[CANCER RESEARCH 50, 4468-4472. August 1, 1990] Development of Drug Resistance to Gallium Nitrate through Modulation of Cellular Iron Uptake1

Christopher R. Chitambar,2 Zorica Zivkovic-Gilgenbach, Jana Narasimhan, and William E. Antholine

Division of Hematology-Oncology, Department of Medicine fC. R. C., Z. Z-G.J, and National BiomédicalESK Center fj. N., W. E. A.], Medical College of Wisconsin, Milwaukee, Wisconsin 53226

ABSTRACT by tumor cells remains a major obstacle to the successful treatment of malignancy. To understand how cells may over We have shown that transferrin-gallium (Tf-Ga) blocks DNA synthe come the cytotoxicity of gallium, we have developed a subline sis through inhibition of cellular iron incorporation and a diminution in the activity of the iron-dependent M2 subunit of ribonucleotide reductase. of HL60 cells which is relatively resistant to growth inhibition by this metal. We have found that the primary mechanism of To examine the mechanisms of drug resistance to gallium, we developed a subline of HL60 cells (R cells) which is 29-fold more resistant to drug resistance to gallium nitrate involves the ability of cells to growth inhibition by gallium nitrate than the parent line (S cells). R cells overcome the gallium-induced block in iron uptake. As a result, displayed a 2.5-fold increase in transferrin (Tf) receptor expression, gallium-resistant cells are able to acquire sufficient iron for without a change in receptor affinity for Tf. The uptake and release of DNA synthesis. 67Ga were similar for both S and R cells. The uptake of 59Fe-Tf by S cells was inhibited by gallium nitrate over 24-48 h of incubation. In contrast, "Fe-Tf uptake by R cells, although initially inhibited by gallium MATERIALS AND METHODS nitrate at 24 h, was no longer inhibited at 48 h of incubation. 59FeQ3 Chemicals. Gallium nitrate and human Tf were obtained from Alfa uptake by R cells was significantly greater than that of S cells, regardless Products (Danvers, MA) and Sigma Chemical Co. (St. Louis, MO), of the time in culture. Despite the increase in 59Feuptake by R cells, the respectively. Sodium [12SI]iodideand "FeCl.i were obtained from New ferritin content of these cells was lower than that of S cells. The England Nuclear (Boston, MA). Saturation of Tf with 59Fe and iodi- ribonucleotide reductase electron spin resonance signal of R cells was nation of "Fe-Tf were performed as described previously (11). Hydrox comparable to that of S cells. R cells were not cross-resistant to Adria- yurea and vincristine were obtained from Calbiochem-Behring Corp. mycin, vincristine, c/s-platinum or hydroxyurea. Resistance to gallium (San Diego, CA) and LyphoMed, Inc. (Rosemont, IL), respectively. nitrate in this subline of III .6(1cells results primarily from the ability of Adriamycin and c/s-platinum were purchased from Adria Laboratories cells to overcome the gallium-induced block in iron incorporation. In (Columbus, OH) and Bristol Laboratories (Bristol Myers, Evansville, addition, intracellular iron in R cells appears to traffic preferentially to a IN), respectively. non-ferritin compartment. Cells and Cell Growth Studies. Human promyelocytic leukemic HL60 cells were obtained from American Type Culture Collection (Rockville, MD) and were maintained in RPMI 1640 containing 10% fetal calf INTRODUCTION serum with 200 units/ml of penicillin, 0.2 mg/ml of streptomycin, and 50 fig/'ml of gentamicin (complete medium). A gallium nitrate-resistant Gallium, a group 111A metal, is currently undergoing clinical HL60 subline (R cells) was developed over a period of 9-12 months by investigation as a chemotherapeutic agent for the treatment of growing cells in increasing concentrations of gallium nitrate. Incre certain malignancies (gallium nitrate, NSC 15200) (1). While ments in the concentration of gallium nitrate in the medium were made the antitumor activity of this agent has been documented for only when the growth of R cells was equivalent to that of gallium some time, the mechanism of cytotoxicity of gallium has only nitrate-sensitive cells (S cells). Once a resistant subline had been estab recently been elucidated (2, 3). The avid binding of gallium to lished, R cells were routinely subcultured with 84 ^M gallium nitrate in the iron transport protein Tf3 results in the formation of stable the medium. For studies of growth kinetics, confluent cells were plated Tf-Ga complexes (4) which are incorporated into cells through at 5 x IO5 cells/ml in multiwell plates in the presence of varying Tf receptor-mediated endocytosis (5, 6). We have shown that concentrations of gallium nitrate, Adriamycin (0.01-1.0 MM),vincris exposure of HL60 cells to Tf-Ga results in a decrease in iron tine (1-10 nM), c/s-platinum (0.5-2.5 pM) or hydroxyurea (25-300 MM). Cell counts were performed after 72 h of incubation and cell viability uptake, an increase in cellular Tf receptor number (resulting was determined by trypan blue exclusion. from intracellular iron deficiency), and a subsequent arrest in I25l-Tf Binding Studies. Cell surface Tf receptor density was deter the growth of these cells (7). The inhibition of cellular prolif mined by I25l-Tf binding to intact cells as described previously (11). S eration is the result of a diminution in the activity of the iron- and R HL60 cells plated with or without 84 MMgallium nitrate were dependent M2 subunit of ribonucleotide reductase (2). This harvested after 24, 48, and 72 h of incubation. Cell counts at these time enzyme is responsible for the reduction of ribonucleotides to points were approximately 0.8, 1.4, and 2.2 x 106/ml, respectively. 125I- deoxyribonucleotides, a rate-limiting step in DNA synthesis Tf binding studies were performed on 5 x 10scells at 4°Cand maximal (8). The inhibitory effects of gallium on cell growth can be Tf binding was determined according to the method of Scatchard (12). reversed by iron salts (9), Tf-Fe (7), hemin (2), or iron-pyridoxal 67Ga Uptake and Release Studies. Confluent S and R cells were replated in complete medium (5 x IO5cells) in 1-ml multiwell plates. isonicotinoylhydrazone (10). 67Ga citrate, 0.5 ¿iCi,wasadded to each culture. After 24 and 48 h of The development of resistance to chemotherapeutic agents incubation, cells were harvested by centrifugation and the radioactivity in cell pellets was counted to determine the amount of 67Gaincorporated Received 10/27/89; revised 1/16/90. The costs of publication of this article were defrayed in part by the payment by cells. To examine the release of 67Ga from cells, R and S cells which of page charges. This article must therefore be hereby marked advertisement in had incorporated 67Ga over 24 or 48 h were washed twice and reincu- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. bated at 37°Cin fresh medium. Aliquots of cell suspension were 1This work was supported by USPHS Grant ROI CA41740 from the National Cancer Institute to C. R. C. removed after 2, 4, 6, and 24 h of incubation and centrifuged (2000 2To whom requests for reprints should be addressed, at Division of Hematol- rpm for 10 min). The radioactivity in the cell pellet and supernatant ogy/Oncology, Medical College of Wisconsin, 8700 W. Wisconsin Avenue, was counted to determine the fraction of 67Ga released from cells over Milwaukee, WI 53226. 3The abbreviations used are: Tf, transferrin; Tf-Ga, transferrin-gallium; S time. 59FeUptake Studies. Confluent S and R cells were replated (5 x 10s cells, gallium nitrate-sensitive cells; R cells, gallium nitrate-resistant cells; ESR, electron spin resonance; PBS, phosphate-buffered saline. cells) in 1-ml multiwell plates in the absence or presence of 84 MM 4468

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1990 American Association for Cancer Research. GALLIUM RESISTANCE AND IRON UPTAKE gallium nitrate. 59Fe-Tf (approximately 2 ^g Tf/ml) was added to each Table 1 '"I-Tfbinding to gallium nitrate-sensitive and -resistant HL60 cells well and cells were harvested after 24 and 48 h of incubation at 37°C I2!l-Tf binding studies were performed after 24 h of replating cells in fresh in a CO2 incubator. Cells were washed with PBS by centrifugation at medium. R cells used were continuously grown in medium containing 84 MM gallium nitrate (GN) and were replated in the presence of 84 pM gallium nitrate. 1000 rpm for 10 min and the radioactivity in the cell pellets was S cells »erereplated in the absence or presence of 84 jiM gallium nitrate as shown. counted. In a separate experiment, S and R cells which had been Values represent the mean ±SE (n = 4). subcultured for 0, 24, or 48 h in their respective media (without or with Maximal transferrin binding 84 UM gallium nitrate) were harvested, washed, and reincubated in Cells Additive (ng/10'cells) corresponding fresh media (5 x IO5cells/ml) with 59FeCl3(10 ng 59Fe, Sensitive 26.2 ±4.2 115,000 cpm/ml) for an additional 24 h. Cells were harvested by GN 28.8 ±3.5 centrifugation and the radioactivity in the cell pellet was counted. Measurement of Cellular Ferritin Content. S and R cells were har Resistant GN vested, washed twice with PBS containing 0.1% bovine serum albumin and were resuspended in the same buffer containing 0.1% Triton X- 100. After disruption of cells by sonication, cellular debris was removed by centrifugation (10,000 x g for 30 min) and the cytoplasmic super 0.30 natant was assayed for ferritin using a radioimmunoassay kit from Bio- Rad (Richmond, CA). ESR Spectroscopy Studies. Studies of the tyrosyl free radical of ribonucleotide reducíasewere performed on HL60 cells as previously if 0.20 - described by us (2). Approximately 5 x IO8S and R cells were incubated for 24 h in medium containing 0-960 n\t gallium nitrate. Cells were o harvested and washed with PBS and direct ESR measurements were co performed on frozen samples at -196°C in quartz finger Dewar flasks. 0.10 - ESR spectra were recorded nine times and averaged by computer.

RESULTS 0.00

Growth of S and R Cells in the Presence of Gallium Nitrate. BOUND (ng/106 cells) The dose-response curves of S and R cells to gallium nitrate Fig. 2. Scatchard analysis of 125I-Tfbinding to S and R HL60 cells. S and R are shown in Fig. 1. The concentration of gallium nitrate cells were harvested after 48 h of growth in their respective media and were assayed for '25I-Tf binding. Inset, binding of 12!I-Tf to R cells at 72 h relative to required to inhibit cell growth by 50% was 79.6 juMfor the S S cells at the same time point. Points and bars, mean ±SEM (n = 7). cells and 2295.8 ßMforthe R cells. Hence, the R cells were approximately 29 times more resistant to growth inhibition by Table 2 Uptake of"Ga by gallium nitrate-sensitive and -resistant HL60 cells gallium nitrate than the S cells. "Ga, 0.5 lid, was added to S and R cells plated at 5 x 10! cells in 1-ml Transferrin Receptor Expression. In previous studies, we have multiwell plates. Values shown represent the total "Ga cpm, corrected for decay, shown that the uptake of 67Ga by HL60 cells is mediated by the in the cell pellets after 24 and 48 h of incubation. Data represent the mean binding of 67Ga-Tf complexes to cellular Tf receptors and that ±95% confidence limits. Statistical analysis is discussed in the text. "Ga cpm incorporated by cells the uptake of this metal closely correlates with the density of Time cell surface Tf receptors (6). Therefore, to determine whether (h) Sensitive Resistant R cells expressed altered numbers of Tf receptors, cells were 24 21,374± 3,046 27,076 ±6,334 48 35.611 ±13,000 assayed for Tf binding following various times in subculture. 30,484 ±6,087 As shown in Table 1, at 24 h R cells in gallium nitrate expressed a markedly greater number of Tf receptors than S cells exposed shown in Fig. 2, R cells displayed almost twice the number of to the same concentration of gallium. At 48 h, R cells were Tf receptors as S cells, without a change in receptor affinity for compared with S cells plated without gallium, so as to examine the ligand. At 72 h of subculture, when cells had attained Tf receptor expression under conditions of equal growth. As confluency, Tf receptor expression on R cells was approxi mately 2.5 times that of S cells (Fig. 2, inset). 2.5 67Ga Uptake by Cells. To examine whether resistance could 100 ÕL be explained on the basis of differences in the cellular incor -, 2.0 z poration of gallium, 67Ga uptake by R and S cells was compared CD 80 3 (Table 2). The 67Ga cpm incorporated by S cells at 24 and 48 h I er O -iID was not significantly different from the 67Ga cpm incorporated 1.5 60 -D X by R cells at the same time points (P = 0.14 and 0.5 by 2-tailed -i(D o 0 0.5 24 h to 48 h, this increase failed to reach statistical significance •o—o 20 o (P = 0.063). Likewise, the increase in 67Ga uptake by R cells D from 24 h to 48 h was not significant (P = 0.46). In additional 0 l experiments, cells which had incorporated 67Ga over 24 or 48 10 100 1000 h were washed and allowed to release 67Ga in fresh medium. Gallium nitrate concentration (uW) No differences were noted in the release of 67Ga from R and S Fig. 1. Effects of gallium nitrate on the growth of sensitive (S) and resistant (R) HL60 cells. S and R cells were plated at 5 x 10' cells/ml in the presence of cells (not shown). Hence, resistance to gallium does not appear increasing concentrations of gallium nitrate. Cell counts were performed at 72 h to result from changes in the uptake and release of gallium of incubation. S cells (O); R cells (•).Data shown represent the mean ±SD from cells. (bars) of three experiments. Cell number in the absence of gallium nitrate (100%, control) was 2.28 ±0.27 x 10' cells/ml for S cells and 2.29 ±0.19 x 10' cells/ 59Fe Uptake by Cells. We have shown previously that the ml for R cells. growth-inhibitory effects of Tf-Ga result from an inhibition of 4469

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1990 American Association for Cancer Research. GALLIUM RESISTANCE AND IRON UPTAKE iron incorporation into HL60 cells (7). Incubation of S cells up-regulation of the Tf receptor coupled with an increase in the with 84 /IM gallium nitrate resulted in a marked inhibition of incorporation of Fe. 59Feuptake (as 59Fe-Tf) at 24 and 48 h of incubation (Fig. 3A). Cellular Ferritin Content. Table 3 shows that regardless of This decrease in iron uptake resulted in an arrest in the prolif the time in subculture, the ferritin content of R cells grown eration of these cells. As shown in Fig. 3B, 59Fe uptake by R continuously in the presence of 84 ¿¿Mgalliumnitrate was cells at 24 h was also inhibited by 84 /IM gallium nitrate. markedly lower than that of S cells. From the data shown in However, at 48 h, 59Fe uptake by these cells was no longer Figs. 3 and 4, it should be appreciated that R cells contained inhibited by gallium nitrate and as a result, R cells proliferated less ferritin than S cells despite the fact that the uptake of 59Fe normally. (from either 59Fe-Tf or 59FeCl3)was greater in R cells. As stated previously, resistance to gallium was developed Effects of Gallium Nitrate on the Tyrosyl Free Radical of through routine growth of R cells in the presence of gallium Ribonucleotide Reducíase.Ribonucleotide reducíaseconsists of nitrate in medium supplemented with 10% fetal calf serum an effector-binding Ml subunil and an iron-conlaining M2 (containing bovine TO- To exclude any influence that exoge- subunit (8). The M2 subunit also conlains a lyrosyl free radical nously added human Tf may have had on the uptake of 59Fe in which produces a characteristic signal on ESR spectroscopy, the above studies, S and R cells growing in their usual culture and Ihe aclivily of Ihis subunit correlates closely wilh ihe medium were pulsed with 59FeCl3at different times of subcul magnilude of Ihis ESR signal (13, 14). Exposure of HL60 cells ture. As shown in Fig. 4, the uptake of 59Fe from 59FeCl3was lo Tf-Ga resulls in a marked decrease in ihe amplilude of this always greater in the R cells regardless of the time points ESR signal and a decrease in deoxyribonucleolide synlhesis (2). examined. The "Fe uptake studies suggest strongly that R cells As shown in Fig. 5, Ihe ESR speclra of S and R cells were overcome the growth-inhibitory effects of gallium by a sustained examined following incubalion of cells with increasing concen- Iralions of gallium nitrale. Consislent with our earlier studies (2), increasing concentralions of gallium nilrate resulted in a marked suppression in the ESR signal of S cells. In contrast, 14 Sensitive B Resistant this effect was not seen with R cells. In the absence of gallium, the ESR signals of S and R cells were of comparable magnilude, 12 ihereby suggesling thai under conditions of equal cellular pro liferation, the aclivily of Ihe M2 subunit of ribonucleolide o 10 reducíasewas similar in bolh cell populations. Lack of Cross-Resistance belween Gallium Nilrate and Olher Anlineoplastic Agents. The sensitivilies of S and R cells lo "o.I 8 Adriamycin, vincrisline, m-plalinum and hydroxyurea were compared. Drug resislance lo Adriamycin and vincrisline (mul- lidrug resislance) is mediated by a cell membrane-based P- 01 m glycoprolein which facililates drug efflux from cells (15). The melai c/s-plalinum has been reported lo bind to Tf (16), and 24 h 48 h 24 h 48 h resislance lo Ihis drug is relaled lo an overexpression of metal-

TIME lothionein (17). Mouse L-cells have been shown lo develop Fig. 3. Uptake of "Fe-Tf by S and R HL60 cells. Cells were plated in the resislance lo hydroxyurea ihrough an increase in ihe M2 sub absence or presence of 84 /UMgallium nitrate. "Fe-Tf was added to each well at the onset of incubation and "Fe cpm in the cell pellets were counted after 24 and unil of ribonucleolide reducíaseresulling from gene amplifica 48 h of incubation. A, "Fe uptake by S cells in the absence (O), or presence of 84 tion (18). No differences in the sensilivily of R and S cells lo f.M gallium nitrate (•)./'. "Fe uptake by R cells in the absence (A) or presence the above antineoplaslic agenls were seen (dala noi shown). of 84 (/\i gallium nitrate (A). Data represent mean ±SD (bars) of three experi These sludies indicale lhal Ihe mechanism of resislance lo cell ments. growlh inhibilion by gallium is unique and is independenl of olher known mechanisms of drug resislance. 100 DISCUSSION Allhough ihe mechanisms of lumor cell resislance to many 80 drugs are noi known, receñísludieshave defined Ihe basis of drug resistance to certain antineoplastic agenls. These mecha nisms include decreased drug Iransporl inlo Ihe cell, increased 60 drug efflux from Ihe cell, alleralions in or overproduclion of inlracellular largel proleins, gene amplificalion, and increased drug degradalion (19). Our present study describes an addi- 40 Table 3 Ferritin content of gallium nitrate-sensitive and -resistant HL60 cells S and R cells were analyzed for ferritin content at the times shown following 20 subculture in fresh media. Growth medium for R cells contained 84 ,/\i gallium 0-24 24-48 48-72 nitrate. Data represent the mean ±SD of three experiments. content 59 Time of FeClj Pulse (h) protein)Sensitive18.2(¿ig/mg Time in Fig. 4. Uptake of "FeCl3 by S and R HL60 cells. Following 0, 24, or 48 h of subculture(h)24 replating in fresh media, S and R cells were harvested, replated in fresh media and pulsed for 24 h with "FeCl3. d, S cells in complete medium; B, represent R + 3.8 ±0.5 4872Ferritin 17.6 ±1.1 5.7 ±0.3 cells in complete medium containing 84 >IMgallium nitrate. Data shown represent 20.0 ±2.7Resistant5.0 6.0 ±0.6 the mean ±SD (bars) of three experiments. 4470

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SENSITIVE RESISTANT

Fig. 5. ESR spectra of the tyrosyl free radical of ribonu- cleotide reducíase.Gallium nitrate-sensitive (S) and -resistant (R) HL60 cells were harvested after 24 h of exposure to 0- 960 /JMgallium nitrate. The ESR spectra of gallium nitrate- sensitive (S) and -resistant (R) cells are shown in the left and right columns, respectively. Gallium nitrate (GN) concentra tions are shown in the middle column. ESR measurements were performed at -196°Con frozen samples containing 5 x 10* cells. Spectrometer conditions: microwave frequency, 9.098 GHz; modulation amplitude, 5 Gauss; modulation frequency, 100 kHz; incident microwave power, 100 mW; gain, 8 X IO4;and "temperature, —196°C.Data represent the signal average of nine scans.

3150 3230 3350 3150 3250 3350 MAGNETIC FIELD (G) MAGNETIC FIELD (G) tional mechanism in which drug resistance is mediated by the subunit of ribonucleotide reducíaseduring the S phase of the combination of sustained up-regulation of a cellular receptor cell cycle. The activity of this enzyme, as determined by ESR and enhanced incorporation of a nutrient essential for cell spectroscopy and measurements of cellular deoxyribonucleotide growth. content, is decreased following exposure of HL60 cells to Tf- Since drug resistance can develop on the basis of alterations Ga (2). While the inhibitory effect of gallium on ribonucleotide in the intracellular accumulation of drug, it was essential for us reducíaseis believed to be secondary to a decrease in intracel to examine the uptake and release of 67Ga by R cells. Although lular iron, it is possible that gallium may also have a more the uptake of 67Ga correlates closely with the density of cellular direct effect on this enzyme. Cells resistant to agents known to Tf receptors (6), the increase in Tf receptors in the R cells did act specifically on the M2 subunit (e.g., hydroxyurea) display not lead to a corresponding increase in the uptake of 67Ga. an overproduction of the M2 subunit and an increase in its Furthermore, the release of intracellular 67Ga from R and S ESR signal (13). In our studies, the ESR signals from S and R cells was similar. Resistance to gallium nitrate therefore does cells were found to be similar when examined at 24 h, under not appear to result from decreased cellular accumulation or conditions of equal growth rate (i.e., in the absence of gal- enhanced release of this metal from cells. lium).On further comparison of the ESR spectra from S and R Although R cells displayed more Tf receptors than S cells, it cells, it is obvious that in the presence of increasing concentra should be appreciated that it is not the increase in Tf receptors tions of gallium, the signal from S cells was diminished while perse which results in resistance to gallium. In previous studies signal from R cells was relatively unchanged, even at 960 ¿¿M utilizing a defined serum-free system, we have clearly shown of gallium nitrate. We interpret these results in the following that acute exposure of (sensitive) HL60 cells to Tf-Ga results way: In the presence of 84 ¡J.Mgallium,iron uptake by S cells is in an inhibition of cellular iron incorporation and a subsequent inhibited and thus less intracellular iron is available to support increase in cellular Tf receptors over 24-48 h. However, despite the activity of the iron-dependent M2 subunit of ribonucleotide the increase in Tf receptors, iron uptake by S cells is consistently reducíase.Accordingly, the ESR signal of S cells diminishes in inhibited by gallium at all stages of proliferation (7). The key the presence of gallium. In contrasl, iron uplake by R cells is difference between R and S cells is the finding that iron uptake noi inhibiled by gallium; therefore sufficient inlracellular iron by R cells is not continuously inhibited by gallium. R cells at is available lo support the activily (ESR signal) of Ihe M2 confluency and replating do have an increased number of cell subunil. Since Ihe ESR signals from S and R cells are similar surface Tf receptors (in contrast to S cells which have low under condilions of equal proliferalion, il would appear lhal R numbers of Tf receptors at replating). However, it is the increase cells are noi resislanl lo gallium by virtue of increased aclivily in Tf receptors coupled with enhanced iron uptake which en of Ihe M2 subunit of ribonucleotide reducíaseduring the first ables R cells to incorporate iron in sufficient amounts to support 24 of subcullure. Clearly, further invesligations involving direct DNA synthesis and other critical iron-dependent processes. assay of ribonucleotide reducíase are needed lo assess Ihe Cellular requirements for iron during DNA synthesis appear aclivily of Ihis enzyme in R cells. to be related to the increased activity of the iron-containing M2 In addilion lo developing resislance lo gallium ihrough an 4471

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1990 American Association for Cancer Research. GALLIUM RESISTANCE AND IRON UPTAKE increase in Tf receptors and iron uptake, our studies also gallium transferrin. Biochemistry, 22: 292-299, 1983. 5. Larson, S. M., Rasey, J. S., Allen, D. R., Nelson, N. J., Grunbaum, Z., Harp, provide indirect evidence that R cells may utilize iron more G. D., and Williams, D. L. Common pathway for tumor cell uptake of efficiently than S cells. This conclusion is based upon a com galIium-67 and iron-59 via a transferrin receptor. J. Nati. Cancer Inst., 64: 41-53, 1980. parison of the ferritin content of the two cell populations. 6. Chitambar, C. R., and Zivkovic, Z. Uptake of gallium-67 by human leukemic Ferritin synthesis is directly stimulated by iron and is increased cells: demonstration of transferrin receptor-dependent and transferrin-inde- or decreased when iron delivery to cells is increased or decreased pendent mechanisms. Cancer Res., 47: 3929-3934, 1987. 7. Chitambar, C. R., and Seligman, P. A. Effects of different transferrin forms (20, 21). Since iron uptake by R cells was greater than S cells, on transferrin receptor expression, iron uptake and cellular proliferation of both cell populations would be expected to contain at least human leukemic HL60 cells: mechanisms responsible for the specific cyto equivalent amounts of ferritin. However, the ferritin content of toxicity of transferrin-gallium. J. Clin. Invest., 78: 1538-1546, 1986. 8. Thelander, L., and Reichard, P. Reduction of ribonucleotides. Annu. Rev. R cells was less than one-third that of S cells. This suggests Biochem., 48: 133-158, 1979. that a significant portion of iron incorporated by R cells is not 9. Rasey, J. S., Nelson, N. J., and Larson, S. M. Tumor cell toxicity of stable gallium nitrate: enhancement by transferrin and protection by iron. Eur. J. available to stimulate ferritin synthesis and is instead utilized Cancer Clin. Oncol., 18: 661-668, 1982. more efficiently for cell growth. 10. Chitambar, C. R., and Zivkovic, Z. Inhibition of hemoglobin production by Gallium has been shown to share several properties with iron transferrin-gallium. Blood, 69: 144-149, 1987. 11. Chitambar, C. R., Massey, E. J., and Seligman, P. A. Regulation of transfer with respect to Tf binding (4), cellular uptake by Tf receptors rin receptor expression on human leukemic cells during proliferation and (5, 6), and incorporation into ferritin (6, 22). While Tf-Ga and induction of differentiation. Effects of gallium and dimethylsulfoxide. J. Clin. Tf-Fe compete for initial entry into the cell via Tf receptor- Invest., 72: 1314-1325, 1983. 12. Scalchimi. G. The attractions of proteins for small molecules and ions. Ann. mediated endocytosis, subsequent intracellular interactions be NY Acad. Sci., 5/: 660-672, 1949. tween the two metals remain to be defined. Gallium and iron 13. Eriksson, S., Graslund, A., Skog, S., Thelander, L., and Tribukait, B. Cell present in intracellular "pools" may compete for binding to cycle-dependent regulation of mammalian ribonucleotide reducíase.J. Biol. Chem., 259: 11695-11700, 1984. macromolecules essential for cellular function. Since the incor 14. Graslund, A., Shalin, M., and Sjoberg, B-M. The tyrosyl free radical in poration of 67Ga into S and R cells was similar, it could be ribonucleotide reducíase.Environ. Health Perspect., 64: 139-149, 1985. 15. Pastan, I., and Gottesman, M. Multiple-drug resistance in human cancer. N. postulated that R cells overcome the cytotoxicity of gallium Engl. J. Med., 316: 1388-1393, 1987. through enhanced incorporation of iron and expansion of the 16. Sykes, T. R., Stevens-Newsham, L. G., and Noujaim, A. A. In vitro binding of platinum compounds to human transferrin. Res. Commun. Chem. Pathol. iron pool relative to the gallium pool. 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Christopher R. Chitambar, Zorica Zivkovic-Gilgenbach, Jana Narasimhan, et al.

Cancer Res 1990;50:4468-4472.

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