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Stable transfection of a new, chimeric reporter in the human malaria parasite Plasmodium falciparum confers green fluorescence and methotrexate resistance that can be quantitated by Western blotting and flow cytometry. This provides a sensitive, live reporter for exploitation of genomic and high-throughput assays for the identification of new pathogenic determinants.

Characterization of non-transferrin (non-Tf) iron transport by K562 cells has revealed unique properties relative to iron uptake mechanisms present in other cell types (Inman, R. S., and Wessling-Resnick, M. (1993) J. Biol. Chem. 268, 8521-8528). Since treatment of K562 cells with phorbol esters promotes stable megakaryocytic differentiation, we examined the uptake of non-Tf iron in response to protein kinase C activation. Treatment of K562 cells with phorbol esters increased the cellular uptake of 55Fe 4-6-fold compared with untreated cells. The phorbol ester-induced stimulation of 55Fe uptake was time- and dose-dependent, with significantly enhanced transport observed only after prolonged administration of 50 nM phorbol 12,13-dibutyrate (> 8 h). These effects can be attributed to an increased Vmax of transport (14.0 +/- 5 versus 0.6 +/- 0.2 pmol/min/10(6) cells) as well as a 6-fold increase in the apparent Km (1.2 +/- 0.4 versus 0.2 +/- 0.06 microM). It is thought that the reduction of Fe3+ to Fe2+ is required as a first step in the uptake mechanism, and the associated ferrireductase activity of K562 cells is also enhanced with phorbol ester treatment by 5-10-fold (337 +/- 53 versus 43 +/- 3 pmol/min/10(6) cells). Bryostatin-1, a protein kinase C activator that fails to induce differentiation of K562 cells, did not promote this effect, indicating that the enhanced transport activity is dependent on the differentiation response. The idea that synthesis of a new class of transporters is responsible for this effect is supported by the observation that actinomycin D blocks up-regulation of non-Tf iron transport. The increased transport and ferrireductase activity induced upon differentiation also correlate with the appearance of saturable iron-binding sites on the surface of K562 cells with Kd approximately 0.4 microM. These results indicate that non-Tf iron transport activity and the expression of cell-surface iron-binding proteins can be controlled by environmental factors that promote megakaryocytic differentiation of K562 cells.

Treatment with phorbol esters increases endocytosis of the transferrin receptor in K562 cells (Klausner, R. D., Harford, J., and van Renswoude, J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3005-3009). In this report, we demonstrate that this effect is reversible within early times of protein kinase C activation (< 2 h) but that prolonged exposure to phorbol esters results in a net loss of receptors. These effects are not due to the differentiation response of K562 cells to phorbol esters since bryostatin-1 also down-regulates the endocytosis of the transferrin receptor and shut downs receptor synthesis, but does not induce differentiation (Hocevar, B. A., Morrow, D. M., Tykocinski, M. L., and Fields, A. P. (1992) J. Cell Sci. 101, 671-679). We have characterized the early stages of receptor down-regulation which occur due to stimulation of receptor internalization from the cell surface. The fact that fluid-phase pinocytosis is also enhanced upon protein kinase C activation indicates that this effect is not specific for the transferrin receptor itself, but is a rather general cellular response to tumor-promoting phorbol esters. The fate of down-regulated transferrin receptors was followed in morphological and subcellular fractionation studies that demonstrate localization of this pool of receptors in early endocytic and recycling compartments. Our results exclude the possibility that transferrin receptor down-regulation results in trafficking of the receptor to lysosomal compartments for degradation. This idea is consistent with the observations that the time course of transferrin receptor degradation is not enhanced in stimulated K562 cells, while transferrin receptor synthesis is shut down. Our results rigorously demonstrate that activation of protein kinase C down-regulates the K562 cell transferrin receptor in two stages: acute regulation of early steps in endocytosis that results in an immediate reduction of approximately 40% in cell surface number of receptors and a more chronic reduction in transferrin receptor synthesis upon prolonged exposure to phorbol esters (> 15 h).