the dose dependent effect of cyclic amp on ribonucleotide reductase in mitogen stimulated...

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 concentrations 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.

0006-291XBO $1.50

383 Copyright 0 1990 by Academic Press. Inc.

All rights of reproduction in any form reserved.


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%

384


[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.

385


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 concentrations 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.

386


-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

387


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

388


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


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.

1. 2.

3.

4.

lhelander, L., and Reichard, P. (1979) Annu. Rev. Bicchem. 48,133. Eriksson, S., Graslund, A., Skcg, S., Thelander, L., and

Tribukait, B., (1984) J. Biol. them. 259, 11695. Coffino, P., Gray, J.W., and Tomkins, G.M. (1975) Proc. Natl.

Acad. Sci. USA 72, 878. Albert, D.A., and Ndzenski, E. (1989) J. Cell. Physiol. 138,

129. 5.

6. 7.

8.

9. ,lO.

Albert, D.A., Ncdzenski, E., Yim, G., and Kowalski, J., (in press J. Cell Physiol.). Boyum, A. (1968) Scan. J. Clin. Lab. Invest. 21, Supp1.97, 77. Albert, D.A., and Bluestein, H.G. (1984) Int. J. Immunophannacol

6, 147. Albert, D.A., Gudas, L.J., and Ncdzenski, E. (1987) J. Cell.

Physiol. 130, 262. Taylor, I.W., (1980) J. Histochem. Cytcchem 28, 1021. Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982) Cold Spring

Harbor, N.Y. 11. Kafatos, F.C., Jones, C-W., and Efstratiadis, A. (1979) Nut. Acid.

12. 13. 14. 15.

Res. 7, 1541. Thelander, L., and Berg, P. (1986) Mol. and Cell. Biol. 6, 3433. Crabtree, G.R. (1989) Science 243, 355. Reichard, P., and Ehrenbery, A. (1983) Science 221, 514. Ergstrom, Y., Eriksson, S., Jildevik, I., Skcg, S., Thelander, L.,

16. 17.

and Tribukait, B., (1985) J. Biol. Chem. 260, 9114. LeMaire, I., and Coffino, P. (1977) Cell 11, 149. Russell, J.L., and Steinberg, R.A. (1987) J. Cell. Physiol. 130, 203.

390

the dose dependent effect of cyclic amp on ribonucleotide reductase in mitogen stimulated...

Documents