Separation of cellular iron containing compounds by electrophoresis.

High resolution separation of metalloproteins and other iron compounds based on native gel electrophoresis followed by 59Fe autoradiography is described. Lysates of mouse spleen erythroid cells metabolically labeled with 59Fe-transferrin were separated on 3-20% polyacrylamide gradient gels in the presence of Triton X100 and detected by autoradiography. In addition to ferritin and hemoglobin, several compounds characterized by their binding of iron under different conditions were described. Iron chelatable by desferrioxamine migrated in the region where several high-molecular weight compounds were detected by silver staining. The technique is nondissociative, allowing identification of iron compounds with the use of specific antibodies. Cellular iron transport and the action of iron chelators on specific cellular targets can be investigated in many small biological samples in parallel.

Transferrin and iron distribution in subcellular fractions of K562 cells in the early stages of transferrin endocytosis.

Iron distribution in subcellular fractions was investigated at different times after a single cohort of 59Fe-125 I-labeled transferrin (Tf) endocytosis in K562 cells. Cell homogenates prepared by hypotonic lysis and deoxyribonuclease (DNAase) treatment were fractionated on Percoll density gradients. Iron-containing components in the postmitochondrial supernatant were further fractionated according to their molecular weight using gel chromatography and membrane filtration. In the initial phases of endocytosis, both iron and Tf were found in the light vesicular fraction. After 3 min the labels diverged, with iron appearing in the postmitochondrial supernatant and Tf in the heavy fraction containing mitochondria, lysosomes and nuclei. Iron released from Tf-containing vesicles appeared both in low- and high-molecular-weight fractions in the postmitochondrial supernatant. After 5 min of endocytosis 59Fe activity in the low-molecular-weight fraction remained constant and 59Fe accumulated in a high-molecular-weight fraction susceptible to desferrioxamine chelation. After 10 min, 59Fe radioactivity in this fraction decreased and a majority of cytosolic 59Fe was found in ferritin. These results do not support the concept of the cytosolic low-molecular-weight iron pool as a kinetic intermediate between transferrin and ferritin iron in K562 cells.

Inhibition of heme synthesis decreases transferrin receptor expression in mouse erythroleukemia cells.

The coordination of transferrin receptor (TfR) expression and heme synthesis was investigated in mouse erythroleukemia (MEL) cells of line 707 treated with heme synthesis inhibitors or in a variant line Fw genetically deficient in heme synthesis. Cells of line 707 were induced for differentiation by 5 mM hexamethylene bisacetamide (HMBA). TfR expression increased in the course of induction, as judged by increased TfR mRNA synthesis, increased cytoplasmic TfR mRNA level, and by the increased number of cellular 125I-Tf binding sites. Addition of 0.1 mM succinylacetone (SA) decreased cellular TfR to the level comparable with the uninduced cells. The decrease was reverted by the iron chelator desferrioxamine (DFO) but not by exogenous hemin. In short-term (1-2 hours) incubation, SA inhibited 59Fe incorporation from transferrin into heme, whereas total cellular 59Fe uptake was increased. A decrease in TfR mRNA synthesis was apparent after 2 hours of SA treatment. Conversely, glutathione peroxidase mRNA synthesis, previously shown to be inducible by iron, was increased by SA treatment. Cells of heme deficient line Fw did not increase the number of Tf binding sites after the induction of differentiation by 5 mM sodium butyrate. SA had no effect on TfR expression in Fw cells. The results suggest that the depletion of cellular non-heme iron due to the increase in heme synthesis maintains a high level of transferrin receptor expression in differentiating erythroid cells even after the cessation of cell division.

The relationship between heme synthesis and iron uptake in erythroid cells.

Hemin inhibited 59Fe uptake from transferrin (Tf) by mouse erythro-leukemia cells (MELC) induced for differentiation by hexamethylene bisacetamide (HMBA), but the rate and the extent of 125I-Tf endocytosis was unaffected. Hemin inhibited 59Fe incorporation into heme by a greater proportion than the overall uptake of 59Fe from Tf. Desferrioxamine (DFO) reverted the inhibition of 59Fe transport into MELC caused by hemin. Exogenous 5-aminolevulinic acid (ALA) stimulated 59Fe utilization for heme synthesis in MELC but did not revert inhibition induced by hemin. No evidence for a direct effect of heme on the Tf cycle or iron release was found.

Inhibition of cellular iron uptake by haem in mouse erythroleukaemia cells.

Haemin inhibited iron uptake from transferrin (Tf) by mouse erythroleukaemia cells (MELC) induced for differentiation by hexamethylene bisacetamide (HMBA). The rate of 59Fe internalization was decreased, but the rate and the extent of 125I-Tf endocytosis was unaffected by the addition of haemin. Haemin inhibited 59Fe incorporation into haem by a greater proportion than the overall uptake of 59Fe from Tf. The reduction of total cellular 59Fe uptake was more pronounced at 59Fe-Tf concentrations closer to saturation. Exogenous 5-aminolaevulinic acid stimulated 59Fe utilization for haem synthesis in MELC but did not revert the inhibition induced by haemin. Haem synthesis measured by 14C-glycine incorporation into haem was maintained for at least 1 h without an external transferrin iron source and was inhibited by the addition of haemin equally over the whole range of Tf concentrations studied. Desferrioxamine (DFO) stimulated cellular uptake of 59Fe by the uninduced cells and reverted the inhibition of 59Fe transport into HMBA treated cells caused by haemin. Addition of DFO within a short-term incubation had no effect on haem synthesis measured by 14C-glycine incorporation into haem. No evidence for a direct effect of haem on the transferrin cycle or iron release was found. It was concluded that the reduction of iron uptake by haemin treated MELC is secondary to the decrease in iron utilization for haem synthesis.

Non-transferrin donors of iron for heme synthesis in immature erythroid cells.

The mechanism of iron uptake from several iron-containing compounds by transferrin-depleted rabbit reticulocytes and mouse spleen erythroid cells was investigated. Iron complexes of DL-penicillamine, citrate and six different aroyl hydrazones may be utilized by immature erythroid cells for hemoglobin synthesis, although less efficiently than iron from transferrin. HTF-14, a monoclonal antibody against human transferrin, reacts with rabbit transferrin and inhibits iron uptake and heme synthesis by rabbit reticulocytes. HTF-14 had no significant effect on iron uptake and heme synthesis when non-transferrin donors of iron were examined. Ammonium chloride (NH4Cl) increases intracellular pH and blocks the release or utilization of iron from the internalized transferrin. NH4Cl only slightly affected iron incorporation and heme synthesis from non-transferrin donors of iron. Hemin inhibited transferrin iron uptake and heme synthesis, but had a much lesser effect on iron incorporation and heme synthesis from non-transferrin donors of iron. These results allow us to conclude that transferrin-depleted reticulocytes take up iron from all of the examined non-transferrin iron donors without the involvement of the transferrin/transferrin receptor pathway.

Iron uptake from transferrin and transferrin endocytic cycle in Friend erythroleukemia cells.

Several aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of hemoglobin synthesis by dimethyl sulfoxide. The maximal rate of iron uptake from 59Fe-labeled transferrin, 1.5 X 10(6) atoms of Fe/cell per 30 min in uninduced cells, increased to 3 X 10(6) atoms/cell after 5 days of induction. The increase in iron uptake was not accompanied by a proportional increase in the number of transferrin receptors detected by 125I-labeled transferrin binding, suggesting a more efficient iron uptake by transferrin receptors in induced cells, with the rate of about 26 iron atoms per receptor per hour, compared to 15 atoms in uninduced cells. In agreement with this conclusion are results of the study of cellular 125I or 59Fe labeled transferrin kinetics. In the induced cells transferrin endocytosis and release proceeded with identical rates and all the endocytosed iron was retained inside the cell. On the other hand, transferrin release by uninduced cells was significantly slower and a substantial part of internalized 59Fe was released. On the basis of these results, different efficiency of iron release from internalized transferrin, accompanied by changes in cellular transferrin kinetics, is proposed as one of the factors determining the rate of iron uptake by developing erythroid cells.

Regulation of iron uptake in erythroid and non-erythroid cells.

Iron uptake and the transferrin endocytotic cycle were studied in Friend erythroleukemia 707 cells cultured over a period of 5 days after induction with dimethylsulphoxide, or without it. The increase in iron uptake observed 5 days following induction with dimethylsulphoxide is not associated with a corresponding increase in the number of transferrin receptors. While the uptake rate of induced cells is 26 atoms of iron per one transferrin receptor per hour, in non-induced cells, the rate is only 15 atoms of iron. Induced cells retain all iron, with endocytosis and transferrin release occurring at the same rate. By contrast, the rate of iron release from non-induced cells is slower and part of the iron bound to transferrin returns from the cell. Haem inhibits iron uptake in induced cells and has no effect on non-induced cells. Its regulatory role is apparently confined to the erythroid cell with massive haemoglobin synthesis. The differences in the efficacy of iron release from induced and non-induced erythroleukemia cells are, possibly, apart from the changes in the number of transferrin receptors, an additional factor involved in the control of cellular iron uptake.

Iron uptake and transferrin endocytosis in undifferentiated and differentiated erythroid cells.

Iron uptake from transferrin (Tf) by Friend erythroleukemia cells was studied before and after induction of the cells by dimethyl sulfoxide (DMSO) in culture. The increase in iron uptake after 5 days of induction was not accompanied by a proportional increase in the number of transferrin receptors detected by 125I-labeled Tf binding, resulting in DMSO induced cells taking up iron with the rate of about 26 iron atoms per receptor per hour, compared to 15 atoms in uninduced cells. In agreement with this finding are results of the study of cellular 125I or 59Fe-labeled Tf kinetics. In the induced cells Tf endocytosis and release proceeded with identical rates and all the endocytosed iron was retained inside the cell. Tf release by uninduced cells was slower and a part of internalized 59Fe was released. Hemin inhibits Tf-bound iron uptake by DMSO induced cells but not by the uninduced cells. Correspondingly, addition of hemin had little effect on Tf kinetics in the uninduced cells, while the release of Tf was slower in hemin-treated DMSO induced cells. Different efficiency of iron release from internalized Tf, accompanied by changes in cellular Tf kinetics is therefore proposed as a factor determining the rate of iron uptake by developing erythroid cells.

Iron uptake by MOLT 3 cells from transferrin/monoclonal antitransferrin antibody complexes.

The ability of seven monoclonal antibodies (MAbs) reactive with human transferrin (Tf) to inhibit iron uptake from Tf by cells of the human lymphoid line MOLT 3 was compared with the effect of these MAbs on Tf binding to MOLT 3 surface Tf receptors. MAbs HTF-01, HTF-06, HTF-07, HTF-11 and HTF-14 inhibited both iron uptake and Tf binding. Complexing Tf with MAbs HTF-04 or HTF-05 resulted in an increased association of Tf with the cells but iron uptake was diminished. Following the intracellular kinetics of Tf/HTF-04 complex has shown that the whole complex is endocytosed and Tf iron is retained inside the cell, but Tf release from both the cell surface and the intracellular compartment is slower when compared to Tf alone. Iron uptake inhibition was therefore attributed to lengthening of the Tf cell cycle after complexing of Tf with HTF-04 and it is suggested that the rate of cellular Tf turnover might, together with the number of functioning Tf receptors, determine the rate of iron uptake by cells.

Effect of hemin on poly (A)-containing RNA synthesis and transport from nucleus into cytoplasm in Friend cells of the Fw line.

The effect of hemin on the transcription and post-transcriptional events (processing and transport of globin mRNA and other poly(A)-containing RNAs to the cytoplasm) was investigated in murine erythroleukemia Friend cells (MELC) of the Fw line. Uninduced Fw cells or Fw cells induced to differentiation by butyric acid were incubated in vitro without or with exogenous hemin. Short labelling periods (5-20 min) with (3H) uridine were used. The synthesis of globin mRNA and other poly(A)-containing RNAs at the level of their precursors in the nucleus and at the level of mature forms in the cytoplasm was measured. Our results indicate that hemin mainly affects the processing or transport of globin mRNA and other poly(A)-containing RNAs from the nucleus to the cytoplasm. The effect of exogenous hemin is quite different in uninduced and induced Fw cells. Exogenous hemin stimulates accumulation of poly(A)-containing RNA in nuclei of uninduced Fw cells. On the other hand exogenous hemin causes an increase in cytoplasmic globin mRNA and other cytoplasmic poly(A)-containing RNA in Fw cells induced by sodium butyrate.

Effect of hemin and heme synthesis inhibitors on cellular protein synthesis.

Inhibitors of heme synthesis, 2,2'-bipyridyl, isonicotinic acid hydrazide (INH), and D,L-penicillamine markedly inhibited only the synthesis of hemoglobin and had no effect on the synthesis of the bulk of nonhemoglobin proteins in spleen cells of anemic mice. Exogenous hemin stimulated the synthesis of hemoglobin as well as the synthesis of the bulk of nonhemoglobin proteins. However, by further analysis of nonhemoglobin proteins it was possible to detect intermediates of hemoglobin synthesis, globin chains with highly specific radioactivity of L-[4,5-3H]leucine which were eluted together with nonhemoglobin proteins during the chromatography on CM-Sephadex C-50. Protein synthesis in Friend erythroleukemia cells of the Fw line which have the genetic defect of heme synthesis was resistant to D,L-penicillamine and Desferal; 2,2'-bipyridyl had an inhibitory effect. Hemin was without effect on protein synthesis in these neoplastic cells.

The effect of heme on intracellular iron pools during differentiation of Friend erythroleukemia cells.

The inhibition of cellular iron uptake by hemin described previously in reticulocytes was studied in murine erythroleukemia (Friend) cells that can be induced to differentiate in culture by dimethyl sulfoxide (DMSO). Hemin had no effect on iron uptake into noninduced cells. After the induction by DMSO, hemin inhibited iron uptake into Friend cells and this effect of hemin became more pronounced with the further progress of differentiation. The reduction of cellular iron accumulation was caused mainly by inhibition of iron incorporation into heme, iron uptake into the non-heme pool was little influenced by hemin treatment. Inhibition of heme synthesis by isonicotinic acid hydrazide (INH) caused an accumulation of iron in mitochondria in DMSO-induced cells, but not in uninduced cells. On the basis of these results, a specific system transporting iron to mitochondria induced by DMSO treatment is suggested as a target for the inhibitory action of hemin. In Friend cells of the Fw line which are deficient in ferrochelatase, heme has no effect on iron uptake. The addition of INH to the Fw cells does not enhance the iron accumulation in mitochondria.

The role of heme in the regulation of iron uptake during Friend cell differentiation.

The inhibitory effect of hemin on iron uptake and heme synthesis was investigated in Friend erythroleukemia cells. In cells of line 707 the inhibition of iron uptake became more pronounced after the induction of erythroid differentiation by dimethyl sulfoxide. No such rise in sensitivity to hemin was found in cells of line Fw, which are deficient in ferrochelatase activity and do not synthesize hemoglobin after induction of differentiation by sodium butyrate. The possibility that hemin influences mainly the uptake of iron used directly for heme synthesis, and to a lesser extent intracellular storage of iron is discussed.

Effect of heme on globin messenger RNA synthesis.

Erythroid spleen cells from anemic mice were incubated with various inhibitors of heme synthesis. All the effective inhibitors of heme synthesis decreased the incorporation of labelled leucine into protein in spleen erythroid cells. Hemin completely restored protein synthesis to the control levels in isonicotinic acid hydrazide (INH) and penicillamine treated erythroid cells. These two specific inhibitors of heme synthesis were used for the study of the effect of heme on globin mRNA synthesis. Newly synthesized [3H]-uridine labelled globin mRNA was isolated by hybridization to globin cDNA covalently bound to the cellulose column. INH and penicillamine inhibited [3H] uridine incorporation into globin mRNA. The addition of hemin to INH or penicillamine treated cells almost completely restored globin mRNA synthesis. Moreover, hemin alone slightly increased the incorporation of [3H] uridine into globin mRNA. Similar results were obtained in experiments with Friend erythroleukemia cells of the Fw line. These cells are deficient in ferrochelatase enzyme activity and heme synthesis is not significantly increased after induction of differentiation by chemical inducers. The cells induced by butyric acid were incubated for 2 h with or without 100 muM hemin. The incorporation of [3H] uridine into globin mRNA was measured. A 38% increase in globin mRNA synthesis was seen in cells incubated with hemin as compared with cells incubated without hemin. These results seem to indicate that heme affects transcription or processing of globin mRNA precursors. Heme inhibitors also reduce [3H] uridine incorporation into other poly(A)-containing RNA in erythroid spleen cells incubated in vitro. In agreement with these results hemin causes increase of synthesis of some other poly(A)-containing RNA in Friend cells described above.