Role of the acidic receptosome in the uptake and retention of 67Ga by human leukemic HL60 cells.

The uptake of 67Ga by HL60 cells requires binding of 67Ga-transferrin (Tf) to cell surface Tf receptors. To further examine this process, we have studied early events in the cellular uptake of 67GaTf. Cell surface-bound 67GaTf and 59FeTf displayed similar kinetics during the first 10 min of uptake. Thereafter, approximately 10% of intracellular 67Ga was released by cells while 59Fe internalization continued to increase with time. In pulse-chase studies of 125I-Tf-67Ga uptake, internalized 125I-Tf, but not 67Ga, was chased out of cells by nonradioactive Tf-Ga. Exposure of cells to monensin, a carboxylic ionophore, during initial uptake decreased the internalization of both 125I-Tf and 67Ga. Exposure to monensin at a later time, after cells had incorporated 125I-Tf-67Ga or 59FeTf, caused an increase in the release of 67Ga and 59Fe with a decrease in the release of 125I-Tf. Ammonium chloride inhibited the internalization of both 67Ga and 59Fe. 67GaTf uptake by HL60 cells involves initial internalization into an acidic receptosome. This is followed by dissociation of 67Ga and Tf and subsequent trafficking of each to separate intracellular compartments. Disruption of this process by monensin results in the release of 67Ga from cells.

Development of drug resistance to gallium nitrate through modulation of cellular iron uptake.

We have shown that transferrin-gallium (Tf-Ga) blocks DNA synthesis through inhibition of cellular iron incorporation and a diminution in the activity of the iron-dependent M2 subunit of ribonucleotide reductase. 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 growth inhibition by gallium nitrate than the parent line (S cells). R cells displayed a 2.5-fold increase in transferrin (Tf) receptor expression, without a change in receptor affinity for Tf. The uptake and release of 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, 59Fe-Tf uptake by R cells, although initially inhibited by gallium nitrate at 24 h, was no longer inhibited at 48 h of incubation. 59FeCl3 uptake by R cells was significantly greater than that of S cells, regardless of the time in culture. Despite the increase in 59Fe uptake by R cells, the ferritin content of these cells was lower than that of S cells. The ribonucleotide reductase electron spin resonance signal of R cells was comparable to that of S cells. R cells were not cross-resistant to Adriamycin, vincristine, cis-platinum or hydroxyurea. Resistance to gallium nitrate in this subline of HL60 cells results primarily from the ability of cells to overcome the gallium-induced block in iron incorporation. In addition, intracellular iron in R cells appears to traffic preferentially to a non-ferritin compartment.

Influence of cellular iron status on the release of soluble transferrin receptor from human promyelocytic leukemic HL60 cells.

We have previously shown that human leukemic HL60 cells release from their surface a soluble form of the transferrin receptor. Because of the regulatory role of iron in transferrin receptor expression, we have now examined the relationship between iron and the release of soluble transferrin receptor from HL60 cells. Cells grown in serum-free, transferrin-free medium containing iron-pyridoxal isonicotinoyl hydrazone (Fe-PIH) displayed approximately 70% less iodine 125-labeled transferrin surface binding and released 60% less soluble transferrin receptor than cells grown in serum-supplemented medium. Incubation of cells with increasing concentrations of Fe-PIH resulted in a progressive decrease in the release of soluble transferrin receptor over 18 hours of incubation. In contrast, receptor release was increased after incubation of cells with the iron chelator deferoxamine. This effect was completely blocked by cycloheximide. Transferrin receptor release from cells over 2 hours was unaffected by the presence of transferrin-iron, suggesting that transferrin receptor release occurs independent of the cellular handling of its ligand. Exposure of cells to phorbol myristate acetate resulted in a decrease in cell surface transferrin receptor and a decrease in the release of soluble transferrin receptor. Our studies show that transferrin receptor release from HL60 cells changes during iron excess or iron deficiency and that these changes are the result of alterations in cell surface transferrin receptor density. Our studies suggest that elevated serum transferrin receptor levels seen in clinical iron deficiency reflect corresponding increases in transferrin receptors at the cellular level.