effects of guanine ribonucleotide accumulation on the … · containing 1 mg adenine. fifty-eight %...

[CANCER RESEARCH 45. 4940-4945, October 1985]

Effects of Guanine Ribonucleotide Accumulation on the Metabolism and CellCycle of Human Lymphoid Cells1

Yechezkel Sidi,2 Jerry L. Hudson, and Beverly S. Mitchell3

Departments of Internal Medicine [Y. S., B. S. M.] and Pathology [J. L H.] and Simpson Memorial Research Institute. University of Michigan, Ann Arbor, Michigan 48109

ABSTRACT

A deficiency of purine nucleoside phosphorylase activity isassociated with marked depletion of T-lymphocytes which is felt

to be mediated by accumulation and further metabolism of thepurine nucleoside phosphorylase substrate, 2'-deoxyguanosine.Human T-lymphoblasts incubated in the presence of 2'-deoxy-

guanosine and the purine nucleoside phosphorylase inhibitor 8-aminoguanosine accumulate deoxyguanosine 5'-triphosphatewhereas B-lymphoblasts and mature T4+-cell lines accumulate

GTP under identical conditions. We have compared the effectsof guanine ribo- and deoxyribonucleotide accumulation on the

metabolism and cell cycle of the respective cell lines. Deoxyguanosine 5'-triphosphate elevations in T-lymphoblasts are associated with inhibition of [3H]uridine incorporation into DNA and a

complete block at the Gi-S interface of the cell cycle. In contrast3- to 5-fold increases in guanosine 5'-triphosphate pools in B-lymphoblasts and mature T-cell lines do not inhibit [3H]uridine

incorporation into DNA or RNA but do cause a pronouncedslowing in the progression of cells through S phase. B-lympho

blasts deficient in the salvage enzyme hypoxanthine guaninephosphoribosyltransferase do not accumulate guanosine 5'-tri-phosphate from 2'-deoxyguanosine and progress normally

through the cell cycle, demonstrating a requirement for guaninesalvage to inhibit cell growth. Guanine ribonucleotide accumulation was also associated with inhibition of de novo purine biosynthesis and a moderate decline in adenine nucleotide poolsbut not with inhibition of protein synthesis or alterations in basallevels of 3':5'-cyclic adenosine monophosphate or 3':5'-cyclic

guanosine monophosphate. We conclude that the accumulationof guanine ribonucleotides by actively cycling human lymphoidcells is associated with an increase in S-phase cells and inhibition

of growth. This effect is distinctly different from that producedby 2'-deoxyguanosine 5'-triphosphate and should be taken intoaccount in pharmacological studies with 2'-deoxyguanosine and

its analogues.

INTRODUCTION

Inherited deficiency of PNP4 results in a severe immunodefi

ciency syndrome characterized by T-)ymphocyte depletion (1).PNP catalyzes the conversion of guanosine and 2'-deoxygua-

1This investigation was supported by Grant CA 34085 awarded by the National

Cancer Institute, Department of Health and Human Services.2Present address: Department of Medicine D, Beilinson Medical Center, Petah

Tikva, Israel.3Scholar of the Leukemia Society of America. To whom requests for reprints

should be addressed, at Simpson Memorial Institute, 102 Observatory, Ann Arbor,Ml 48109.

'The abbreviations used are: PNP, purine nucleoside phosphorylase; PCA,perchloric acid; TCA, trichloroacetic acid; 6-MMPR, 6-mercaptopurine riboside;HPRT, hypoxanthine guanine phosphoribosyltransferase; PBS, phosphate-bufferedsaline; CAMP, 3':5'-cyclic AMP; cGMP, 3':5'-cydic GMP.

Received 1/8/85; revised 5/22/85; accepted 6/27/85.

nosine to guanine and the toxicity to lymphoid cells in this diseasestate has been attributed to the accumulation of 2'-deoxygua-

nosine and its selective metabolism to dGTP in T-cells. Culturedhuman and murine T-lymphoblasts exposed to 2'-deoxyguano

sine alone accumulate large amounts of dGTP which is felt toinhibit the enzyme ribonucleotide reducÃase,deplete dCTP pools,and consequently inhibit DNA synthesis (2-6). The addition tosuch cultures of 8-aminoguanosine, a relatively weak competitiveinhibitor of PNP (7), markedly potentiates both the toxicity of 2'-

deoxyguanosine and the accumulation of dGTP by these cells(8). '

In contrast to these data, recent studies have indicated thatcultured B-lymphoblasts and mature T4+-cell lines, as well as

peripheral blood mitogen-stimulated lymphocytes, accumulateGTP rather than dGTP when exposed to 2'-deoxyguanosine and

8-aminoguanosine (9, 10). Guanine ribonucleotide synthesis in

these cells requires HPRT activity and is apparently mediated bythe prolonged availability of intracellular guanine resulting from aslower rate of 2'-deoxyguanosine phosphorolysis in the pres

ence of a weak PNP inhibitor; more potent inhibition of PNPactivity completely abolishes both guanine and guanine ribonucleotide formation (9). Of particular interest, however, is theobservation that guanine ribonucleotide accumulation is directlyassociated with inhibition of cellular proliferation (9,10). Becauseof the increasing evidence that guanine ribonucleotides may beimportant regulators of cell growth (11-13), we have investigated

the effects of guanine ribonucleotide accumulation generated bythe exposure of cells to 2'-deoxyguanosine and 8-aminoguano

sine on the metabolism and cell cycle of cultured human lymphoidcell lines.

MATERIALS AND METHODS

Chemicals. 2'-Deoxyguanosine, guanosine, 6-methylmercaptopurine

riboside and azaserine were purchased from Sigma (St. Louis, MO). 8-

Aminoguanosine was a gift from Dr. L. Townsend, Department of Medicinal Chemistry, University of Michigan. [5,6-3H]Uridine (40 Ci/mmol) waspurchased from New England Nuclear (Boston, MA). Sodium [14C]for-mate (40-60 Ci/mmol) was purchased from ICN (Irvine.CA). i_-[4,5-3H]-

leucine (46 Ci/mmol) was purchased from Amersham, Arlington Heights,IL.

Cell Lines. MOLT-4 and RPMI-8402 T-cell lymphoblastic leukemialines were obtained from HEM Research, Rockville, MD. The MGL-8 B-

lymphoblast line was originally obtained from Dr. John Ã¼tttefield,JohnsHopkins University, Baltimore, MD. B-lymphoblast lines GM-558, GM-2292, and GM-1899 were obtained from the Human Genetic Mutant CellRepository, Bethesda, MD. The mature T-cell lines HUT 78 and HUT 102

lines were obtained from Dr. P. Bunn, National Cancer Institute, Washington, DC (14,15). The cells were grown in RPM11640 medium (GrandIsland Biological Co. Laboratories, Grand Island, NY) containing 10%horse serum. All incubations were performed at a concentration of 2 x105 cells/ml in a 5% CO2 humidified incubator.

CANCER RESEARCH VOL. 45 OCTOBER 1985

4940

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GTP TOXICITY FOR LYMPHOID CELLS

Nucleotide Pool Measurements. Nucleotide pools were quantitatedusing high pressure liquid chromatography on a Partisil-10 SAX aniÃ³nexchange column (Whatman, Clifton, NJ). Following incubations 0.5-1x 107cells were washed once, resuspended in 150 n\ PBS and extractedwith 170 i-l ice cold 1 N PCA. After 5 min at 4Â°C the extract was

neutralized with 1 N KOH. Fifty to 200 n\ of the supernatant were injectedfor each run. All ribo- and deoxyribonucleoside triphosphates were

completely separated using a linear gradient from 50% Buffer A (0.002M NH4HPO4, pH 2.8) to 60% Buffer B (0.75 M NH4HPO4, pH 3.9) over 30min at a flow rate of 2 mi/min. Mono-, di-, and trinucleotides were

separated using a stepwise gradient of the same buffers begmnning at100% Buffer A and ending at 60% Buffer B over 80 min. IMP, GMP, andxanthosine 5'-phosphate were not well separated from each other under

these conditions. The individual nucleotide concentrations were calculated by comparing areas of peaks absorbing at 254 nm to the areasgenerated by nmol amounts of pure standards using a 3390A HewlettPackard integrator.

3':5'-Cydic AMP and 3':5'-cydic GMP were measured in 100-^1 cell

extracts using radioimmunoassay kits for acetylated cyclic nucleotides(New England Nuclear, Boston, MA). Cell pellets containing 2-4 x 107

cells were suspended in buffered salt solution consisting of 138 mMNaCI, 2.7 mM KCI, 8.1 HIM NaiHPO4, 1.5 rriM KH2PO4, 1 M MgCb, and0.6 mM CaCI2, pH 7.4, and 0.2-ml aliquots were added to 1 ml boiling

0.5 mu theophyllin in PBS. The samples were diluted in 2 ml of 0.05 Msodium acetate buffer, pH 6.2, sonicated for 5 s, and centrifugea at 4000x g for 15 min. The standard curve for cAMP was linear in the range of0.01-0.20 pmol/sample. The standard curve for cGMP was linear in therange of 0.005-0.250 pmol. The recovery of standard cAMP and cGMP

was over 95% using the above extraction procedure. GTP in concentrations up to 10,000 pmol/100 n\ did not change the values obtained fromstandard cGMP samples.

De Novo Furine Biosynthesis Determinations. The effects of nucleo-sides and 6-MMPR on purine biosynthesis were measured by a modifi

cation of the method of Hershfield and Seegmiller (16) after preincubationof the cells with the specified additives at a cell concentration of 2 x 105/

ml. Two h before ending the incubation the cells were pelleted and thesuspension volume was adjusted to 2 ml RPM11640 medium plus 10%horse serum at a concentration of 10s cells/ml. Twenty nC\ of sodium[14C]formate were added for the final 2-h incubation at 37Â°C. The

samples were centrifugea and the cells were washed in cold phosphate-

buffered saline and resuspended in 1 ml of cold RPMI 1640 medium.Four ml of 1.25 N PCA were added to the cell suspensions and to 1 mlof the supematants. The samples were covered with aluminum foil,boiled for 1 h, and cooled. The supematants were transferred to tubescontaining 1 mg adenine. Fifty-eight % NH4OH (1.8 ml) was added, the

tubes were vortexed, and 0.5 ml 10% AgNO3 was added. The tubeswere left at 4Â°Covernight. The sediment was washed 6 times with cold

water until the supematants contained no radioactivity and resuspendedin 0.5 ml of 0.1 N HCI. The samples were boiled for 1 h and thesupematants were counted in Bray's scintillation fluid. Cell suspensions

and supematants pretreated with PCA before the addition of [14C]formate

served as blanks.DNA, RNA, and Protein Synthesis. Undine incorporation into DNA

and RNA was measured in cells incubated for different times and withthe specified additives by adding 1 Â¿iCiof [3H]uridine to 2-ml aliquots for

the last 2 h of incubation. The cell pellet was washed twice with coldPBS and 1 ml 0.5 N cold PCA was added. The sediment was washedconsecutively with etherethanol (1:1) solution and 10% TCA at 4Â°Cand

suspended in 0.4 ml of 0.3 N KOH. The tubes were incubated for 20 hat 37Â°C under parafilm cover. The solution was cooled and 10 ^l 10 N

PCA were added. The supernatant was neutralized by 0.2 ml 0.7 N KOHand the clear supernatant containing the RNA hydrolysate was counted.The sediment was washed with 10% cold TCA, suspended in 0.6 ml 10N TCA, and dissolved by heating for 1 h at 70Â°C. The samples werecounted using Bray's scintillation fluid. Blanks were obtained by the

addition of the same amount of [3H]uridine to cell pellets pretreated with

cold 0.5 N PCA.Protein synthesis was measured by incubating 2-ml aliquots of cell

suspensions with 2 >iCi of [3H]leucine for the last 2 h of incubation. The

cells were washed 2 times in cold phosphate buffered saline and 1 ml10% TCA was added at 4Â°C.The sediment was filtered on 24-mm GF/

A fiberglass filters (Whatman, Clifton, NJ), washed with cold 10% TCAand counted.

DNA Content Determinations. DNA content analyses were performedon 1-3 x 105 cells following the specified incubations. The cells were

washed twice with cold PBS. Chicken RBC suspension (200 ^l) wasadded as an internal standard and 1 ml of freshly made propidium iodidestain consisting of 75 Ã•/Mpropidium iodide: 10 mM NaCI:0.01 MTris:RNase(700 /<g/liter):NP40 (1 ml/liter), pH 8.0, was added and the samples werestirred and incubated for 45 min at 4Â°C.Then 100 Â¡A2% paraformalde-

hyde were added while stirring. Analysis of DNA content was done usinga Coulter EPICS V flow cytometer (17). Cell cycle phase estimationswere obtained by analyzing the averaged DNA histograms using thePARA 1 program running on an E.A.SY/Terak computer (EPICS Division,Coulter Corporation, Hialeah, FL).

RESULTS

Nucleotide Pool Alterations. The effects of incubations of B-lymphoblasts, mature T-cell lines, and T-lymphoblasts with 2'-

deoxyguanosine and 8-aminoguanosine on nucleotide pools areshown in Chart 1. Following 24-h incubations, B-lymphoblasts(HPRT positive) and mature T-cell lines accumulated 3- to 5-foldthe baseline level of GTP. In contrast T-lymphoblasts showed noincrement in GTP but a striking increase in dGTP. B-lymphoblasts

which are HPRT negative did not accumulate either GTP ordGTP. These data are consistent with previously describedexperiments (8,9). In the presence of elevated intracellular GTPlevels, ATP pools decreased by means of 17 and 15% for thetwo HPRT positive B-lymphoblast lines and 42 and 21% for themature T-cell lines. LTTPpods were similarly reduced by 24 and18% in B-lymphoblasts and 32 and 29% in mature T-cells, whileCTP levels were unaffected. The accumulation of dGTP in T-lymphoblasts was also associated with reductions in ATP levelswhich were most pronounced in the RPMI 8402 cell line.

In order to see whether the changes in nucleoside triphosphatepools reflect similar changes in nucleoside mono- and diphos-phate pool sizes, we measured the changes in adertine andguanine mono-, di-, and triphosphate pools in B-lymphoblastsand mature T-cells which resulted from incubation with 8-aminoguanosine and 2'-deoxyguanosine (data not shown). In both

cell types the marked increase in GTP levels was associatedwith an approximately 4-fold increase in GDP and minimal ele

vation of GMP. These alterations in guanine nucleotide poolswere associated with divergent effects on adenine nucleotidepools. In the B-lymphoblast line there was a small drop in ATP

levels with no significant change in AMP or ADP pools in fourseparate experiments. In contrast there was a 5- to 6-foldreduction in AMP and ADP in the HUT-78 line while ATP levelsfell to less than 50% of control values in two experiments.Despite these fluctuations the energy charge for adenine ribo-nucleotides remained constant for both cell types.

The effect of increases in intracellular GTP on cyclic nucleotidepools in the MGL-8 B-lymphoblast line is shown in Table 1.Despite a mean 5-fold increase in GTP levels, cAMP levelsremained unchanged. The cGMP concentration increased 2-fold


4941



GIP TOXICITY FOR LYMPHOID CELLS

IO5

0IO5Ã¨

io4."5

oIO5

0201GTP_

r^ J^ATP1LT

TIE

FÃ‰n^nÂ»UTP-^

r1^ r-r^r"^ n*, r3^ ^*,,->.CTP-â€¢â€¢^^j

j bxiÃ¬ | "* Â£?ÃŒ1 i ~ijr r""~Li* â€¢ n~ndGTP.

yIML86M998 6MI899 6M2292 HUT78 HUTI02 MOLT4 RPMI8402

n-18) (n-12) (n-4) (n-6) (n-13) (n-6) in. e) (n-4)

) LYMPHOBLASTS B LYMPHOBLASTS MATURE T CELL T LYMPHOBLASTS(HPRTt) (HPRT-) LINES

Chart 1. Effects of 2'-deoxyguanosine and 8-aminoguanosine on nucteoside

triphosphate pools. Measurements were done after 24-h incubations in the presence of 100 MM8-aminoguanosme alone (Q) or 50 MM2 ' -deoxyguanosine plus 100

Â»IM8-aminoguanosine (i i), n. number of independent experiments which wereperformed with each cell line; oars, SD.

Table 1

Effects of GTP elevations on cyclic nucleotide poolsMGL-8 B-lymphoblasts were incubated for 24 h in the presence of 100 Â«M 8-

aminoguanosine alone or 50 Â¡M2 ' -deoxyguanosme plus 100 Â¡M8-aminoguanosine.Nucleotide pool sizes were measured as described in 'Materials and Methods."

Additives

GTP CAMP cGMP(pmol/10Â«cells) (pmol/107 cells) (pmol/107 cells)

n = 6 n = 4 n = 6

Control8-Aminoguanosine+

dGuo1574

Â±360* (6)"

7966 Â±1822 (6)8Â±3 (4)

11 Â±1 (4)0.4Â±0.1 (6)

0.8 Â±0.2 (6)

' Mean Â±1 SD." Numbers in parentheses, number of experiments performed in duplicate.

but this difference was not statistically significant (0.05 < P <0.1).

Effects of Guanine Ribonucleotide Accumulation on DMA,RNA, and Protein Synthesis and the Cell Cycle. We haveshown previously that dGTP-mediated toxicity for T-lympho-blasts is associated with a marked decrease in [3H(uridine incor

poration into DNA which is most probably due to inhibition ofribonucleotide reducÃaseactivity (6). Since GTP is an essentialprerequisite for both RNA and protein biosynthesis, we compared the effects of GTP and dGTP accumulation on [3H]uridine

incorporation into DNA and RNA and [3H]leucine incorporationinto protein. Chart 2 demonstrates a lack of inhibition of [3H]-uridine incorporation into DNA in B-lymphoblasts (A, HPRT positive; B, HPRT negative) and mature T-cells (C), whereas DNAsynthesis in T-lymphoblasts accumulating dGTP is markedly

impaired (D). Similarly there is no demonstrable inhibition of RNAor protein synthesis in the B- or mature T-cells; the apparent risein the incorporation of [3H]uridine into RNA in B-lymphoblasts

and to a lesser extent in the HUT 78 cells was a consistentfinding in these experiments perhaps explained by the decreaseÂ¡nUTP pool sizes and an increase Â¡nUTP specific activity.

We then examined the effects of GTP accumulation on theDNA content of the cell lines. The progression of normal B-lymphoblasts through S phase was slowed during incubationswith 50 Â»Mguanosine or 2'-deoxyguanosine in the presence of

8-aminoguanosine (Chart 3) whereas no change in DNA contentoccurred in B-lymphoblasts deficient in HPRT activity (Chart 4).

The changes in cell cycle were maximal after 24 h of incubationand returned to the control pattern after 72 h. The time courseof these changes was parallel to the time course of GTP accumulation which was also maximal after 24 h in the HPRT-positiveB-lymphoblasts. Similar cell cycle changes were observed duringincubations with 300 UMguanosine or 2'-deoxyguanosine in the

absence of 8-aminoguanosine. In contrast to these findings, T-lymphoblasts incubated with 3 MM 2'-deoxyguanosine plus 8-

aminoguanosine demonstrated a very early block in S-phaseleading to complete absence of S-phase cells after 24 h (Chart

5)-Effects of Guanine Ribonucleotide Accumulation on de

Novo Purine Biosynthesis. The moderate depletion of adeninenucleotides in cells accumulating GTP led us to ask whetherGTP was significantly inhibiting de novo purine biosynthesis. Wetherefore measured the rate of incorporation of [14C]formate into

intracellular and extracellular purines during incubations of B-lymphoblasts with 2'-deoxyguanosine, guanosine, or 6-MMPR,

a known inhibitor of de novo purine synthesis. Chart 6 demonstrates that the rate of formate incorporation was markedlyreduced by either Quo or dGuo alone at 4 h and by the combination of Quo or dGuo and 8-AGuo at 24 h. The magnitude ofthe inhibition at 24 h was similar to that caused by 6-MMPR. 6-

MMPR and another inhibitor of de novo purine biosynthesis,azaserine, also produced an accumulation of cells in S phase(Chart 7) which was somewhat similar to the effects of 2'-

deoxyguanosine or guanosine on these cells.

DISCUSSION

We have demonstrated previously that a 3- to 5-fold elevationin intracellular GTP pools which is sustained over a 24-h period

in lymphoid cells lines causes marked inhibition of cellular proliferation, whereas transient increases in GTP of less than 12 hduration have no effect on cell growth (9). Our current studiesdocument that GTP accumulation at 24 h is associated wtih (a)a mild to moderate decline in ATP levels, more pronounced inmature T-cell lines than in B-lymphoblasts; (b) inhibition of de

novo purine biosynthesis; (c) no significant alterations in intracellular cyclic AMP or GMP levels; (d) no direct inhibition ofincorporation of [3H]uridine into DNA or RNA, or demonstrable

inhibition of protein synthesis; and (e) accumulation of cells in Sphase.


4942




Chart 2. Effects of 2'-deoxyguanosine and

8-aminoguanosine on RNA. DNA, and proteinsynthesis. RNA (â€¢)and DNA (O) synthetic rateswere measured by the incorporation of [3H]uridine into acid-insoluble material as describedin "Materials and Methods." The protein syn

thetic rate (G) was measured by the incorporation of [3H]leucine into acid-insoluble material.

Incubations were done in the presence of 50pin 2'-deoxyguanosine and 100 Â¡M8-amino

guanosine and results were compared to control incubations with 100 /IM 8-aminoguanosinealone. The data are the results from a singleexperiment; similar results were obtained in asecond experiment. A, B-lymphoblast line GM-558 (HPRT positive); B, B-tymphoblast line GM-1899 (HPRT negative); C, mature T-cell lineHUT-78; D, T-lymphoblast line MOLT-4.

OÃœ200

OOC lOO

LO 12 24 O 12

HOURS OF INCUBATION

24

H. B.

dtiuo * B-RGuo Quo t 8-fÃ¬Guo

Charta. Effect of 2'-deoxyguanosine (dGuo) and 8-aminoguanosine (8-AGuo)

on the DNA content of GM-558 cells. The histograms were obtained from quadruplicate determinations in a single experiment. Similar data were obtained in twoadditional experiments. Values above the figures are estimations of the percentageof cells with Go-G,, S-phase and M DNA content, respectively. A, 8-aminoguanosinealone; B, 8-aminoguanosine plus 2'-deoxyguanosine; C, 8-aminoguanosine plus

guarÃosme(Quo).

The combinationof pronouncedinhibitionof [14C]formateincorporationinto total purines with decreasesin the adeninenucleotidepoolprovidesgoodevidencethat increasesinguanineribonucleotidesinhibit de novo purine biosynthesisin theselymphoidcells.This effect is most probablyattributableto theallostericinhibitionof glutaminephosphoribosylpyrophosphateamidotransferase,an enzyme pivotal to purine biosynthesiswhichis allostericallyinhibitedby GMPandto a lesserextentbyGDPandGTP(18).

It remainsunclear,however,whetherthis degreeof adenineribonucleotidedepletionin the presenceof excessguanineribonucleotidesis sufficientto inhibitcell growth. We attemptedin additionalexperimentsto increasethe adeninenucleotidepoolswhilemaintainingelevatedguaninenucleotidelevels.However,the additionof adenineimmediatelyreducedGTPpoolsbycompeting for 5-phosphoribosyl1-pyrophosphate.Similarly,

i

L53-36-31

8-flQuo dGuo+B-flCiuo

58-39-33

Guo + B-flGuo

Chart 4. Effect of 2'-deoxyguanosine (dGuo) and 8-aminoguanosine (8-AGuo)

on the DNA content of HPRT-deficient cells. The histograms were obtained fromquadruplicate determinations in a single experiment. Similar data were obtained intwo additional experiments. Numerical values are estimations of the percentage ofcells with GO-G,, S-phase and M DNA content. A, 8-aminoguanosine alone; 8, 8-aminoguanosine plus 2'-deoxyguanosine; C, 8-aminoguanosine plus guarÃosme.

coincubationof cells with either adenosineand an inhibitorofadenosinedeaminaseor 5-amino-4-imidazolecarboxamideri-boside,an intermediatein de novopurinebiosynthesis,resultedin increasesin ATPlevelsbut markedlypreventedthe accumulationof GTP.Hencewe were unableto dissociatethe growthinhibitorypropertiesof guaninenucleotideaccumulationfromtheconcomitantadeninenucleotidedepletion.

Recentwork with the S-49 murineT-lymphomalinehas addressedthe relativecontributionsof adenineand guanineribonucleotidedepletionto cellulartoxicity inducedby inhibitionofde novo purinebiosynthesis.The toxic effects of 6-MMPR,aknown inhibitorof the amidotransferaseenzyme,have beendemonstratedto result predominantlyfrom guaninenucleotidedepletion(19,20).Theuseof alanosine,an inhibitorof adenylo-succinicacid synthetase,to deplete specificallythe adeninenucleotidepool while leavingGTPlevelsunaffectedresultedinminimalinhibitionof isotopeincorporationinto RNAor DNAbut


4943




fl.

5<-i-33-t 3

CDNTRDL CONTROL

B-flGuo dtÃ uo * B-flQuo B-nnPR RZHSERINE

57-39-13

Chart 7. Effects of 2'-deoxyguanosine (c/Guo), fr-MMPR, or azaserine on the

ONA content of B-lymphoblasts. Quadruplicate determinations were performed in5 g â€”2 E â€”-1 5 each of two independent experiments. Incubations were carried out with 50 UM2'-

deoxyguanosine plus 100 MM8-aminoguanosine (8-AGuo), 2 MM6-methylmercap-topurine riboside or 10 MMazaserine.

hrs g L, hrs.

185-13-3 L.3-L.8--1B

ai-i hrs Â« a"-i hrs .Charts. Effect of 2'-deoxyguanosine and 8-aminoguanosine on DNA content

of T- and B-lymphoblasts. The histograms were obtained from quadruplicatedeterminations. A. T-lymphoblasts (MOLT 4) incubated in the presence of 3 MM2'-

deoxyguanosine plus 100 MM8-aminoguanosine; 8, B-lymphoblasts (MGL 8) incubated in the presence of 50 MM2 ' -deoxyguanosine and 100 MM8-aminoguanosine.

1IMI1-4lu 24 hr 4 hr 24 hr 4 hr 24 hr

2' DEOXYGUANOSINE GUANOSINE 6 MMPR

Oif

Charts. Effects of 2'-deoxyguanosine. guanosme. and 8-aminoguanosine on

punne biosynthesis in B-lymphoblasts. Punne biosynthesis was measured by theincorporation of [14C)formate into total cellular and extracellular purines. The data

are the mean of two experiments. Incubations were done in the absence (LI) orpresence P) of 100 MM8-aminoguanosine. 2'-Deoxyguanosine, 50 MM;guanosine,

50 MM;6-methylmercaptopurine riboside, 2 MM.

did result in a block within early S phase of the cell cycle. Incontrast specific depletion of GTP by an inhibitor of IMP dehy-drogenase resulted in marked inhibition of DNA synthesis and ablock at the Gi-S interface. It appears from these studies that

adenine nucleotide depletion plays only an ancillary role in thetoxicity induced by inhibiting purine biosynthesis and may notsolely account for the GTP-mediated inhibition of growth which

we have observed. It should also be noted that UTP pools weredepressed in the face of GTP elevation, suggesting an additionalinhibitory effect of GTP elevation on pyrimidine biosynthesis.However, the addition of uridine did not prevent growth inhibition,making it unlikely that pyrimidine starvation played a role ininhibiting cell growth.

We then explored the effects of guanine nucleotide elevationson cyclic nucleotide levels. GTP, in conjunction with a membrane-bound regulatory protein, modifies the activity of adenylate cy-clase and hence is an important potential regulator of intracellularcAMP levels (12). In addition elevation of GTP, a substrate forguanlyate cyclase, could result in increases in intracellular cGMPlevels. However, we were unable to document any significantchange in either cAMP or cGMP in cultured lymphoid cells.Although other investigators have also failed to document effectsof fluctuating GTP concentrations on steady state cAMP levelsin other cell types (21, 22), it remains possible that effects oncompartmentalized cyclic nucleotide pools might not be detectedby these methods. For example low GTP levels have shown toimpair the p'-adrenergic responsiveness of adenylate cyclase

activity in intact cells in the absence of any detectable effect onbasal cAMP levels (21).

The differential effects of 2'-deoxyguanosine plus 8-amino

guanosine on the cell cycle of T-lymphoblasts as compared toB-lymphoblast and mature T-cell lines reflect the different bio

chemical bases underlying the cytotoxicity to these cells. dGTPaccumulation in the T-lymphoblasts is associated with a pronounced block at the G,-S interface, analogous to that seen incells incubated with 2'-deoxyadenosine and an inhibitor of aden-

osine deaminase but distinctly different from the later S-phase

block induced by hydroxyurea and thymidine (23, 24). GTPaccumulation in B-lymphoblasts and mature T-cells on the other

hand causes a slowing of progression through S phase which issimilar to that described in HL-60 cells exposed to high 2'-

deoxyguanosine concentrations (25). Although our metabolicstudies have not elucidated the mechanism underlying accumu-


4944




lation of cells in S phase, it is clear that the effects of 2'-

deoxyguanosine on the cell cycle of leukemic cells cannot simplybe attributed to changes in the deoxyribonudeotide pools. Theuse of 2'-deoxyguanosine in the absence of a potent inhibitor of

either its degradation to guanine or guanine salvage by HPRTactivity will result in effects mediated by GTP as well. Thereforethe biochemical basis for potential synergism between 2'-

deoxyguanosine and other chemotherapeutic agents must becarefully analyzed to include the effects of ribonudeotide as wellas deoxyribonudeotide pool fluctuations.

It is becoming increasingly apparent that guanine nucleotideshave multiple regulatory functions within the cell and recentevidence indicates that GTP-binding proteins may play a majorrole in regulating cell growth and transformation (11-13). Theability to increase GTP levels selectively within malignant lymph-

oid cells by pharmacological means may provide a new approachfor elucidating the functions of such proteins in human cells.

ACKNOWLEDGMENTS

We thank Robert Conn and Larry Kahn for their assistance in the ceÂ«cyclestudies and Sue Grech for typing the manuscript.

REFERENCES

1. Gibtett, E. R., Ammarai,A. J., Wara, D. W., Sandman,R., and Diamond,L K.Nucleoside-phosphorylasedeficiency in a child with severely defective T-cellimmunity and normal B-cell immunity. Lancet, 1:1010-1013,1975.

2. Carson, D. A., Kaye, J., Matsumoto, S.. SeegmiHer,J. E., and Thompson, L.Biochemical basis for the enhanced toxicity of deoxyribonudeosides towardmalignant human T cell lines. Proc. Nati. Acad. Sci. USA, 76: 2430-2433,1979.

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1985;45:4940-4945. Cancer Res Yechezkel Sidi, Jerry L. Hudson and Beverly S. Mitchell Metabolism and Cell Cycle of Human Lymphoid CellsEffects of Guanine Ribonucleotide Accumulation on the

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