Vol. 167, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
March 16, 1990 Pages 383-390
UANIELALBERT,JANICEKOWALSKI, EIWAFDINKNODZENSKI, MARKMICEK, AND
Department of Medicine, University of Chicago Medical Center
Box 74, 5841 South Maryland Avenue, Chicago, IL 60637
Received January 16, 1990
Cyclic adenosine monophosphate arrests proliferating T lymphccytes in the Gl phase of the cell cycle. Herewedemonstratethat excqenous and endcgenous elevations in cyclic AMP concentration result in diminished mitcqen stknulation, cell cycle arrest, and decreased ribonucleotide reduotase messenger RNA concentra- tions in peripheral blood mononuclear cells. Atlowerconcentrations (less than UWI) of dibutyryl cyclic AMP that do not generate cell cycle arrest there is inhibition of ribonucleotide reductase actiirity without decreased messenger RNA concentration for the ML? subunit of rihmucleotide reductase. However, at higher concentrations of dibuty?zyl cyclic AMP there is Gl cell cycle arrest and reduced M2 specific messenger RNA concentration. Thus, cyclic AMP inhibition of lymphccyte activation may occur by different m that are dose dependent. Q 1990 Academic
mess, Inc.
Ribonucleotide reductase is a cell cycle regulated enzyme responsible
for the in vivo production of all four deoxynucleotides for DNA synthesis
and is necessary for cell proliferation(l). Enzyme activity is low in Gl
phase but rises in S and G2/M phase and correlates with the increase in
one of the two subunits of the enzyme-M2(2). Exposure of proliferating
lymphocytes to pharmacologic concentrations of cyclic AMP results in Gl
phase arrest(3). Data from lymphoblastoid cell lines suggests that cyclic
AMP may inhibit ribonucleotide reductase activity by post-translational
modification (phosphorylation) of the M2 subunit protein with a diminution
of catalytic activity of the enzyme(4). However, observations on the cell
cycle regulation of M2 messenger RNA concentrations suggest that cyclic
AMP induced Gl phase arrest is associated with a diminution of M2 specific
message suggesting a possible pre-translational effect of cyclic AMP(5)
as well. The experiments reported here address whether cyclic AMP
diminishes ribonucleotide reductase activity by post translational
modification or by reducing M2 specific messenger RNA or both.
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Vol. 167, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
CfzllCulh.re
Human peripheral blocd mononuclear cells were obtained by Sepracell (Sepratech Corp., Oklahoma City, OK) density gradient centrifugation of heparinized blood from normal volunteers(6). Cells were washed twice - first in phosphate buffered saline, then in RPMI 1640 media supplemented with 0.1% fetal calf serum and 0.4% albumin as previously described (7) in 0.2ml volume 96 well flat bottomed tissue culture plates at lx106 cells/ml or in larger flasks when indicated. Cultures were stimulated with phytohemagglutinin (PI-IA) (Sigma, St. Iouis, MO) at a final concentration of lm/rnl and incubated for 72 hours at 37°C in a humidified 5% Co2 in-air atmosphere. Mitcgen stimulation was assayed in 200~1 aliguots by 3H thymidine uptake (&Ci) during a four hour pulse before termination at 72 hours. Cells were harvested by an automated harvester on glass fiber filters and counted in a Packard scintillation counter using scintillation fluid (Aquasol, NEN Research Products, Boston, MA). Results reported are the means of triplicate determinations plus or minus the standard deviation in cpm/2x105 cells.
Ribonucleotide -WY Ribonucleotide reductase activity was measured by the conversion of CDP
to deoxyCDP in cell sonic&es as previously described (8).
Cells were prepared for cytometry by centrifugation, at the times indicated, then DNA was stained by resuspendiq the cells in hypotonic propidium iodide supplemented by Triton X 100 (1%) by the method of Taylor (9) - Cell cycle analysis was performed on a FACS IV flow microfluorimeter.
RNAExtraction Cellswerewashed in PBS and resuspended inaguanidinemixthat
consists of 3.5M guanidine hydrochloride, .02m potassium acetate, and 1OmM EDTA (with a 1O:l guanidine mix to pellet volume ratio) and RNA was precipitated with absolute ethanol at -2O"C, and then reprecipitated in guanidine and extracted with phenol:&lorofonn:isoamylalcohol(lO).
DotBlot RNA was denatured in 50% deionized formamide and 6% formaldehyde at
50°C for 1 hour. Samples containing 1OcLg total cellular RNA were applied to Gene Screen Plus (New England Nuclear, Boston, MA) nylon membranes using a dot blot (Hybri-Dot) manifold (Bethesda Research Laboratories, Gaithersburg, MD) (11). The filter was dried and baked for 2 hours at 8O'C.
Nick'lmnslationardmnbing l-21.(4 of cDNA (12) was nick translated using a Nick Translation Reagent
Kit (Bethesda Research Laboratories, Gaithersburg, MD). Hybridization was performed at 65°C overnight with salmon sperm DNA to reduce background. Blots were washed in .lxSSC at 25°C for 30 minutes and 2xSSC with 1% SES at 65°C for 30 minutes and then exposed to autoradiographic film.
Results
Inhibition of Mitogen Stinulation by Cyclic AMP
Figurelshowstheeffectof increasing concentrations ofdibutyryl cyclic AMP added at time 0 to mitogen stimulation at 72 hours. About 90%
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Vol. 167, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
[Bt, CAMP]
Figure l.Inhibition of Mitqen Stimlation by Cyclic AMP. Normal hman peripheral blood lymphocytes were cultured as described in Methods. In the presence of increasing concentrations of dibutyql cyclic AMP for 72 hours ENA synthesis was inhibited as measured by 3H- thymidineuptake.
inhibition is achieved at doses of 1 to 5nM dibutyryl cyclic AMP and is
similar /in magnitude to the inhibition of proliferation seen in the
transformed muse T lynqqhoblast cell line S49(8). A phosphodiesterase
inhibitor, R0-20-1794, was used to elevate endogenous CAMP levels and the
dose response in mitogen stimulated peripheral blood lymphocytes (Figure
2) is also similar to that seen in S49 tells(8). In both sets of
exp&ments, cytofluorqraphs did not show cell cycle arrest without
substantial diminution of 3H-thymidine uptake (>lmM dibutyryl cAMP and
>lOw RO-20-1794-data not shown). Thus, both exogenous and endogenous
elevations in cyclic AMP result in diminution of mitogen responsiveness as
measured by 3H-thymidine uptake at 72 hours.
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II::! , , , , i 2 6 IO 14 I8 22 26 30
RO-20- 1794 @MI
Figure Z.Inhibition of Mitagen Stimulation by RC-20-1794. Cells oultured as in Figure 1 in the presence of increasing concen- trations of RC-20-1794, an inhibitor of cyclic AMP phosphodiesterase.
Cyclic AMP diminishes FGbonucleoti* R&uctse Activity
The addition of cyclic AMP to peripheral blood mononuclear cells in
concentrations that do not generate Gl cell cycle arrest diminishes
ribenuclectide reductase activity. In Table 1 we show that cells exposed
to lower concentrations of cyclic AMP (5OOm) for 72 hours have reduced
rtinucleotide reductase activity. In multiple experiments,
concentrations of dibutyryl cyclic AMP between lO@l and 500@ reduced 3H-
thymidine uptake 10% to 70% without generating actual Gl cell cycle
arrest. This suggests that at concentrations of cyclic AMP that are
insufficient to generate cell cycle arrest there is still diminution of
ribonucleotide reductase activity and some decrement in 3H-thymidine
uptake.
TABLE 1. Ribonucleotide Reduotase Adivity
-Pi-IA mA PHA+oyclic AMP
CDP Reduotase Activity* 138 3435 639
*in CFN/m protein in cell sonioates
Rikonucleotide reduotase (CDP reductase) activity was assayed as described in the Methods section. For this experiment cells were cultured with or without phytohemagglutinin (PHA-lug/M) and dibutyryl cyclic AMP (50op~) for 72 hours. In this experiment there was a 10% reduction in both 3H- thymidine uptake and proportion of cells in S phase.
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-PHA
I
+ PHA
24 hr 48 hr 72 hr
PHA +
1imM CAMP
/
426!148 6.552?756 9.417?258
: / L 4
3022165 1.928?342 5.764?462
Figure 3.Cyclic AMP inhibits the increase in M2 Specific Messenger RNA concemtrations that a cxmnpanies mitqen stimulation. Cells cultured for 24, 48 and 72 hours in the presence or absence of phytohemagglutinin with or without lmM dibutyql cyclic AMP. In each row cytofluorqrams, dot blots and 3H-thymidine uptake (in CPM) are shown. Proportion of S phase cells in each culture is 0% in all cultures at 24 hours, 40% in F+L+stimulated cells at 72 hours and approximately 20% in FWA stimulated cells cultured with lmM dibutyryl CAMFJ.
The Effect of Cyclic AMP on Cell Cycle Distributicm ard M2 specific
I%sxrqerRNAConcentration
In Figure 3 we show the time cmrse, cell cycle distribution, 3H-
thymidine uptake and M2 specific messenger F?NA concentrations of
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Vol. 167, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
unstimulated peripheral blood mononuclear cells, mitcgen stimulated cells
and stimulated cells that were exposed to l.mM dibutyryl cyclic AMP.
Unstimulated cells remain in Gl phase over 72 hours and the M2 specific
message concentration diminishes over tin-e. Not shown is the 0 time
point which is identical to the 24 hour -pHA data in terms of cell cycle
distribution, 3H-thymidine uptake and dot intensity. Mitogen stimulated
cells shm a progression to exponential distribution beginning between 24
and 48 hours. This is a ccompaniedby increasing 3H-thymidineuptake and
increasing dot intensity. Dibutyryl cyclic AMP (ImM) addition resulted in
an approximte 50% reduction in 3H-thymidine uptake in this experiment and
diminution in massage intensity at 72 hours. The dot intensity of cyclic
AMP treated cells is always less than in mitogen stimulated cells and
similar to unstimulated cellswhen significant cell cycle arrest occurred.
Disaxssion
Mitcqen stimulation of cells results in an extensively characterized
seguence of biochemical events that eventuates in cell proliferation(l3).
my enzyme systems of intermediary metabolism are activated during this
process including those involved in DNA synthesis(13). tibonucleotide
reductase is a critical enzyme in the biosynthetic pathway of DNA
synthesis. This enzyme is responsible for the in vivo production of all
four deoxynucleoside triphosphates utilized by lXA polymerase, and is thus
a potential regulatory site for cell proliferation.
Theenzyn-teiscmposedoftwodissimilarsubunits. TheMlsubunit
contains the birding sites for the allosteric effector molecule that
regulate the enzyme's substrate specificity and overall activity. The M2
subunit contains the tyrosyl radicals that form the catalytic site for the
reduction reaction as well as an non-h- iron moiety that is necessary
for enzyme activity. The holoenzyme is most likely a dimer of Ml and M2
pairs(l4). The Ml subunit appears to be constitutive in fibroblast and
lmhoblast cell lines whereas the M2 subunit activity and protein
concentration appears to be cell cycle regulated and rate-limiting(2,15).
Thus, the enzyme activity is cell cycle regulated with its greatest
activity in S and G2/M phase which correlates with increased M2 activity
in those portions of the cell cycle(2,15).
Cyclic AMP appears to be a negative regulator of lymphocyte
proliferation, arrestimg cells in the Gl phase of the cell cycle(l6). 1ts
mechanism of action requires cyclic AMP dependent protein kinase (Pm-a
serine threonine kinase) which phosphorylates multiple target proteins,
including its own regulatory subunit(l7). However, phosphorylation of the
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Vol. 167, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
M subunit of ribonucleotide reductase is an unlikely explanation for
cyclic AMP induced cell cycle arrest because there is very little M2
protein in Gl phase cells Which is the point at Which cyclic AMP arrests
cells. Eurthermore, the cell cycle regulation of ribonucleotide reductase
activity is normal in PKA minus cells suggesting PKA is not involved in
the normal variation in enzyme activity.
The experiments presented here demonstrate that resting peripheral
blood mononuclear cells have moderate concentrations of M2 specific
messenger RNA, but if unstimulated cells are left in culture, this message
wncentration diminishes over time. Mitogen stimulation increases IQ
specific messenger RNA wncentration and this appears to roughly
correlate with the amount of cells in S phase as judged by cytofluorometry
and 3H-thymidine uptake. Cyclic AMP treatment clearly diminishes the
number of cells in S phase as reflected by both the cytofluorqrams and
3H-thymidine uptake and diminishes M2 specific messenger RNA
wncentration.
Ribonucleotide reductase activity is very low in unstimulated cells
both at time 0 and after 72 hours in culture, Whereas ETIA stimulated cells
have a 3O-fold increase in activity. This increased activity is
diminished by cyclic AMP even in concentrations too low to generate actual
Gl cell cycle arrest. The reduced 3H-thymidineuptake andmodest
diminution of cells in S phase at lower concentrations of cyclic AMP could
be the result of inhibition of ribonucleotide reductase activity without
Gl arrest or decreased M2 message. By wntrast, cyclic AMP at high
concentration (>lmM dibutyryl CAMP) inhibits cell cycle progression in Gl
and is accompanied by diminution of IQ specific messenger RNA
concentration. The mechanism of diminished messenger RNA concentration
for the M2 subunit of ribonucleotide reductase in Gl arrested cells is
unclear. It is not a toxic mechanism since cells can be washed out of
cyclic ANP and recover. E'urthermore, the cytofluorcqraphic patterns of Gl
arrested cells shows no increased debry as is seen in cultures of dying
cells exposed to toxic concentrations of various reagents. More likely
this is the result of a specific effect of cyclic AMP on cell metabolism
that causes Gl arrest and decreased messenger RNA concentrations. The
mechanism probably involves protein kinase A phosphorylation of a cellular
protein but Whether the decrease in message is the cause or the result of
Gl arrest is unknown at present.
WethankIzu-sThelander forthegift ofM2 probe. Robert Kimand
Hsien-Yi Iu provided technical assistance.
389
Vol. 167, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Support for these studies was provided by the Arthritis Foundation of
Illinois and through an Arthritis Investigator Award (D.A.A.) from the
Arthritis Foundation.
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