[CANCER RESEARCH 46, 6165-6168, December 1986]
Differential Turnover of the Subunits of Ribonucleotide ReducÃ-asein Synchronized
Leukemia LI 210 Cells1
Eric H. Rubin and Joseph G. Cory2
Department of Biochemistry, University of South Florida College of Medicine, Tampa, Florida 33612
molecular weight of 41,000) subunit allhough no measuremenls
were made of the "larger" subunil. Cory and Fleischer (13),
ABSTRACT
Ribonuleotide reducÃ-asecatalyzes the rate-limiting step in the de novo
synthesis of 2'-deoxyribonucleoside 5'-t r¡phosphatesthat is required for
DNA replication. The mammalian enzyme consists of two nonidentical
protein subunits that are both required for enzyme activity. In leukemia
1.1210 cells, enriched in Ci-phase cells by centrifugal elutriation, it was
found that ribonucleotide reducÃ-aseactivity increased as the cells pro
gressed to S-phase. The two subunits making up the holoenzyme did not
increase coordinate!)'. The nonheme iron subunit increased much more
rapidly than the effector-binding (EB) subunit. The activity of the holo
enzyme paralleled the level of the EB subunit, which was limiting. The
half-lives of the holoenzyme and its subunits were determined in S-phase
cells by treatment with cycloheximide. The half-lives of the holoenzyme
and the nonheme iron and EB subunits, as determined by enzyme activity,
were 3.5, 7.6, and 4 h, respectively. These half-lives are consistent with
the data that indicate that the EB subunit is the limiting component in
LI210 cells.
INTRODUCTION
Ribonucleotide reducÃ-ase(EC 1.17.4.1 ) catalyzes the reaction
in which 2'-deoxyribonucleoside 5'-diphosphates are generated
by the direct reduction of the corresponding ribonucleoside 5'diphosphates. This reaction is the rate-limiting step in the de
novo synthesis of the 2'-deoxyribonucleoside
5'-triphosphates
required for DNA replication. The mammalian enzyme consists
of two nonidentical protein subunits that are both required for
enzymatic activity (1, 2). One of these subunits contains a
nonheme iron and free radical and the other subunit contains
the effector-binding site(s). The enzyme is strongly regulated
by nucleoside 5'-triphosphates (2) and nucleoside 5'-diphosphates (3), which are obligatory positive effectors and can also
serve as negative effectors.The enzyme activity has been shown
to be cell cycle dependent (4-6) and to be highest in tumors
with the greatest proliferation rate (7, 8).
There is general agreement that ribonucleotide reducÃ-ase
activily is cell cycle dependent Several groups have reponed
lhal Ihe Iwo subunils making up Ihe holoenzyme are noi
coordinalely altered during Ihe rise and fall of reducÃ-aseactivity
during the cell cycle (9-14). There is, however, published disagreemenl as lo which subunil is Ihe controlling or limiting
componenl for enzyme activity. Eriksson and Martin (9), Er
iksson et al. (10), and Engstrom et al. (11) report that, in S49
mouse lymphoma cells, mouse mammary lumor TA3 cells, and
bovine kidney MDBK cells, the level of ribonucleotide reducÃ-ase
aclivity is controlled by the level of the nonheme iron (M2)
subunit, and that the level of the effector-binding (Ml) subunit
is not changed during the cell cycle. Standard et al. (12) report
thai clam and sea urchin ribonucleolide reducÃ-ase aclivily is
relaled to the synthesis of the nonheme iron (a protein with a
Received 3/20/86; revised 7/24/86; accepted 8/21/86
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 CA 27398 from the USPHS, National
Cancer Institute.
2To whom requests for reprints should be addressed, at Department of
Biochemistry, University of South Florida College of Medicine, Box 7, 12901
North 30th Street, Tampa, FL 33612.
Youdale et al. (14), and Youdale et al. (15) reporl lhal, in
Ehrlich tumor cells (13) and regeneraling ral liver (13-15), Ihe
level of ribonucleolide reducÃ-aseactivily is dependent on the
limiting level of the effeclor-binding (Ml) subunil as Ihe non
heme iron subunil is in excess. In Chinese hamsler ovary cells,
Ihe level of Ihe nonheme iron componenl was also reported lo
be in excess (16). On Ihe olher hand, Alberi and Gudas (17)
reporl lhal in S49 cells Ihe increase in ribonucleolide reducÃ-ase
seen as the cells transverse the cell cycle was not due to increased
levels of either subunit but due to changes in the allosteric
regulation of the enzyme.
In ihis reporl, we present data obtained from leukemia LI 210
cells in which the levels of the two ribonucleotide reducÃ-ase
subunits were determined as the cells passed from G! to Sphase of the cell cycle and correlated with the level of ribonu
cleolide reducÃ-aseaclivity. Further, the half-life of each com
ponent was determined in S-phase LI210 cells to which cyclo
heximide was added.
MATERIALS
AND METHODS
Growth of LI 210 Cells. The L1210 cells were grown in culture in
RPMI 1640 culture medium, supplemented with 10% horse serum,
sodium bicarbonate (2 g/liter), and gentamicin sulfate (50 mg/liter).
The cells were grown at 37°C.
Elutriation of I 1210 Cells. The cells were separated by centrifugal
elutriation using the Beckman elutriation rotor. The method was essen
tially that of Mitchell and Tupper (18). The rotor speed was maintained
at 2200 rpm and the flow rate of the medium was varied. The eluant
was passed through an Isco absorbance monitor at 340 nm to follow
the elution of the cells from the rotor. The initial flow rate was 10 ml/
min. Minimum essential medium culture medium was used during the
elutriations. The flow rate was increased by increments of 2 ml/min
and was changed only after the cell peak was completely eluted at the
particular flow rate. The Gìcells eluted at a flow rate of 12 ml/min.
The S-phase cells were eluted at flow rates between 18 and 30 ml/min.
For these experiments, four or five separate elutriations had to be
carried out to obtain sufficient GI or S-phase cells for the particular
experiments. The collection of either the GI or S-phase cell fractions
was completed within 3 h of the multiple elutions. The cells were kept
on ice during this period. The cells were collected by centrifugation and
resuspended at a density of 1 x IO6cells/ml. Cell counts were made in
a model ZBI Coulter Counter.
Cell Cycle Analysis. Cell cycle analysis of the cell fractions from the
elutriation runs were made on a Coulter EPICS. The cells were treated
with RNase in sodium citrate and the DNA stained with propidium
iodide according to the method of Vindelov (19).
Preparation of Cell-free Extracts. After the particular incubation
period, the cells were collected by centrifugation. The cells were resus
pended in phosphate-buffered saline and recentrifuged. The cell pellet
(approximately 1 x IO8 cells) was homogenized (20 strokes) in 1.0 ml
of 0.02 M Tris-HCl, pH 7.0, containing 1 mM DTE.3 A motor-driven
Teflon pestle-glass homogenizer, immersed in ice, was used for the
homogenization step. The homogenate was centrifuged at 27,000 x g
for 50 min. The supernatant fluid was passed over Dowex-1-acetate
columns (0.9 ml packed to volume) in the cold and columns washed
3The abbreviation used is: DTE, dithioerythritol.
6165
Downloaded from cancerres.aacrjournals.org on April 15, 2017. © 1986 American Association for Cancer Research.
TURNOVER
OF RIBONUCLEOTIDE
REDUCÕASE
with an additional 1.0 ml of Tris-HCl buffer, pH 7.0, containing 1 mM
DTE. This step removed the endogenous nucleotides from the extract.
The cell-free extracts were quick frozen in an acetone-dry ice bath and
stored at -90°C.This extract was used for the measurement of enzyme
(Grand Island, NY). The unlabeled nucleotides and other biochemicals
were purchased from Sigma Chemical Company (St. Louis, MO).
activity.
Incubation of GI Cells. The L1210 cells in the d
incubated in RPMI 1640 culture media with 10% horse
bicarbonate, and gentamicin sulfate. The culture flasks
an initial cell concentration of 1 x IO6 cells/ml. There
RESULTS
fraction were
serum, sodium
were set up at
were triplicate
flasks for each time point. At various timed intervals, cells were col
lected by centrifugation and cell-free extracts prepared as described.
Incubation of S-Phase Cells. The L1210 cells (1 x IO6cells/ml) were
incubated in RPMI 1640 culture medium containing horse serum,
sodium bicarbonate, and gentamicin sulfate. Cycloheximide was added
at a concentration of 10 ^g/ml. Cells were collected at regular timed
intervals and cell-free extracts prepared as indicated previously. Tripli
cate flasks were set up for each time point.
I h>inidim- and Leucine Incorporation Studies. 1,1210 cells from the
fractions eluting at the various flow rates were collected by centrifuga
tion and resuspended in complete RPMI 1640 culture medium at 1 x
IO6 cells/ml. [3H]Thymidine (6.25 MCi/flask, 50 Ci/mmol) was added
to each flask and the cells incubated for 30 min at 37°C.The cell pellet
was collected by centrifugation, homogenized in 6% perchloric acid (1
ml), and centrifuged. The insoluble pellet was extracted two additional
times with 1 ml of 6% perchloric acid each time. The DNA pellets were
solubilized in 0.1 M NaOH (1 ml) and aliquots taken for radioactivity
measurements.
LI210 cells in log phase growth were incubated at 37°Cin the
presence of various concentrations of cycloheximide for 2 h. [3H]Leucine (0.2 /¿Ci/ml,140 Ci/mmol) was added to each flask and the
cells incubated an additional 30 min. The cell pellet was extracted with
6% perchloric acid (1 ml x 3). The protein pellets were solubilized in
0.1 M NaOH (1 ml) and aliquots taken for radioactivity measurements.
Preparation of Nonheme Iron and Effector-Binding Subunits. The
nonheme iron and effector-binding subunits were prepared from the
ammonium sulfate fraction of Ehrlich tumor cells (20) by chromatography on blue dextran-Sepharose (21). The nonheme iron subunit was
eluted with Tris-HCl, 0.02 M, containing 1 mM DTE, while the effectorbinding subunit was eluted with 0.25 M NaCl in 0.02 M Tris-HCl,
containing 1 mM DTE.
The nonheme iron subunit was further purified by chromatography
on Whatman DEAE-52-cellulose. The fraction from the blue-dextran
column was loaded onto a DEAE-cellulose column (1.5 x 12 cm) which
had been equilibrated with 0.02 M Tris-HCl containing 1 mM DTE.
After the protein peak was washed off with the 0.02 M Tris-HCl, the
buffer was changed to 0.09 mM Tris-HCl, containing 1 mM DTE, to
elute the active fraction. This was concentrated by ultrafiltration on an
Amicon PM-10 membrane in a stirring cell. The concentrate was
dialyzed against 0.02 M Tris-HCl with 1 mM DTE and then quick
frozen (acetone-dry ice) in small aliquots and stored at —90°C.
These fractions of the nonheme iron and effector-binding subunits
did not contain any nucleotidases or nucleotide kinases that would alter
either the substrate or nucleotide effector concentrations as determined
by high-performance liquid chromatography.
Assay of CDP ReducÃ-aseActivity. CDP reduction was measured by
the procedure of Steeper and Steuart (22) using Dowex-1-borate col
umns. The assay mixture contained in a final volume of 0.15 ml: [14C]CDP (0.10 /¿Ci,0.05 mM); DTE (1 mM); magnesium acetate (4 HIM);
sodium phosphate (16 mM); enzyme extract (75 n\, 3-4 mg/ml); and
exogenous nonheme iron or effector-binding components as indicated.
In place of ATP, 5'-adenylylimido-diphosphate,
1 mM, was used as
positive effector of CDP reducÃ-aseactivity. All assays were carried out
for 30 min al 37°Cin triplicale.
Wilh Ihe use of Ihe crude extracts passed over the Dowex-1-acetate
columns and 5'-adenylylimido-diphosphate
inslead of ATP, Ihe reac
tion was linear for al leasl 40 min.
Malcriáis. [14C]CDP (350 mCi/mmol) was purchased from Research
Products International Corporation (Mount Prospect, IL). RPMI and
minimum essential medium culture media, horse serum, and sodium
bicarbonate were purchased from Grand Island Biological Company
Ribonucleotide ReducÃ-aseActivity in (,, Cells. LI210 cells
were enriched for the GI phase of the cell cycle by centrifugal
elutriation. The cell fraction which eluted at a flow rate of 12
ml/min had 82% G, cells, 16% S-phase cells, and 2% G2/M
cells as determined by cell cycle analysis by cytofluorometry.
This cell fraction incorporated [3H]lhymidine into DNA at only
15% of the maximum rate seen for the S-phase cells. As seen
in Fig. 1, ribonucleotide reducÃ-aseholoenzyme activity as mea
sured by CDP reduction was extremely low in the cell-free
extracts prepared from the GI cells. The addition of exogenous
effeclor-binding subunit to this fraction stimulaled CDP reduc
tion 3.6-fold. As the GI cells were incubated at 37°C,Ihere was
an increase in CDP reducÃ-aseactivily of approximalely 4-fold
wilh the peak of aclivily at 10 h. By 24 h, the reducÃ-aseaclivily
had decreased slighlly. When exogenous effeclor-binding subunil was added lo Ihe cell-free exlracls prepared al Ihe various
lime poinls, Ihere was a slriking increase in CDP reducÃ-ase
aclivily. Al 8 h Ihere was a 4-fold increase in reducÃ-aseaclivily
over Ihe t = 0 fraclion wilh exogenous effeclor-binding subunil.
The addilion of exogenous nonheme iron subunil lo Ihe cellfree exlracl had no effecl on Ihe level of CDP reducÃ-aseaclivily.
In ihis lime frame the GI, S- and G2/M cell cycle distributions
in 6-, 9-, and 24-h samples were : 26, 57, and 17; 20, 45, and
36; and 54, 38, and 8%, respectively. [3H]Thymidine incorporalion inlo DNA slarled lo increase belween 0 and 3 h and
reached a maximum belween 6 and 9 h. [3H]Tnymidine incorporalion inlo DNA correlaled wilh Ihe percenlage of cells in Sphase.
Half-Life of Holoenzyme and Its Subunits. L1210 cells were
enriched for S-phase cells by centrifugal elutrialion. The cell
fraclions lhal eluted belween Ihe flow rales of 18 and 30 ml/
min were used in these studies. By cell cycle analysis, the
enrichment of S-phase cells was between 70 (fraclion al 30 ml/
min) and 90% (fraclion at 18 ml/min). These cells were incu
bated in culture at 37°Cin the presence of cycloheximide (10
iig/ml). At Ihis concentralion of cycloheximide, [3H]leucine
incorporalion
inlo Ihe prolein of Ihe cells was inhibited 87%.
0.2
E
o
•=
O.I
Q
U
e
e
io
INCUBATION TIME.hr
24
Fig. 1. Ribonucleotide reducÃ-aseactivity in synchronized 1,1210 cells. LI210
cells, enriched for Ci-phase cells by centrifugal elutriation, were incubated in
culture. At t = 0, and at other time intervals, the cells were collected by
centrifugation and cell-free extracts prepared as described in "Materials and
Methods." CDP reducÃ-asewas measured directly in the cell-free extracts (•)and
after the addition of exogenous nonheme iron subunit, 20 ¿ig(•),or effectorbinding subunit, 50 /<y(A), prepared from Ehrlich tumor cells. The data points
are the average of triplicate determinations.
6166
Downloaded from cancerres.aacrjournals.org on April 15, 2017. © 1986 American Association for Cancer Research.
TURNOVER
OF RIBONUCLEOTIDE
REDUCÕASE
was al leasl a 4-fold increase in holoenzyme as measured by
ribonucleolide reducÃ-ase assays. The level of ribonucleolide
reducÃ-asemeasured in ihe G, cell population is an overestimalion as these cells were contaminated with S-phase cells (16%)
as determined by flow cylomelry. However, wilh Ihe addilion
of exogenous effeclor-binding subunil, there was a marked
increase in Ihe level of CDP reducÃ-ase activily, indicaling an
excess of Ihe nonheme iron subunil and a limiling amount of
the effector-binding subunit. In the LI210 cells, the produclion
of Ihe nonheme iron subunit peaked at 8 h. The level of Ihe
enzyme activity paralleled the level of Ihe effeclor-binding sub
unil. Further, when Ihe half-lives of the holoenzyme and the
individual subunils were delermined, il was found thai Ihe halflife of Ihe holoenzyme was 3.5 h while Ihe half-lives of Ihe
nonheme iron and effector-binding subunits were 7.6 and 4 h,
respectively. The shorter half-life of the effeclor-binding subunil
correlaled wilh ihe overall loss of reducÃ-ase aclivily and is
consislenl wilh Ihe effeclor-binding subunit being the limiling
subunil in LI210 cells.
The disparily in ihe results from Ihis laboralory and Ihe work
of olhers previously published may relale lo the different pa
rameters chosen for the measuremenls of Ihe iwo subunils. In
some
aspects, we are in agreemenl wilh Ericksson and Martin
DISCUSSION
(9), Ericksson et al. (10), and Engslrom et al. (11). Our dala,
In ihe presenl sludy, Ihe levels of ribonucleolide reducÃ-ase as well as Iheirs, show lhal Ihe Iwo subunils making up ribo
activity and the levels of the subunils were delermined in LI 210 nucleolide reducÃ-ase are noi coordinalely conlrolled and Ihe
nonheme iron subunil increases as Ihe cells pass from G, lo Scells as Ihey passed from G, Ihrough ihe cell cycle. It was found
thai, as Ihe cells passed from G, Ihrough ihe cell cycle, there
phase. Our dala show lhal in LI210 cells Ihe nonheme iron
subunil is in excess while Ihe effeclor-binding subunil is limil
ing. These resulls are similar lo Ihe dala oblained for regenerHoloenzyme
aling rat liver and Ehrlich lumor cells (13, 14). Those data
indicale that the level of the effeclor-binding subunil is in excess
and conslanl during Ihe cell cycle Iransverse in ihe MDBK cells
(11). In hydroxyurea-resislanl TA3 cells, il was shown lhal Ihe
10.5
conlenl of free radical of Ihe nonheme iron subunil increased
as Ihe cells passed from GÌ
lo S-phase. Measuremenls of neilher
ihe enzyme aclivily nor Ihe level of Ihe effeclor-binding subunil
were delermined in ihis sludy (10). Albert and Gudas (17)
concluded lhat the changes in reducÃ-aseaclivily during Ihe cell
O.I
cycle were relaled lo allosleric effecls as a resull of changes in
4
8
INCUBATION TIME, hr
the deoxyribonucleoside triphosphate pools and not due to
Fig. 2. Half-life of ribonucleotide reducÃ-ase.L1210 cells, enriched for S-phase
changes in ihe synlhesis of either the effeclor-binding (Ml) or
cells by centrifugal elutrialion, were incubated in culture in the presence of
nonheme iron (M2) subunil of ribonucleolide reducÃ-ase.How
cycloheximide (10 ng/ml). At t = 0, and at other time intervals, the cells were
ever, compuler modeling by Jackson (23) indicaled lhat Ihe
collected by centrifugation and cell-free extracts were prepared as described in
the "Materials and Methods." CDP reducÃ-asewas assayed directly in the cell-free
aclivily of ribonucleolide reducÃ-asewas conlrolled by Ihe level
extracts. The data shown by the O, D, and A each represent a separate experiment.
of ihe enzyme protein and not by allosleric effeclors. Based on
The data are shown on a semilog plot assuming the loss of activity is a first-order
Ihe reaclivity of a protein in clam and sea urchin eggs, which
decay. The line drawn through the data points was Titled by least squares. The
activity in the / = 0 sample was 0.35 nmol/30 min/mg of protein.
bound to monoclonal antibody againsl yeasl lubulin (24) and
which also inhibiled ribonucleolide reducÃ-aseaclivily, Slandarl
et al. (12) arrived al Ihe conclusion lhal ferlilizalion of Ihe clam
NHI
EB
and sea urchin eggs leads lo a marked increase in a prolein with
t I0
>
a molecular weight of 41,000, which Ihey identified as Ihe small
<05
subunil of ribonucleotide reducÃ-ase. They concluded lhal Ihe
0.5
unfertilized oocyles conlained an excess of Ihe effector-binding
¡
subunit and that the nonheme iron subunit was limiting, al
though no measurement of Ihe effeclor-binding subunil was
made.
INCUBATION
TIME , hr
Turner et al. (4) had earlier reported a half-life for ribonucle
INCUBATION
TIME, hr
Fig. 3. Half-life of nonheme iron (NHI) and effector-binding (EB) subunits.
olide reducÃ-aseof 2 h in synchronized L-cells. For ihe half-life
The cell-free extracts prepared and described in Fig. 2 were supplemented with
of Ihe holoenzyme in L1210 cells, we delermined a value of 3.5
excess exogenous NHI or EB subunit. For measurement of the half-life of the
h. It is possible thai the difference in half-lives of the subunils
NHI subunit, excess EB subunit (SO ¿ig)was added; for the half-life of the EB
subunit, excess NHI subunit (20 /ig) was added. The data shown by O, D, and A
for LI210 cells and Ihose reported for Ihe M2-overproducing
represent three separate experiments. The data are shown on a semilog plot. The
mouse mammary lumor TA3 cells (10) and Ihe MDBK cells
activities in the t = 0 samples for the assays to which exogenous NHI or EB
subunit was added were 0.35 and 0.94 nmol/30 min/mg, respectively.
(11) is due lo Ihe paramelers measured. In our experimenls, we
After the 10-h incubation period with cycloheximide, cell via
bility, as measured by trypan dye exclusion, was 95% or greater.
Cells were collected at specific time intervals and cell-free
extracts prepared and assayed for CDP reducÃ-aseactivity. As
seen in Fig. 2, when the cells were incubated in the presence of
cycloheximide, the holoenzyme as measured by CDP reducÃ-ase
activity had a half-life of 3.5 h. The points on ihis graph presenl
dala from three separate experimenls and each point is the
average of triplicate flasks, each run in triplicate. The line
drawn through the data points is fitted by least squares. The
correlation coefficienl of this line is -0.91, which is significant
al the/>< 0.001 level.
The decay rates of the nonheme iron and effector-binding
subunits were determined by adding an excess of the complemenlary subunil lo Ihe assay mixture. These dala are shown in
Fig. 3. From ihese dala, Ihe half-life of ihe effeclor-binding
subunil was 4 h while ihe half-life of Ihe nonheme iron subunil
was 7.6 h. The dala in ihese figures represenl Ihree separale
experimenls that correlate wilh Ihe dala for ihe decay of ihe
holoenzyme. The correlalion coefficienls for bolh of Ihese lines
were significanl at the level of P < 0.001.
6167
Downloaded from cancerres.aacrjournals.org on April 15, 2017. © 1986 American Association for Cancer Research.
TURNOVER
OF RIBONUCLEOTIDE
measured directly the loss of biologically active protein through
the measurement of enzyme activity. It is known that cycloheximide treatment can result in the altered rates of degradation
of various proteins and this must be considered. However, as
we showed in the regenerating rat liver system (13), the effectorbinding subunit decreased at a more rapid rate than did the
nonheme iron subunit, in agreement with the studies carried
out in the LI210 cells with cycloheximide. In the experiments
of Eriksson et al. (10) and Engstrom et al (11), the loss of the
free-radical signal was measured to determine the half-life of
the nonheme iron (M2) subunit. It was found, however, that
the level of the tyrosyl free radical could be variable and that
there was no absolute correlation obtained between radical
content and enzyme activity during purification (25). Lassmann
et al. (26) have shown that the electron spin resonance signal
for ribonucleotide reducÃ-asecan be measured in intact prolif
erating Ehrlich tumor cells. Their data showed that ribonucle
otide reducÃ-aseactivity did not decrease in cells taken 3 and 7
days after transplantation, while there was a 2-fold decrease in
the concentralion of Ihe lyrosine free radicals during the same
period. This would suggest that the nonheme iron subunit which
contains the free radical is not the limiling subunil of mam
malian ribonucleolide reducÃ-ase,in agreement with our conclu
sions.
For measurement of the half-life of the effector-binding sub
unil, a radioimmunoassay procedure was used (11). A direcl
assay of ihe biological aclivily was noi reported. In a recent
paper by Shirahala and Pegg (27), il was shown lhal Ihe halflife of S-adenosylmelhionine decarboxylase differed depending
on whelher biological aclivity or immunoreactivily was Ihe
parameler measured. As measured by biological aclivily (en
zyme aclivily or titralion of ihe aclive sile), the half-life of Sadenosylmelhionine decarboxylase was 35 min. However, when
measured by ihe loss of immunoreaclive prolein, Ihe half-life
was delermined lo be 139 min. Thal is, allhough immunoreac
live protein species remained for a longer period, biologically
aclive enzyme was losl 4 limes fasler. Perhaps Ihis is Ihe case
wiih ihe subunils of ribonucleolide reducÃ-ase as reported by
Eriksson et al. (10) and Engslrom et al. (11).
For an enzyme as crilical for DNA replicalion as ribonucle
olide reducÃ-ase,il would be expecled lhal ihe nalure of regulalion of ihe iwo subunils would be ihe same in ihe different
mammalian cell lines. In Escherichia coli, Ihe Iwo subunils are
encoded for by Iwo genes on Ihe same operon and hence are
coordinalely increased and decreased (28). The underslanding
of ihe overall conlrol of ribonucleolide reducÃ-ase activity
Ihrough regulalion of Ihe levels of Ihe individual subunils is
essenlial lo underslanding the key role the enzyme plays in
DNA replication. Since it has been shown thai Ihe individual
subunils of lumor cell ribonucleolide reducÃ-asecan be specifi
cally and independenlly inhibiled (29), ihe varialion in ihe levels
of ihe individual subunils may also be an imporlanl factor in
devising successful chemotherapy protocols directed at this site.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
REFERENCES
1. Cory, J. G., and Sato, A. Regulation of ribonucleotide reducÃ-aseactivity in
mammalian cells. Mol. Cell. Biochem., 53/54: 257-266, 1983.
2. Nutter, L. M.. and Cheng. Y.-C. Nature and properties of mammalian
ribonucleoside diphosphate reducÃ-ase. Pharamacol. Ther., 26: 191-207,
1984.
3. Cory, J. G., Rey. D. A., Bacon, P. E., and Carter, G. L. Nucleoside 5'-
28.
29.
REDUCÕASE
diphosphates as effectors of mammalian ribonucleotide reducÃ-ase.J. Biol.
Chem., 260: 12001-12007, 1985.
Turner, M. K., Abrams, R., and Lieberman, I. Levels of ribonucleotide
reducÃ-aseaclivily during Ihe division cycle of the L cell. J. Biol. Chem., 243:
3725-3728,1968.
Murphree, S., Stubblefield, E., and Moore, E. C. Synchronized mammalian
cell cullures. Exp. Cell Res., 58: 118-124, 1969.
Kucera. R., Brown, C. L., and Paulus, H. Cell cycle regulalion of ribonucle
otide reducÃ-aseactivity in permeable mouse L cells and in extraéis.J. Cell
Physiol., 117: 158-168, 1983.
Elford, H. L., Freese, M., Passamani, E., and Morris, H. P. Ribonucleolide
reducÃ-aseand cell proliferation. I. Varialions of ribonucleolide reducÃ-ase
aclivily wilh lumor growlh rale in a series of ral hepatomas. J. Biol. Chem.,
245:5228-5233, 1970.
Takeda, E., and Weber, G. Role of ribonucleolide reducÃ-asein expression of
Ihe neoplaslic program. Life Sci., 28: 1007-1014, 1981.
Eriksson, S., and Marlin, D. W., Jr. Ribonucleotide reducÃ-asein cultured
mouse lymphoma cells. Cell cycle-dependent variation in the activity of the
subunil prolein M2. J. Biol. Chem., 256: 9436-9440, 1981.
Eriksson, S., Graslund, A., Skog, S., Thelander, L., and Tribukail, B. Cell
cycle-dependenl regulalion of mammalian ribonucleotide reductase. The S
phase-correlaled increase in subunil M2 is regulaled by de novo prolein
synthesis. J. Biol. Chem., 259: 11695-11700, 1984.
Engstrom, Y., Eriksson, S., Jildevik, I., Skog, S., Thelander, L., and Tribukail, B. Cell cycle-dependenl expression of mammalian ribonucleolide reduc
Ã-ase.Differential regulalion of the Iwo subunits. J. Biol. Chem., 260: 91149116, 1985.
Slandarl, N. M., Bray, S. J., George, E. L., Hunl, T., and Ruderman, J. V.
The small subunit of ribonucleolide reducÃ-aseis encoded by one of Ihe most
abundant Iranslationally regulaled malernal RNAs in clam and sea urchin
eggs. J. Cell Biol., 100: 1968-1976, 1985.
Cory, J. G., and Fleischer, A. E. Noncoordinale changes in ihe componenls
of ribonucleolide reducÃ-asein mammalian cells. J. Biol. Chem., 257: 12631266, 1982.
Youdale, T., Frappier, L., Whitfield. J. F., and Rixon, R. H. Changes in the
cyloplasmic and nuclear adivines of ihe ribonucleotide reducÃ-aseholoenzyme
and ils subunils in regeneraling liver cells in normal and thyroparathyroideclomized rats. Can. J. Biochem. Cell Biol., 62: 914-919, 1984.
Youdale, T., Whitfield, J. F., and Rixon, R. H. l-a.25-Dihydroxyvitamin D3
enables regeneraling liver cells to make functional ribonucleotide reductase
subunits and replicate DNA in thyroparalhyroideclomized
rals. Can. J.
Biochem. Cell Biol., 63: 319-324, 1985.
Wrighl, J. A., and Cory, J. G. Alleralions in Ihe components of ribonucleotide
reducÃ-asein hydroxyurea-resislanl hamsler cells. Biosci. Rep., 3: 741-748,
1983.
Albert, D. A., and Gudas, L. J. Ribonucleotide reductase activily and deoxyribonucleoside Iriphosphate metabolism during the cell cycle of S49 wildtype and mutanl mouse T-lymphoma cells. J. Bioi. Chem., 260: 679-684,
1985.
Milchell, B. F., and Tupper, J. T. Synchronization of mouse 3T3 and SV40
3T3 cells by way of centrifugal elutrialion. Exp. Cell Res., /06: 351-355,
1977.
Vindelov, L. L. Flow microfluoromelric analysis of nuclear DNA in cells
from solid tumors and cell suspensions. A new method for rapid isolation
and slaining of nuclei. Virchows Arch. B. Cell Palhol., 24: 227-242, 1977.
Cory, J. G., and Mansell, M. M. Sludies on mammalian ribonucleolide
reductase inhibition by pyridoxal phosphate and ihe dialdehyde derivatives
of adenosine, adenosine 5'-monophosphate, and adenosine 5'-triphosphale.
Cancer Res., 35: 390-396, 1975.
Cory, J. G., Fleischer, A. E., and Munro, J. B., III. Reconslilulion of Ihe
ribonucleolide reducÃ-aseenzyme from Ehrlich lumor cells. J. Biol. Chem.,
25* 2898-2901, 1978.
Sleeper, J. R., and Sleuarl, C. D. A rapid assay for CDP reductase aclivily
in mammalian cell exlracls. Anal. Biochem., 34: 123-130, 1970.
Jackson, R. C. A kinelic model of regulalion of ihe deoxyribonucleoside
iriphosphale pool composilion. Pharmacol. Ther., 24: 279-301, 1984.
Kilmarlin, J. V., Wright, B., and Milstein, C. Rat monoclonal antilubulin
anlibodies derived by using a new nonsecreling ral cell line. J. Cell Biol., 93:
576-582, 1982.
Thelander, M., Graslund, A., and Thelander, L. Subunit M2 of mammalian
ribonucleotide reductase. Characterization of a homogeneous protein isolated
from M2-overproducing mouse cells. J. Biol. Chem., 260: 2737-2741, 1985.
Lassmann, G., Liermann, B., I .chinami. W., Graelz, H., Koberling, A., and
Langen, P. Ribonucleotide reducÃ-ase in asciles lumor cells delected by
eleclron paramagnelic resonance speclroscopy. Biochem. Biophys. Res. Com
mun., 132:1137-1143, 1985.
Shirahala, A., and Pegg, A. E. Regulalion of S-adenosylmelhionine decar
boxylase activily in rat liver and prostate. J. Biol. Chem., 260: 9583-9588,
1985.
Carson, J., Fuchs, J. A., and Messing, J. Primary structure of the Escherichia
coli ribonucleoside diphosphate reductase operon. Proc. Nati. Acad. Sci.
USA, 81: 4294-4297, 1984.
Cory, J. G., and Chiba, P. Combination chemotherapy directed at the
components of nucleoside diphosphate reducÃ-ase.Pharmacol. & Ther., 29:
111-127, 1985.
6168
Downloaded from cancerres.aacrjournals.org on April 15, 2017. © 1986 American Association for Cancer Research.
Differential Turnover of the Subunits of Ribonucleotide
Reductase in Synchronized Leukemia L1210 Cells
Eric H. Rubin and Joseph G. Cory
Cancer Res 1986;46:6165-6168.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/46/12_Part_1/6165
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on April 15, 2017. © 1986 American Association for Cancer Research.