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D R Richardson

Publications and source records attributed to D R Richardson.

At least 73 records · Page 4Linked to original sources
PubMed1998

Function and regulation of transferrin and ferritin.

Iron represents a paradox for living systems by being essential for a wide variety of metabolic processes (oxygen transport, electron transport, DNA synthesis, etc) but also having the potential to cause deleterious effects. Because of Iron's virtual insolubility and potential toxicity under physiological conditions, specialized molecules for the acquisition, transport, and storage of iron in a soluble, nontoxic form have evolved to meet cellular and organismal iron requirements. Physiologically, the majority of cells in the organism acquire iron from a well-characterized plasma glycoprotein, transferrin. Iron uptake from transferrin is reasonably well understood, and involves the binding of transferrin to the transferrin receptor, internalization of transferrin within an endocytic vesicle by receptor-mediated endocytosis, and the release of iron from the protein by a decrease in endosomal pH. Most of the transferrin-bound iron is used for the synthesis of hemoglobin by developing erythroid cells. Senescent erythrocytes are internalized by the macrophages that liberate hemoglobin iron and release it back to plasma transferrin at a rate that normally matches the rate of iron transport for erythropoiesis. Unfortunately, the mechanisms and controls involved in the release of iron from macrophages have not been defined. After iron release from transferrin within endosomes, iron passes through the endosomal membrane by ill-understood mechanisms and then enters the poorly characterized intracellular labile pool. Iron in the labile pool that exceeds requirement for the synthesis of functional heme and nonheme iron-containing proteins is stored within the iron-storage protein, ferritin. Evidence in vitro indicates that relatively soluble ferrous iron can enter or be released from ferritin. However, we know virtually nothing about the exchange of iron with ferritin in intact cells, and some evidence indicates that the degradation of the ferritin protein may be an important mechanism for the release of iron within the cell. Cellular iron uptake and storage are coordinately regulated through a feedback control mechanism mediated at the post-transcriptional level by cytoplasmic factors know as iron-regulatory proteins 1 and 2. These proteins "sense" levels of iron in the transit pool and, when iron in this pool is scarce, they bind to stem-loop structures known as iron-responsive elements on the 5' untranslated region of the ferritin mRNA and 3' untranslated region of the transferrin mRNA. Such a binding inhibits translation of ferritin mRNA and stabilizes the mRNA for transferrin receptors. The opposite scenario develops when iron in the transit pool is plentiful. This remarkable regulatory mechanism prevents the expansion of a catalytically active intracellular iron pool, while maintaining sufficient concentrations of the metal for metabolic needs.

Ferritins↗
PubMed1997

Mobilization of iron from neoplastic cells by some iron chelators is an energy-dependent process.

Iron (Fe) chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class may be useful agents to treat Fe overload disease and also cancer. These ligands possess high activity at mobilizing 59Fe from neoplastic cells, and the present study has been designed to examine whether their marked activity may be related to an energy-dependent transport process across the cell membrane. Initial experiments examined the release of 59Fe from SK-N-MC neuroblastoma (NB) cells prelabelled for 3 h at 37 degrees C with 59Fe-transferrin (1.25 microM) and then reincubated in the presence and absence of the chelators for 3 h at 4 degrees C or 37 degrees C. Prelabelled cells released 4-5% of total cellular 59Fe when reincubated in minimum essential medium at 4 degrees C or 37 degrees C. When the chelators desferrioxamine (DFO; 0.1 mM) or PIH (0.1 mM) were reincubated with labelled cells at 4 degrees C, they mobilized only 4-5% of cellular 59Fe, whereas as 37 degrees C, these ligands mobilized 21% and 48% of cell 59Fe, respectively. The lipophilic PIH analogue, 311 (2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone; 0.1 mM), which exhibits high anti-proliferative activity, released 10% and 53% of cellular 59Fe when reincubated with prelabelled cells at 4 degrees C and 37 degrees C, respectively. Almost identical results were obtained using the SK-Mel-28 melanoma cell line. These data suggest that perhaps temperature-dependent mechanisms are essential for 59Fe mobilization from these cells. Interestingly, the metabolic inhibitors, 2,4-dinitrophenol, oligomycin, rotenone, and sodium azide, markedly decreased 59Fe mobilization mediated by PIH, but had either no effect or much less effect on 59Fe release by 311. Considering that an ATP-dependent process was involved in 59Fe release by PIH, further studies examined 4 widely used inhibitors of the multi-drug efflux pump P-glycoprotein (P-gp). All of these inhibitors, namely, verapamil (Ver), cyclosporin A (CsA), reserpine (Res) and quinine (Qui), decreased 59Fe mobilization by PIH but had little or no effect on 59Fe release mediated by analogue 311. Further, both CsA and Ver increased the proportion of ethanol-soluble 59Fe within cells in the presence of PIH, suggesting inhibited transport of the 59Fe complex from the cell. However, when PIH-mediated 59Fe release was compared between a well characterized Chinese hamster ovary cell line (CHRB30) expressing high levels of P-gp and the relevant control cell line (AuxB1), no appreciable difference in the kinetics of 59Fe release were found. In contrast, it was intriguing that the CHRB30 cells released more 59Fe into control medium (i.e., without PIH) than the AuxB1 control line (16.7% compared to 5.9%, respectively). In summary, the results suggest that a temperature- and energy-dependent process was involved in the efflux of the PIH-59Fe complex from the cells. In contrast, 59Fe release mediated by 311 was temperature-dependent but not energy-dependent, and could occur by simple diffusion or passive transport. Further studies investigating the membrane transport of Fe chelators may be useful in designing regimes that potentiate their anti-neoplastic effects.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents II: the mechanism of action of ligands derived from salicylaldehyde benzoyl hydrazone and 2-hydroxy-1-naphthylaldehyde benzoyl hydrazone.

We have recently screened 36 analogues of the lipophilic iron (Fe) chelator, pyridoxal isonicotinoyl hydrazone (PIH), for their antiproliferative effect (Richardson et al, Blood 86:4295, 1995). Of these compounds, 1 chelator derived from salicylaldehyde benzoyl hydrazone (206) and 4 ligands derived from 2-hydroxy-1-naphthylaldehyde benzoyl hydrazone (308, 309, 311, and 315) showed pronounced antiproliferative activity, being far more effective than desferrioxamine (DFO). The present study was designed to investigate in detail the mechanism of action of these PIH analogues in a variety of neoplastic cell lines. This investigation showed that the analogues were far more active than DFO at inhibiting cellular proliferation and 3H-thymidine, 3H-leucine, and 3H-uridine incorporation. Additional experiments showed that, in contrast to DFO, the 5 analogues were potent at preventing 59Fe uptake from transferrin (Tf) and increasing 59Fe release from cells at concentrations as low as 10 micromol/L. Examination of the distribution of 59Fe in neoplastic cells using native polyacrylamide gel electrophoresis (PAGE)/59Fe-autoradiography showed that most of the 59Fe taken up from Tf was incorporated into ferritin, although 3 other previously unrecognized components (bands A, B, and C) were also identified. Band C comigrated with 59Fe-citrate and was chelated on incubation of neuroblastoma cells with DFO, PIH, or the PIH analogues, with this compartment being the main intracellular target of these ligands. Further work showed that the effects of the chelators at inducing characteristics consistent with apoptosis or necrosis were cell line-specific, and while DFO increased the percentage of cells in the G0/G1 phases in all cell types, the effect of analogue 311 on the cell cycle was variable depending on the cell line. This study provides further evidence for the potential use of these Fe chelators as anticancer agents.

Antineoplastic Agents↗
PubMed1997

The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells.

Iron uptake by mammalian cells is mediated by the binding of serum Tf to the TfR. Transferrin is then internalized within an endocytotic vesicle by receptor-mediated endocytosis and the Fe released from the protein by a decrease in endosomal pH. Apart from this process, several cell types also have other efficient mechanisms of Fe uptake from Tf that includes a process consistent with non-specific adsorptive pinocytosis and a mechanism that is stimulated by small-Mr Fe complexes. This latter mechanism appears to be initiated by hydroxyl radicals generated by the Fe complexes, and may play a role in Fe overload disease where a significant amount of serum non-Tf-bound Fe exists. Apart from Tf-bound Fe uptake, mammalian cells also possess a number of mechanisms that can transport Fe from small-Mr Fe complexes into the cell. In fact, recent studies have demonstrated that the membrane-bound Tf homologue, MTf, can bind and internalize Fe from 59Fe-citrate. However, the significance of this Fe uptake process and its pathophysiological relevance remain uncertain. Iron derived from Tf or small-Mr complexes is probably transported into mammalian cells in the Fe(II) state. Once Fe passes through the membrane, it then becomes part of the poorly characterized intracellular labile Fe pool. Iron in the labile Fe pool that is not used for immediate requirements is stored within the Fe-storage protein, ferritin. Cellular Fe uptake and storage are coordinately regulated through a feedback control mechanism mediated at the post-transcriptional level by cytoplasmic factors known as IRP1 and IRP2. These proteins bind to stem-loop structures known as IREs on the 3 UTR of the TfR mRNA and 5 UTR of ferritin and erythroid delta-aminolevulinic acid synthase mRNAs. Interestingly, recent work has suggested that the short-lived messenger molecule, NO (or its by-product, peroxynitrite), can affect cellular Fe metabolism via its interaction with IRP1. Moreover, NO can decrease Fe uptake from Tf by a mechanism separate to its effects on IRP1, and NO may also be responsible for activated macrophage-mediated Fe release from target cells. On the other hand, the expression of inducible NOS which produces NO, can be stimulated by Fe chelators and decreased by the addition of Fe salts, suggesting that Fe is involved in the control of NOS expression.

Antigens, Neoplasm↗
PubMed1997

Cytotoxic analogs of the iron(III) chelator pyridoxal isonicotinoyl hydrazone: effects of complexation with copper(II), gallium(III), and iron (III) on their antiproliferative activities.

This study examined if complexation with metals increased the antiproliferative activities of chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class. Addition of iron(III) to some PIH analogs markedly depressed their activities, whereas it had little effect on others. The gallium(III) complex of PIH, but not its copper(II) complex, was more efficient than the apochelator at inhibiting [3H]thymidine incorporation.

Antineoplastic Agents↗
PubMed1997

Perceptions of rape: the influence of closeness of relationship, intoxication and sex of participant.

Intoxication of the assailant and/or victim is often mentioned in relation to acquaintance rape. The present study tests how independent observers use this information in their perceptions of acquaintance and stranger rape. One hundred and forty-nine females and 104 males read scenarios depicting a sexual assault and made attributions of responsibility for the rape and evaluated the actors. Victims' intoxication consistently influenced perceptions of victim culpability as well as respondent evaluation of her. The impact of closeness of relationship was much more complex and less consistent than the effects of victim intoxication.

Alcohol Drinking↗
PubMed1996

Distribution of iron in reticulocytes after inhibition of heme synthesis with succinylacetone: examination of the intermediates involved in iron metabolism.

Succinylacetone (SA) is an inhibitor of heme synthesis that acts on the enzyme delta-aminolevulinic acid dehydratase. When reticulocytes are incubated with 59Fe-transferrin (59Fe-Tf) in the presence of SA, there is an accumulation of 59Fe in the mitochondrion and in a cytosolic non-heme intermediate that has been described as a putative Fe transporter (Adams et al, Biochim Biophys Acta 1012:243, 1989). Considering these observations, the present study was designed to examine the intermediates of Fe metabolism in control and SA-treated reticulocytes. This investigation showed that in the cytosol of control cells, most 59Fe was incorporated into hemoglobin (Hb) with a minor amount entering ferritin. In addition, a previously unrecognized cytosolic intermediate was identified (band X) that was absent when heme synthesis was inhibited with SA. Upon reincubation of SA-treated reticulocytes with protoporphyrin IX, band X initially increased in intensity and then decreased later in the incubation. In contrast, when 59Fe-labeled control cells were reincubated in the presence of SA and unlabeled diferric Tf, there was a marked decrease in the intensity of band X. These experiments suggest that component X may be an intermediate involved in the transfer of heme in the cytosol. Alternatively, these data could also be interpreted as indicating that band X may be a short-lived hemoprotein. We have confirmed the presence of an 59Fe-containing molecule in the cytosol of SA-treated reticulocytes (band Y) that is not present in control cells. However, when cells were incubated with 59Fe-Tf plus SA and then chased in the presence of SA and unlabeled diferric Tf, there was no decrease in this cytosolic pool of Fe, suggesting that it was not a intermediate supplying Fe for either ferritin or heme synthesis. Finally, there is little low molecular weight (Mr) Fe in reticulocytes, and our studies suggest that the low-Mr Fe present does not behave as an intermediate. Moreover, after inhibition of heme synthesis with SA, 59Fe in the low-Mr compartment was markedly decreased, suggesting that this component may be heme or a low-Mr heme-containing molecule. Considering the apparent lack of a cytosolic Fe transporter in rabbit reticulocytes, an alternative model of intracellular Fe transport is proposed that does not implicate a potentially toxic intermediate pool of low-Mr Fe complexes.

Animals↗
PubMed1996

Intermediate steps in cellular iron uptake from transferrin. II. A cytoplasmic pool of iron is released from cultured cells via temperature-dependent mechanical wounding.

A previous study described a cytoplasmic, transferrin (Tf)-free, iron (Fe) pool that was detected only when cells were mechanically detached from the culture substratum at 4 degrees C, after initial incubation with 59Fe-125I-Tf at 37 degrees C (Richardson and Baker, 1992a). The release of this internalized 59Fe could be markedly reduced if the cells were treated with proteases or incubated at 37 degrees C prior to detachment. The present study was designed to characterize this Fe pool and understand the mechanism of its release. The results show that cellular 59Fe release increased linearly as a function of preincubation time with 59Fe-Tf subsequent to mechanical detachment at 4 degrees C using a spatula. These data suggest that the 59Fe release was largely composed of end product(s) and was not an "intermediate Fe pool." When the Fe(II) chelator, dipyridyl (DP), was incubated with 59Fe-Tf and the cells, it prevented the accumulation of 59Fe that was released following mechanical detachment at 4 degrees C. Other chelators had much less effect on the proportion of 59Fe released. Examination of the 59Fe released showed that after a 4-h preincubation with 59Fe-Tf, approximately 50% of the 59Fe was present in ferritin. These data indicate that mechanical detachment of cells at 4 degrees C resulted in membrane disruptions that allow the release of high M(r), molecules. Moreover, electron microscopy studies showed that detachment of cells from the substratum at 4 degrees C resulted in pronounced membrane damage. In contrast, when cells were detached at 37 degrees C, or at 4 degrees C after treatment with pronase, membrane damage was minimal or not apparent. These results may imply that temperature-dependent processes prevent the release of intracellular contents on membrane wounding, or alternatively, prevent wounding at 37 degrees C. The evidence also indicates that caution is required when interpreting data from experiments where cells have been mechanically detached at 4 degrees C.

2,2'-Dipyridyl↗
PubMed1996

Character and duration of pharmacological effects of intravenous isoxsuprine.

Isoxsuprine is a therapeutic medication used to treat navicular disease and other lower limb problems in horses and is one of the more frequently detected therapeutic agents in racing horses. In a crossover study, horses were administered isoxsuprine i.v. to determine the character and duration of its pharmacological effects. Isoxsuprine significantly increased heart rate 5-150 min following injection. Unrestrained activity following isoxsuprine treatment was significantly greater than control activity for 105 min after treatment. There was an apparent, although statistically nonsignificant, increased cutaneous blood flow resulting in visible water vapour and sweat production 5-60 min after administration. Initially, there was no difference in skin temperature between control and isoxsuprine treatment values; however, skin temperature decreased below control values 45-120 min after injection. Concurrently, there was a significant decrease in rectal temperature reflecting a decrease in body core temperature. Using infrared thermography, a significant decrease in superficial skin temperature of the front legs occurred 30-240 min after treatment. Isoxsuprine also reduced smooth muscle tone, which was apparent by decreased tone of the internal anal sphincter 10-180 min after treatment. It was concluded that the measurable pharmacological effects of i.v. isoxsuprine are short lived, since none of the above responses were apparent 4 h or more after i.v. administration.

Animals↗
PubMed1995

The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents.

Numerous studies have suggested that iron (Fe) chelators such as desferrioxamine (DFO) may be useful antitumor agents (Blatt and Stitely, Cancer Res 47:1749, 1987; Becton and Bryles, Cancer Res 48:7189, 1988). Recent work with several analogues of the lipophilic Fe chelator, pyridoxal isonicotinoyl hydrazone (PIH), indicate that some of these ligands are considerably more efficient than DFO both in terms of their Fe chelation efficacy and at preventing 3H-thymidine incorporation by neuroblastoma (NB) cells (Richardson and Ponka, J Lab Clin Med 124:660, 1994). Considering this fact, the present study was designed to test the antiproliferative effect of a wide range of PIH analogues to identify the most active compounds. A total of 36 ligands have been examined that were synthesized by condensation of three types of aromatic aldehydes (pyridoxal, salicylaldehyde, and 2-hydroxy-1-naphthyladehyde) with a range of acid hydrazides. The effects of these chelators were assessed using the human NB cell line, SK-N-MC. Although PIH was far more effective than DFO at preventing Fe uptake from transferrin, it was less effective than DFO at preventing cellular proliferation (DFO ID50 = 22 mumol/L; PIH ID50 = 75 mumol/L). In contrast, 14 PIH analogues were far more efficient than DFO at preventing proliferation (ID50 = 1 to 7 mumol/L) and may have potential as antitumor agents. The most effective compounds were those hydrazones derived from 2-hydroxy-1-naphthylaldehyde. Most of the PIH analogues were considerably more effective than DFO at both preventing 59Fe uptake from 59Fe-transferrin and in mobilizing 59Fe from prelabeled NB cells. In addition, a linear relationship between Fe chelation efficacy and antiproliferative activity was found only for hydrazones derived from salicylaldehyde. Apart from gallium (Ga) nitrate having an antiproliferative effect by itself, this metal potentiated the antiproliferative effect of PIH but not that of DFO. Spectrophotometric studies showed that PIH could chelate Ga, and it can be suggested that, like the PIH-Fe complex that donates Fe to reticulocytes (Ponka et al, Biochim Biophys Acta 718:151, 1982), the PIH-Ga complex may efficiently bestow Ga to NB cells. The results suggest that analogues of PIH deserve further vigorous investigation because they may be useful therapeutic agents for the treatment of cancer.

Antineoplastic Agents↗
PubMed1995

Identification of a mechanism of iron uptake by cells which is stimulated by hydroxyl radicals generated via the iron-catalysed Haber-Weiss reaction.

Recent studies have demonstrated that preincubation of SK-Mel-28 melanoma cells with ferric ammonium citrate (FAC) resulted in marked stimulation of 59Fe uptake from 59Fe-125I-transferrin (Tf), but only at Tf concentrations above that required for saturation of the Tf receptor (Richardson and Baker (1992) J. Biol. Chem. 267, 13972-13979). The mechanism responsible for this stimulation was unknown and is the subject of the present report. Preincubation of cells with FAC (25 micrograms/ml), followed by a 2 h incubation with 59Fe-125I-Tf (0.1 mg/ml; 1.25 microM), resulted in temperature-dependent 59Fe uptake to approx. 200% of the control value. Furthermore, the effect was not specific for melanoma cells and was also observed in other normal and neoplastic cells. Preincubation of melanoma cells with FAC also stimulated 59Fe uptake from 59Fe-citrate, but to a far greater extent than that observed with 59Fe-125I-Tf (viz., > 20-fold that seen for the control). Interestingly, neither receptor-mediated endocytosis nor the postulated diferric Tf reductase were involved in the FAC-activated Fe uptake process from Tf. However, the addition of free radical scavengers to FAC such as catalase, superoxide dismutase, ceruloplasmin, Hepes, mannitol and high concentrations of BSA or ascorbate, markedly depressed FAC-activated 59Fe uptake from 59Fe-125I-Tf and 59Fe-citrate. These agents when added to control cells had no effect on 59Fe uptake. The addition of superoxide generating agents and hydrogen peroxide to minimum essential medium (MEM) containing FAC but not to MEM alone, also stimulated 59Fe uptake. These data suggest that the initial activation of the FAC-stimulated Fe uptake system was caused by the production of hydroxyl radicals via the Fe-catalysed Haber-Weiss reaction. We propose that this Fe uptake process represents an important cellular defense mechanism against oxidant stress generated in the presence of low-molecular-weight Fe complexes.

Animals↗
PubMed1995

The effect of redox-related species of nitrogen monoxide on transferrin and iron uptake and cellular proliferation of erythroleukemia (K562) cells.

The iron-responsive element-binding protein (IRE-BP) modulates both ferritin mRNA translation and transferrin receptor (TfR) mRNA stability by binding to specific mRNA sequences called iron-responsive elements (IREs). The regulation of IRE-BP in situ could possibly occur either through its Fe-S cluster and/or via free cysteine sulphydryl groups such as cysteine 437 (Philpott et al, J Biol Chem 268:17655, 1993; and Hirling et al, EMBO J 13:453, 1994). Recently, nitrogen monoxide (NO) has been shown to have markedly different biologic effects depending on its redox state (Lipton et al, Nature 364:626, 1993). Considering this fact, it is conceivable that the NO group, as either the nitrosonium ion (NO+) or nitric oxide (NO+), may regulate IRE-BP activity by S-nitrosylation of key sulphydryl groups or via ligation of NO. to the Fe-S cluster, respectively. This hypothesis has been examined using the NO+ generator, sodium nitroprusside (SNP); the NO. generator, S-nitroso-N-acetylpenicillamine (SNAP); and the NO./peroxynitrite (ONOO-) generator, 3-morpholinosydnonimine hydrochloride (SIN-1). Treatment of K562 cells for 18 hours with SNP (1 mmol/L) resulted in a pronounced decrease in both the RNA-binding activity of IRE-BP and the level of TfR mRNA. In addition, Scatchard analysis showed a marked decrease in the number of specific Tf-binding sites, from 590,000/cell (control) to 170,000/cell (test), and there was also a distinct decrease in Fe uptake. Furthermore, SNP did not decrease cellular viability or proliferation. In contrast, the NO. generator, SNAP (1 mmol/L), increased RNA-binding activity of IRE-BP, the level of TfR mRNA, and the number of TfRs in K562 cells. Moreover, both SNAP (1 mmol/L) and SIN-1 (0.5 mmol/L) reduced cellular proliferation. The results are discussed in context of the possible physiologic role of redox-related species of NO in regulating iron metabolism.

Antioxidants↗
PubMed1995

A novel iron uptake mechanism mediated by GPI-anchored human p97.

The established process for iron uptake into mammalian cells involves transferrin and its receptor. Here, the role of the glycosyl-phosphatidylinositol (GPI)-linked transferrin homologue, melanotransferrin or p97, was studied using CHO cell lines defective in the transferrin receptor (TR) and transfected with human TR and/or human p97. The presence of p97 doubled the iron uptake, which could be explained by the binding of one atom of iron to one molecule of p97. The internalization of iron was shown to be temperature sensitive and saturated at a media iron concentration of 2.5 micrograms/ml with a Vmax of 0.1 pmol Fe/10(6) cell/min and a Km of 2.58 microM for p97. Treatment of the cells with either phosphatidylinositol-phospholipase C or monoclonal antibodies against p97 resulted in over a 50% reduction and a 47% increase in the iron uptake respectively. These data identify p97 as a unique cell surface GPI-anchored, iron binding protein involved in the transferrin-independent uptake of iron in mammals.

Animals↗
PubMed1995

Nitrogen monoxide decreases iron uptake from transferrin but does not mobilise iron from prelabelled neoplastic cells.

The effect of congeners of nitrogen monoxide (NO) on iron (Fe) uptake from 59Fe-125I-transferrin (Tf) and release of 59Fe from prelabelled cells have been investigated in SK-MEL-28 human melanoma cells, human K562 cells and mouse MDW-4 cells. These studies have been initiated as it has been suggested that the tumoricidal effects of NO may be mediated by its acting to release Fe from cells (Hibbs et al., 1984 Biochem. Biophys. Res. Commun. 123, 716-723; Hibbs et al., 1988 Biochem. Biophys. Res. Commun. 157, 87-94). The nitrosonium ion (NO+) generator, sodium nitroprusside (SNP), decreased 59Fe uptake by melanoma cells to 57% of the control without decreasing 125I-Tf uptake after a 4-h incubation with 59Fe-125-Tf (1.25 microM). Longer incubations up to 24 h decreased 59Fe uptake and also 125I-Tf uptake. Two breakdown products of SNP, ferricyanide and cyanide, had no effect on 59Fe uptake. In addition, photolysis of the SNP solution prevented the inhibition of 59Fe uptake, suggesting that NO was the active agent. Two nitric oxide (NO.) producing agents, 3-morpholinosydnonimine (SIN), and S-nitroso-N-acetylpenicillamine (SNAP), also decreased 59Fe uptake from 59Fe-125I-Tf. Superoxide dismutase increased the efficacy of SIN, and the NO-scavenger, oxyhaemoglobin, prevented the inhibition of 59Fe uptake mediated by SNAP, again suggesting that NO was the active agent. Furthermore, dialysis studies demonstrated that none of the NO-generating agents could remove 59Fe from 59Fe-125I-Tf, suggesting that the decrease in cellular Fe uptake observed was not due to NO releasing Fe from the Fe-binding sites of Tf. Despite the ability of NO-producing agents at inhibiting 59Fe uptake by cells, they could not remove significant amounts of 59Fe from melanoma cells prelabelled with either 59Fe-citrate or 59Fe-125I-Tf. Similar data were obtained using K562 and MDW-4 cells. Interestingly, the NO+ generating agent, SNP, had no effect on [3H]thymidine uptake. However, when SNP was converted to an NO. generator by the addition of 1 mM ascorbate, its effect was similar to the NO. generator, SNAP, markedly reducing [3H]thymidine incorporation to 33% of the control value. The addition of unlabelled diferric Tf (0.625 microM) to SNAP ameliorated its inhibitory effect on cellular [3H]thymidine uptake, suggesting that the interaction of NO. with Fe was of importance in the inhibition observed. The results are discussed in the context of the cytostatic potential of NO via its binding to Fe.

Animals↗
PubMed1995

Growth of human tumor cell lines in transferrin-free, low-iron medium.

Iron is essential for tumor cell growth. Previous studies have demonstrated that apart from transferrin-bound iron uptake, mammalian cells also possess a transport system capable of efficiently obtaining iron from small molecular weight iron chelates (Sturrock et al., 1990). In the present study, we have examined the ability of tumor cells to grow in the presence of low molecular weight iron chelates of citrate. In chemically defined serum-free medium, most human tumor cell lines required either transferrin (5 micrograms/ml) or a higher concentration of ferric citrate (500 microM) as an iron source. However, we have also found that from 13 human cell lines tested, 4 were capable of long-term growth in transferrin-free medium with a substantially lower concentration of ferric citrate (5 microM). When grown in medium containing transferrin, both regular and low-iron dependent cell lines use transferrin-bound iron. Growth of both cell types in transferrin medium was inhibited to a certain degree by monoclonal antibody 42/6, which specifically blocks the binding of transferrin to the transferrin receptor. On the contrary, growth of low-iron dependent cell lines in transferrin-free, low-iron medium (5 microM ferric citrate) could not be inhibited by monoclonal antibody 42/6. Furthermore, no autocrine production of transferrin was observed. Low-iron dependent cell lines still remain sensitive to iron depletion as the iron(III) chelator, desferrioxamine, inhibited their growth. We conclude that low-iron dependent tumor cells in transferrin-free, low-iron medium may employ a previously unknown mechanism for uptake of non-transferrin-bound iron that allows them to efficiently use low concentrations of ferric citrate as an iron source. The results are discussed in the context of alternative iron uptake mechanisms to the well-characterized receptor-mediated endocytosis process.

Cell Culture Techniques↗
PubMed1994

Two saturable mechanisms of iron uptake from transferrin in human melanoma cells: the effect of transferrin concentration, chelators, and metabolic probes on transferrin and iron uptake.

The mechanisms of iron (Fe) and transferrin (Tf) uptake by the human melanoma cell line, SK-MEL-28, have been investigated using chelators and metabolic probes. These data provide evidence for two saturable processes of Fe uptake from Tf, namely, specific receptor-mediated endocytosis and a second nonspecific, non-receptor-mediated mechanisms which saturated with respect to Fe uptake at a Tf concentration of approximately 0.3 mg/ml. In contrast to Fe uptake, Tf uptake increased linearly up to at least 1 mg/ml. Furthermore, under the culture conditions used, the second nonspecific, non-receptor-mediated mechanism was the most important process in terms of quantitative Fe uptake. Two concentrations of Tf-125I-59Fe (0.01 and 0.1 mg/ml) were used in order to characterise the specific and nonspecific Fe uptake pathways. Membrane permeable chelators were equally effective at both Tf concentrations, whereas membrane impermeable chelators were significantly (P < 0.001) more effective at reducing the internalisation of Fe at the higher Tf concentration, consistent with a mechanism of Fe uptake which occurred at a site in contact with the extracellular medium. The oxidoreductase inhibitor, amiloride, only slightly inhibited Fe uptake at the higher Tf concentration, suggesting that the second nonspecific process was not mediated by a diferric Tf reductase. Three lysosomotrophic agents and the endocytosis inhibitor, phenylglyoxal, markedly reduced Fe uptake at both Tf concentrations, and it is concluded that a saturable process consistent with receptor-mediated endocytosis of Tf occurred at the lower Tf concentration, while the predominant mechanism of Fe uptake at high Tf concentrations was a second saturable process consistent with adsorptive pinocytosis.

Humans↗