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P Ponka

Publications and source records attributed to P Ponka.

At least 55 records · Page 3Linked to original sources
PubMed1996

Mode of action of iron (III) chelators as antimalarials. IV. Potentiation of desferal action by benzoyl and isonicotinoyl hydrazone derivatives.

The antimalarial action of iron chelators is limited by factors related to drug permeation and parasite susceptibility to metal deprivation. In this study we applied iron-chelating isonicotinoyl and benzoyl hydrazones on Plasmodium falciparum cultures and assessed their antimalarial properties. The agents w ere used both individually and in combination with deferoxamine (DFO), a clinically approved iron chelator, and with hydroxyethyl-starch-DFO, a macromolecular carrier of DFO. Salicylaldehyde isonicotinoyl hydrazone (SIH) and 2-hydroxy-1-naphthylaldehyde m-fluorobenzoyl hydrazone (HNFBH) were found to be highly efficient in suppressing parasite growth at all developmental stages (IC50 24 +/- 6 micromol/L and 0.21 +/- 0.04 micromol/L, respectively, in a 36-to-42 hour test). In combination with impermeant DFO, SIH and HNFBH actions on ring forms were significantly potentiated in terms of speed of drug action and extent of inhibition. The combined effect of the hydrazones with DFO was greater than additive. Based on the capacity of SIH to extract iron from infected cells and to transfer the metal to extracellular DFOs, we propose a mechanism for a synergistic action of permeant hydrazones and impermeant (DFO) iron chelators. The application of a combination of iron chelators as antimalarials might be of therapeutic value.

Animals↗
PubMed1996

The role of iron supply in the regulation of 5-aminolevulinate synthase mRNA levels in murine erythroleukemia cells.

Mouse erythroleukemia (MEL) cells transformed by Friend virus and induced to undergo erythroid differentiation by treatment with hexamethylenebisacetamide (HMBA) increase erythroid specific 5-aminolevulinate synthase (ALAS-E) mRNA levels by 4-15-fold and decrease "housekeeping" 5-aminolevulinate synthase (ALAS-N) mRNA levels by 1.2-1.4-fold. Iron affects translation of (ALAS-E) mRNA but nothing is known about its effect at the pretranslational level of the expression of (ALAS-E) mRNA. The aim of this study was to examine the effect of iron on the synthesis of (ALAS-E) mRNA and (ALAS-N) mRNA. This effect was compared with the effect of iron on the iron on the synthesis of H-ferritin and transferrin receptor mRNAs. Incubation of uninduced or induced MEL cells with iron chelator pyridoxal isonicotinoyl hydrazone (PIH) or desferrioxamine (Desferal) and 3H-uridine decreased the level of the 3H-labeled (ALAS-E) mRNA. The treatment with either diferric transferrin or Fe-PIH increased the level of the 3H-labeled (ALAS-E) mRNA. The opposite effect was observed on the level of the 3H-labeled (ALAS-N) mRNA. These findings indicate that iron might play its role also at the pretranslational level of the expression of ALAS-E or in the stability of (ALAS-E) mRNA.

5-Aminolevulinate Synthetase↗
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↗
PubMed1994

Regulation of protein kinase C (PKC) expression by iron: effect of different iron compounds on PKC-beta and PKC-alpha gene expression and role of the 5'-flanking region of the PKC-beta gene in the response to ferric transferrin.

We have studied effects of ferric transferrin (FeTF), ferric lactoferrin (FeLF), ferric complexes of pyridoxal- or salicylaldehyde-isonicotinoyl hydrazone, (Fe-PIH, Fe-SIH), and ferric ammonium citrate (FAC) on expression of protein kinase C (PKC) mRNA transcripts in a variety of cultured cell lines. FeTF supported an increase of PKC-beta mRNA transcripts in T-lymphoblastoid (CCRF-CEM; Jurkat), B-lymphoblastoid (Daudi; Raji), promyelocyte (HL-60), erythroleukemia (K562), and monocyte (U937) cell lines. By contrast, FeLF, Fe-PIH, and Fe-SIH did not support an increase of PKC-beta mRNA transcripts in any of these cell lines. Furthermore, FAC supported an increase of PKC-beta mRNA transcripts in HL-60, K562, and U937 cells only. Preincubation of cells with desferrioxamine (DF), a cell-permeable iron chelator, abolished the increments of PKC-beta mRNA observed in response to FeTF or FAC. In contrast to results with PKC-beta, neither FeTF nor FAC caused an increase of PKC-alpha transcripts in any cell line. To locate iron-responsive DNA regulatory elements of the PKC-beta gene, we prepared genetic constructs containing various portions of the human PKC-beta 5'-flanking DNA linked to the firefly luciferase gene. Constructs were cotransfected with the neomycin resistance plasmid, Pwl-neo, into HRE H9 cells, and stable transfectants were selected in G418. Treatment with FeTF of transfectants bearing chimeric gene constructs with 2,200 bp of the PKC-beta 5'-flanking region increased luciferase activity and mRNA transcripts 2.5-fold. This increase was blocked by DF. Neither luciferase activity nor mRNA increased with FeTF in stable transfectants bearing constructs with 342 bp or 587 bp of the PKC-beta 5'-flanking region. These data provide direct confirmation that iron is involved in regulation of PKC-beta but not PKC-alpha gene expression in many cell lines. The form in which iron is presented to these cell lines appears to affect its availability for this function, and cells vary in their capabilities to use nontransferrin iron to support PKC-beta gene expression. Finally, transcriptional upregulation of PKC-beta by FeTF is mediated by DNA sequences located between -2200 bp and -587 bp in the 5'-flanking region of the human PKC-beta gene.

Animals↗
PubMed1994

Deoxyribose degradation catalyzed by Fe(III)-EDTA: kinetic aspects and potential usefulness for submicromolar iron measurements.

Iron ions play a central role in .OH radicals formation and induction of oxidative stress in living organisms. Iron-catalyzed .OH radical formation degrades deoxyribose to thiobarbituric acid reactive substances (TBA-RS). This paper analyzes kinetic properties of the Fe(III)-EDTA-catalyzed deoxyribose degradation in the presence of ascorbate. The yield of TBA-RS formation in the presence of EDTA was 4-fold higher than in its absence, contrasting with results reported elsewhere, Cu(II)-EDTA and Fe(III)-citrate were unable to catalyze deoxyribose degradation. The dependence on deoxyribose concentration was fitted to a Lineweaver Burk-like plot and it was calculated that approximately 4.5 mM deoxyribose scavenged half of the .OH radicals formed. The data for Fe(III)-EDTA concentration dependence could also be fitted to a rectangular hyperbolic function. This function was linear up to 1 microM added FeCl3 and this property could be utilized as an assay for the estimation of submicromolar iron concentrations. Submicromolar concentrations of iron could induce measurable yields of TBA-RS. Differences of as little as 0.1 microM Fe(III)-EDTA could be reproducibly detected under optimum experimental conditions, above a consistent background absorbance that was equivalent to 0.35 +/- 0.05 microM Fe(III)-EDTA and represented contaminating iron in the reactants that could not be removed with Chelex-100. The low method determination limit makes the deoxyribose degradation reaction potentially useful as a new, highly sensitive and cost effective assay for iron quantification.

Ascorbic Acid↗
PubMed1994

Regulation of transferrin receptor mRNA expression. Distinct regulatory features in erythroid cells.

In proliferating non-erythroid cells, the expression of transferrin receptors (TfR) is negatively regulated by the amount of intracellular iron. Fe-dependent regulation of TfR occurs post-transcriptionally and is mediated by iron-responsive elements (IRE) located in the 3' untranslated region of the TfR mRNA. IREs are recognized by a specific cytoplasmic binding protein (IRE-BP) that, in the absence of Fe, binds with high affinity to TfR mRNA, preventing its degradation. While TfR numbers are positively correlated with proliferation in non-erythroid cells, in hemoglobin-synthesizing cells, their numbers increase during differentiation and are, therefore, negatively correlated with proliferation. This suggests a distinct regulation of erythroid TfR expression and evidence, as follows, for this was found in the present study. (a) With nuclear run-on assays, our experiments show increased TfR mRNA transcription following induction of erythroid differentiation of murine erythroleukemia (MEL) with Me2SO. (b) Me2SO treatment of MEL cells does not increase IRE-BP activity which is, however, increased in uninduced MEL cells by Fe chelators. (c) Following induction of MEL cells, there is an increase in the stability of TfR mRNA, whose level is only slightly affected by iron excess. (d) Heme-synthesis inhibitors, such as succinylacetone and isonicotinic acid hydrazide, which inhibit numerous aspects of erythroid differentiation, also inhibit TfR mRNA expression in induced MEL cells. However, heme-synthesis inhibition does not lead to a decrease in TfR mRNA levels in uninduced MEL cells. Thus, these studies indicate that TfR gene expression is regulated differently in hemoglobin synthesizing as compared to uninduced MEL cells.

Aminolevulinic Acid↗
PubMed1994

The effect of the iron(III) chelator, desferrioxamine, on iron and transferrin uptake by the human malignant melanoma cell.

The mechanism of action of the clinically used iron(III) chelator, desferrioxamine (DFO), on preventing iron (Fe) uptake from transferrin (Tf) has been investigated using the human melanoma cell line SK-MEL-28. This investigation was initiated due to the paucity of information on the mechanisms of action of DFO in neoplastic cells and because recent studies have suggested that DFO may be a useful antitumor agent. The effect of DFO was dependent on incubation time. After a 2-h incubation, DFO acted like the extracellular chelators, EDTA and diethylenetriaminepentaacetic acid, because there was little inhibition of 59Fe uptake from Tf. In contrast, after a 24-h incubation, DFO (0.5 mM) efficiently reduced internalized 59Fe uptake from Tf to 18% of the control value. These observations suggested the existence of a kinetic block to the entry of the apochelator to intracellular Fe pools and/or to the exit of the DFO-59Fe complex. Indeed, cellular fractionation demonstrated that, in contrast to the decrease in the percentage of 59Fe in the ferritin and membrane fractions, a marked increase in the percentage of 59Fe present in the ferritin-free cytosol occurred. These observations suggested an accumulation of the DFO-59Fe complex within the cell. The highly lipophilic Fe chelator, pyridoxal isonicotinoyl hydrazone, was far more effective than DFO at preventing 59Fe uptake from Tf, illustrating the importance of membrane permeability for effective Fe chelation. Desferrioxamine at a concentration of 1 mM decreased internalized 125I-Tf uptake to 70% of the control. However, the decrease in 59Fe uptake observed could only be partially accounted for by a decrease in Tf uptake, and it appeared that DFO was chelating 59Fe at an intracellular site consistent with the transit Fe pool. The results are discussed in the context of the use of Fe chelators as effective antineoplastic agents.

Cell Division↗
PubMed1994

Iron chelators of the pyridoxal isonicotinoyl hydrazone class. Relationship of the lipophilicity of the apochelator to its ability to mobilise iron from reticulocytes in vitro.

Analogues of the iron (III) chelator, pyridoxal isonicotinoyl hydrazone (PIH) show high potential as orally active agents for the treatment of iron-overload diseases, such as thalassemia. In the present study, the n-octanol-water partition coefficients of 30 analogues of PIH were measured by thin-layer chromatography and also calculated using the additive schemes of Rekker. The purpose was to examine the relationship between lipophilicity of the apochelator and its ability to promote the release of 59Fe from reticulocytes loaded with nonheme 59Fe. Interestingly, maximum activity occurred when the partition coefficient of the apochelator was approximately 1 (log P = 0). Considering the results in the context of the design and synthesis of more active analogues of PIH, it can be suggested that before initiating synthesis, a useful indication of biological activity can be determined by examining the lipophilicity of the molecule, using the schemes of Rekker to calculate the partition coefficient. By using this strategy, analogues of PIH with inappropriate lipophilicity can be excluded before initiating the expensive process of screening in biological models.

Animals↗
PubMed1994

The iron metabolism of the human neuroblastoma cell: lack of relationship between the efficacy of iron chelation and the inhibition of DNA synthesis.

The mechanisms of iron uptake from transferrin and the effects of iron chelators on these processes were investigated in human neuroblastoma cells. This study was performed because numerous reports have indicated that neuroblastoma cells contain iron-rich ferritin and are also especially sensitive to iron chelation by deferoxamine. The mechanisms of iron and transferrin uptake were examined in the human neuroblastoma cell line SK-N-MC by using human transferrin labeled with iodine 125 and iron 59. Internalized and membrane-bound 59Fe and 125I-transferrin were separated with the protease pronase. Total internalized and membrane 125I-transferrin uptake was biphasic with time, whereas total and internalized 59Fe uptake was linear. Iron uptake from transferrin was prevented by incubation at 4 degrees C and also by lysosomotrophic agents. In addition, 59Fe uptake occurred by two processes. The first process was consistent with receptor-mediated endocytosis involving internalization of transferrin bound to specific binding sites. Iron uptake also occurred by a second process, which was not saturable up to a transferrin concentration of 0.06 mg/ml. In terms of quantitative iron uptake, however, the second process was far less important than receptor-mediated endocytosis. Deferoxamine (0.25 mmol/L) only slightly increased 59Fe release from prelabeled cells; the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (0.25 mmol/L) was six times more effective. Moreover, when pyridoxal isonicotinoyl hydrazone (0.2 mmol/L) was added together with labeled transferrin over a 2-hour incubation, 59Fe uptake from transferrin decreased to 18% of the control value, whereas deferoxamine (0.2 mmol/L) had no appreciable effect. Even though deferoxamine (0.1 mmol/L) had little effect on 59Fe uptake or release, it reduced uptake of tritiated thymidine to 33% of the control value after a 24-hour incubation. Three analogs of pyridoxal isonicotinoyl hydrazone, pyridoxal benzoyl hydrazone (#101), pyridoxal p-methoxybenzoyl hydrazone (#107), and pyridoxal m-fluorobenzoyl hydrazone (#109), had chelation activities comparable to that of pyridoxal isonicotinoyl hydrazone and were more effective than either deferoxamine or pyridoxal isonicotinoyl hydrazone at preventing tritiated thymidine uptake. These results suggest that the pyridoxal isonicotinoyl hydrazone analogs have potential as effective antiproliferative agents and deserve further investigation.

DNA↗
PubMed1993

Expression of ferrochelatase mRNA in erythroid and non-erythroid cells.

Ferrochelatase, which catalyses the last step in haem biosynthesis, i.e. the insertion of Fe(II) into protophorphyrin IX, is present in all cells, but is particularly abundant in erythroid cells during haemoglobinization. Using mouse ferrochelatase cDNA as a probe two ferrochelatase transcripts, having lengths of 2.9 kb and 2.2 kb, were found in extracts of mouse liver, kidney, brain, muscle and spleen, the 2.9 kb transcript being more abundant in the non-erythroid tissues and the 2.2 kb transcript more predominant in spleen. In mouse erythroleukemia cells the 2.9 kb ferrochelatase transcript is also more abundant; however, following induction of erythroid differentiation by dimethyl sulphoxide there is a preferential increase in the 2.2 kb transcript, which eventually predominates. With mouse reticulocytes, the purest immature erythroid cell population available, over 90% of the total ferrochelatase mRNA is present as the 2.2 kb transcript. Since there is probably only one mouse ferrochelatase gene, the occurrence of two ferrochelatase transcripts could arise from the use of two putative polyadenylation signals in the 3' region of ferrochelatase DNA. This possibility was explored by using a 389 bp DNA fragment produced by PCR with synthetic oligoprimers having sequence similarity with a region between the polyadenylation sites. This fragment hybridized only to the 2.9 kb ferrochelatase transcript, indicating that the two transcripts differ at their 3' ends and suggesting that the 2.2 kb transcript results from the utilization of the upstream polyadenylation signal. The preferential utilization of the upstream polyadenylation signal may be an erythroid-specific characteristic of ferrochelatase gene expression.

5-Aminolevulinate Synthetase↗
PubMed1993

Regulation of heme biosynthesis: distinct regulatory features in erythroid cells.

Our previous research has demonstrated that in hemoglobin-synthesizing cells, as compared with nonerythroid cells, a step in iron transport from transferrin localized between the transferrin receptor and ferrochelatase is rate-limiting for the synthesis of heme. In this communication we report our more recent studies on the mechanisms involved in the regulation of the transferrin receptors and ferrochelatase in differentiating erythroid cells. Our studies indicate that transferrin receptor gene expression is regulated differently in hemoglobin synthesizing as compared with uninduced murine erythroleukemia (MEL) cells: 1) With nuclear run-on assays our experiments showed increased transferrin receptor mRNA transcription cells of MEL following induction of erythroid differentiation with dimethylsulfoxide (DMSO). 2) DMSO treatment of MEL cells does not increase iron-responsive element binding protein (IRE-BP) activity which is, however, increased in uninduced MEL cells by Fe chelators. 3) Following induction of MEL cells there is an increase in the stability of transferrin receptor mRNA whose level is only slightly affected by iron excess. Using murine ferrochelatase cDNA as a probe, two ferrochelatase transcripts having lengths of 2.9 kb and 2.2 kb were found in extracts of mouse liver, kidney, brain, muscle and spleen, the 2.9 kb transcript being more abundant in nonerythroid tissues and the 2.2 more predominant in spleen. In MEL cells, the 2.9 ferrochelatase transcript is also more abundant; however, following induction of erythroid differentiation by DMSO there is a preferential increase in the 2.2 kb transcript which eventually predominates. With mouse reticulocytes, the purest immature erythroid cell population available, over 90% of the total ferrochelatase mRNA is present as the 2.2 kb transcript. Our further experiments indicate that the 2.2 kb transcript results from the utilization of the upstream polyadenylation signal and suggest that the preferential utilization of the upstream polyadenylation signal may be an erythroid-specific characteristic of ferrochelatase gene expression. These results provide further evidence for the idea that iron metabolism and heme synthesis are controlled by distinct mechanisms in erythroid versus nonerythroid cells.

Animals↗
PubMed1992

Evaluation of the iron chelation potential of hydrazones of pyridoxal, salicylaldehyde and 2-hydroxy-1-naphthylaldehyde using the hepatocyte in culture.

A range of new analogues of the promising iron chelator pyridoxal isonicotinoyl hydrazone was prepared and assessed for activity in reducing hepatocyte iron, mechanism of action and potential in iron-chelation therapy. A total of 45 compounds were synthesized by condensation of aromatic aldehydes (pyridoxal, salicylaldehyde and 2-hydroxy-1-naphthylaldehyde) with various acid hydrazides prepared by systematic substitutions on the benzene ring or by the replacement of the ring with an acetyl, pyridyl, furoyl or thiophene moiety. The effects of these compounds on 59Fe uptake and intracellular distribution in hepatocytes in culture and on 59Fe mobilization from prelabeled hepatocytes were assessed. Toxicity, lipophilicity and the ability to chelate plasma transferrin-bound 59Fe were also evaluated. Several compounds were much more active than pyridoxal isonicotinoyl hydrazone and may have clinical potential. These included pyridoxal benzoyl hydrazone, pyridoxal p-methoxybenzoyl hydrazone, pyridoxal m-fluorobenzoyl hydrazone and pyridoxal 2-pyridyl hydrazone. All were more effective at reducing iron uptake than mobilizing hepatocyte iron; they also may act primarily on the transit iron pool rather than on storage iron. Other compounds (e.g., salicylaldehyde p-t-butyl-benzoyl hydrazone) redistributed ferritin-59Fe to different intracellular sites but had little net effect on hepatocyte iron levels.

Animals↗
PubMed1992

Transferrin-receptor-independent but iron-dependent proliferation of variant Chinese hamster ovary cells.

The purpose of this study is to clarify the role of iron, transferrin, an iron-binding protein in vertebrate plasma, and transferrin receptors in cell proliferation. Transferrin, which is indispensable for most cells growing in tissue culture, is frequently referred to as a "growth factor". Proliferating cells express high numbers of transferrin receptors, and the binding of transferrin to their receptors that is needed for cells to initiate and maintain their DNA synthesis is sometimes regarded as analogous to other growth factor-receptor interactions. Although numerous previous experiments strongly indicate that the only function of transferrin in supporting cell proliferation is supplying cells with iron, they did not completely rule out some direct or signaling role transferrin receptors could play in cell proliferation. To address this issue, we exploited transferrin-receptor-deficient mutant Chinese hamster ovary (CHO) cells (McGraw, T. E., Greenfield, L., and Maxfield, F. R., 1987, J. Cell. Biol. 105, 207-214) in which various aspects of iron and transferrin metabolism in relation to their capacity to proliferate were investigated. Variant cells neither specifically bind transferrin nor do their extracts contain any detectable functional transferrin receptors, yet they proliferate and synthesize DNA with rates comparable to those observed with parent CHO cells. Desferrioxamine, an iron chelating agent, inhibits growth and DNA synthesis of both variant and control CHO cells. This inhibition can be fully alleviated, in both cell types, by ferric pyridoxal isonicotinoyl hydrazone, which can supply cells with a utilizable form of iron by a pathway not requiring transferrin and their receptors. Studies of 59Fe uptake and 125I-transferrin binding revealed that parent cells can take up iron by at least three mechanisms: from transferrin by receptor-dependent and -independent (nonspecific, nonsaturable, not requiring acidification) pathways and from inorganic iron salts (initially present in the medium as FeSO4). Although variant CHO cells are unable to acquire transferrin iron via the receptor pathway, two remaining mechanisms provide these cells with sufficient amounts of iron for DNA synthesis and cell proliferation. In conclusion, although transferrin receptors are dispensable in terms of their absolute requirement for proliferating cells, a supply of iron is still needed for their DNA synthesis. Transferrin-receptor-deficient CHO cells may be a useful model for investigating receptor-independent iron uptake from transferrin and nontransferrin iron sources.

Animals↗