Role of the Acidic Receptosome in the Uptake and Retention of 67Gaby Human Leukemic HL60 Cells1

The uptake of 67Gaby HL60 cells requires binding of 67Ga-transferrin (TO to cell surface Tf receptors. To further examine this process, we have studied early events in the cellular uptake of 67GaTf. Cell surfacebound "~(.;i11 and '"loll displayed similar kinetics during the first 10 min of uptake. Thereafter, approximately 10% of intracellular 67Gawas released by cells while 59Fe internalization continued to increase with time. In pulse-chase studies of l25I-Tf-67Gauptake, internalized I25l-Tf, but not 67Ga,was chased out of cells by nonradioactive Tf-Ga. Exposure of cells to monensin, a carboxylic ionophore, during initial uptake de creased the internalization of both 125I-Tfand 67Ga.Exposure to monensin at a later time, after cells had incorporated 125I-Tf-67Gaor "FeTf, caused an increase in the release of 67Gaand 59Fewith a decrease in the release of ' '"I-11. Ammonium chloride inhibited the internalization of both '7Ga and !9Fe. 67GaTf uptake by III 00 cells involves initial internalization into an acidic receptosome. This is followed by dissociation of 67Gaand Tf and subsequent trafficking of each to separate intracellular compart ments. Disruption of this process by monensin results in the release of 67Gafrom cells. INTRODUCTION The preferential uptake of 67Ga by certain tumors has resulted in the use of this radioisotope as a scanning agent for the detection of occult malignancies in vivo (1). Recent clinical studies in patients with lymphoma have shown that high-dose 67Ga scanning can be used to detect recurrent disease and to determine the prognosis and response to ongoing therapy (2). While considerable progress has been made in the utilization of 67Ga in the clinical setting, information regarding its mech anism of intracellular localization is incomplete. Gallium is known to resemble iron with respect to binding to Tf,-' the iron transport protein (3). We and others have shown that the cellular uptake of 67Ga is mediated by cell surface receptors for Tf (4, 5). In addition, a portion of gallium incorporated into human leukemia HL60 cells appears to bind to intracellular ferritin (4). While previous studies have elucidated the role of the Tf receptor in the initial stages of the cellular uptake of gallium, subsequent steps in the internalization and intracellular traf ficking of this metal remain to be defined. To obtain a better understanding of the steps involved in the cellular incorporation of gallium, we have compared the uptake kinetics of gallium, iron, and Tf. In addition, we have investigated the effects of monensin, a carboxylic ionophore (6), on the uptake and reten tion of gallium, iron, and Tf by HL60 cells. MATERIALS AND METHODS Human Tf (substantially iron free) and ammonium chloride were purchased from Sigma Chemical Co. (St. Louis, MO). Monensin Received 8/1/89; revised 11/2/89; accepted 11/27/89. 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 work was supported by USPHS Grant RO l CA41740 from the National Cancer Institute to C. R. C. 1To whom requests for reprints should be addressed, at Division of Hematology/Oncology, Medical College of Wisconsin, 8700 W. Wisconsin Avenue, Milwaukee, WI 53226. 3The abbreviations used are: Tf, transferrin; Tf-Ga, transferrin-gallium. (Calbiochem, La Jolla, CA) was dissolved in ethanol (1 mivi stock solution). 59FeCl5 and 67Ga citrate (carrier free) were obtained from Amersham (Arlington Heights, IL) and Mediphysics (Richmond, CA), respectively. 59FeTfwas prepared according to the method of Bates and Schlabach (7). Na'25l was purchased from Amersham and Tf was iodinated by the chloramine-T method (8), as previously described (9). Tissue Culture. Human promyelocytic leukemic HL60 cells were obtained from American Type Culture Collection (Rockville, MD) and cells were maintained in RPMI 1640 medium containing 10% fetal calf serum, 200 units/ml of penicillin, 0.2 mg/ml of streptomycin, and 50 Mg/ml of gentamicin. Cells in log growth phase were used for the experiments described below. Internalization of Surface-bound 67GaTf and "FeTf by HL60 Cells. The rate of internalization of cell surface Tf receptor-bound 67Ga-Tf or 125I-Tf-67Ga(dual label) was examined using a modification of a previ ously described method which involves washing cells in acidic buffer to remove noninternalized radioactivity (10). To bind 67GaTf to cell sur face Tf receptors, IO7HL60 cells were incubated with 10 ^Ci of 67Ga citrate and 25 Mg(0.625 MM)of Tf in serum-free medium for 90 min at 4°C(total incubation volume, 500 M').In studies utilizing the dual label, cells were incubated with 67Ga and Tf, with 125I-Tfadded as a trace label (IO6 cpm 125I-Tf/20 Mgof TO. Unbound 67GaTf or 125I-Tf-67Ga was removed by washing cells twice with ice-cold medium. Cells were then resuspended in 1 ml of warm medium at 37°Cand aliquots of cell suspension were removed after 1-45 min of incubation. The aliquots were promptly transferred to microfuge tubes containing 2-3 volumes of 20 mM acetic acid, 150 mivi NaCl, pH 3.5 (acid wash) buffer to dissociate cell surface (noninternalized) radioactivity from cells, and the tubes were centrifuged for l min in a microfuge centrifuge at full power. The pellet and supernatant were separated and the radioactivity in each fraction was counted with an LKB Compugamma gamma counter. The acid-resistant cpm in the cell pellet was taken to represent 67Ga which had been internalized by cells during the respective incu bation period. In a parallel experiment, the internalization of cell surface Tf receptor-bound 59Fe-Tf was examined. Experimental condi tions were identical to those described for 67Ga, except that cells were incubated at 4°Cwith 25 Mg(0.625 MM)of Tf saturated with 59Fe (37 ng 59Fe). Pulse-Chase Studies. Cells (106/ml) were incubated in serum-free medium with 125I-Tf-67Gaat 37°Cfor l h (pulse). Cells were then washed and replated in 1 ml of fresh medium alone or the same medium containing 250 Mg/ml of Tf-Ga (chase medium). Aliquots of cell sus pension were removed and centrifuged in a microfuge centrifuge after 5-90 min of incubation at 37"C. 67Ga and 125Icpm in the cell pellet and supernatant were counted simultaneously by using a gamma coun ter dual channel setting. Effect of Monensin on 67Ga Uptake and Release. Prior to the use of monensin in these studies, HL60 cells were exposed to different con centrations of monensin for varying lengths of time to determine the effect on cell viability. Exposure of cells to 100 MMmonensin for 2 h resulted in a significant loss of viability as determined by trypan blue exclusion. Cells exposed to 10 MMmonensin for 2-3 h retained a viability equivalent to control cells (>90%), while longer periods of exposure to monensin resulted in a progressive loss of viability. There fore, all the experiments utilizing monensin were performed by using a subtoxic concentration (10 /JM)of this agent, (a) To examine the effect of monensin on the internalization of a single complement of cell surface Tf receptor-bound 67GaTf or 125I-Tf-67Ga,internalization stud ies described above were performed in the presence of 10 MMmonensin. (¿>) To examine the effects of monensin on the release of 125I-Tf-67Ga from cells, HL60 cells were plated (IO6 cells/ml) in two separate flasks in serum-free medium containing 67Ga citrate (0.5 MCi/ml) and 5 Mg/ ml of Tf with 12SI-Tf(IO6 cpm/20 Mgof TOAfter l h of incubation at 1484 on April 14, 2017. © 1990 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from "Ga UPTAKE AND RECEPTOSOMAL pH 37°Cin a CO.. incubator, 10 Õ<Mmonensin was added to one flask and the incubation was continued for l h more. Cells were then washed twice with ice-cold medium to remove unincorporated '25I-Tf-67Gaand were resuspended at 37°Cin their corresponding medium (with or without monensin). One-mi aliquots of cell suspension were harvested after 5-90 min of incubation and the cells were removed by centrifugation. The radioactivity in the cell pellet and supernatant was counted to determine the fraction of 67Ga and I2'l-Tf retained by cells. The effect of monensin on the cellular retention of "Fe was examined by using similar experimental conditions, except that cells were incubated with approximately 50,000 cpm/ml of 5*FeTf instead of "Ga. Effect of Ammonium Chloride on Internalizaron of '' (.a and "Fe. HL60 cells were incubated at 37°Cfor l h in serum-free medium containing 30 miviammonium chloride. 67GaTf or "FeTf was bound to cell surface Tf receptors at 4"C and the internalization of "Ga and "Fe was examined as described above, in the absence or presence of 30 mM ammonium chloride. Cells exposed to ammonium chloride for up to 4 h retained a viability equivalent to control cells.