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

Home , Deoxyadenosine, Deoxyguanosine, Deoxyribonucleoside, Deoxyribonucleotide, Guanosine monophosphate, Nucleoside triphosphate

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

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 nosine to guanine and the toxicity to lymphoid cells in this disease state has been attributed to the accumulation of 2'-deoxygua- A deficiency of purine nucleoside phosphorylase activity is nosine and its selective metabolism to dGTP in T-cells. Cultured associated with marked depletion of T-lymphocytes which is felt human and murine T-lymphoblasts exposed to 2'-deoxyguano to be mediated by accumulation and further metabolism of the purine nucleoside phosphorylase substrate, 2'-deoxyguanosine. sine alone accumulate large amounts of dGTP which is felt to Human T-lymphoblasts incubated in the presence of 2'-deoxy- inhibit the enzyme ribonucleotide reducÃase,deplete dCTP pools, and consequently inhibit DNA synthesis (2-6). The addition to guanosine and the purine nucleoside phosphorylase inhibitor 8- such cultures of 8-aminoguanosine, a relatively weak competitive aminoguanosine accumulate deoxyguanosine 5'-triphosphate inhibitor of PNP (7), markedly potentiates both the toxicity of 2'- whereas B-lymphoblasts and mature T4+-cell lines accumulate (8).deoxyguanosine ' and the accumulation of dGTP by these cells GTP under identical conditions. We have compared the effects of guanine ribo- and deoxyribonucleotide accumulation on the In contrast to these data, recent studies have indicated that metabolism and cell cycle of the respective cell lines. Deoxygua cultured B-lymphoblasts and mature T4+-cell lines, as well as nosine 5'-triphosphate elevations in T-lymphoblasts are associ peripheral blood mitogen-stimulated lymphocytes, accumulate ated with inhibition of [3H]uridine incorporation into DNA and a GTP rather than dGTP when exposed to 2'-deoxyguanosine and complete block at the Gi-S interface of the cell cycle. In contrast 8-aminoguanosine (9, 10). Guanine ribonucleotide synthesis in 3- to 5-fold increases in guanosine 5'-triphosphate pools in B- lymphoblasts and mature T-cell lines do not inhibit [3H]uridine these cells requires HPRT activity and is apparently mediated by the prolonged availability of intracellular guanine resulting from a incorporation into DNA or RNA but do cause a pronounced slower rate of 2'-deoxyguanosine phosphorolysis in the pres slowing in the progression of cells through S phase. B-lympho ence of a weak PNP inhibitor; more potent inhibition of PNP blasts deficient in the salvage enzyme hypoxanthine guanine phosphoribosyltransferase do not accumulate guanosine 5'-tri- activity completely abolishes both guanine and guanine ribonu phosphate from 2'-deoxyguanosine and progress normally cleotide formation (9). Of particular interest, however, is the observation that guanine ribonucleotide accumulation is directly through the cell cycle, demonstrating a requirement for guanine associated with inhibition of cellular proliferation (9,10). Because salvage to inhibit cell growth. Guanine ribonucleotide accumula of the increasing evidence that guanine ribonucleotides may be tion was also associated with inhibition of de novo purine bio important regulators of cell growth (11-13), we have investigated synthesis and a moderate decline in adenine nucleotide pools the effects of guanine ribonucleotide accumulation generated by but not with inhibition of protein synthesis or alterations in basal the exposure of cells to 2'-deoxyguanosine and 8-aminoguano levels of 3':5'-cyclic adenosine monophosphate or 3':5'-cyclic sine on the metabolism and cell cycle of cultured human lymphoid guanosine monophosphate. We conclude that the accumulation cell lines. of guanine ribonucleotides by actively cycling human lymphoid cells is associated with an increase in S-phase cells and inhibition of growth. This effect is distinctly different from that produced MATERIALS AND METHODS by 2'-deoxyguanosine 5'-triphosphate and should be taken into account in pharmacological studies with 2'-deoxyguanosine and Chemicals. 2'-Deoxyguanosine, guanosine, 6-methylmercaptopurine its analogues. riboside and azaserine were purchased from Sigma (St. Louis, MO). 8- Aminoguanosine was a gift from Dr. L. Townsend, Department of Medic inal Chemistry, University of Michigan. [5,6-3H]Uridine (40 Ci/mmol) was INTRODUCTION purchased from New England Nuclear (Boston, MA). Sodium [14C]for- Inherited deficiency of PNP4 results in a severe immunodefi mate (40-60 Ci/mmol) was purchased from ICN (Irvine.CA). i_-[4,5-3H]- ciency syndrome characterized by T-)ymphocyte depletion (1). leucine (46 Ci/mmol) was purchased from Amersham, Arlington Heights, PNP catalyzes the conversion of guanosine and 2'-deoxygua- IL. Cell Lines. MOLT-4 and RPMI-8402 T-cell lymphoblastic leukemia 1This investigation was supported by Grant CA 34085 awarded by the National lines were obtained from HEM Research, Rockville, MD. The MGL-8 B- Cancer Institute, Department of Health and Human Services. lymphoblast line was originally obtained from Dr. John Ã¼tttefield,Johns 2 Present address: Department of Medicine D, Beilinson Medical Center, Petah Hopkins University, Baltimore, MD. B-lymphoblast lines GM-558, GM- Tikva, Israel. 2292, and GM-1899 were obtained from the Human Genetic Mutant Cell 3 Scholar of the Leukemia Society of America. To whom requests for reprints Repository, Bethesda, MD. The mature T-cell lines HUT 78 and HUT 102 should be addressed, at Simpson Memorial Institute, 102 Observatory, Ann Arbor, Ml 48109. lines were obtained from Dr. P. Bunn, National Cancer Institute, Wash 'The abbreviations used are: PNP, purine nucleoside phosphorylase; PCA, ington, DC (14,15). The cells were grown in RPM11640 medium (Grand perchloric acid; TCA, trichloroacetic acid; 6-MMPR, 6-mercaptopurine riboside; HPRT, hypoxanthine guanine phosphoribosyltransferase; PBS, phosphate-buffered Island Biological Co. Laboratories, Grand Island, NY) containing 10% saline; CAMP, 3':5'-cyclic AMP; cGMP, 3':5'-cydic GMP. horse serum. All incubations were performed at a concentration of 2 x Received 1/8/85; revised 5/22/85; accepted 6/27/85. 105 cells/ml in a 5% CO2 humidified incubator.

Nucleotide Pool Measurements. Nucleotide pools were quantitated addition of the same amount of [3H]uridine to cell pellets pretreated with using high pressure liquid chromatography on a Partisil-10 SAX aniÃ³n cold 0.5 N PCA. exchange column (Whatman, Clifton, NJ). Following incubations 0.5-1 Protein synthesis was measured by incubating 2-ml aliquots of cell x 107 cells were washed once, resuspended in 150 n\ PBS and extracted suspensions with 2 >iCi of [3H]leucine for the last 2 h of incubation. The with 170 i-l ice cold 1 N PCA. After 5 min at 4Â°C the extract was cells were washed 2 times in cold phosphate buffered saline and 1 ml neutralized with 1 N KOH. Fifty to 200 n\ of the supernatant were injected 10% TCA was added at 4Â°C.The sediment was filtered on 24-mm GF/ for each run. All ribo- and deoxyribonucleoside triphosphates were A fiberglass filters (Whatman, Clifton, NJ), washed with cold 10% TCA completely separated using a linear gradient from 50% Buffer A (0.002 and counted. M NH4HPO4, pH 2.8) to 60% Buffer B (0.75 M NH4HPO4, pH 3.9) over 30 DNA Content Determinations. DNA content analyses were performed min at a flow rate of 2 mi/min. Mono-, di-, and trinucleotides were on 1-3 x 105 cells following the specified incubations. The cells were separated using a stepwise gradient of the same buffers begmnning at washed twice with cold PBS. Chicken RBC suspension (200 ^l) was 100% Buffer A and ending at 60% Buffer B over 80 min. IMP, GMP, and added as an internal standard and 1 ml of freshly made propidium iodide xanthosine 5'-phosphate were not well separated from each other under stain consisting of 75 Ã•/Mpropidium iodide: 10 mM NaCI:0.01 MTris:RNase these conditions. The individual nucleotide concentrations were calcu (700 /nucleotides PARA 1 program running on an E.A.SY/Terak computer (EPICS Division, (New England Nuclear, Boston, MA). Cell pellets containing 2-4 x 107 Coulter Corporation, Hialeah, FL). cells were suspended in buffered salt solution consisting of 138 mM NaCI, 2.7 mM KCI, 8.1 HIM NaiHPO4, 1.5 rriM KH2PO4, 1 M MgCb, and 0.6 mM CaCI2, pH 7.4, and 0.2-ml aliquots were added to 1 ml boiling RESULTS 0.5 mu theophyllin in PBS. The samples were diluted in 2 ml of 0.05 M sodium acetate buffer, pH 6.2, sonicated for 5 s, and centrifugea at 4000 Nucleotide Pool Alterations. The effects of incubations of B- x g for 15 min. The standard curve for cAMP was linear in the range of lymphoblasts, mature T-cell lines, and T-lymphoblasts with 2'- 0.01-0.20 pmol/sample. The standard curve for cGMP was linear in the deoxyguanosine and 8-aminoguanosine on nucleotide pools are range of 0.005-0.250 pmol. The recovery of standard cAMP and cGMP shown in Chart 1. Following 24-h incubations, B-lymphoblasts was over 95% using the above extraction procedure. GTP in concentra (HPRT positive) and mature T-cell lines accumulated 3- to 5-fold tions up to 10,000 pmol/100 n\ did not change the values obtained from the baseline level of GTP. In contrast T-lymphoblasts showed no standard cGMP samples. increment in GTP but a striking increase in dGTP. B-lymphoblasts De Novo Furine Biosynthesis Determinations. The effects of nucleo- sides and 6-MMPR on purine biosynthesis were measured by a modifi which are HPRT negative did not accumulate either GTP or cation of the method of Hershfield and Seegmiller (16) after preincubation dGTP. These data are consistent with previously described of the cells with the specified additives at a cell concentration of 2 x 105/ experiments (8,9). In the presence of elevated intracellular GTP ml. Two h before ending the incubation the cells were pelleted and the levels, ATP pools decreased by means of 17 and 15% for the suspension volume was adjusted to 2 ml RPM11640 medium plus 10% two HPRT positive B-lymphoblast lines and 42 and 21% for the horse serum at a concentration of 10s cells/ml. Twenty nC\ of sodium mature T-cell lines. LTTPpods were similarly reduced by 24 and [14C]formate were added for the final 2-h incubation at 37Â°C. The 18% in B-lymphoblasts and 32 and 29% in mature T-cells, while samples were centrifugea and the cells were washed in cold phosphate- CTP levels were unaffected. The accumulation of dGTP in T- buffered saline and resuspended in 1 ml of cold RPMI 1640 medium. lymphoblasts was also associated with reductions in ATP levels Four ml of 1.25 N PCA were added to the cell suspensions and to 1 ml which were most pronounced in the RPMI 8402 cell line. of the supematants. The samples were covered with aluminum foil, In order to see whether the changes in nucleoside triphosphate boiled for 1 h, and cooled. The supematants were transferred to tubes containing 1 mg adenine. Fifty-eight % NH4OH (1.8 ml) was added, the pools reflect similar changes in nucleoside mono- and diphos- tubes were vortexed, and 0.5 ml 10% AgNO3 was added. The tubes phate pool sizes, we measured the changes in adertine and were left at 4Â°Covernight. The sediment was washed 6 times with cold guanine mono-, di-, and triphosphate pools in B-lymphoblasts water until the supematants contained no radioactivity and resuspended and mature T-cells which resulted from incubation with 8-ami noguanosine and 2'-deoxyguanosine (data not shown). In both in 0.5 ml of 0.1 N HCI. The samples were boiled for 1 h and the supematants were counted in Bray's scintillation fluid. Cell suspensions cell types the marked increase in GTP levels was associated and supematants pretreated with PCA before the addition of [14C]formate with an approximately 4-fold increase in GDP and minimal ele served as blanks. vation of GMP. These alterations in guanine nucleotide pools DNA, RNA, and Protein Synthesis. Undine incorporation into DNA were associated with divergent effects on adenine nucleotide and RNA was measured in cells incubated for different times and with pools. In the B-lymphoblast line there was a small drop in ATP the specified additives by adding 1 Â¿iCiof[3H]uridine to 2-ml aliquots for levels with no significant change in AMP or ADP pools in four the last 2 h of incubation. The cell pellet was washed twice with cold separate experiments. In contrast there was a 5- to 6-fold PBS and 1 ml 0.5 N cold PCA was added. The sediment was washed consecutively with etherethanol (1:1) solution and 10% TCA at 4Â°Cand reduction in AMP and ADP in the HUT-78 line while ATP levels suspended in 0.4 ml of 0.3 N KOH. The tubes were incubated for 20 h fell to less than 50% of control values in two experiments. at 37Â°C under parafilm cover. The solution was cooled and 10 ^l 10 N Despite these fluctuations the energy charge for adenine ribo- PCA were added. The supernatant was neutralized by 0.2 ml 0.7 N KOH nucleotides remained constant for both cell types. and the clear supernatant containing the RNA hydrolysate was counted. The effect of increases in intracellular GTP on cyclic nucleotide The sediment was washed with 10% cold TCA, suspended in 0.6 ml 10 pools in the MGL-8 B-lymphoblast line is shown in Table 1. N TCA, and dissolved by heating for 1 h at 70Â°C. The samples were Despite a mean 5-fold increase in GTP levels, cAMP levels counted using Bray's scintillation fluid. Blanks were obtained by the remained unchanged. The cGMP concentration increased 2-fold

incorporation into DNA and RNA and [3H]leucine incorporation into protein. Chart 2 demonstrates a lack of inhibition of [3H]- IO5 uridine incorporation into DNA in B-lymphoblasts (A, HPRT pos itive; B, HPRT negative) and mature T-cells (C), whereas DNA synthesis in T-lymphoblasts accumulating dGTP is markedly ^ATP1LT r^ J impaired (D). Similarly there is no demonstrable inhibition of RNA 0IO5Ã¨ or protein synthesis in the B- or mature T-cells; the apparent rise in the incorporation of [3H]uridine into RNA in B-lymphoblasts and to a lesser extent in the HUT 78 cells was a consistent finding in these experiments perhaps explained by the decrease TIE Â¡nUTPpool sizes and an increase Â¡nUTPspecific activity. nÂ»UTP-^ FÃ‰n^ We then examined the effects of GTP accumulation on the DNA content of the cell lines. The progression of normal B- lymphoblasts through S phase was slowed during incubations io4."5 with 50 Â»Mguanosine or 2'-deoxyguanosine in the presence of 8-aminoguanosine (Chart 3) whereas no change in DNA content ,->.CTP-â€¢â€¢^^jr1^ r-r^r"^ n*, r3^ ^*, occurred in B-lymphoblasts deficient in HPRT activity (Chart 4). oIO5 The changes in cell cycle were maximal after 24 h of incubation and returned to the control pattern after 72 h. The time course of these changes was parallel to the time course of GTP accu mulation which was also maximal after 24 h in the HPRT-positive B-lymphoblasts. Similar cell cycle changes were observed during incubations with 300 UMguanosine or 2'-deoxyguanosine in the ~ndGTP. j bxiÃ¬ | "* Â£?ÃŒ1 i ~ijr r""~Li* â€¢ n absence of 8-aminoguanosine. In contrast to these findings, T- 0201GTP_ lymphoblasts incubated with 3 MM 2'-deoxyguanosine plus 8- aminoguanosine demonstrated a very early block in S-phase leading 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 adenine yIML86M998 6MI899 6M2292 HUT78 HUTI02 MOLT4 RPMI8402 n-18) (n-12) (n-4) (n-6) (n-13) (n-6) in. e) (n-4) nucleotides in cells accumulating GTP led us to ask whether ) LYMPHOBLASTS B LYMPHOBLASTS MATURE T CELL T LYMPHOBLASTS GTP was significantly inhibiting de novo purine biosynthesis. We (HPRTt) (HPRT-) LINES therefore measured the rate of incorporation of [14C]formate into Chart 1. Effects of 2'-deoxyguanosine and 8-aminoguanosine on nucteoside intracellular and extracellular purines during incubations of B- triphosphate pools. Measurements were done after 24-h incubations in the pres ence of 100 MM8-aminoguanosme alone (Q) or 50 MM2 ' -deoxyguanosine plus 100 lymphoblasts with 2'-deoxyguanosine, guanosine, or 6-MMPR, Â»IM8-aminoguanosine (i i), n. number of independent experiments which were a known inhibitor of de novo purine synthesis. Chart 6 demon performed with each cell line; oars, SD. strates that the rate of formate incorporation was markedly reduced by either Quo or dGuo alone at 4 h and by the combi Table 1 nation of Quo or dGuo and 8-AGuo at 24 h. The magnitude of Effects of GTP elevations on cyclic nucleotide pools the inhibition at 24 h was similar to that caused by 6-MMPR. 6- MGL-8 B-lymphoblasts were incubated for 24 h in the presence of 100 Â«M8- aminoguanosine alone or 50 Â¡M2' -deoxyguanosme plus 100 Â¡M8-aminoguanosine. MMPR and another inhibitor of de novo purine biosynthesis, Nucleotide pool sizes were measured as described in 'Materials and Methods." azaserine, also produced an accumulation of cells in S phase (Chart 7) which was somewhat similar to the effects of 2'- GTP CAMP cGMP (pmol/10Â«cells) (pmol/107 cells) (pmol/107 cells) deoxyguanosine or guanosine on these cells. Additives n = 6 n = 4 n = 6 Â±360* (6)" Control Â±3 (4) Â±0.1 (6) 8-Aminoguanosine+ 7966 Â±1822 (6)8 11 Â±1 (4)0.4 0.8 Â±0.2 (6) DISCUSSION dGuo1574 ' Mean Â±1 SD. We have demonstrated previously that a 3- to 5-fold elevation " Numbers in parentheses, number of experiments performed in duplicate. in intracellular GTP pools which is sustained over a 24-h period in lymphoid cells lines causes marked inhibition of cellular prolif but this difference was not statistically significant (0.05 < P < eration, whereas transient increases in GTP of less than 12 h 0.1). duration have no effect on cell growth (9). Our current studies Effects of Guanine Ribonucleotide Accumulation on DMA, document that GTP accumulation at 24 h is associated wtih (a) RNA, and Protein Synthesis and the Cell Cycle. We have a mild to moderate decline in ATP levels, more pronounced in shown previously that dGTP-mediated toxicity for T-lympho- mature T-cell lines than in B-lymphoblasts; (b) inhibition of de blasts is associated with a marked decrease in [3H(uridine incor novo purine biosynthesis; (c) no significant alterations in intra poration into DNA which is most probably due to inhibition of cellular cyclic AMP or GMP levels; (d) no direct inhibition of ribonucleotide reducÃase activity (6). Since GTP is an essential incorporation of [3H]uridine into DNA or RNA, or demonstrable prerequisite for both RNA and protein biosynthesis, we com inhibition of protein synthesis; and (e) accumulation of cells in S pared the effects of GTP and dGTP accumulation on [3H]uridine phase.

CANCER RESEARCH VOL. 45 OCTOBER 1985 4942

Chart 2. Effects of 2'-deoxyguanosine and 8-aminoguanosine on RNA. DNA, and protein synthesis. RNA (â€¢)andDNA (O) synthetic rates were measured by the incorporation of [3H] uridine into acid-insoluble material as described in "Materials and Methods." The protein syn thetic rate (G) was measured by the incorpora tion of [3H]leucine into acid-insoluble material. Incubations were done in the presence of 50 pin 2'-deoxyguanosine and 100 Â¡M8-amino guanosine and results were compared to con O trol incubations with 100 /IM 8-aminoguanosine Ãœ alone. The data are the results from a single 200 experiment; similar results were obtained in a second experiment. A, B-lymphoblast line GM- 558 (HPRT positive); B, B-tymphoblast line GM- O 1899 (HPRT negative); C, mature T-cell line OC HUT-78; D, T-lymphoblast line MOLT-4. lOO

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8-flQuo dGuo+B-flCiuo Guo + B-flGuo dtiuo * B-RGuo Quo t 8-fÃ¬Guo Chart 4. Effect of 2'-deoxyguanosine (dGuo) and 8-aminoguanosine (8-AGuo) Charta. Effect of 2'-deoxyguanosine (dGuo) and 8-aminoguanosine (8-AGuo) on the DNA content of HPRT-deficient cells. The histograms were obtained from on the DNA content of GM-558 cells. The histograms were obtained from quadru quadruplicate determinations in a single experiment. Similar data were obtained in plicate determinations in a single experiment. Similar data were obtained in two two additional experiments. Numerical values are estimations of the percentage of additional experiments. Values above the figures are estimations of the percentage cells with GO-G,, S-phase and M DNA content. A, 8-aminoguanosine alone; 8, 8- of cells with Go-G,, S-phase and M DNA content, respectively. A, 8-aminoguanosine aminoguanosine plus 2'-deoxyguanosine; C, 8-aminoguanosine plus guarÃosme. alone; B, 8-aminoguanosine plus 2'-deoxyguanosine; C, 8-aminoguanosine plus guarÃosme(Quo). coincubationof cells with either adenosineand an inhibitorof The combinationof pronouncedinhibitionof [14C]formatein adenosinedeaminaseor 5-amino-4-imidazolecarboxamideri- corporationinto total purines with decreasesin the adenine boside,an intermediateinde novopurinebiosynthesis,resulted nucleotidepoolprovidesgoodevidencethatincreasesinguanine in increasesinATPlevelsbut markedlypreventedtheaccumu ribonucleotidesinhibit de novo purine biosynthesisin these lationof GTP.Hencewe were unableto dissociatethe growth lymphoidcells.This effect is most probablyattributableto the inhibitorypropertiesofguaninenucleotideaccumulationfromthe allostericinhibitionof glutaminephosphoribosylpyrophosphateconcomitantadeninenucleotidedepletion. amidotransferase,an enzyme pivotal to purine biosynthesis Recentwork with the S-49 murineT-lymphomalinehas ad whichis allostericallyinhibitedbyGMPandto a lesserextentby dressedthe relativecontributionsof adenineandguanineribo GDPandGTP(18). nucleotidedepletionto cellulartoxicity inducedby inhibitionof It remainsunclear,however,whetherthis degreeof adenine de novo purinebiosynthesis.Thetoxic effects of 6-MMPR,a ribonucleotidedepletionin the presenceof excessguanineri known inhibitorof the amidotransferaseenzyme,have been bonucleotidesissufficientto inhibitcell growth. We attempted demonstratedto result predominantlyfromguaninenucleotide in additionalexperimentsto increasethe adeninenucleotide depletion(19,20).Theuseof alanosine,aninhibitorof adenylo- poolswhilemaintainingelevatedguaninenucleotidelevels.How succinicacid synthetase,to deplete specificallythe adenine ever,the additionof adenineimmediatelyreducedGTPpoolsby nucleotidepoolwhileleavingGTPlevelsunaffectedresultedin competing for 5-phosphoribosyl1-pyrophosphate.Similarly, minimalinhibitionof isotopeincorporationintoRNAor DNAbut

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Chart 7. Effects of 2'-deoxyguanosine (c/Guo), fr-MMPR, or azaserine on the ONA content of B-lymphoblasts. Quadruplicate determinations were performed in 57-39-13 5 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 MM 6-methylmercaptopurine riboside or 10 MMazaserine.

did result in a block within early S phase of the cell cycle. In hrs g L, hrs. contrast specific depletion of GTP by an inhibitor of IMP dehy- drogenase resulted in marked inhibition of DNA synthesis and a block at the Gi-S interface. It appears from these studies that adenine nucleotide depletion plays only an ancillary role in the toxicity induced by inhibiting purine biosynthesis and may not 85-13-3 L.3-L.8--1B solely account for the GTP-mediated inhibition of growth which we have observed. It should also be noted that UTP pools were depressed in the face of GTP elevation, suggesting an additional 1 inhibitory effect of GTP elevation on pyrimidine biosynthesis. ai-i hrs Â« a"-i hrs . However, the addition of uridine did not prevent growth inhibition, Charts. Effect of 2'-deoxyguanosine and 8-aminoguanosine on DNA content making it unlikely that pyrimidine starvation played a role in of T- and B-lymphoblasts. The histograms were obtained from quadruplicate inhibiting cell growth. determinations. A. T-lymphoblasts (MOLT 4) incubated in the presence of 3 MM2'- We then explored the effects of guanine nucleotide elevations deoxyguanosine plus 100 MM8-aminoguanosine; 8, B-lymphoblasts (MGL 8) incu bated in the presence of 50 MM2 ' -deoxyguanosine and 100 MM8-aminoguanosine. on cyclic nucleotide levels. GTP, in conjunction with a membrane- bound regulatory protein, modifies the activity of adenylate cyclase and hence is an important potential regulator of intracellular cAMP levels (12). In addition elevation of GTP, a substrate for guanlyate cyclase, could result in increases in intracellular cGMP levels. However, we were unable to document any significant change in either cAMP or cGMP in cultured lymphoid cells. Although other investigators have also failed to document effects of fluctuating GTP concentrations on steady state cAMP levels in other cell types (21, 22), it remains possible that effects on compartmentalized cyclic nucleotide pools might not be detected by these methods. For example low GTP levels have shown to impair the p'-adrenergic responsiveness of adenylate cyclase activity in intact cells in the absence of any detectable effect on basal cAMP levels (21). The differential effects of 2'-deoxyguanosine plus 8-amino guanosine on the cell cycle of T-lymphoblasts as compared to B-lymphoblast and mature T-cell lines reflect the different bio chemical bases underlying the cytotoxicity to these cells. dGTP accumulation in the T-lymphoblasts is associated with a pro

O nounced block at the G,-S interface, analogous to that seen in if cells incubated with 2'-deoxyadenosine and an inhibitor of adenosine deaminase but distinctly different from the later S-phase lu 24 hr 4 hr 24 hr 4 hr 24 hr block induced by hydroxyurea and thymidine (23, 24). GTP 1IMI1-42' DEOXYGUANOSINE GUANOSINE 6 MMPR Charts. Effects of 2'-deoxyguanosine. guanosme. and 8-aminoguanosine on accumulation in B-lymphoblasts and mature T-cells on the other punne biosynthesis in B-lymphoblasts. Punne biosynthesis was measured by the hand causes a slowing of progression through S phase which is incorporation of [14C)formate into total cellular and extracellular purines. The data similar to that described in HL-60 cells exposed to high 2'- are the mean of two experiments. Incubations were done in the absence (LI) or presence P) of 100 MM8-aminoguanosine. 2'-Deoxyguanosine, 50 MM;guanosine, deoxyguanosine concentrations (25). Although our metabolic 50 MM;6-methylmercaptopurine riboside, 2 MM. studies have not elucidated the mechanism underlying accumu-

CANCER RESEARCH VOL. 45 OCTOBER 1985 4944

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1985 American Association for Cancer Research. GTP TOXICITY FOR LYMPHOID CELLS lation of cells in S phase, it is clear that the effects of 2'- Inhibitors of purine nucleoside phosphorylase. C(8) and C(5') substitutions. Btochem.Pharmacol.,37:163-171,1982. deoxyguanosine on the cell cycle of leukemic cells cannot simply 8. Kazmers, I. S., Mitchell, B. S., Daddona, P. E., Wotring, L. L., Townsend, L. be attributed to changes in the deoxyribonudeotide pools. The B., and Kelley, W. N. Inhibition of purine nucleoside phosphorylase by 8- use of 2'-deoxyguanosine in the absence of a potent inhibitor of aminoguanosine:selective toxicity for T lymphoblasts. Science (Wash. DC), 274:1137-1139,1981. either its degradation to guanine or guanine salvage by HPRT 9. SkJi,Y., and Mitchell, B. S. Deoxyguanosinetoxicity for B and mature T cell activity will result in effects mediated by GTP as well. Therefore lines is mediated by guanine ribonucleotideaccumulation. J. Clin. Invest., 74: the biochemical basis for potential synergism between 2'- 1640-1648,1984. 10. Spaapen, L. J. M., Rijkers, G. T., Staal, G. E. J., Rijksen, G., Wadman, S. K., deoxyguanosine and other chemotherapeutic agents must be Stoop. J. W., and Zegers, B. J. M. The effect of deoxyguanosine on human carefully analyzed to include the effects of ribonudeotide as well lymphocyte function. J. Immunol.. 732:2311-2323,1984. 11. Gibbs, J. B., Sigal, I. S., Poe, M., and Scolnick, E. M. Intrinsic GTPaseactivity as deoxyribonudeotide pool fluctuations. distinguishesnormal and oncogene ras p21 molecules. Proc. Nati. Acad. Sd. It is becoming increasingly apparent that guanine nucleotides USA,87: 5704-5708,1984. 12. Oilman,A. G. Guaninenucleotide-bindmgregulatory proteins and dual control have multiple regulatory functions within the cell and recent of adenylatecydase. J. Clin. Invest., 73:1-4,1984. evidence indicates that GTP-binding proteins may play a major 13. Hughes, S. M. Are guanine nucleotide binding proteins a distinct dass of role in regulating cell growth and transformation (11-13). The regulatory proteins? FEBS Lett., 764:1-8,1983. ability to increase GTP levels selectively within malignant lymph- 14. Gazdar,A. F.,Carney,D.N., Bum, P.A., Russen,E.K.,Jaffe,E.S., Schlechter, G. P., and Guccion, L. G. Mitogen requirementsfor the in vitro propagation of oid cells by pharmacological means may provide a new approach cutaneous T-cell lymphoma. Blood, 55:409-417,1980. for elucidating the functions of such proteins in human cells. 15. Poiesz. B. J., Ruscetti, F. W., Gazdar, A. F., Bunn. P. A., Minna, J. D., and Gallo, R. C. Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc. Nati. Acad. Sd. USA, 77: 7415-7419,1980. ACKNOWLEDGMENTS 16. Hershfield.M. 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CANCER RESEARCH VOL. 45 OCTOBER 1985 4945 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1985 American Association for Cancer Research. Effects of Guanine Ribonucleotide Accumulation on the Metabolism and Cell Cycle of Human Lymphoid Cells

Yechezkel Sidi, Jerry L. Hudson and Beverly S. Mitchell

Cancer Res 1985;45:4940-4945.

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