Characterization of the defect in a variant of HL-60 promyelocytic leukemia cells with reduced transferrin receptor expression

Abstract

The mechanism by which a clone of HL-60 human promyelocytic leukemia cells designated Tf-Gel-1 expresses reduced levels of the transferrin receptor (TfR) was investigated. Tf-Gel-1 was developed by continuous exposure of HL-60 cells to human iron-saturated transferrin covalently linked to the plant toxin gelonin (Tf-Gel); this variant was five- to sixfold more resistant to Tf-Gel than parental HL-60 cells. The amount of cell surface, as well as of solubilized, TfR and the cycling pools of TfR in Tf-Gel-1 cells, as measured by the binding of [125I]Tf, were all decreased to 20-30% of the levels present in parental cells. The growth of Tf-Gel-1 cells was independent of exogenous Fe3+ and was comparable to that of parental HL-60 cells. Despite the lower levels of TfRs, the Tf-Gel-1 clone retained the capacity to alter receptor expression, depending upon the phase of growth and the intracellular iron concentration, and to down-regulate TfRs in response to inducers of differentiation. Southern hybridization of cellular DNA with TfR cDNA did not reveal differences between parental and Tf-Gel-1 cells in the level and arrangement of the TfR gene. Basal and inducible (repressible) levels of TfR mRNA from Tf-Gel-1 cells, as measured by northern hybridization of cellular RNA with TfR cDNA, were comparable to those of parental cells. Metabolic labeling of cells with [35S]methionine, followed by immunoprecipitation of TfRs, demonstrated that the amount of radioactivity incorporated into TfRs in Tf-Gel-1 cells was reduced to a degree that approximated the decrease in [125I]Tf binding. Cell surface TfRs prepared from exponentially growing parental cells labeled with 125I by the solid-phase lactoperoxidase-glucose oxidase method existed as a doublet, with one form being phosphorylated and the other not phosphorylated. In contrast, Tf-Gel-1 cells not only contained diminished amounts of TfRs but also contained only the phosphorylated form of TfRs in the surface membrane. The decrease in the surface membrane concentration of the TfR in Tf-Gel-1 cells was specific for this glycoprotein, since the levels of other cell surface antigens, such as CD13, CD15 and CD45, were normal in Tf-Gel-1 cells. A reduction in the incorporation of [3H]mannose into the acid-insoluble fraction of cells and an increase in sensitivity to ricin suggested that Tf-Gel-1 cells possessed an aberration in carbohydrate metabolism.