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[CANCER RESEARCH 48, 839-843, February 15, 1988] Leukemia LI 210 Cell Lines Resistant to Ribonucleotide ReducÃ-aseInhibitors1 Joseph G. Cory2 and Gay L. Carter Division of Medical Oncology, Department of Internal Medicine, University of South Florida College of Medicine, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612 determination of the |1( '|,,,s, ' at least five drug concenlrations were ABSTRACT used. Cell aliquols were laken after 3 days for cell counls. Cell counts were determined in a ( 'miller Counter, Model ZBI. Leukemia LI 210 cell lines, EDI and ED2, were generated which were resistant to the cytotoxic effects of deoxyadenosine/erylhro-9-{2hydroxyl-3-nonyl)adenine and deoxyadenosine/erythro-9-(2-hydroxyl-3non)Hack-nine plus 2,3-dihydro-l//-pyrazole|2,3al¡midazole/Desferal, re spectively. The EDI and ED2 were characterized to show that these cell lines had increased levels of ribonucleotide reducÃ-ase as measured by CDP reduction. The reducÃ-ase activity in crude cell-free extracts from the EDI and ED2 cells was not inhibited by dATP. For CDP reducÃ-ase, the activation by adenylylimido diphosphate and inhibition by dGTP and d'lTP in these extracts from the EDI and ED2 cells were the same as Generalion of EDI and ED2 Cell Lines. LI210 cells were incubaled in Ihe presence of deoxyadenosine, 30 MM/EHNA, 5 MMplus IMPY, 100 MM/Desferal, 30 MMfor 24 h. The cells (75 cells/dish) were Ihen plaled on sofl agar (Sea-Kern agar, 0.275% with 20% horse serum). Colonies which grew out were picked after 21 days and cultured in liquid medium in increasing concentrations of dAdo/EHNA plus IMPY/Df. After inilial studies utilizing [l4C]cylidine conversion to | "C'jdeovyeylidinc and incorporalion inlo DNA indicated that mosl of Ihe resistance was to dAdo/EHNA, Ihe cells were divided inlo iwo groups. The EDI cells were generated by growing the cells in liquid medium in which Ihe concentrâtion of dAdo was increased al a conslanl concentration of EHNA (5 MM).The ED2 cells were generated by growing Ihe cells in liquid medium in which Ihe concentrane us of dAdo and IMPY were bolh increased al conslanl concentrations of EHNA (5 MM)and Df (48 MM).The cells were periodically replated on soft agar and the surviving colonies plucked and recultured in liquid medium. The mosl resislani cell lines were mainiained as Ihe slock EDI and ED2 cell lines. Preparai inn of Crude Cell-free Exlracls. Equal numbers of wild-lype EDI, and ED2 cells in midlog phase were laken for Ihe preparation of Ihe crude exlracts. The cells were pelleted by centrifugation at 500 x g for 10 min. The cells were then washed two times with cold phosphatebuffered saline and recenlrifuged. The cell pellets were resuspended in Tris-HCl buffer, 0.02 M, pH 7.0, conlaining dilhioerylhrilol, 1 min al a concentration of 5 x 10" cells/ml. After swelling for 5 min on ice, Ihe cell suspension was homogenized wilh a motor-driven Teflon pesile (20 slrokes). The homogenale was centrifuged for 1 h at 21,000 x g al 4"( '. for the wild-type L1210 cells. The EDI and ED2 cells were highly crossresistant, as measured by growth inhibition, to deoxyguanosine/8-aminoguanosine, 2-fluorodeoxyadenosine, and 2-fluoroadenine arabinoside. While the ED2 cells showed resistance to 2,3-dihydro-lff-pyrazole[2,3a)-imidazole/Desferal (6-fold), the EDI and ED2 cell lines showed less resistance to hydroxyurea, 4-methyl-S-amino-l-formylisoquinoline thiosemicarbazone, and the dialdehyde of inosine. These data indicate that the mechanisms of resistance to the ribonucleotide reducÃ-ase inhib itors are related to the increased level of ribonucleotide reducÃ-aseactivity and to the decreased sensitivity of the effector-binding subunit to dATP. INTRODUCTION In earlier studies, it was shown that combinations of drugs directed at the non-heme iron and effector-binding subunits of ribonucleotide reducÃ-asegave synergistic inhibilion of L1210 cell growth in culture (1-3). Further studies using colony form ing assays were carried out to show that these combinations of drugs directed at Ihe subunils of ribonucleolide reducÃ-asealso resulted in synergistic cytotoxicity (4). In the course of these studies several colonies grew out in the presence of drug con centrations which killed greater than 99% of the cells. These clones were picked and cultured in liquid medium in the pres ence of the same drugs. Selection of the specific clones was based on the resislance of the cell lines to growth inhibition and to resistance to inhibition of [14C]cytidine conversion to The supernalanl fluid was applied lo a Dowex-1 -acelale column which had been prewashed wilh Tris-HCl buffer conlaining dilhioerylhrilol, 1 HIM.The enzyme exlract was washed from the column with 2 volumes of Tris-HCl/dithioerylhrilol. The eluanl was Ihen irealed with ammo nium sulfate to 80% saturation. The precipitate was collected by cen trifugation, dissolved in Tris-HCl/dithioerylhritol, and dialyzed againsl Ihe same buffer. The purpose of Ihe Dowex-1-acelale chromalography was lo remove endogenous nucleolides which mighl interfere wilh Ihe assay. The ammonium sulfate step al 80% saturation was done lo concenlrate the protein after Ihe Dowex step. The recovery of aclivily was in excess of 100% due lo Ihe removal of low molecular u eight substances. Aliquots of Ihe dialyzed extracts were quick frozen in an acelone-dry ice balh and slored al -90°C. No loss of CDP reducÃ-ase deoxycytidine and incorporalion inlo DNA in inlacl cells in Ihe presence of the ribonucleotide reductase inhibitors. In this paper, we describe iwo of ihese cell lines in lerms of their sensitivily to a variety of ribonucleotide reductase inhibi tors. MATERIALS AND METHODS Growth of LI210 Cells in Culture. LI 210 cells were grown in suspen sion cuiiure in RPMI 1640 culture medium supplemented with 10% horse serum, gentamicin sulfate (50 mg/liter), and sodium bicarbonate (2 g/liler). The cells were seeded al 1.75 x IO5cells/ml in Ihe presence and absence of drugs. The cells were grown at 37°Cin a humidified incubator gassed with 90% air/10% CO2. All cell cultures were set up in triplicale. Aliquols (1.0 ml) were taken daily for growth curves. For Received 8/3/87; revised 10/16/87; accepted 11/13/87. 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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This research was supported by grants CA 27398 and CA 42070 from the USPHS, National Cancer Institute, and the Grand Chapter of the Eastern Star. 2To whom requests for reprints should be addressed, at Department of Internal Medicine, MDC Box 19, USF College of Medicine, 12901 Bruce B. Downs Blvd., Tampa, FL 33612. aclivily was observed in the frozen samples. Assay of Ribonucleotide ReducÃ-aseAclivily. CDP reducÃ-aseaclivily was assayed by Ihe method of Sleeper and Steuart (5) using Dowex-1borale columns lo separate deoxycytidine from cylidine. The assay mixlure for CDP reducÃ-aseconlained in a final volume of 0.15 ml [14C]-CDP, 0.03 MCi, 1.87 nmol; AMP-PNP, 1500 nmol; magnesium acelale, 600 nmol; sodium phosphate buffer, pH 7.0, 1000 nmol; and enzyme extract. After a 30-min reaction period, Ihe reducÃ-aseassay was slopped by healing for 4 min in a boiling waler bath. The samples were cooled and treated with snake venom (6) as previously described. All reductase assays were carried out in triplicate. The specific act ivities were compared on a per mg protein basis ralher lhan on a per cell basis. The assay was linear wilh time for at least 40 min. The activily measured 3The abbreviations used are: [ICjjo, concentration of drug required to inhibit cell growth or reductase activity by 50%; AMP-PNP, adenylylimido diphosphate; AGuo, 8-aminoguanosine; Df, Desterai: EHNA, erythro-9-(2-hydroxyl-3nonyl)adenine; F-araA, 2-fluoroadenine arabinoside; F-dAdo, 2-fluoro-2'-deoxyadenosine; IMPY, 2,3-dihydro-l//-pyrazole[2,3ajimidazole; Inox, dialdehyde de rivative of inosine; MAIQ, 4-methyl-S-amino-l-formylisoquinoline thiosemicar bazone; dAdo, deoxyadenosine; dGuo, deoxyguanosine; F-dATP, 2-fluoro-dATP. 839 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1988 American Association for Cancer Research. DRUG-RESISTANT L1210 CELL LINES was linear with respect to the protein added when the protein concen tration was in excess of 600 Mg/rnl (90 (¿g/tube)in the reaction mixture. This was observed with extracts from the wild-type, EDI, and ED2 cells. The differences in enzyme activities in the extracts from the wild type, EDI, and ED2 cells were observed using either equal volumes of extracts or equal amounts of protein in the assays. ("•qCytidineMetabolism in L1210 Cells. L1210 cells were collected by centrifugation and resuspended in fresh culture medium (10 ml) at a concentration of 1.6 x IO6cells/ml. The cells were incubated in the presence and absence of drugs for 30 min at 37°Cin air/CO2 (95/5%). [14C]Cytidine (0.5 MCi, 350 mCi/mmol) was added to each flask and the cells were incubated for an additional 60 min. The cells were collected and subjected to the Schmidt-Thannhauser procedure (7) to separate the acid-soluble, RNA, and DNA fractions. The acid-soluble fraction was neutralized, lyophilized, and treated with snake venom (8). The [14C)deoxycytidine was separated from the [14C]cytidine by chromatography on Dowex-1-borate columns. All flasks were set up in triplicate. Adenosine Deaminase and Kinase Activities. Adenosine deaminase activity was determined using a continuous spectrophotometeric assay at 25°C.The conversion of adenosine to inosine was measured at 265 nm. The assay mixture consisted of adenosine, 0.063 mM in 0.02 M Tris-HCl, pH 7.O. The reaction was started by the addition of the crude cell-free extract which had been passed over a Dowex-1-acetate column and dialyzed against 0.02 M Tris-HCl, pH 7.O. The assays were run with equal amounts of extract protein (0.3 mg/tube). Kinase activity was determined using [8-'4C]deoxyadenosine (45 mCi/mmol) as the substrate. The crude cell-free extracts after passage over Dowex-1acetate columns and dialysis against 0.02 M Tris-HCl, pH 7.0 were incubated with deoxycoformycin (57 fig/ml) for 1 h on ice. The reaction mixture contained in a final volume of 0.095 ml [14C]deoxyadenosine (0.2 ^Ci); ATP (285 nmol); magnesium acetate (190 nmol); sodium phosphate buffer, 950 nmol, pH 7.0; and deoxycoformycin-treated enzyme extract, 0.3 mg protein/tube). Aliquots (5 fil) were taken at 10, 20, and 30 min of incubation at 25°Cand spotted directly on thin-layer cellulose plates. These reaction aliquots were spotted on the thin-layer cellulose plates over carrier dAMP and deoxyadenosine standards. The thin-layer cellulose plates were developed with a butanol:water (86:14) solvent. In this system dAMP and other nucleotides remain at the origin while deoxyadenosine moves with an Rr of 0.5. The dAMP (origin) and deoxyadenosine areas were scraped directly into scintilla tion vials and counted for the radioactivity. The reactions were linear over the 30-min period measured. All assays were carried out in triplicate. Materials. The LI210 cell line was purchased from the American Type Culture Collection (Rockville, MD). The RPMI 1640 culture medium, horse serum, and sodium bicarbonate were purchased from Grand Island Biological Co. (Grand Island, NY). IMPY, Inox, and MAIQ were obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute through the assistance of Dr. Nancita R. Lomax. EHNA was purchased from Burroughs-Wellcome, Research Triangle Park, NC. Desierai was a gift from Ciba-Geigy Corp., Summit, NJ. Deoxycoformycin was a gift from Warner-Lambert Co., Ann Arbor, MI. Hydroxyurea, AMP-PNP, deox yadenosine, and gentamicin sulfate were purchased from Sigma Chem ical Co., St. Louis, MO. 8-Aminoguanosine was purchased from Calbiochem-Behring, La Jolla, CA. 2-F-Deoxyadenosine and 2-fluoroadenine arabinoside were gifts from Dr. John A. Montgomery, Southern Research Institute, Birmingham, AL. 1-Isoquinolylmethylene-A'-hydroxy-/V"-aminoguanidine-tosylate was obtained from Dr. Eric J. Lien, University of Southern California, Los Angeles, CA. [14C]Cytidine (350 mCi/mmol) and ['4C]CDP (513 mCi/mmol) were purchased from determine if combinations of inhibitors directed at the nonheme iron and effector-binding subunits of ribonucleotide reductase resulted in synergistic cell kill, it was observed that several different colonies grew out in the presence of cytotoxic doses of dAdo/EHNA or dAdo/EHNA plus IMPY/Df combi nations. The LI210 cell line which was selected for resistance to dAdo/EHNA was then grown in increasing concentrations of dAdo/EHNA. This cell line was called EDI. Another cell line was selected for further study because of its resistance to the combination dAdo/EHNA plus IMPY/Df. This cell line was also subjected to increasing concentrations of dAdo and IMPY with the EHNA and Df concentrations being held con stant. This cell line was designated as ED2. As seen in Fig. 1, the combination of dAdo/EHNA which caused essentially complete inhibition of wild-type LI210 cell growth had no effect on the growth of EDI cells. Likewise, the ED2 cell line was refractory to the growth-inhibitory effects of the combination dAdo/EHNA plus IMPY/Df. The doubling times of the EDI and ED2 cell lines were the same as for the wild-type LI210 cells (10 to 12 h). The resistance traits in the EDI and ED2 cell lines were stable. Even after passage for over a month in drug-free medium, the resistance phenotypes in the EDI cells to dADo/EHNA and in the ED2 cells to dAdo/ EHNA plus IMPY/Df were as strong as the cells maintained in the presence of these drug combinations. Ribonucleotide ReducÃ-aseActivity in LI 210 Cell Lines. CDP reducÃ-aseactivity was determined in the wild-type L1210 cells and in the resistant cell lines, EDI and ED2. Cell-free extracts, after passage over Dowex-1-acetate, precipitation with ammo nium sulfate (80%), and dialysis were used as the source of enzyme. Increases in specific activities as large as 9-fold were seen in the EDI and ED2 cells compared with the wild-type cells. As seen in Fig. 2, the CDP reducÃ-ase activily in ihe exlracls from Ihe EDI and ED2 cells was much less sensilive lo feedback inhibilion by dATP. The [IC]50s for dATP for Ihe reducÃ-aseaclivity in the exlracls from Ihe EDI and ED2 cells were grealer lhan 150 UMcompared wilh ihe [IC]50of less than 20 fiM for dATP for reducÃ-aseaclivity from wild-type LI210 cells. On the other hand, the [IC]50s for dGTP were essentially the same for the reducÃ-aseaclivities from these three LI210 cell lines although some differences were seen in the percentage of conlrol versus Ihe [dGTP] curves. The Kas for Ihe aclivalion of CDP reducÃ-aseby AMP-PNP were Ihe same (0.4 to 0.5 mM) EDI. dAdo/EHNA DAYS IN CULTURE Research Products Int. Co., Mount Prospect, IL and New England Nuclear Corp., Boston, MA, respectively. [8-14C]-Deoxyadenosine (45 mCi/mmol) was purchased from Moravek Biochemicals, City of Indus try, CA. RESULTS Resistance of LI 210 Cell Lines to Ribonucleotide ReducÃ-ase Inhibitors. During the soft-agar studies being carried out to E02 .dAdo/EHNA/IMPY/Df DAYS IN CULTURE Fig. 1. Growth of L1210 cells in culture. LI210 cell lines were grown in culture in the presence and absence of drugs. Each control or drug-treated cell condition was set up in triplicate. Aliquots were taken at daily intervals for cell counts. Left, wild-type LI 210 cells in the absence of drugs (O) and in the presence of dAdo, 400 pM/EHNA, 5 »M(•);EDI cells in the absence of drugs (D) and in the presence of dAdo. 800 ^M/EHNA, 5 >iM(•);right, wild-type L1210 cells in the absence of drugs (O) and in the presence of dAdo, 400 ^M/EHNA, 5 MM/ IMPY. 450 fiM/Df, 48 JÕM (•);ED2 cells in the absence of drugs (D) and in the presence of dAdo/EHNA/IMPY/Df at the same concentrations as for wild-type cells (•). 840 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1988 American Association for Cancer Research. DRUG-RESISTANT LI210 CELL LINES IK' ED2. HU . MAIO 2.0 ID o l.O • WT o EOI • ED2 X ËO.S E05 o 50 u a« \ 2 4 DAYS IN CULTURE DAYS IN CULTURE dATP.uM UGTP.jjM Fig. 2. Effect of dATP and dGTP on CDP reducÃ-aseactivities in L1210 cell lines. Crude extracts were prepared from wild-type (WT), EDI, and ED2 L1210 cell lines. CDP reducÃ-aseactivily was delermined as described under "Malcriáis and Methods" excepl lhat dATP or dGTP was added at the final concentralions as indicaled. The control activilies for Ihe wild-lype. EDI, and ED2 cell lines were (left), 0.08, 0.43, and 0.29 and (right), 0.06, 0.32, and 0.46 nmol/30 min/ mg prolein, respectively. Fig. 3. Growth of LI210 cells in cullure. LI210 cell lines were grown in cullure in the presence and absence of drugs. Each conlrol or drug-treated condition was set up in triplicate. Aliquots were taken at daily intervals for cell counts. Left, wild-type cells in ihe absence of drugs (O) and in the presence of hydroxyurea (HU), 400 ßM (•)and ED2 cells in the absence of drugs (D) and in the presence of hydroxyurea, 400 ^M (B); righi, wild-type cells in the absence of drugs (O) and in the presence of MAIQ, 3 /<M(•)and ED2 cells in the absence of drug (D) and in the presence of MAIQ, 3 /iM (•). Table 1 Concentralions of drugs required to inhibit LI 210 cell growth in culture „MEDI>1000* for the reducÃ-aseactivities in the extracts from these three cell lines (data not shown). In cell-free extracts prepared from the EDI and ED2 cell lines, ribonucleotide reducÃ-aseactivily showed Ihe same degree of sensilivily to hydroxyurea and IMPY as did the reducÃ-ase activily in Ihe wild-lype LI210 cells (dala noi shown). Cross-Resistance of EDI and ED2 Cell Lines to Ribonucleo tide ReducÃ-aseInhibitors. The EDI and ED2 cell lines, selecled for resislance lo dAdo/EHNA and dAdo/EHNA plus IMPY/ Df, respeclively, were sludied for iheir sensitivities lo a wide range of ribonucleolide reducÃ-aseinhibilors. A combinalion of dGuo and AGuo would be expecled lo generate intracellular levels of dGTP lo inhibil reducÃ-aseaclivily (9); F-dAdo would lead to the formalion of F-dATP (10). The ED2 cell line was highly resislanl lo reducÃ-aseinhibilors direcled al Ihe effeclorbinding subunil (F-dAdo or dGuo/AGuo) and Ihe cells grew as well as Ihe conlrol cells. Sludies wilh Ihe EDI cell line gave very similar dala in lerms of resislance lo these agenls (dGuo/ AGuo or F-dAdo). The EDI and ED2 cell lines also showed resislance lo F-araA (dala noi shown). On Ihe olher hand, the EDI and ED2 cell lines were less resistanl lo ribonucleolide reducÃ-aseinhibilors direcled al Ihe non-heme iron subunil. Fig. 3 shows ihe dala for Ihe effecls of hydroxyurea or MAIQ on Ihe growlh curves for ihe ED2 cell line. Essentially the same data were oblained for ihe EDI cell line. The [IC]50s were delermined for Ihese and olher reducÃ-aseinhibilors and Ihese dala are summarized in Table 1. The EDI and ED2 cell lines showed Ihe grealesl degrees of resislance lo inhibilors direcled at the effector-binding subunit (13- to >25-fold). Resistance to Inox was minimal. The increases in [IC]50s for the inhibilors directed at the non-heme iron subunit were much less, ranging from a 1.7-fold increase for MAIQ in ED2 cells lo a 6.8-fold increase for IMPY/Df in ihe ED2 cells. Adenosine Deaminase and Deoxyadenosine Kinase Activities in 1,1210 Cell Lines. The levels of adenosine deaminase aclivily were essentially the same in the wild-type, EDI, and ED2 cell lines (46, 47, and 53 nmol/min/mg protein, respeclively). Like wise Ihere was no change in Ihe kinase aclivily using deoxyadenosine as Ihe substrate. The kinase studies were carried out with exlracls preincubaled wilh deoxycoformycin to inaclivale Ihe deaminase aclivily. These values were 18.4, 20.0, and 22.0 DrugdAdo/EHNA" 175* >1000* dGuo/AGuo" F-dAdo" 2.3 30 Inox" 125 340 Hydroxvurea'iMPY/br1-isoquinolylmethylene-yV-hydroxy420 98 160* 640* >1000* 37 160 475 1090* 2.51.2[IC|,„,6.83.1ED21000* 6.02.0 W-aminoguanidine-losylate' MAIQrWildtype40* " Inhibitors direcled al the effector-binding subunit. * These [IC]50s are with respect to dAdo, dGuo, or IMPY; in each case the concentralion of EHNA, AGuo, or Df was held conslant at 5, 25. and 40 /JM, respectively. f Inhibitors direcled al Ihe non-heme subunit. Table 2 Metabolism off'CJcytidine in LI210 cell lines lypeEDICytidine Drug (UM)Wild conversion)Conlrol IMPY/Df (360/40) dAdo/EHNA (600/5) to deoxycytidine (% 1.0 2.3(230)° 0.5(50) 0(0) (290) 1.1 (110) 0.7(70) 3.1(310)ED22.9 3.8 (380) Cytidine to deoxycytidine to DNA (cpm/IO6 cells) Conlrol 1640 3010(184) 3730(227) IMPY/Df (360/40) 183(11) 572(35) 1004(61) dAdo/EHNA (600/5) 0(0) 1114(68) 1190(73) °Numbers in parentheses, percentage of control of wild-lype LI 210 cells. pmol/min/mg prolein for ihe wild-type, EDI, and ED2 cells, respeclively. [l4C]Cytidine Metabolism in LI 210 Cell Lines. The conversion of [uC]cylidine lo deoxycylidine and incorporalion inlo DNA was delermined in the wild-type, EDI, and ED2 cell lines. In addilion, Ihe effecls of Ihe drug combinalions IMPY/Df and dAdo/EHNA on [14C]cylidine metabolism in these cells were sludied. As seen in Table 2, Ihe EDI and ED2 cells showed increases (2- lo 3-fold) in CDP reducÃ-aseaclivily in ihe inlacl cells as measured by Ihe increased formation of deoxycytidine. Wilh Ihe drug combinalion dAdo/EHNA, Ihe formalion of deoxycylidine was complelely blocked in Ihe wild-lype cells, while Ihe ED 1 and ED2 cells were noi inhibiled, and if anylhing, Ihe formalion of deoxycylidine was increased in the EDI and ED2 cells relalive lo Iheir conlrols. The incorporalion of cylidine inlo DNA via deoxycylidine was also increased in Ihe EDI and ED2 cells. The incorporalion of label inlo DNA in ihe 841 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1988 American Association for Cancer Research. DRUG-RESISTANT L1210 CELL LINES niKNA for Ihe non-heme iron subunit with gene amplification (27, 28). As can be seen from Ihe dala in Table 2, ribonucleotide reducÃ-aseaclivity as measured in intacl cells was elevated 2- lo 3-fold in Ihe EDI and ED2 cells compared lo Ihe wild-lype 1.1210 cells. On Ihe other hand, the incorporation of [I4C]- EDI and ED2 cells was less sensitive to the inhibition by the IMPY/Df or dAdo/EHNA combination. There was no change in the incorporation of cytidine into the RNA of these cells. DISCUSSION One of the major problems which arises in the chemotherapy of human tumors is the development of drug-resistant clones. There are multiple biochemical bases for the development of drug resistance to a single agent (11-16). In the studies pre sented here, clones of LI210 cells grew out in the presence of inhibitors directed at ribonucleotide reducÃ-ase.The biochemical bases for the resistance to dAdo/EHNA in the EDI cell line and the resistance to dAdo/EHNA plus IMPY/Df in the ED2 cell line appear to be due to the increased levels of ribonucleo tide reducÃ-aseactivity in the EDI and ED2 cell lines and to the altered effector-binding subunit which renders the ribonucleo tide reducÃ-aseinsensitive to the negative effector, dATP (Fig. 2). While Ihe enzyme in Ihe extracts from the EDI and ED2 cell lines was not inhibited by dATP, the apparent A', for the activalor, AMP-PNP was Ihe same as for Ihe wild-lype cells. This would appear lo be an anomalous finding as il has been reported that ATP and dATP both bind to the activity and specificity siles (17). Consequently, il would be expected that an alteration in the dATP binding site or siles would likewise alter the ATP-binding sites(s). However, this kind of alteration (altered dATP binding with no corresponding effect on ATP binding) has been reported previously (18,19). In some of these mutant cell lines the sensitivity lo dATP was decreased while the sensitivity to dGTP and dTTP remained unchanged (18, 19) or conversely in which the sensilivily lo dATP was unaltered but the sensitivity to dGTP and dTTP was decreased (19). The reducÃ-asefrom another mutant cell showed decreased sensitiv ity to dATP but increased binding of the positive effeclor, ATP (20). As seen from the data in Table 1, Ihe grealer drug resistances were seen for Ihose agents directed at the effeclorbinding subunit. The exception to this was in the resistance to Inox. However, previous studies have shown lhat as an inhibitor of ribonucleotide reducÃ-ase,Inox specifically inhibils the effec tor-binding subunit (21) bul il also has oilier siles of action (22, 23). The ED2 cell line showed a relatively high degree of resistance to IMPY/Df and ihis was expecled as ihese cells had been grown in increasing concenlralions of IMPY. The EDI and ED2 cell lines showed cross-resislance to F-dAdo and FAraA, consistent wilh altered dATP-binding sile(s). Ribonucle otide reducÃ-aseas measured by CDP reducÃ-aseaclivity in the crude extracts from the EDI and ED2 cell lines showed the same degree of sensitivity to dGTP as did Ihe enzyme from Ihe wild-type cells (Fig. 2); similar data were obtained with dTTP (data not shown). The observed cross-resislance lo dGuo/AGuo in the intact cells was therefore unexpected, based on the sensitivity of reducÃ-aseactivily in crude exlracls lo dGTP (Fig. 2). Il is possible that the increased | l('|M,s determined for the cylidine inlo DNA via deoxycylidine nucleolides in Ihe dAdo/ EHNA-irealed EDI and ED2 cells was reduced lo only 30 lo 40% of Ihe incorporation in Ihe untreated EDI and ED2 cells. However, compared lo Ihe conlrol value (1640 cpm/106 cells) for Ihe wild-lype LI210 cells, Ihe level of incorporation in the drug-treated EDI and ED2 cells was 68 to 73% of control value in the wild-type cells. This level of DNA synlhesis may be sufficient to support cell division under these condilions ren dering Ihese cell lines resislanl lo growth inhibilion by Ihese drugs. Since Ihe levels of deoxyadenosine kinase and adenosine (learninase activities were Ihe same in Ihe wild-lype L1210 cells and Ihe EDI and ED2 cells lines, Ihe potential mechanisms of lack of activation or increased inactivalion could be ruled oui. These sludies show lhat mechanisms of resistance to drugs directed at Ihe subunils of ribonucleolide reducÃ-ase can be multiple and can be due lo increased levels of reductase activity and to alterations in the sensitivily lo negative effectors. Studies are being direcled al determining Ihe levels of Ihe individual subunils and Ihe nature of the insensilivily to dATP in the resistanl EDI and ED2 cell lines. REFERENCES various agenls in the intacl EDI and ED2 cell lines result from a combination of the increased levels of reducÃ-ase aclivity (dGuo/8AGuo; hydroxyurea; IMPY/Df; 1-isoquinolylmelhylene-TV-hydroxy-yV-aminoguanidine-tosylate; MAIQ) and the altered effector-binding subunil (dAdo/EHNA; F-dAdo; FaraA). Il has been shown lhal the resistance to hydroxyurea is related to increased levels of ribonucleotide reducÃ-aseactivity in the cells rather than lo marked alterations in the sensitivily of the enzyme to hydroxyurea (24-26). It has been reported that Ihe hydroxyurea-resislanl cells have elevaled levels of 1. Sato, A., Carter, G. L., Bacon, P. E., and Cory, J. G. Effects of combinations of drugs having different modes of action at the ribonucleotide reductase site on growth of LI 210 cells inculture. Cancer Res., 42:4353-4357, 1982. 2. Sato, S., Bacon, P. E., Schneller, S. W., and Cory, J. G. Effect of combina tions of deoxyguanosine and 8-aminoguanosine with 2,3-dihydro-lH-imidazo[ 1,2-b]pyrazole on LI210 cell growth in culture. Biochem. Pharmacol., 33:689-691, 1984. 3. Sato, A., Montgomery, J. A., and Cory, J. G. Synergistic inhibition of leukemia L1210 cell growth in vitro by combinations of 2-fluoroadenine nucleosides and hydroxyurea or 2,3-dihydro-lH-pyrazole[l,2-a]imidazole. Cancer Res., 44: 3286-3290, 1984. 4. Cory, J. G., Sato, A., Carter, G. L., Bacon, P. E., Montgomery, J. A., and Brown, N. C. The utility of combinations of drugs direcled at specific sites of the same target enzyme-ribonucleotide reductase as the model. Adv. Enzyme Regul., 23: 181-192, 1985. 5. Steeper, J. R., and Steuart, C. E. A rapid assay for CDP reductase activity in mammalian cell extracts. Anal. Biochem., 34: 123-130, 1970. 6. Cory, J. G., and Whitford, T. W., Jr. Ribonucleotide reductase and DNA synthesis in Erlich ascites tumor cells. Cancer Res., 32: 1301-1306, 1972. 7. Schmidt, G., and Thannhauser, S. J. A new method for the determination of deoxyribonucleic acid, ribonucleic acid and phosphoproteins in animal tis sues. J. Biol. Chem., 161: 83-89, 1945. 8. Cory, J. G., Mameli. M. M., George, C. B., and Wilkinson, D. S. Inhibition of nucleic acid synthesis in Erlich tumor cells by periodate-oxidized adenosine and adenylic acid. Arch. Biochem. Biophys., 160: 495-503, 1974. 9. Kazmers, I. S., Mitchell, B. S., Dadonna, P. E., Wotring, L. L., Townsend, L. B., and Kelley, W. N. Inhibition of purine nucleoside phosphorylase by 8aminoguanosine: selective toxicity for T lymphoblasts. Science (Wash. DC), 214: 1137-1139, 1981. 10. Carson, D. A., Wasson, D. B., Kaye, J., Ullman, B., Martin, D. W., Jr., Robins, R. K., and Montgomery, J. A. Deoxycytidine kinase-mediated tox icity of deoxyadenosine analogues toward malignant human lymphoblasts in vitro ana toward murine LI 210 leukemia in vivo. Proc. Nati. Acad. Sci. USA, 77:6865-6899, 1980. 11. Curt, G. A., Clendeninn, N. J., and Chabner, B. A. Drug resistance in cancer. Cancer Treat. Rep., 68: 87-99, 1984. 12. Berlino, J. R., Mini, E., Sobrero, A., Moroson, B. A., Love, T., Jastreboff, M., Carmen, M . Srimatkandada, S., and Dube, S. Methotrexate resistant cells as targets for selective chemotherapy. Adv. Enzyme Regul., 24: 3-11, 1985. 13. Reed, E., and Chabner, B. A. Clinical cancer treatment and drug resistance: the interface of the clinic and the laboratory. In: 3. G. Cory and A. Szentivanlyi, (eds.). Cancer Biology and Therapeutics, pp. 173-179. New York: Plenum Press, 1987. 14. Beidler, J. L., Chang, T., Meyers, M. B., Peterson, R. H. F., and Spengler, B. A. Drug resistance in Chinese hamster lung and mouse tumor cells. Cancer Treat. Rep., 67: 859-867, 1983. 842 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1988 American Association for Cancer Research. DRUG-RESISTANT LI210 CELL LINES 15. Ling, V., Kartner, N., Sudo, T., Siminovitch, L., and Riordan, J. R. Multidrug-resistance phenotype in Chinese hamster ovary cells. Cancer Treat. Rep., 67: 869-874, 1983. 16. Beck, W. T. Vinca alkaloid-resistant phenotype in cultured human leukemic lymphoblasts. Cancer Treat. Rep., 67:875-882, 1983. 17. Eriksson, S., Thelander, L., and Ackerman, M. Allosteric regulation of calf thymus ribonucleoside diphosphate reducÃ-ase.Biochemistry, 18:2948-29S2, 1979. 18. Ayusawa, IX. Iwata, K.. and Seno, T. Alteration of ribonucleotide reducÃ-ase in aphidicolin-resistant mutants of mouse FM 3A cells with associated resistance to arabinosyladenine and arabinosylcytosine. Somatic Cell Genet., 7:27-42, 1981. 19. Eriksson, S., Gudas, L. J., Ullman, B., Clift, S. M., and Martin, D. W., Jr. DeoxyATP-resistant ribonucleotide reducÃ-aseof mutant mouse lymphoma cells. J. Biol. Chem., 256:10184-10188, 1981. 20. Waddell, D., and Ullman, B. Characterization of a cultured human I -cell line with genetically altered ribonucleotide reducÃ-aseactivity. J. Biol. Chem., 25«:4226-4231, 1983. 21. Cory, J. G., and Fleischer, A. E. Specific inhibitors directed at the individual components of ribonucleotide reducÃ-aseas an approach to combination chemotherapy. Cancer. Res., 39:4600-4604, 1979. 22. Cory, J. G., Mansell, M. M., and Whitford, T. W., Jr. Inhibition of ribonu 23. 24. 25. 26. 27. 28. cleotide reducÃ-aseactivity and nucleic acid synthesis in tumor cells by the dialdehyde derivatives of inosine (NSC 118994) and inosinic acid. Cancer Res., 56:3166-3170, 1976. Cory, J. G., Parker, S. H., and Fox, C. S. Inhibition of RNA synthesis in Ehrlich tumor cells by the dialdehyde derivative of inosine (NSC 118994). Cancer Res., 38: 815-822, 1978. Lewis, W. H., and Wright, J. A. Isolation of hydroxyurea-resistant CHO cells with altered levels of ribonucleotide reducÃ-ase.Somatic Cell Genet., 5: 83-96, 1979. Akerblom, L., Ehrenberg, A., Graslund, A., Lankinen, H., Reichard, P., and Thelander, L. Overproduction of the free radical of ribonucleotide reducÃ-ase in hydroxyurea-resistant mouse fibroblast 3T6 cells. Proc. Nati. Acad. Sci. USA, 78:1159-2163, 1981. Dick, J. E., and Wright, J. A. Human diploid fibroblasts with alterations in ribonucleotide reducÃ-aseactivity, deoxynucleotide pools and in vitro life span. Mech. Ageing Dev., 13: 119-126, 1980. Thelander, L., and Berg, P. Isolation and characterization of expressible cDNA clones encoding the Ml and M2 subunits of mouse ribonucleotide reducÃ-ase.Mol. Cell. Biol., 6: 3433-3442, 1986. Wright, J. A., Alam, T. G., McClarty, G. A., Tagger, A. Y., and Thelander, L. Altered expression of ribonucleotide reductase and role of M2 gene amplification in hydroxyurea-resistant hamster, mouse, rat, and human cell lines. Somatic Cell Genet., 13:155-165, 1987. 843 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1988 American Association for Cancer Research. Leukemia L1210 Cell Lines Resistant to Ribonucleotide Reductase Inhibitors Joseph G. Cory and Gay L. Carter Cancer Res 1988;48:839-843. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/48/4/839 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1988 American Association for Cancer Research.