Download Mechanisms of Inhibition of DNA Synthesis by 2

(CANCER RESEARCH 49. 6923-6928. December 15. I9S9]
Mechanisms of Inhibition of DNA Synthesis by 2-Chlorodeoxyadenosine
Lymphoblastic Cells1
in Human
Johannes Griffig, Rainer Koob, and Raymond L. Blakley2
Department oj Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101 [J. G„R. A'.. R. L. B.J, and Department of
Pharmacology, L'nirersity of Tennessee College of Medicine-Memphis, Memphis, Tennessee 38163 ¡R.L. B.J
the intracellular inhibition of ribonucleolide reduction in cells
exposed to CldAdo was not investigated. Hirota et al. (7)
showed that FM3A mouse mammary cells exposed to CldAdo
inhibitor of the reduction of ADP, CDP, UDP and GDP by ribonucleotide
for 8 h showed a decline in intracellular dATP and dGTP but
reducÃ-asein extracts of CCRF-CEM with 50% inhibition at concentra
not in dCTP or dTTP. Moreover, at concentralions near the
tions of 0.1 to 0.3 MM.In cells exposed to 0.3 UM2-chloro-2'-deoxyadcnosine (CldAdo), the intracellular concentration of CldATP reaches 2 MM IC50 for growth, dNTP pool changes were very small.
There is also growing evidence that CldTP direclly interwithin 15 min, and DNA synthesis by the cells is inhibited 90% within
feres wilh DNA synlhesis. Carson et al. (2) reporled thai CCRF30 min. At concentrations of extracellular CldAdo that inhibit DNA
synthesis, there is also marked inhibition of intracellular conversion of CEM cells exposed lo 0.2 MMCldAdo incorporate some of the
analogue inlo DNA over a 24-h period, but kinetics, concentra
cytidine to deoxycytidine nucleotides indicating significant intracellular
inhibition of ribonucleotide reducÃ-ase.Exposure of cells to 0.3 MMCldAdo
tion dependence, and relation to total DNA synthesis were not
decreases dCTP by 63% in 30 min, dATP and d FTP by 20%, and dGTP
investigaled. Parker et al. (9) found lhal DNA polymerases a,
0, and 7 are inhibiled by CldATP wilh apparent A'¡
values in
by a smaller amount. Similar decreases in these pools occur when other
ABSTRACT
2-Chloro-2'-deoxyadenosine
5'-triphosphate
(CldATP) ¡sa strong
inhibitors of ribonucleotide reducÃ-aseare present at concentrations caus
ing similar inhibition of DNA synthesis. Deoxycytidine treatment of cells
inhibited by CldAdo restores dCTP and other pools, but restoration of
DNA synthesis is incomplete, indicating that there is another mechanism
for inhibition of DNA synthesis in addition to depletion of deoxyribonucleotide pools. This alternate mechanism is probably related to the
incorporation of CldAdo into DNA that occurs despite a 25-times lower
intracellular level of CldATP than dATP.
INTRODUCTION
The 2-chloro- and 2-bromo- analogues of deoxyadenosine
are much more cytotoxic to T-lymphoblastic, B-lymphoblastic,
and myeloblastic cell lines than dAdo1 (1-5), due to their
resistance to adenosine deaminase (6). Cytotoxicity is closely
related to inhibition of DNA synthesis with little effect on RNA
or DNA synthesis (2, 4). Cells exposed to either analogue
accumulate in early S phase or, at higher drug concentration,
at the G,-S border (4, 7), suggesting a blockade of initiation of
DNA synthesis.
We originally predicted that these nucleoside analogues, after
intracellular conversion to analogues of dATP, might be potent
inhibitors of ribonucleotide reducÃ-ase(8). However, it is still
unclear to what degree intracellular ribonucleotide reduclion is
inhibited by cells exposed to CldAdo and whether there are
other mechanisms of cytotoxicity. Although Parker et al. (9)
reported that ADP reduction by crude extraéisof K562 cells is
50% inhibited by aboul 130 niviCldATP or BrdATP, reduction
of CDP, GDP, and UDP was noi invesligaled. Furthermore,
Received 5/22/89; revised 9/1/89; accepted 9/21/89.
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.
1Supported in part by USPHS Grant ROI CA 39242 from the National
Cancer Institute and by American Lebanese Syrian Associated Charities.
2 To whom requests for reprints should be addressed, at Department of
Biochemical and Clinical Pharmacology. St. Jude Children's Research Hospital.
332 North Lauderdale. P. O. Box 318. Memphis. TN 38101.
'The abbreviations used are: dAdo. deoxyadenosine; dGuo. deoxyguanosine;
dCyd. deoxycytidine; dUrd, deoxyuridine; dThd, thymidine: dNMP. deoxynucleotidc monophosphate; dNTP. deoxynucleotide triphosphate; CldAdo, 2chloro-2'-deoxyadcnosine; CldAMP, 2-chloro-2'-deoxyadenosine 5'-phosphate;
CldATP. 2-chloro-2'-deoxyadenosine
5'-triphosphate;
BrdAdo. 2-bromo-2'deoxyadenosine; BrdATP. 2-bromo-2'-deoxyadenosine 5'-triphosphatc; HEPES.
4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acid; HPLC. high-performance
liquid chromatography: PEI cellulose, polyethylenimine cellulose; ara-C. l-ji-Darabinofuranosylcytosinc; azido-C. 2'-azido-2'-deoxycytidine; EHNA. erythro-9(2-hydroxy-3-nonyl)adcnine; ICM. concentration for 50rc inhibition of growth.
the range 3 lo 21 MM.Our own invesligalions wilh defined
primers and lemplales indicate that CldATP interaclion wilh
DNA polymerases is complex even in ihe absence of olher
proleins,4 so that it is difficult to interpret apparent K, values
obtained in reactions using gapped genomic DNA as lemplate
and primer. It is difficult to assess on ihe basis of Ihese resulls
how much CldATP interference with the action of DNA polymerase contributes to CldAdo cylotoxicity for human lymphoblasts.
In the present study, we have endeavored to obtain evidence
of the relative imporlance in CldAdo aclion on cells of ribonu
cleolide reducÃ-aseinhibition versus interference with DNA rep
lication. Such information is of interest because CldATP pro
duces clinical responses in advanced chronic lymphocytic leu
kemia and auloimmune hemolylic anemia (10) and in pedialric
acute monocytic leukemia.5
MATERIALS
AND METHODS
Materials. (l/-14C]CDP (438 mCi/mmol), [methyl-'H]lkymidine (84
Ci/mmol), [i/-'4C]ADP (550 mCi/mmol), [8-'4C]ATP (51 mCi/mol),
[8-'4C]GTP (52 mCi/mmol), organic counting scintillant, and aqueous
counting scintillant were from Amersham. [8-'4C)GMP (60 mCi/
mmol), [2-14C]UMP (60 mCi/mmol), and [S-'HJCIdAdo (7 Ci/mmol)
were from Moravek Biochemicals.
ATP nucleoside monophosphate kinase, snake venom (Crotatm adamanteus), unlabeled nucleotides, proteinase K, DNase I, RNase A,
bacterial alkaline phosphatase and PEI cellulose were obtained from
Sigma. Guanosine monophosphate kinase was from Boehringer Mann
heim.
[8-'4C]GDP and |2-I4C]UDP were synthesized from the monophosphates by the method of Cory and Bacon (11) with purification of the
products by HPLC on a Whatman Partisi! 10 SAX column (4.6 x 250
mm), with gradient elution. Solvent A: 20 ITIMammonium phosphate,
pH 2.8; solvent B: 500 mw ammonium phosphate, pH 4.8. A linear
gradient from 25% B to 100% B over 25 min was used, with a flow
rate of 1 ml/min and detection by absorbance at 254 nm. Yields were:
[2-14C]UDP, 70%; [8-'"C]GDP, 90%.
Since '4C-labeled cytidine was not commercially available, it was
prepared by the action of snake venom (250 ug) on [Õ/-I4C)CDP(50
fiCi) in 100 ITIMTris-HCl, pH 8.5, containing 5 HIMmagnesium acetate
in a volume of 0.5 ml. After incubation at 37°Cfor 40 min, the product
4 P. Hentosh. R. Koob. and R. L. Blakley. unpublished results.
5 V. Santana and R. L. Blakley. unpublished results.
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INHIBITION
was isolated by reversed-phase HPLC
OF DNA SYNTHESIS BY CldAdo
on a Beckman Altex d» 5-nm
ODS column (4.6 x 250 mm) with water as an eluant. Elution times
were: CDP, 3 min; cytidine, 11 min.
Assay of Ribonucleotide ReducÃ-ase.All assay reaction mixtures con
tained 60 mM HEPES buffer (pH 7.2), 6 IHM magnesium acetate, 6
mM DTT, 4 mM NaF, and 0.1 mM Fed, and cell extract (0.7 mg of
protein), in a total volume of 180 ^1. In addition, reaction mixtures for
assays with specific substrates were supplemented as follows: for CDP
reduction, 5 mM ATP and 0.5 mM [t/-'4C]CDP; for UDP reduction, 5
IHM ATP and 0.5 mM [2-'4C]UDP; for ADP reduction, 0.5 mM [U-'4C]
ADP. 2 mM [8-14C)ATP, 0.2 mM dGTP, and 10 MM EHNA; for GDP
reduction, 0.5 mM |8-14qGDP, 2 mM [8-14C)GTP, 0.5 mM dTTP, and
2 mM ATP. The specific radioactivity of all labeled nucleotides was
1320 dpm/nmol.
The reactions were at 37°C and were started by
addition of cell extract and terminated after 30 min by heat (4 min,
95°C). In preliminary experiments to establish the suitability of these
assay conditions, concentrations of residual nucleotides at the end of
incubation were determined by HPLC on an SAX column as described
for preparation of labeled UDP and GDP except that the gradient was
Oto 100% B over 30 min.
To the cooled reaction mixtures were added 250 ¿ig
of carrier dNMP
corresponding to the substrate, and nucleotides were then dephosphorylated by addition of 250 iA of a solution containing snake venom (2.5
mg), Tris-HCI (pH 8.8; 40 ¿iinol),and magnesium acetate (1 ftmoi).
After incubation at 37"C for 90 min, the reaction was terminated by
heating at 95°Cfor 4 min, and precipitated material was removed in
the Eppendorf centrifuge. In the case of assays of ADP and GDP
reduction, relatively large amounts of purine bases were present in the
samples. These were separated from nucleosides by reversed-phase
HPLC on an Altex Ultrasphere C,»5-^m ODS column (4.6 x 250 mm)
with dimoii by a gradient formed from water (solvent A) and 80%
methanol (solvent B). A linear gradient from 0 to 30% B over 30 min
was used, with a flow rate of 1 ml/min and detection at 254 nm.
Guanine and adenine eluted with retention times of 11 and 18 min and
guanosine. dGuo, adenosine, and dAdo with times of 17, 19, 27, and
29 min, respectively. The effluent was collected in fractions of 1 ml,
and those in the nucleoside region containing radioactivity were pooled.
Boric acid was added to nucleoside samples to give a final concentra
tion of 25 mM, and deoxyribonucleosides were separated from ribonucleosides by chromatography on the borate form of PEI cellulose at
4°Caccording to Sato et al. (12) except that the bed size was 10x1
cm. dCyd, dUrd. and dAdo were eluted with 10 ml of 25 mM boric
and dGuo with 20 ml. Samples (1 ml) of eluate were counted,
counts were corrected for those obtained with reaction mixtures
taining no enzyme.
Growth of Cells and Preparation of Extracts. All experiments
performed with a tetraploid subline of CCRF-CEM cells.
acid
and
con
were
For the preparation of cell extracts for experiments on ribonucleotide
reducÃ-ase,cells were grown in roller bottles (Falcon 3027) in minimal
essential medium (Eagle) with L-glutamine and spinner salts (Hazelton
Research Products) supplemented with 25 mM NaHCOj, 10% newborn
calf serum, penicillin (100 units/ml) and streptomycin (1 mg/ml). Cells
were seeded at 2.5 x 105/ml and incubated at 37°Cunder 95% air-5%
CO2. Cell density was determined with a model ZB1 particle counter
(Coulter Electronics, Inc.) fitted with a C-1000 Coulter channelyzer.
Cells were harvested in log phase of growth at a density of about 7.5 x
lO'/ml. After centrifugaron at 4°C,cells from 3 liters of medium were
washed with cold 100 mM HEPES, pH 7.2, containing
threitol and 1 mM MgCl; and then suspended
of the same composition. The suspension was
sonication was performed with a type CS-75
periods of 10 s were used, interspersed with
2 mM dithio-
in 2 ml of cold solution
kept in an ice bath while
Branson Sonifier. Four
1-min cooling intervals.
The supernatant after centrifugation at 100,000 g was frozen rapidly in
liquid nitrogen and stored at —¿70°C.
was a modification of that of Garret and Santi (13). To a suspension
of log phase cells at a density of 3 x lOVml were added 0.05 volume
of 0.5 M HEPES buffer (pH 7.2) and [8-'H]CldAdo
(0.33 Ci/mmol) to
give a final concentration of 0.3 ^M. The culture (total volume. 1 liter)
was incubated in a Bélicospinner flask in a water bath at 37°C.After
various time intervals,
two 35-ml samples were withdrawn
and har
vested by centrifugation for 5 min at 600 g. and the tubes containing
the pellet were chilled on ice. Each pellet was dispersed in 325 /tl of
ice-cold 0.5 M HC1O4, and the suspension was transferred to an Eppen
dorf centrifuge tube and kept on ice for 10 min. After pelleting in a
centrifuge in a cold room (4°C),each supernatant was transferred to a
clean Eppendorf tube, and perchloric acid was extracted by extensive
vortexing with 325 n\ of 0.5 M tri-n-octylamine
in 1.1,2-trichlorotrifluoroethane. After centrifugation. the upper (aqueous) layer was trans
ferred to a clean tube and treated for oxidative degradation of ribonucleotides. To the chilled extract were added 10 ^1 of 0.5 M NaIO4,
the mixture was kept on ice for 2 min. Methylamine (12.5 p\ of
concentration, slowly adjusted to pH 7.5 with phosphoric acid)
added, and the mixture was incubated for 30 min at 37°C.Finally,
and
4 M
was
2.5
p\ of l M rhamnose were added to remove excess periodate, and the
samples were immediately put on ice or stored frozen at -20°C.
Concentrations of dNTPs were determined by HPLC chromatography
on a Whatman Partisi! 10 SAX column (4.6 x 250 mm) with isocratic
elution by 0.3 M ammonium phosphate, pH 3.2, contained 8.6%
acetumi rile. The flow rate was 2 ml/min, and detection was by absorbance at 270 nm. Retention times for dCTP, dTTP, dATP, CldATO,
and dGTP were 15, 21, 26, 40, and 45 min, respectively. Peaks from
the elution profile were integrated by a cut and weigh procedure, which
was found to give results that were more reproducible and exhibited
better proportionality
between measured area and nucleotide amount
than with electronic integration. Peak areas were converted to amounts
of nucleotide by comparison with peak areas of 0.6-nmol samples
chromatographed under the same conditions. Effluent from the column
was collected in 1-ml fractions, and the amount of CldATP was deter
mined by scintillation
counting of fractions in a peak with similar
elution time to authentic CldATP. Intracellular concentrations were
calculated from the amount of nucleotide in the sample chromato
graphed, the number of cells extracted, and the cell volume (obtained
from the Coulter channelyzer). The mean cell volume for these cultures
was 1420fl.
Incorporation of Labeled Thymidine and CldAdo into DNA. The cell
suspension was the same as that used in the dNTP pool study. At
intervals, duplicate 1-ml samples of suspension were withdrawn for
measurement of incorporation
of [8-'H]CldAdo
into DNA. Before
addition of [8-'H]CldAdo
to the culture, immediately after its addition,
and 30 min, 2 h, and 4 h later, duplicate 1-ml samples were removed
for 60-min incubation with 0.5 ^M [me//i>7-'H]thymidine
(0.2 Ci/mmol)
at 37°C.These samples were used for estimation of the rate of thymidine
incorporation. The cells were collected from each 1-ml sample of both
series by centrifugation, washed twice with ice-cold 50 mM Tris-HCI,
pH 7.2. containing 100 mM NaCl (Tris-saline), and extracted overnight
with 60% methanol at -20°C. The suspension from each sample was
centrifuged,
the pellet was washed with ice-cold 0.3 M HC1O4 and
suspended in water (1 ml), and the suspension was made slightly
alkaline with a few microliters of 0.5 N NaOH in order to dissolve the
pellet. After addition of 10 ^1 of calf thymus DNA solution (2 mg/ml)
as a carrier, the nucleic acid was precipitated by addition of 250 ¿jlof
ice-cold 50% trichloroacetic acid containing 0.5 M sodium pyrophosphate, collected on a Whatman GF/C filter, washed with 20 ml of icecold 5% trichloroacetic acid containing 0.1 M sodium pyrophosphate
and with 5 ml absolute ethanol, and air dried. The material on the filter
was dissolved by heating with 2 ml of NCS tissue solubilizer (Amersham) and 200 /¿Iof water at 50°C for 20 min and counted in a
For other experiments, cells were grown in 750-ml flasks (Falcon
3028) in RPMI 1640 medium supplemented with 2 mM i.-glutamine,
scintillation counter after addition of 200 f. I of acetic acid and 10 ml
of organic counting scintillant (Amersham).
For calculation of the amount of [mefA^V-'Hlthymidine incorporated,
10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (1
mg/ml). Cells were harvested in log phase at a density of 3 to 6 x IO5/
total counts in the DNA samples were first corrected for the relatively
small amount of counts due to [8-'H]CldAdo incorporation. In addition,
ml.
Measurement of Pools of dNTPs and of CldATP. The method used
the specific radioactivity of the dTTP pool rather than of added thy
midine was used for calculation of incorporation. For this purpose. 35-
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INHIBITION OF DNA SYNTHESIS BY CldAdo
ml portions of cell suspension were removed at the same time as samples
for measurement of incorporation and incubated with 0.5 MM[methyl'Hjthymidine (0.2 Ci/mmol) for 60 min at 37°C.Cells were washed
and extracted and intracellular dTTP was measured as in the dNTP
pool part of the study. The effluent from the HPLC column was
collected in 1-ml fractions, and radioactivity in the dTTP fractions was
determined.
Incorporation of CldAdo Exclusively into DNA as CldAMP. Cells
were incubated with 0.3 MMCldAdo as in the above experiments, and
macromolecular components were prepared from cell samples. Proteins
and RNA were hydrolyzed enzymatically, and DNA was reprecipitated
with acid. A sample of the DNA was used for determination of radio
activity, and the remainder was hydrolyzed with DNase I, snake venom
phosphodiesterase, and bacterial alkaline phosphatase according to the
method of Spriggs et al. (14). Deoxyribonucleotides were separated by
reversed-phase HPLC on an Altex Ultrasphere Ci»ODS 5-Mmcolumn
(4.6 x 250 mm) with isocratic elution with 50 m\t potassium phosphate
buffer, pH 2.85. Retention times for dCyd, dGuo, dThd. dAdo, and
CldAdo were: 7, 13, 14, 20, and 29 min. Effluent from the column was
collected in 1-ml fractions, and radioactivity was determined.
Conversion of [i/-'4C|Cytidine lo Deoxyribonucleotides and Its Incor
poration into DNA. Incubations of cells were performed as previously
described. Drugs at the indicated concentrations were added to each 3ml culture at the beginning of the incubation at 37°C.After 3 h, \U'4C]cytidine (438 mCi/mmol) was added to a final concentration of 0.5
MM,and incubation continued for 1 h. Cells were washed twice with
Tris-saline and extracted overnight with 1 ml of 60/¿methanol at
—¿20°C.
The suspension was centrifuged, and the pellet was used for
determination of incorporation into DNA as previously described. The
methanolic supernatant was evaporated to dryness. and nucleotides in
the residue were dephosphorylated by dissolving in 200 M!of a solution
of 50 mM Tris-HCI, pH 8.5, containing 5 mM magnesium acetate. 5
mM dCMP, and 4 mg of snake venom. The mixture was incubated at
37°Cfor 1 h before inactivating the venom by boiling for 4 min. After
addition of 800 M' of 25 niM boric acid, deoxyribonucleosides were
separated from ribonucleosides on PEI cellulose as described, and
radioactivity was determined in ACS.
HPLC. All analyses involving this technique were performed with a
system consisting of two Beckman 110A pumps, an Altex 420 solvent
programmer, an LKB 2151 variable wavelength detector, and a HewlettPackard 3390A recording integrator.
In order to avoid changes in specific radioaclivily of ihe sub
strate, [8-'4C]ATP was used in the assay of ADP reduction and
[8-'4C]GTP in ihe assay of GDP reduclion. The specific radioactivity of the Iriphosphale was adjusted to that of the diphosphate (1320 dpm/nmol). Under these conditions, the rate of
reduction of each of the substrates was constant through the
30-min reaction period. l4C-labeled substrates were used in all
cases because 'H-labeled substrates gave high blanks in noenzyme controls.
ICso values for inhibition of each of the four substrales by
CldATP and BrdATP fell wilhin a narrow range (0.11 lo 0.28
UM) and were about 100 times lower lhan values for inhibition
by dATP in the presence of EHNA (Table 1).
Comparison of the Inhibition of Ribonucleotide Reduction and
of DNA Synthesis. A melhod used by Äkerblom and Reichard
(16) lo examine whelher inhibilion of DNA synlhesis occurs by
way of ribonucleolide reducÃ-aseinhibition was adaplcd to our
system. We compared the relative effects of four inhibilors on
cytidine conversion to dCyd nucleotides and its incorporation
into DNA. The inhibitors chosen were: ara-C, which acts pri
marily on DNA polymerase (17); hydroxyurea and azido-C,
which are inhibitors of ribonucleotide reducÃ-ase(16, 18-20);
and CldAdo. The results (Fig. 1) are clearcul for ara-C where
ihe concenlralion lo inhibil dCyd nucleolide formation is 100
limes higher lhan for inhibilion of incorporation inlo DNA.
Concenlralions of Ihe olher compounds causing inhibilion of
dCyd nucleolide formation were severalfold higher than re
quired for inhibition of DNA synthesis. Il is apparenl that
CldAdo, as well as azido-C and hydroxyurea, markedly inhibits
Table I Inhibition hy ( IdATP. HrdATP. and dATP ofrihonucleotidc reducÃ-asein
cell extracts ofCCRF-CEM cells
The rates of reduction (pmol/min/mg) in uninhibited controls were: ADI', 23:
GDP. 50; CDP, 35; UDP. 13.
(n\JCldATP0.18
SubstrateADP
(+10 /IM
EHNA)35
GDP
O.M
CDP
0.28
UDPBrdATP0.25 0.20IC50
RESULTS
0.11
0.28
0.16I)dATP
55
45
10
Inhibition of Ribonucleotide Reduction by CldATP. Unfraclionaled exiracls from CCRF-CEM cells were used for ihese
sludies. A valid assay requires lhat the concentrations of nucleolide subslrales and aclivalors remain above iheir A'mvalues
ihroughoul ihe reaclion period (30 min). Nucleolide concentra
tions remaining after incubation under assay conditions were
determined by HPLC on an SAX column, and ihe final specific
activity of radiolabeled nucleotides was also determined. When
0.5 HIMCDP or UDP was incubated in the presence of 5 m,\i
ATP (required as aclivator), ihe final concenlralion of ihe
subslrale was 143 and 167 MM,respectively, which is satisfaclory
since A',,,values reporled for ihe Moll 4F reducÃ-aseare 7 and
50 MM.respectively (15).
In the case of ADP and GDP reduclion, ATP is not needed
for activation bul is necessary for regeneralion of substrate,
since without its addilion ADP was 90% depleled in 5 min and
GDP was 98% depleled in 30 min. When 0.5 mM (t/-'4C]ADP
was incubaled in a reaclion mixlure conlaining 2 m\i ATP and
10 MMEHNA (included lo inhibil adenosine deaminase), Ihe
ADP concentration after 30 min was 395 MM.compared with a
A"nlof 80 MMfor the Molt 4F reducÃ-ase.Similarly, afler incubalion of 0.5 niM [8-'4C]GDP for 30 min in a reaclion mixlure
containing 2 mM ATP and 2 mM GTP, and GDP level was still
230 MM.compared with a A'mof 33 MMfor Molt 4F reducÃ-ase.
Extracellular Inhibitor Concentration
Fig. I. Inhibition of intracellular ribonucleotide reduction and DNA synthesis
by various drugs. CCRF-CEM cells were incubated in the presence of various
concentrations of CldAdo (A). azido-C' (B). hydroxyurea ((') or ara-C (¡))for 3
h. After addition of 0.5 ^M [i"-14C]cytidine. cell suspensions were incubated at
37°Cfor an additional hour. Conversion of cytidine to deoxyribonucleotides (•)
and its incorporation into DNA (A) were then determined as described in
"Materials and Methods."
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INHIBITION
OF DNA SYNTHESIS BY CldAdo
intracellular ribonucleotide reduction at concentrations that
inhibit DNA synthesis.
Changes in dNTP Pools in Cells Treated with CldAdo, Hy
droxyurea, or Azido-C. Intracellular effects of the inhibition of
23.
ribonucleotide reducÃ-aseare reflected to some extent by dNTP
pools in cells exposed to inhibitors (Fig. 2). Cells were incubated
in the presence of 0.3 p\t CldAdo, a concentration that reduces
•¿O
clonogenicity to about 60% of controls in 4 h and to about 10%
"o
in 18 h (4). Intracellular CldATP rose to 2 ßM
in 15 min and
by 4 h had reached 4 ¡¡M
(Fig. 2A). dNTP pools were decreased
e 60
modestly by CldAdo within 30 min, and thereafter the response
o
did not greatly increase. dCTP decreased 63% (from 16 MMto
40
o
C
6 n\i), but for dATP and dTTP the decrease was about 20%
o
Ü
and for dGTP it was still less.
20
Azido-C and hydroxyurea act primarily or solely by inhibition
of ribonucleotide reducÃ-ase( 16,18-20). At concenlralions caus
ing 90% inhibilion of ihymidine incorporalion inlo DNA by
1234
cells exposed lo ihe drugs for 4 h, ihe major effecl was also lo
Incubation Time (hr)
decrease dCTP (Fig. 3). The olher dNTP pools also decreased
Fig. 3. Intracellular concentration of dNTPs in CCRF-CEM cells treated with
(excepl for dTTP in ihe presence of hydroxyurea) bui lo a lesser hydroxyurea or azido-C. Cell suspension were incubated with 0.5 M hydroxyurea
exlenl and with higher minimum concenlralions. The drop in (A) or 20 n\t azido-C (B) at 37"C. At various time intervals, duplicate samples
Ihe dCTP pool was similar lo lhal caused by 0.3 //M CldAdo in of the suspension were withdrawn, and cell extracts were subjected to HPLC for
the determination of dATP (•).dTTP (D), dGTP (A), and dCTP (A).
ihe case of 0.5 HIMhydroxyurea, but more profound in the case
of 20 n\i azido-C.
500
Inhibition of DNA Synlhesis. The effecls of various concentrations of CldAdo on DNA synthesis closely parallel those on
growth and viability (4). Exposure of cells to 0.3 //M CldAdo
caused thymidine incorporation into DNA lo drop very rapidly,
falling lo a mean of 10% of ihe uninhibited value over ihe firsl
30 min (Fig. 4). Thereafter, inhibilion remained al 95%. Thus,
Ihe inhibilion of ribonucleotide reduclion and Ihe consequenl
changes in dNTP pools were associated with a rapid and
profound inhibilion of DNA synthesis.
Chlorodeoxyadenosine Incorporation into DNA. The resulls
00
1234
in Fig. 2 indicale lhal for most of ihe incubalion period ihe
Incubation Time (hr) with CldAdo
inlracellular concenlralion of CldATP is only aboul 3 ¿IM,
Fig. 4. Incorporation of [8-'H]CldAdo into DNA and inhibition of DNA
whereas dATP is presenl al about 50 JJM.Despite this unfavorsynthesis by CldAdo. At various time intervals, samples were withdrawn from the
same cell suspension containing 0.3 >i\t |8-'H]CldAdo described in Fig. 2 for
measurement of CldAdo incorporalion into DNA (O). and for incubation with
[mtV/i.v/-'H]lhymidinc for I h at 37°Cfor measurement of DNA synthesis (A). In
the second experiment. 0.5 n\\ dCyd was added to the suspension after I h of
incubation with CldAdo. and CldAdo incorporation into DNA (•)and DNA
synthesis (A) were subsequently determined. DNA synthesis resulls are plotted at
the midpoint of each 60-min period of incubation with ('Hlthymidine.
able ratio, CldATP was incorporaled inlo DNA al a significant
and constanl rale ihroughoul the incubation period (Fig. 4).
The level of this incorporalion, 3.2 pmol/h/106 cells, is still
much less lhan ihe inhibited rate of thymidine incorporation
(40 lo 60 pmol/h/106 cells), and in fact, the ratio of these rates
approaches the ralio of the intracellular concenlralions of
CldATP and dATP.
When [S-'HJCldAdo is incubaled wilh cells and Ihe cell
01234
Incubation Time (hr) with CldAdo
Fig. 2. Inlracellular concenlralions of dNTPs and of CldATP in CCRF-CEM
cells treated with CldAdo. A. cell suspension was incubated with 0.3 JJM|8-'H|CldAdo at 37°C.At various time intervals, duplicate samples of the suspension
were withdrawn, and cell extracts were subjected to HPLC for the determination
of dATP (•).dTTP (D). dGTP (A). dCTP (A), and CldATP (O). B, 0.5 MM
deoxycytidine was added to the medium at the time indicated.
exlracls are subsequenlly subjecled to HPLC in a system thai
separates Ihe nucleoside and Ihe mono-, di-, and Iriphosphate,
the only major radioactive peaks appearing in ihe citiate cor
respond to these species (5). It seems highly probable, iherefore,
lhat CldAdo is incorporated into DNA withoul loss of the
chlorine or other melabolic modificalions. To confirm this, we
harvested cells that had been incubated with [8'H]CldAdo,
isolated macromolecular components, digested RNA and prolein, and reprecipitaled the DNA. Since nearly all of the counts
remained in the DNA, there was no significant labeling of
RNA. By enzymatic hydrolysis of the DNA to deoxyribonucleosides and separation of the latter by HPLC, it could be
6926
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INHIBITION OF DNA SYNTHESIS BY CïdAdo
demonstrated that all of the radioactivity appeared in a peak
with the same elution time as CldAdo, well separated from the
normal deoxyribonucleosides.
Effect of Deoxycytidine on dNTP Pools and Inhibition of DNA
Synthesis. Deoxycytidine specifically relieves the inhibition of
the growth of CCRF-CEM cells by CldAdo,6 whereas a com
bination of similar concentrations of dAdo, dGuo, and dThd
has no significant effect. When 0.5 UMdCyd was added to the
medium of cells incubated with 0.3 MM[8-'H]CldAdo for l h
under the conditions in Figs. 2 and 3, the intracellular concen
tration of dCTP rapidly increased, and after a further hour had
returned to control levels (Fig. 2B). The dTTP pool also re
turned to control levels, but the dGTP level did not regain its
small loss, and dATP continued to decline slightly.
Despite the fact that, within l h of dCyd addition, dCTP,
dTTP, and dGTP pools were close to control levels and dATP
was 80% of control, DNA synthesis levels had only returned to
40% of control after 2 h of incubation with dCyd and did not
increase any further (Fig. 3). It is also important to note that
the presence of dCyd caused a marked and rapid decrease in
intracellular CldATP to 25% or less of the level attained in the
absence of dCyd (Fig. 2B). This drop in CldATP was accom
panied by a marked decrease in the rate of its incorporation
into DNA (Fig. 4).
DISCUSSION
Inhibition of Ribonucleotide ReducÃ-aseby CldATP. Since in
our hands the published procedures for purification of ribonucleotide reducÃ-ase(21-23) did not result in significant purifi
cation of enzyme from CCRF-CEM cells, we used unfractionated cell extracts. We established assay conditions for these
extracts such that concentrations of substrates and activators
remained high relative to reported Kmvalues, the specific activ
ity of substrates remained constant, and the rate of reduction
remained constant through the incubation period (30 min).
Such conditions were not established in some earlier work with
crude extracts. With this system, the rate of CDP reduction
was 35 pmol/min/mg protein in the CCRF-CEM cell extracts.
This is higher than CDP reducÃ-aseactivily (convened lo Ihe
same unils) reporled by olhers for cell extraéiseven from similar
sources: Moll 4F, 2 (23); calf Ihymus, 7 (21); Ehrlich ascites, 5
(22); Novikoff rat lumor, 22 (24); and rabbil bone marrow, 1.3
(25). The use of the unfractionaled cell exlracl in ihis assay
syslem improved Ihe likelihood thai any relevanl regulatory
proteins would be retained in the system. In this assay system,
BrdATP and CldAdo have similar IC50 values (Table 1), in
contrasl lo Ihe observation thai BrdAdo is less loxic than
CldAdo in vivo (4). Inhibition is similar for all four substrates
with either BrdATP or CldATP and is 50- lo 500-fold grealer
than for dATP in the presence of the mÃ-enosmedeaminase
inhibitor EHNA. The IC5(, values for CldATP (0.11 to 0.28
n\i) are only aboul 10% of Ihe inlracellular concenlralion of
Ihis nucleolide found in cells Ireated with 0.3 ¿¿M
CldAdo (Fig.
2). Similar inhibition of ADP reduction in crude extracts of
K562 cells by CldATP and BrdATP has been reported by Parker
et al. (9).
Ribonucleolide Reductase Inhibition in Inlacl Cells. In cells
exposed for 3 h to 0.05 n\i CldAdo, DNA synthesis, as meas
ured by cytidine incorporation inlo DNA via CMP, CDP,
dCDP, and dCTP, was inhibiled 90% (Fig. 1). This irealmenl
also caused 70% inhibilion of ribonucleolide reducÃ-ase (as
judged by cytidine conversion to dCyd nucleotides). This resull
is consislenl with the view thai inhibilion of the reducÃ-aseat
least contribules lo inhibilion of DNA synlhesis. Similar resulls
were obtained wilh equiloxic concentrations of azido-C and
hydroxyurea. However, in the case of ara-C, inhibilion of DNA
synlhesis occurred at a concentralion 100 times lower lhan lhat
required to inhibit ribonucleolide reduclion, as expecled in view
of Ihe faci lhal inlerference with DNA polymerase by incorporalion of ara-C inlo DNA is the primary mechanism by which
ara-CTP interrupls DNA synlhesis (26). Nevertheless, in cells
exposed to 0.3 ^M CldAdo for various periods, the decrease in
dNTP pools was rapid but small, except in Ihe case of dCTP.
Hirota et al. (7) found that in FM3A mouse mammary cells Ihe
greatest drop was in the dATP pool, followed by dGTP, and
Ihere was no effect on dCTP. Because of the small and unequal
drop in dNTP pools produced by CldATP, we examined the
effect of Iwo other inhibitors of ribonucleotide reducÃ-ase, hy
droxyurea and azido-C. At concenlralions producing Ihe same
inhibilion of DNA synlhesis as 0.3 ¿IM
CldAdo (90% after 4 h),
Ihe effecl on pools (Fig. 3) was similar. These resulls for
hydroxyurea and azido-C are similar lo Ihose of Akerblom and
Reichard with 3T6 cells (16) except thai in Ihe laller sludy Ihe
decrease in dATP was much grealer, and higher concentrations
of inhibitors were used.
The unequal changes in pool size produced by inhibitors of
ribonucleotide reducÃ-asein our study and those of others (7,
16) are apparently inconsistenl wilh Ihe observed similar inhi
bilion of Ihe reduclion of all four subslrates in cell extraéis.
However, lighl conlrol of relalive pool sizes by regulalory
mechanisms probably accounls for Ihe unequal effecls on differenl pools seen in Figs. 2 and 3. Since pool sizes represenl
Ihe balance belween synlhesis and ulilizalion, decreased ulilizalion due to inhibition of DNA synthesis explains the small
decreases in pool size despile significanl reducÃ-aseinhibilion.
The similar effecls on dTNP pools produced by azido-C, hy
droxyurea, and CldAdo are a slrong argumenl lhal Ihe latter
does produce intracellular inhibition of ribonucleolide reduc
Ã-ase.
Addition of dCyd to the medium of cells exposed to CldAdo
restores dTTP and dCTP pools to normal levels and decreases
CldATP by 70% (Fig. 2) but reslores DNA synlhesis lo only
40% of ils normal level (Fig. 4). This suggesls lhal CldATP
can inlerfere wilh DNA synlhesis by anolher mechanism in
addilion lo deplelion of dNTP pools. This mechanism is noi
classical inhibilion of DNA polymerase (for example, compelilive wilh dATP) bul is more likely relaled lo Ihe incorporation
of CldATP into DNA at a constant rate about one-twenly-fiflh
that of dThd incorporation. This incorporalion persisls even in
the presence of dCyd although al a rale decreased by a faclor
of 3. Such incorporation of CldAdo incorporation of CldAdo
into DNA by human DNA polymerases causes major disruplion
of DNA synlhesis4 and al leasl partly accounts for accumulation
of DNA strand breaks (7, 27).
ACKNOWLEDGMENTS
We thank Vicki Gray for skillful typing of the manuscript.
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INHIBITION OF DNA SYNTHESIS BY CldAdo
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Mechanisms of Inhibition of DNA Synthesis by
2-Chlorodeoxyadenosine in Human Lymphoblastic Cells
Johannes Griffig, Rainer Koob and Raymond L. Blakley
Cancer Res 1989;49:6923-6928.
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