polymerase a or &by Ihese agents would
interfere with ihe exlension of Ihe primer RNA inlo Okazaki
fragmenls and, Ihus, ihe conversion of single-slranded DNA al
the replication fork inlo double-slranded DNA. The persislence
of Ihese single-slranded DNA regions could increase ihe num
ber of available binding siles on DNA from which ihe primase
could initiate Okazaki fragment synthesis. One consequence of
enhanced binding of the primase to DNA could be replicón
misfiring and ihe rereplication of cerlain chromosomal DNA
segmenls lhat are seen following treatment of cells with either
ara-C or other inhibitors (40-42). Fara-ATP, by inhibiting
DNA primase and not Ihe polymerases, would seem less likely
lhan ara-C to induce "replicón misfiring" and gene amplifica
tion.
This study provides evidence lhat Fara-AMP has a novel
mechanism of action in exponentially growing CCRF-CEM
leukemia cells, i.e., blockade of DNA primase. Since DNA
primase is an RNA polymerase, it is possible thai Ihis anlicancer agent also inhibits other RNA polymerases. Allhough ihe
major melabolic effecl of Fara-AMP in rapidly growing human
leukemic cells is directed at DNA synthesis (43), the drug is
also incorporated into RNA (44) and inhibits RNA synthesis
at cytotoxic concentralions (43). In human hemalological ma
lignancies thai have a low growlh fraction, such as chronic
lymphocytic leukemia, these other RNA effects could also
contribule lo Ihe clinical efficacy of Fara-AMP. Further sludies
wilh chronic lymphocytic leukemia cells oblained from palienls
are planned in order to address the importance of Ihese possible
RNA effecls to Ihe aclivily of Fara-AMP in Ihis disease.
ACKNOWLEDGMENTS
The authors wish to acknowledge the generous gift of Fara-ATP
from Dr. William B. Parker of the Southern Research Institute, Bir
mingham, AL. We also thank Dr. Fred Ferrino of the Bowman Gray
School of Medicine for providing us with BuPdGTP, the SJK 132-20
monoclonal antibody to DNA polymerase «,and a sample of DNA
polymerase a, which was immunoaffinity purified from human Tleukemia cells.
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Inhibition of Primer RNA Formation in CCRF-CEM Leukemia
Cells by Fludarabine Triphosphate
Carlo V. Catapano, Kimberley B. Chandler and Daniel J. Fernandes
Cancer Res 1991;51:1829-1835.
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