Effect of Gallium on DNA Synthesis by Human T-Cell Lymphoblasts David W

[CANCER RESEARCH 48, 3014-3018, June 1, 1988] Effect of Gallium on DNA Synthesis by Human T-Cell Lymphoblasts David W. Hedley,1 Edith H. Tripp, Peter Slowiaczek, and Graham J. Mann2

Ludwig Institute for Cancer Research (Sydney Branch), Blackburn Building, University of Sydney, Sydney, N.S. W. 2006, Australia

ABSTRACT (8). Iron is required for a range of cell processes, but surface transferrin receptor expression is markedly enhanced prior to We have studied the antiproliferative effects of gallium nitrate in the initiation of S-phase (9), suggesting that the receptor is cultured CCRF-CEM lymphoblasts. The 50% inhibitory dose for these cells was 120 MM,and after 24 h at a cytostatic concentration (480 MM) regulated to meet specificiron requirements for DNA synthesis. S-phase arrest was observed by DNA flow cytometry. Deoxyribonucleo- One of the most significant requirements would be incorpora side triphosphate pools were all reduced (dA IP, dGTT, and dCTP by tion into the non-heme iron-containing (M2) subunit of ribo 50%, d'lTP by 25%), suggesting inhibition of ribonucleotide reducÃase. nucleotide reducÃase,the enzyme catalyzing the first unique, Administration of tracer amounts (0.5 MM)of either | ÃŒl|uridincor ['I I|- rate-limiting step in DNA synthesis (10). Although it has been deoxyuridine confirmed that DNA synthesis had been inhibited to 20% previously suggested that gallium might inhibit this enzyme (4), of control rates by gallium. Further, the flow of the ribonucleoside into this has not been tested experimentally. Indeed, to our knowl the dTTP pool and DNA was selectively reduced compared to that of the edge the only published investigations of gallium's mode of deoxyribonucleoside. Gallium decreased the specific activity of dTTP action have implicated either DNA polymerase (11) or compe labeled from uridine by 50%, whereas the specific activity of dTTP labeled from deoxyuridine was increased 2.5-fold. Thus counts in DNA tition with divalent cations (12) as possible targets. As an derived from [3H]uridine were decreased by more than 80%, while counts extension of some earlier work suggesting that transferrin in DNA derived from [3H]deoxyuridine were virtually unaltered. Uridine receptor is specifically regulated to meet an iron requirement incorporation into RNA was not affected. Gallium did not significantly for DNA synthesis (13) we investigated the effects of growth alter the capacity of permeabilized naive cells to incorporate | 'I I|dTI P inhibitory concentrations of gallium on both replicative and into DNA, while 24-h gallium pretreatment (which increased the per repair DNA synthesis. The results shown here suggest that centage of S-phase cells) produced a modest increase in pHJd'l'lP ribonucleotide reductase is the principal enzyme target. incorporation, indicating that any effect of gallium on DNA polymerase a is minor. Gallium treatment did not induce or inhibit the repair of DNA single strand breaks. These data demonstrate that gallium inhibits rep MATERIALS AND METHODS licative DNA synthesis, with the major specific enzyme target probably being ribonucleotide reducÃase. Chemicals and Isotopes. Gallium nitrate 99.999% pure was obtained from Aldrich Chemicals (Milwaukee, WI); aphidicolin, polydeoxyadenylic-deoxythymidylic and polydeoxyinosinic-deoxycytidylic acids, un- INTRODUCTION labeled ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, and dTTP were A number of Phase II trials have shown that nonradioactive from Sigma (St. Louis, MO); Micrococcus luteus DNA polymerase from Miles Laboratories (Ekhart, IN) and all radiolabeled nucleosides gallium has activity against some human cancers, particularly and nucleotides were purchased from the Radiochemical Centre, Amer- lymphomas (1). The earlier animal studies were prompted both sham, England. by the antineoplastic properties of compounds of another heavy Cell Culture. The human T-lymphoblast cell line CCRF-CEM (14) metal, platinum, and by the observation that 67Gais selectively was grown as a static suspension culture in RPMI 1640 tissue culture concentrated in tumor masses (2). The basis of gallium's tumor medium supplemented with 10% fetal calf serum and 2 mM gÃ¬marnine. selectivity is not fully understood. Its ionized form binds to These cells have a doubling time of approximately 20 h and were used transferrin and it is thought that the surface transform receptor during exponential growth. is involved in its cellular uptake (3, 4). Unlike iron, however, Measurement of DNA and RNA Synthesis. After 24-h exposure to gallium, cells were spun down, resuspended in their used media in 1- gallium is not greatly concentrated in reticulocytes, and it has ml amounts at 1 x IO6cells/ml, and incubated at 37Â°Cfor 2.5 h with been proposed that its uptake into tumor masses involves 1/iCi [3H]thymidine (45 Ci/mmol) and 0.05 nC\ [14C]uridine (450 mCi/ transferrai to a pool of tissue transferrin, perhaps mediated by mmol). After the cells were washed, nucleic acids were precipitated reduced affinity of transferrin for gallium under the more acidic with 0.5 M perchloric acid and washed once in 10% trichloroacetic acid conditions often found in tumors (5). The tissue iron binding and once in diethyl ethenethanol (1:1). RNA was hydrolyzed by over protein lactoferrin has been proposed as an alternative acceptor night incubation with 0.3 M KOH at 37*C, and DNA was precipitated molecule (6). by cooling to 4'C and addition of 10 M perchloric acid. The DNA was Although it possesses an atomic radius similar to iron, Ga1' then separated from the RNA-containing supernatant by centrifugation, does not undergo the ready transition between divalent and washed in 10% cold TCA3, and solubilized in 0.6 ml 10% TCA at 70"C trivalent oxidation states that makes iron so well suited as a for 45 min. The RNA-containing supernatant was neutralized with 0.7 cofactor in biological redox reactions. Poisoning of an iron- M KOH and recentrifuged. The RNA and DNA extracts were counted in Aqueous Counting Scintillant (Amersham) using an LKB 1215 Rack- containing enzyme through competitive substitution for iron Beta scintillation counter (Wallac oy, Turku, Finland) programmed for would therefore be a plausible mechanism of gallium toxicity. dual 'H and MCcounting. However, Chitambar and Seligman (7) have reported that gal In addition to measuring ['Hjthymidine uptake, DNA flow cytometry lium may block transferrin receptor-mediated iron uptake into was used to investigate the cell cycle effects of gallium. Briefly, cells cells, and an analogous effect caused by monoclonal antibodies were stained with the DNA fluorochromes ethidium bromide and to transferrin receptor can cause arrest of growth in S-phase mithramycin, using the method of Taylor (15). Cellular DNA content was then measured using an ICP22 Flow Cytometer (Ortho Instru Received 11/16/87; revised 2/1/88; accepted 2/22/88. ments, Westwood, MA), and the results displayed as frequency distri 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 bution histograms. Cell cycle phase distribution was calculated plani- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. metrically using a computer program (16). ' To whom requests for reprints should be addressed. 2Present address: Department of Physiological Chemistry, University of 3The abbreviations used are: TCA, trichloroacetic acid; dNTP, deoxynuclec- Umea, S-901 87, Sweden. side triphosphate. 3014

Measurement of dNTP Pools. This measurement was done using the dry. The filters were washed extensively (6x for 15 min) in 5% TCA DNA polymerase method (17). Cells were washed once in phosphate and counted as described above. buffered saline (Dulbecco's formula without Mg2* or Ca2+) containing Cells were either pretreated with gallium for 24 h, or gallium was 2 HIMEDTA, extracted in 1 ml 60% ethanol at -20'C, incubated at added at the time of permeabilization, and [3H]dTTP incorporation 30*C for 10 min then at -20Â°C overnight. The ethanol extract was was compared to that of untreated control cells. separated by centrifugation, the precipitate further extracted with 0.5 Measurement of DNA Repair. The measurement of DNA strand ml ethanol at 30'C for 10 min, and the ethanol supernatants pooled breaks following T-irradiation was done fluorimetrically using a modi and stored at -20"C. To measure dNTP pools, the ethanol extracts fication of the method of Birnboim and Jevcak (20, 21). Gallium- were lyophilized, taken up in 500 /il of 10 HIMTris-HCl, pH 7.85, and treated cells and controls were exposed to 1000 cGy ""Co -y-rays while spun at 30,000 x g at 4Â°Cfor 10 min. Fifty-M!aliquots of the superna on ice and then incubated at 37V in order to allow DNA repair to tants, or appropriate standard solutions of dNTPs, were assayed in occur. At intervals 1 x IO1cells were placed on ice to stop repair. These triplicate in a reaction mixture containing 0.75 HIM MgClj, 40 HIM samples were centrifuged, resuspended in 3 ml cold phosphate buffered Tris-HCl, pH 7.85, 2 HIM dithiothreitol, and 25 ni M. luteus DNA saline, and divided into 0.2-ml aliquots which then were lysed on ice polymerase and an appropriate template in a total volume of 200 ui. by the addition of 0.2 ml 9 M urea IO mM NaOH 2.5 mM diaminocy- d'l'TP and dATP pools were assayed using a polydeoxyadenylic-deox- clohexane-tetraacetate-0.1% sodium dodecyl sulfate. To one control set ythymidylic acid template (0.002 unit/assay) and ['HJdATP or |'H| of triplicates from each time point, 0.4 ml of l M glucose-14 mM 2- dTTP, respectively (0.2 ud/500 pmol/assay). Similarly, dGTP and mercaptoethanol was added to prevent DNA unwinding, subsequently dCTP were assayed using a polydeoxyinosinic-deoxycytidylic acid tem providing a measure of total fluorescence in the assay system. Another plate and [3H]dCTP or [3H]dGTP, respectively. After 60 min incubation set of triplicates was sonicated to induce complete DNA unwinding at 37'C, the reaction was stopped with 0.1 ml 0.1 M tetrasodium (background fluorescence) and a further set of triplicates served as the test samples. After 10 min at 4Â°C0.2 ml of 0.2 M NaOH was added to pyrophosphate and 1 ml 10% TCA. The TCA precipitate was filtered using a l'iter tok Seatron (Flow Laboratories), washed in 5% TCA, and all tubes. The samples were incubated on ice for a further 30 min and then placed at 15'C for 45 min. The reaction tubes were then returned the glass fiber filters dried and counted as above. In Vivo Labeling of dNTP Pools and DNA. Cells (5 x 10") re-sus to 4V and 0.4 ml of l Mglucose containing 14 mM 2-mercaptoethanol pended at 1 x 10'/ml were incubated at 37Â°Cwitheither 0.5 MM[6-3H] was added to each tube to stop further DNA unwinding. All tubes were deoxyuridine (final specific activity, 2 Ci/mmol) or 0.5 Â¿IM[6-3H]uridine then briefly sonicated to disperse the DNA and then 2 ml ethidium (2 Ci/mmol). When deoxyuridine was labeled, 0.5 MMunlabeled uridine bromide in 13.3 mM NaOH were added to give a final concentration of was included, and vice versa, while a third sample was incubated in cold 6.7 fig/ml. The fluorescence of each sample was measured using a deoxyuridine and uridine, both 0.5 Â¿tM.Afterl h ethanol extracts were Perkin-Elmer 3000 spectrofluorimeter, excitation wavelength 520 nm, prepared as described above. The residual cell pellet containing labeled emission 580 nm. nucleic acids was digested with 1.2 ml 0.6 M NaOH at 37V overnight, The percentage of double stranded DNA remaining after alkaline precipitated with 1.5 ml 10% TCA and 0.1 ml 0.1 M tetrasodium hydrolysis was calculated by subtracting the background fluorescence pyrophosphate on ice, and after 20 min collected onto glass fiber filters from sample fluorescence and then dividing by the corrected total using 5% TCA and 95% ethanol washes, and counted as described fluorescence. The results were expressed as mean percentage double stranded DNA at time intervals following â€¢>-irradiaiion. above. Aliquots of ethanol extracts from the unlabeled cells were assayed for dNTP as described above. Extracts from labeled cells were subjected RESULTS to the same DNA polymerase dNTP assay procedure except that only cold complementary dNTP was used. The label accruing to the template Effects of Gallium on Cell Growth. As has been reported by thus represented the total label in the dNTP pool being tested. As others (4), the effects of gallium were potentiated by addition previously described, these measurements allow calculation of the spe of 80 Mg/ml human apotransferrin, but much less so by iron- cific activity of the dNTP pool of interest (18). This value, in turn, is loaded transferrin (data not shown). Gallium plus apotransfer used to evaluate the counts incorporated into extracted DNA. Thus, in rin inhibited growth over the range 120-480 Â¿Â¿M,theformer "Results" we have divided DNA counts by the specific activity of the dTTP pool to estimate the "absolute rate of DNA synthesis." This producing approximately 50% growth inhibition at 24 h and the latter almost complete growth arrest. These inhibitory figure should not be taken literally because the rate of isotope equili bration into the dTTP pool may not have been the same in control and effects were more marked with further exposure, 480 /XMcaus ing cell death after a further 24 h and 120 MMafter 48-72 h. drug treated cells, and some label from uridine was found to enter DNA from the dCTP as well as the dTTP pool (data not shown). However, Most experiments were done after 24 h gallium treatment. these uncertainties are small and do not affect the interpretation of our Effects of Gallium on Nucleic Acid Synthesis. Cells were central data. exposed to either 120 or 480 /tM Ga3+ plus 80 /ig/ml apotrans DNA Polymerase Assay. In situ DNA polymerase activity in lysolec- ferrin for 24 h, and then incubated for 2.5 h with [3H]thymidine ithin-permeabilized cells was measured using a modification of the and [14C]uridine. DNA and RNA were then extracted and method of Miller et al. (19). Cells were washed twice in 150 HIM counted (Table 1). Gallium had essentially no effect on [I4C] sucrose, 80 mM KC1, 35 HIM 4-(2-hydroxyethyl)-l-piperazineethane- uridine incorporation into RNA and produced a slight increase sulfonic acid, 5 m\i potassium phosphates (pH 7.4), 5 mM MgCI2, and in |â€¢'H11bymidine incorporation, especially at the higher concen 0.5 mM CaCl2 (Solution A). They were then suspended in cold solution A at 1 x I0*/ml, and one-third volume of l mg/ml lysolecithin (type tration. 1; Sigma) in Solution A was added for 1 min at 4V. The cells were DNA flow cytometry, however, showed that gallium does then diluted into Solution A at 37V and incubated for 5 min. Permea- retard DNA synthesis (Fig. 1). There was a 50% increase in the bilization of 90-100% cells was confirmed by using try pan blue exclu sion. A DNA synthesis reaction mixture was then added to give a final Table 1 Incorporation off'HJthymidine and {"CJuridine into nucleic acids of volume of 2 x IO7 cells/ml and a final concentration of 150 mM gallium treated cells sucrose-80 mM KC1-35 mM 4-(2-hydroxyethyl)-l-piperazineethanesul- dpm fonic acid-5 mM potassium phosphates (pH 7.4)-5 mM MgCl2-0.5 mM DNA550,472into intoRNA3,354 CaCh-1.5 mM phosphoenol pyruvate-1.25 mM ATP-0.1 mM CTP, Â±52,820" GTP, and UTP-0.25 mM [3H]dTTP (0.4 Ci/mmol), dATP, dCTP, and Control Â±500 597,033 Â±38,902 2,998 Â±474 dGTP, final pH 7.4. At various time points, 25-ul triplicate aliquots 480 MMGa3"[3H]Thymidine676,564 Â±39,150[14C]Uridine 3,358 Â±510 were applied to Whatman No. 3 filters which had been previously "Mean Â±SD of five measurements. soaked in 5% TCA plus 0.2 M sodium pyrophosphate and allowed to * Pretreated with Ga3* for 24 h. 3015

*G1 se. i to be converted successively into dTMP, dTTP, and DNA. SS â€¢M.2 17.6 Deoxyuridine may be phosphorylated directly to dUMP c.v. - s.ex whereas uridine requires the sequential action of kinases and ribonucleotide reductase. The relative incorporation of these two nucleosides into the dTTP pool and DNA can therefore indicate whether the balance between de novo and salvage synthesis of deoxyribonucleotides has been affected by a partic ular drug treatment. Results obtained after 24-h exposure to a LA growth inhibitory gallium concentration (480 UM)are shown in Table 3. It should first be noted that estimation of the absolute rate of DNA synthesis confirmed that gallium inhibits it. Whether label came from deoxyuridine or uridine the values obtained were very similar and were approximately 30% of control in O gallium pretreated cells. Because the gallium treated cells in O cluded 1.5 times as many S-phase cells (Fig. 1) DNA synthesis was actually proceeding at 20% of the control rate. Note also U o that the total counts incorporated into DNA from deoxyuridine x as with the thymidine incorporation data shown in Table 1 Â« i were not depressed. The reduction in DNA synthesis was O masked by the increase in dTTP specific activity. The specific activity of the dTTP pool, when labeled by uridine, fell by one-half after 24-h gallium and this was reflected in the markedly reduced counts appearing in DNA from this source. In contrast, the specific activity of the dTTP pool, when labeled by deoxyuridine, increased 2.5-fold. Thus the dTTP pool of gallium pretreated cells labeled more efficiently from deoxyuridine and less efficiently from uridine than did the dTTP pool of control cells. Effects of Gallium on in Situ DNA Polymerase a Activity. The experiments described above suggest that gallium inhibits ribonucleotide reductase but does not significantly inhibit the 10B lSB 200 250 conversion of dUMP to thymidine. They do not, however, CHANNEL NUMBER exclude an additional effect of gallium on DNA polymerase a. Fig. l. Flow cyto met rie frequency-distribution histograms of cellular DNA The activity of this enzyme was measured by permeabilizing content of control (.-I)and CCRF-CEM cells treated for 24 h with either 120 (Â«) or 480 ((') Â»IMgallium plus 80 fig/ml apotransferrin. Channel number is in cells using lysolecithin and then incubating in a mix of riho- arbitrary units directly proportional to DNA content. Cell cycle phase distribution and deoxyribonucleoside triphosphates, containing [3H]dTTP. is given for each histogram. C.V., coefficient of variation of the G, peak. The This effectively bypasses all enzyme pathways leading up to leftmost peak is an internal biological standard. final incorporation into DNA and allows the effects of drugs Table 2 Intracellular deoxyribonucleoside triphosphate pools following on in situ DNA polymerase a to be measured. Cells were gallium treatment pretreated with 120 or 480 MMgallium for 24 h or only at the pmol/10* cells time of permeabilization. Fig. 2 shows the time course for incorporation of [3H]dTTP into DNA of control and gallium- dATP dCTP dGTP dTTP treated cells. For comparison, the effects of the classical DNA Control62.5 Â±3.5Â° Â±1.4 Â±0.032.3 Â±0.7 30.3 Â±1.142.0 20.3 Â±1.565.0 Â±2.979.562.0 Â±5.3 polymerase a inhibitor aphidicolin, 50 MM,and the ribonucle Â°Mean Â±SD of three observations. otide reductase inhibitor hydroxyurea, 1 mivi, added at the time * Twenty-four-h treatment. of permeabilization, are also shown. Because gallium was used as the nitrate salt, the effects of nitrate ion as NaNO3 were also proportion of cells in S-phase following 24-h exposure to 480 measured and shown to be identical to the control (data not UM gallium. Although this effect is conceivably the result of shown). gallium selectively killing cells in G, and G2 plus mitosis, it is Acute exposure to gallium at the time of permeabilization much more likely that the rate of DNA synthesis was being produced a modest decrease in DNA synthesis similar to that slowed, increasing the probability of a cell being in S-phase at seen with hydroxyurea, but much less than that with aphidi the time of sample preparation. The apparent normality of colin. In contrast, pretreatment for 24 h with gallium, which thymidine incorporation is explained below. increased the percentage of cells in S-phase, produced a slight Effects of Gallium on Deoxyribonucleoside Triphosphate increase in rate of [3H]dTTP incorporation. Pools. Deoxyribonucleotide triphosphate levels following treat Effects of Gallium on DNA Repair. The results shown so far ment with 480 UM gallium are shown in Table 2. Gallium suggest that the major effect of gallium is to inhibit ribonucle halved intracellular pools of dATP, dCTP, and dGTP, but had otide reductase. This enzyme is primarily involved in replicative little effect on dTTP. These effects are similar to those previ DNA synthesis, but its classical inhibitor hydroxyurea may also ously reported using the classical ribonucleotide reductase in affect DNA repair (23). Because we were interested in gallium hibitor hydroxyurea (22). as an agent for use in conjunction with radiotherapy or other Selective Inhibition of Ribonucleoside Flow to dTTP and DNA. cytotoxic drugs, many of which cause cell death through pro Both exogenous deoxyuridine and undine enter the dUMP pool duction of DNA strand breaks, we nevertheless investigated its 3016

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1988 American Association for Cancer Research. EFFECT OF GALLIUM ON DNA SYNTHESIS Table 3 Uptake, conversion to [^HldTTP ana incorporation off'H/deoxyuridine and f'HJuridine into DNA

synthesisi dTTP(dpm/pmol)1693050014.5IncorporationintoDNA(dpm/ pumithymidine/h/104 cells)79.5 10'cells)13,412 10'cells)86,760 cells)513474152167 Control['HjDeoxyuridineI'HIUridine480 Â±0.7Â°79.5 Â±1.6932,358 Â±8,67114,220 Â±0.1Â°62 Â±20131,011 Â±99476,276 MMGa5*[ JH|Deoxyuridine|3HlUridinedTTP(pmol/10'Â±5.362 Â±4,665896 Â±4,8702,427 Â±5.3dTTP(dpm/ Â±29Specificactivityof Â±432DNA ' Mean Â±SD of triplicate samples.

gallium nitrate (NSC-1S200) has significant activity in malig MOO nant lymphomas, and responses have also been noted in bladder Gallium120|iM 24hr 1200 and small cell lung cancers (1). Its major toxicity is to the 24ht E kidney, studies in the rat suggesting that this is due to deposition 1000 in the renal tubules of a gallium calcium phosphate complex Bydroiyurea ImM t (24). Toxicity to bone marrow and mucous membranes is 800 Gallium 480um 5min probably at most minor although difficult to assess because of the effects of heavy pretreatment received by most patients. 600 Because it spares tissues damaged by conventional cytotoxic Aphidicolin50uM 400 drugs gallium should be suitable for inclusion in combination chemotherapy schedules. For a rational design of treatment 200 protocols some idea of its mode of action is required, but surprisingly few studies have been made of this. Chitambar et 10 15 20 al. (25) showed that growth-arresting concentrations of gallium Minutes produced a build up in S-phase, a finding similar to that Fig. 2. Incorporation of [3H)dTTP into permeabilized cells. Control cells were reported in the present paper, while Rasey et al. (4) pointed out compared with those treated at the time of permeabilization with gallium. 480 that gallium may mimic iron in some important cell processes liM, hydroxyurea, 1 HIM.or aphidicolin, 50 UM.In addition, cells were pretreated for 24 h with gallium, 120 and 480 I.M. Means of duplicate samples. and suggested the key iron-dependent enzyme ribonucleotide reducÃaseas a possible target. An early paper by Waalkes et al. (11) showed inhibition of DNA polymerases extracted from 100 Walker 256 carcinosarcoma, although some of this activity may in fact have been directed at the reverse transcriptase of a contaminating type C retrovirus. The present study shows that, at least in CCRF-CEM lymphoblasts, gallium inhibits DNA synthesis with a consequent accumulation of cells in S-phase. Pools of dATP, dGTP, and dCTP are significantly reduced, and the dTTP pool is main tained at near normal levels by increased deoxyribonucleoside salvage, while the contribution of ribonucleoside to the dTTP pool falls. All these data point to decreased ribonucleotide reducÃaseactivity in gallium treated cells. The results resemble the effects of hydroxyurea in our own, unreported, experiments and in similar albeit more exhaustive studies by others (22, 26). 60 They are quite unlike the effects of specific inhibitors of dihy- drofolate reducÃase,thymidylate synthetase, or DNA polymer- Fig. 3. Repair of single-stranded DNA breaks. Results are given as a percent age of double-stranded DNA after alkali treatment of aliquots of cells taken at ase a (22, 27). Gallium did not significantly depress the incor intervals following -, radutimi. â€¢,controls; â€¢pretreatment for 24 h with gallium poration of [3H]dTTP inlo DNA of permeabilized cells. 120 Â»IM.Percentageof double stranded DNA of nonirradiated cells treated with We cannol conclude lhal the effects of gallium, allhough alkali was 84.9 and 89.8% for controls and gallium-treated cells, respectively. similar to those of hydroxyurea, are mediated by the same direcl mechanism (inactivation of the active site free radical), effect on DNA repair using a simple fluorimetrie method to bul we postulate thai either depletion of iron or substitution measure resistance to alkaline induced unwinding during recov for it decreases the availability of functional ribonucleotide ery from 7-radiation. Results are shown in Fig. 3. Pretreatment with 120 /IMGa3+ did not by itself cause detectable single strand reducÃaseenzyme. It is also possible thai the effects of gallium are less specific to begin with. For example, ribonucleolide breaks and had no significant effect on their rate of repair reducÃaseaclivity may have been decreased via protein-prolein following 1000 cGy 7-rays. interactions within the reputase complex (28), ihese in iurn due lo gallium having affecled the conformation of olher replilase, DISCUSSION or olher nucleoprolein, components. However, we can say lhal our dala are againsl direcl inhibilion of the enzyme activily of Gallium as a simple salt is probably unique among the olher replilase components and lhal ribonucleotide reductase elements in exerting a significant anticancer effect in humans. aclivily has noi been secondarily, alloslerically inhibiled, such Although it has not yet been fully evaluated in Phase II trials, as occurs when dTTP rises following aphidicolin exposure (26). 3017

blocks transferrin binding and inhibits tumor cell growth in vitro. Proc. Nati. Although our experiments were largely involved with repli Acad. Sci. USA, 79: 1175-1179, 1982. cative DNA synthesis, we were interested in the possibility that 9. Larrick, J. W., and Cresswell, P. Modulation of cell surface iron transferrin gallium could also have an effect on DNA repair synthesis receptors by cellular density and state of activation. J. Supramol. Struct., II: 579-586, 1979. because such an action could make gallium a more generally 10. Reichard, P., and Ehrenberg, A. Ribonucleotide reducÃ•aseâ€”aradicalenzyme. useful agent in the treatment of the common epithelial cancers. Science (Wash. DC), 221: 514-519, 1983. 11. Waalkes, T. P., Sanders, K., Smith, R. G., and Adamson, R. H. DNA The fluorimetrie DNA unwinding assay is a simple and repro polymerases of Walker 256 carcinosarcoma. Cancer Res., 34:385-391,1974. ducible method for investigating the rate of repair of single 12. Anghileri, L. J. Effects of gallium and lanthanum on experimental tumor strand breaks caused by 7 radiation. Results showed that gal growth. Eur. J. Cancer, 15:1459-1462, 1979. 13. Hedley, D. W., Rugg, C. A., Musgrove, E. A., and Taylor, I. W. Modulation lium at growth inhibitory concentrations had no significant of transferrin receptor expression by inhibitors of nucleic acid synthesis. J. effects on this process. It should be noted, however, that al Cell. Physiol., 124:61-66, 1985. though high ribonucleotide reducÃase activity may not be re 14. Foley, G., Lazarus, H., Farber, S., Uzman, B., Boone, B., and McCarthy, R. Continuous culture of human lymphoblasts from peripheral blood of a child quired for DNA repair synthesis, its classical inhibitor hydrox- with acute leukemia. Cancer (Phila.). 18: 522-529, 1965. yurea has apparently been successfully combined with radio 15. Taylor, I. W. A rapid single step staining technique for DNA analysis by flow microfluorimetry. J. Histochem. Cytochem., 2Â«:1021-1024,1980. therapy for the treatment of cervical cancer (29). Our in vitro 16. Milthorpe, B. K. FMFPAK 1: a program package for routine analysis of data should not therefore necessarily discourage clinical evalu single parameter flow microfluorimetric data on a low cost mini computer. ation of gallium as a radiation potentiator. Comp. Biomed. Res., 13:417-429, 1980. 17. Fox, R. M., Kefford, R. F., Tripp, E. H., and Taylor, I. W. G,-phase arrest Because of its lack of major toxicity to bone marrow and gut, of cultured human leukemic T-cells induced by deoxyadenosine. Cancer Res., we suggest that the most useful clinical role for gallium might 41: 5141-5150, 1981. be as a substitute for hydroxyurea. Although the latter has 18. Hellgren, D., Nilsson, S., and Reichard, P. Effects of arabinosyl-cytosine on thymidine triphosphate pools and polyoma DNA replication. Biochem. Bin limited use as a single agent, there is evidence that it can act phys. Res. Commun., 88:16-22, 1979. synergistically either with other ribonucleotide reducÃaseinhib 19. Miller, M. R., Castello!, J. J., and Pardee, A. B. A permeable animal cell preparation for studying macromolecular synthesis. DNA synthesis and the itors (30) or with 1-0-D-arabinofuranosylcytosine (31). These role of deoxyribonucleotides in S-phase initiation. Biochemistry, 17: 1073- combinations do, however, show increased toxicity to normal 1080, 1978. tissues, and this limitation may be offset by using gallium. 20. Birnboim, H. C., and Jevcak, J. J. Fluorometric method for rapid detection of DNA strand breaks in human white blood cells produced by low doses of radiation. Cancer Res., 41: 1889-1892, 1981. 21. Seto, S., Carrera, C. J., Kobota, M., Wasson, B., and Carson, D. A. Mecha ACKNOWLEDGMENTS nism of deoxyadenosine and 2-chlorodeoxyadenosine toxicity in nondividing human lymphocytes. J. Clin. Invest., 75: 377-383, 1985. We thank Judy Hood for her excellent help in preparing this manu 22. Bianchi, V., Pontis, E., and Reichard, P. Changes of deoxyribonucleoside triphosphate pools induced by hydroxyurea and their relation to DNA script. synthesis. J. Biol. Chem., 261:16037-16042, 1986. 23. Harris, A. L. DNA repair: relationship to drug and radiation resistance, metastasis and growth factors. Int. J. RadiÃ¢t.Biol., 48:675-690, 1985. REFERENCES 24. Newman, R. A., Brody, A. R., and Krakoff, I. H. Gallium nitrate (NSC- 15200) induced toxicity in the rat. A pharmacologie, histopathologic and 1. Foster, B. J., Clagett-Carr, K.. Hoth, I)., and Leyland-Jones, B. Gallium microanalytical investigation. Cancer (Phila.), 44:1728-1740, 1979. nitrate: the second metal with clinical activity. Cancer Treat. Rep., 70:1311- 25. Chitambar, C. R., Massey, E. J., and Seligman, P. A. Regulation of transfer rin receptor expression on human leukemic cells during proliferation and 1319, 1986. induction of differentiation: effects of gallium and dimethylsulphoxide. J. 2. Hart, M. M., Smith, C. F., Yancey, S. T., and Adamson, R. H. Toxicity and Clin. Invest., 72: 1314-1325, 1983. antitumor activity of gallium nitrate and periodically related metal salts. J. Nati. Cancer Inst., 47:1121-1127, 1971. 26. Nicander, B., and Reichard, P. Relations between synthesis of deoxyribonu 3. Harris, A. W., and Sephton, R. G. Transferrin promotion of 67Ga and "Fe cleotides and DNA replication in 3T6 fibroblasts. J. Biol. Chem., 260: 5376- uptake by cultured mouse myeloma cells. Cancer Res., 37:3634-3638,1977. 5381, 1985. 27. Nicander, B., and Reichard, P. Evidence for the involvement of substrate 4. Rasey, J. S., Nelson, N. J., and Larson, S. M. Relationship of iron metabolism cycles in the regulation of deoxyribonucleoside triphosphate pools in 3T6 to tumor cell toxicity of stable gallium salts. Int. J. NucÃ.Med. Biol., 8:303- cells. J. Biol. Chem., 260:9216-9222, 1985. 313, 1981. 28. Noguchi, H., veer Reddy, G. P., and Pardee, A. B. Rapid incorporation of 5. Vallabhajosula, S. R., Harwig, J. F., and Wolf, W. The mechanism of tumor label from ribonucleoside Â¿Â¡phosphatesintoDNA by a cell-free high molec localisation of gallium-67 citrate: role of transferrin binding and effect of ular weight fraction from animal cell nuclei. Cell, 32:443-451, 1983. tumor pH. Int. J. NucÃ.Med. Biol., Â«:363-370, 1981. 29. Piver, M. S., Varlow, J. J., Vongtama, V., and Webster, J. Hydroxyurea and 6. Weiner, R. E., Schreiber, G. S., Hoffer, P. B., and Shannon, T. The relative radiation therapy in advanced cervical cancer. Am. J. Obstet. Gynecol., 120: stabilities of 67Ga complexes of lactoferrin and transferrin at various pH's. 969-972, 1974. Int. J. NucÃ.Med. Biol., 8: 371-378, 1981. 30. Sato, A., Carter, G. L., Bacon, P. E., and Cory, J. G. Effects of combinations 7. Chitambar, C. R., and Seligman, P. A. Effects of different transferrin forms of drugs having different modes of action at the ribonucleotide reducÃasesite on transferrin receptor expression, iron uptake, and cellular proliferation of on growth of LI 210 cells in culture. Cancer Res., Â¥2:4353-4357, 1982. human leukemic HL60 cells. Mechanisms responsible for the specific cyto- 31. Walsh, C. T., Craig, R. W., and Agarwal, R. P. Increased activation of 1-0- toxicity of transferrin-gallium. J. Clin. Invest., 78:1538-1546, 1986. D-arabinofluranosylcytosine by hydroxyurea in LI210 cells. Cancer Res., 40: 8. Trowbridge, I. S., and Lopez, F. Monoclonal antibody to transferrin receptor 3286-3292, 1980.

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David W. Hedley, Edith H. Tripp, Peter Slowiaczek, et al.

Cancer Res 1988;48:3014-3018.

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