Upregulation Of Transferrin Receptor Research Articles

The effect of enterotoxigenic Escherichia coli F4ab,ac on early-weaned piglets: A gene expression study

Diarrhoea in neonatal and early-weaned piglets due to enterotoxigenic Escherichia coli-F4 (ETEC-F4) is an important problem in the pig farming industry. There is substantial evidence for a genetic basis for susceptibility to ETEC-F4 since not all pigs suffer from diarrhoea after an ETEC-F4 infection. A region on SSC13 has been found to be in close linkage to the susceptibility of piglets for ETEC-F4ab,ac. Potential candidate genes on SSC13 have been examined and although some polymorphisms were found to be in linkage disequilibrium with the phenotype, the causative mutation has not yet been found. In this study we are looking at the expression of porcine genes in relation to ETEC-F4ab,ac. With the aid of the Affymetrix GeneChip Porcine Genome Array we were able to find differentially expressed genes between ETEC-F4ab,ac receptor positive (Fab,acR+) piglets without diarrhoea and F4ab,acR+ piglets with diarrhoea or F4ab,acR− animals. Since the susceptibility to ETEC-F4ab,ac was described as a Mendelian trait, it is not so surprisingly that only two differentially expressed genes, transferrin receptor (TFRC) and trefoil factor 1 (TFF1), came out of the analysis. Although both genes could pass for functional candidate genes only TFRC also mapped to the region on SSC13 associated with susceptibility for ETEC-F4, which makes TFRC a positional functional candidate gene. Validation by qRT-PCR confirmed the differential expression of TFRC and TFF1. In piglets without diarrhoea, the expression of both genes was higher in F4ab,acR+ than in F4ab,acR− piglets. Similarly, TFRC and TFF1 expression in F4ab,acR+ piglets without diarrhoea was also higher than in F4ab,acR+ piglets with diarrhoea. Consequently, although both genes might not play a role as receptor for F4 fimbriae, they could be of great importance during an ETEC-F4 outbreak. An upregulation of TFRC can be a consequence of the piglets ability to raise an effective immune response. An elevation of TFF1, a protein involved in mucin formation, may also affect the piglet's capability to cope with ETEC bacteria, rather than being a receptor for its fimbriae.

173 Synergistic toxicity of tyrosine kinase inhibitors with Hsp90 inhibitor 17-AAG in lung cancer cell line

We screened small molecules to identify two compounds, which we named RSL3 and RSL5, that have increased lethality in the presence of oncogenic RAS. Counter screening with biologically active compounds defined aspects of the mechanism of action for RSL3 and RSL5, such as a nonapoptotic, MEK-dependent, and iron-dependent oxidative cell death. Erastin, a previously reported compound with RAS-selective lethality, showed similar properties. RNA interference experiments targeting voltage-dependent anion channel 3 (VDAC3), a target of erastin, demonstrated that RSL5 is a scaffold that acts through VDACs to activate the observed pathway. RSL3 activated a similar death mechanism but in a VDAC-independent manner. We found that cells transformed with oncogenic RAS have increased iron content relative to their normal cell counterparts through upregulation of transferrin receptor 1 and downregulation of ferritin heavy chain 1 and ferritin light chain.

Brain and retinal ferroportin 1 dysregulation in polycythaemia mice

Disruption of iron homeostasis within the central nervous system (CNS) can lead to profound abnormalities during both development and aging in mammals. The radiation-induced polycythaemia (Pcm) mutation, a 58-bp microdeletion in the promoter region of ferroportin 1 (Fpn1), disrupts transcriptional and post-transcriptional regulation of this pivotal iron transporter. This regulatory mutation induces dynamic alterations in peripheral iron homeostasis such that newborn homozygous Pcm mice exhibit iron deficiency anemia with increased duodenal Fpn1 expression while adult homozygotes display decreased Fpn1 expression and anemia despite organismal iron overload. Herein we report the impact of the Pcm microdeletion on iron homeostasis in two compartments of the central nervous system: brain and retina. At birth, Pcm homozygotes show a marked decrease in brain iron content and reduced levels of Fpn1 expression. Upregulation of transferrin receptor 1 (TfR1) in brain microvasculature appears to mediate the compensatory iron uptake during postnatal development and iron content in Pcm brain is restored to wild-type levels by 7 weeks of age. Similarly, changes in expression are transient and expression of Fpn1 and TfR1 is indistinguishable between Pcm homozygotes and wild-type by 12 weeks of age. Strikingly, the adult Pcm brain is effectively protected from the peripheral iron overload and maintains normal iron content. In contrast to Fpn1 downregulation in perinatal brain, the retina of Pcm homozygotes reveals increased levels of Fpn1 expression. While retinal morphology appears normal at birth and during early postnatal development, adult Pcm mice demonstrate a marked, age-dependent loss of photoreceptors. This phenotype demonstrates the importance of iron homeostasis in retinal health.

Abnormal brain iron homeostasis in human and animal prion disorders.

Neurotoxicity in all prion disorders is believed to result from the accumulation of PrP-scrapie (PrPSc), a β-sheet rich isoform of a normal cell-surface glycoprotein, the prion protein (PrPC). Limited reports suggest imbalance of brain iron homeostasis as a significant associated cause of neurotoxicity in prion-infected cell and mouse models. However, systematic studies on the generality of this phenomenon and the underlying mechanism(s) leading to iron dyshomeostasis in diseased brains are lacking. In this report, we demonstrate that prion disease–affected human, hamster, and mouse brains show increased total and redox-active Fe (II) iron, and a paradoxical increase in major iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) at the end stage of disease. Furthermore, examination of scrapie-inoculated hamster brains at different timepoints following infection shows increased levels of Tf with time, suggesting increasing iron deficiency with disease progression. Sporadic Creutzfeldt-Jakob disease (sCJD)–affected human brains show a similar increase in total iron and a direct correlation between PrP and Tf levels, implicating PrPSc as the underlying cause of iron deficiency. Increased binding of Tf to the cerebellar Purkinje cell neurons of sCJD brains further indicates upregulation of TfR and a phenotype of neuronal iron deficiency in diseased brains despite increased iron levels. The likely cause of this phenotype is sequestration of iron in brain ferritin that becomes detergent-insoluble in PrPSc-infected cell lines and sCJD brain homogenates. These results suggest that sequestration of iron in PrPSc–ferritin complexes induces a state of iron bio-insufficiency in prion disease–affected brains, resulting in increased uptake and a state of iron dyshomeostasis. An additional unexpected observation is the resistance of Tf to digestion by proteinase-K, providing a reliable marker for iron levels in postmortem human brains. These data implicate redox-iron in prion disease–associated neurotoxicity, a novel observation with significant implications for prion disease pathogenesis.

High concentrations of ascorbic acid induces apoptosis of human gastric cancer cell by p38-MAP kinase-dependent up-regulation of transferrin receptor

We investigated the molecular mechanism by which ascorbic acid (AA) induces apoptosis in human gastric cancer cells, AGS cells. High concentration (more than 5 mM) of AA increased cellular iron uptake by increasing transferrin receptor (TfR) expression and induced AGS cell apoptosis which was inhibited by catalase. Interestingly, p38 mitogen-activated protein kinase (MAPK) inhibitor inhibited the upregulation of TfR and increased cell survival by AA. TfR-siRNA-transfected cells reduced apoptosis by AA. H 2O 2 increased TfR expression in AGS cells. Taken together, we concluded that high concentration of AA, through H 2O 2, induces apoptosis of AGS cells by p38-MAPK-dependent upregulation of TfR.

Transferrin Receptor-Targeted Lipid Nanoparticles for Delivery of an Antisense Oligodeoxyribonucleotide against Bcl-2

Antisense oligonucleotide G3139-mediated down-regulation of Bcl-2 is a potential strategy for overcoming chemoresistance in leukemia. However, the limited efficacy shown in recent clinical trials calls attention to the need for further development of novel and more efficient delivery systems. In order to address this issue, transferrin receptor (TfR)-targeted, protamine-containing lipid nanoparticles (Tf-LNs) were synthesized as delivery vehicles for G3139. The LNs were produced by an ethanol dilution method, and lipid-conjugated Tf ligand was then incorporated by a postinsertion method. The resulting Tf-LNs had a mean particle diameter of approximately 90 nm and G3139 loading efficiency of 90.4%. Antisense delivery efficiency of Tf-LNs was evaluated in K562, MV4-11, and Raji leukemia cell lines. The results showed that Tf-LNs were more effective than nontargeted LNs and free G3139 (p < 0.05) in decreasing Bcl-2 expression (by up to 62% at the mRNA level in K562 cells) and in inducing caspase-dependent apoptosis. In addition, Bcl-2 down-regulation and apoptosis induced by Tf-LN G3139 were shown to be blocked by excess free Tf and thus were TfR-dependent. Cell lines with higher TfR expression also showed greater Bcl-2 down-regulation. Furthermore, up-regulation of TfR expression in leukemia cells by iron chelator deferoxamine resulted in a further increase in antisense effect (up to 79% Bcl-2 reduction in K562 at the mRNA level) and in caspase-dependent apoptosis (by approximately 3-fold) by Tf-LN. Tf-LN-mediated delivery combined with TfR up-regulation by deferoxamine appears to be a potentially promising strategy for enhancing the delivery efficiency and therapeutic efficacy of antisense oligonucleotides.

Synthetic Lethal Screening Identifies Compounds Activating Iron-Dependent, Nonapoptotic Cell Death in Oncogenic-RAS-Harboring Cancer Cells

We screened small molecules to identify two compounds, which we named RSL3 and RSL5, that have increased lethality in the presence of oncogenic RAS. Counter screening with biologically active compounds defined aspects of the mechanism of action for RSL3 and RSL5, such as a nonapoptotic, MEK-dependent, and iron-dependent oxidative cell death. Erastin, a previously reported compound with RAS-selective lethality, showed similar properties. RNA interference experiments targeting voltage-dependent anion channel 3 (VDAC3), a target of erastin, demonstrated that RSL5 is a scaffold that acts through VDACs to activate the observed pathway. RSL3 activated a similar death mechanism but in a VDAC-independent manner. We found that cells transformed with oncogenic RAS have increased iron content relative to their normal cell counterparts through upregulation of transferrin receptor 1 and downregulation of ferritin heavy chain 1 and ferritin light chain.

Transferrin Receptor Upregulation: In Vitro Labeling of Rat Mesenchymal Stem Cells with Superparamagnetic Iron Oxide

To prospectively evaluate the influence of superparamagnetic iron oxide (SPIO) or ultrasmall SPIO (USPIO) particles on the surface epitope pattern of adult mesenchymal stem cells (MSCs) by regulating the expression of transferrin receptor and to prospectively evaluate the influence of transfection agents (TAs) on the uptake of SPIO or USPIO particles in MSCs. The study was approved by the institutional animal care committee of the University of Tübingen. MSCs were isolated from the bone marrow of four rats. To obtain highly homogeneous MSC populations, MSCs from one rat were single-cell cloned. One MSC clone was characterized and selected for the labeling experiments. The MSCs, which were characterized with flow cytometry and in vitro differentiation, were labeled with 200 microg/mL SPIO or USPIO or with 60 microg/mL SPIO or USPIO in combination with TAs. Aggregations of labeled cells were accommodated inside a defined volume in an agar gel matrix. Magnetic resonance (MR) imaging was performed to measure SPIO- or USPIO-induced signal voids. Quantification of cellular total iron load (TIL) (intracellular iron plus iron coating the cellular surface), determination of cellular viability, and electron microscopy were also performed. Labeling of MSCs with SPIO or USPIO was feasible without affecting cell viability (91.1%-94.7%) or differentiation potential. For MR imaging, SPIO plus a TA was most effective, depicting 5000 cells with an average TIL of 76.5 pg per cell. SPIO or USPIO particles in combination with TAs coated the cellular surface but were not incorporated into cells. In nontransfected cells, SPIO or USPIO was taken up. MSCs labeled with SPIO or USPIO but without a TA showed enhanced expression of transferrin receptor, in contrary to both MSCs labeled with SPIO or USPIO and a TA and control cells. SPIO or USPIO labeling without TAs has an influence on gene expression of MSCs upregulating transferrin receptor. Furthermore, SPIO labeling with a TA will coat the cellular surface.

GPI valence and the fate of secretory membrane proteins in African trypanosomes

Progression of GPI-anchored proteins in bloodstream African trypanosomes correlates with GPI-valence: homodimeric VSG (2 GPI) is a surface protein; heterodimeric transferrin receptor (1 GPI) localizes in the flagellar pocket; homodimeric GPI-minus VSG (0 GPI) is rapidly degraded in the lysosome. We test this relationship using three native secretory/endocytic proteins as monomeric GPI-plus and -minus reporters. GPI-minus procyclin trafficks to the lysosome and is degraded. GPI-plus procyclin trafficks to the flagellar pocket/cell surface and is released (approximately 50%) with an intact anchor, the remainder (approximately 50%) is degraded in the lysosome. GPI-plus BiPNHP, derived from the ER marker BiP, is released quantitatively (>80%), while GPI-plus p67HP, derived from the lysosomal marker p67, turns over by both release (approximately 15%) and lysosomal degradation (>50%). Turnover of endogenous transferrin receptor occurs primarily by lysosomal degradation (>90%). Thus shedding of monovalent GPI reporters correlates inversely with lysosomal targeting. We propose that mono-GPI reporters cycle through the flagellar pocket and endosome until they are disposed of by either shedding or lysosomal targeting. Partitioning between these fates may be a function of individual physical properties. Release is likely due to the exclusive use of C-14:0 myristate in the bloodstream stage GPI anchor. Up-regulation of transferrin receptor by culture in dog serum resulted in prominent cell surface localization, but not in elevated release. Surface receptor was non-functional for ligand binding suggesting that it may be bivalent homodimers of the GPI-anchored ESAG6 receptor subunit.

Up-regulation of transferrin receptor 1 in chronic hepatitis C: Implication in excess hepatic iron accumulation

: To clarify the mechanism of excess hepatic iron accumulation in chronic hepatitis C, we investigated the expressions of transferrin receptor 1 and divalent metal transporter 1 in hepatocytes, both of which are involved in cellular iron uptake, in relation to the degree of hepatic iron accumulation and hepatic fibrosis by immunohistochemistrical study. : Forty-six hepatic tissues with chronic hepatitis C and five normal hepatic tissues were examined. Chemical detection of hepatic iron accumulation was performed by Perl's Prussian blue stain. The immunohistochemistrical study was performed by avidin-biotin complex method with alkaline phosphatase. : In chronic hepatitis C: (1) Hepatic iron accumulation was significantly increased in relation to the advance of the fibrosis. (2) Divalent metal transporter 1 decreased significantly in relation to the advance of hepatic fibrosis. (3) Transferrin receptor 1 expression was always detected, although not in normal hepatic tissues; there was no relation between expression levels and the degree of hepatic fibrosis. : These data demonstrated that the transferrin receptor 1 expression was up-regulated irrespective of the degree of hepatic iron accumulation, suggesting that the up-regulation of transferrin receptor 1 might act as one of the key mechanisms implicated in the accumulation of hepatic iron in chronic hepatitis C.

Upregulation of transferrin receptor 2 and ferroportin 1 mRNA in the liver of patients with chronic hepatitis C

Iron accumulation has been reported to be associated with progression of liver injury. The mechanism of iron accumulation in the liver is not known. In the present study, hepatic messenger RNA (mRNA) expression of transferrin receptor (TfR)1, TfR2, and ferroportin (FP)1 was measured in patients with chronic hepatitis (CH). Eleven patients with CH-B and 43 patients with CH-C were enrolled. All patients underwent liver biopsy. Hepatic expression of TfR1, TfR2 and FP1 mRNA was analyzed using a real-time polymerase chain reaction. Total hepatic iron score (THIS) was evaluated by Prussian blue staining. Serum ferritin concentration is significantly higher in CH-C than in CH-B. Values of THIS of >/=5 were observed only in CH-C patients (44% of CH-C patients). The expression level of TfR2 mRNA was 10-26-fold higher than the TfR1 mRNA expression level. The TfR2 and FP1 mRNA expression was significantly higher in CH-C than in CH-B patients. Hepatic expression of TfR2 and FP1 mRNA was well correlated with THIS. Hepatic iron accumulation is more severe in patients with CH-C. Upregulation of hepatic iron transporters may contribute to the hepatic iron accumulation in CH-C.

Altered expression of iron transport proteins in streptozotocin-induced diabetic rat kidney

Diabetes mellitus is associated with altered iron homeostasis in both human and animal diabetic models. Iron is a metal oxidant capable of generating reactive oxygen species (ROS) and has been postulated to contribute to diabetic nephropathy. Two proteins involved in iron metabolism that are expressed in the kidney are the divalent metal transporter, DMT1 (Slc11a2), and the Transferrin Receptor (TfR). Thus, we investigated whether renal DMT1 or TfR expression is altered in diabetes, as this could potentially affect ROS generation and contribute to diabetic nephropathy. Rats were rendered diabetic with streptozotocin (STZ-diabetes) and renal DMT1 and TfR expression studied using semi-quantitative immunoblotting and immunofluorescence. In STZ-diabetic Sprague-Dawley rats, renal DMT1 expression was significantly reduced and TfR expression increased after 2 weeks. DMT1 downregulation was observed in both proximal tubules and collecting ducts. Renal DMT1 expression was also decreased in Wistar rats following 12 weeks of STZ-diabetes, an effect that was fully corrected by insulin-replacement but not by cotreatment with the aldose reductase inhibitor, sorbinil. Increased renal TfR expression was also observed in STZ-diabetic Wistar rats together with elevated cellular iron accumulation. Together these data demonstrate renal DMT1 downregulation and TfR upregulation in STZ-diabetes. Whilst the consequence of altered DMT1 expression on renal iron handling and oxidant damage remains to be determined, the attenuation of the putative lysosomal iron exit pathway in proximal tubules could potentially explain lysosomal iron accumulation reported in human diabetes and STZ-diabetic animals.

Transferrin receptor induction in Toxoplasma gondii-infected HFF is associated with increased iron-responsive protein 1 activity and is mediated by secreted factors.

Infection with the apicomplexan parasite Toxoplasma gondii results in a significant alteration of the host-cell transcriptional profile. We have previously shown that the transferrin receptor (TfR) is specifically up-regulated in T. gondii-infected human fibroblasts but not in host cells infected with the bacterial pathogens Salmonella Typhimurium and Chlamydia trachomatis. In this report, we describe the prerequisites and physiological conditions that are associated with this pathogen-specific gene induction. Band-shift assays revealed that T. gondii infection resulted in increased activity in the iron response protein IRP1, which, in this state, stabilizes TfR mRNA from degradation. Although T. gondii depends on host-cell iron as demonstrated by sensitivity to deferoxamine, a parasite-induced iron starvation is not responsible for TfR up-regulation. The increased iron availability due to treatment with holotransferrin and FeNTA did not prevent TfR induction nor was the transferrin-independent iron-transporter NRAMP2 up-regulated in infected host cells. In addition, inhibition of parasite replication by drug treatment did not prevent TfR up-regulation. Instead, TfR induction was sensitive to cycloheximide and could be induced by treatment with conditioned media from infected human fibroblasts. Together our findings suggest that the T. gondii-specific TfR up-regulation is not due to a direct interaction of parasitic factors with the iron-uptake machinery of the host cell but is instead mediated indirectly as a result of secreted host cell- or parasite-derived factors.

Oxidant-Induced Iron Signaling in Doxorubicin-Mediated Apoptosis

Publisher Summary This chapter discusses the methodological aspects linking Doxorubicin (DOX)-induced intracellular oxidative stress, iron signaling, and apoptosis. Pertinent redox parameters measured are the following: intracellular glutathione (GSH) levels, aconitase activity, IRP–IRE interaction, transferrin receptor (TfR) expression, cellular iron uptake, DCFH oxidation to DCF, and apoptosis. DOX or adriamycin, a quinone-containing anthracycline antibiotic, is a widely used chemotherapeutic drug for treating leukemia, breast cancer, Hodgkin's disease, or sarcomas. The clinical efficacy of this drug is greatly restricted because of the development of a severe form of cardiomyopathy or congestive heart failure in cancer patients treated with this drug. Current evidence indicates that DOX-mediated cardiotoxicity may be caused by increased generation of reactive oxygen species (ROS) such as superoxide, hydrogen peroxide (H2O2), or hydroxyl radicals (·OH) through redox-activation of DOX. The proapoptotic effect of DOX in myocytes and endothelial cells is attributed to intracellular iron and H2O2 formation. Recently, it is showed that intracellular iron plays a critical role in initiating oxidant-induced apoptosis through upregulation of TfR. Transferrin receptor synthesis is regulated by interaction of the iron regulatory protein (IRP) with the iron-responsive element (IRE) present on the 30-untranslated region of TfR mRNA. The oxidant-induced iron signaling mechanism is a new perspective that should be more fully explored in DOX cardiotoxicity.

Transferrin receptor gene expression and transferrin-bound iron uptake are increased during postischemic rat liver reperfusion

Iron-catalyzed production of reactive oxygen species is a cause of liver injury after ischemia/reperfusion (I/R). The aim of the present study was to address the regulation of transferrin receptor (TfR), which mediates cellular iron uptake, during I/R. The molecular mechanisms controlling TfR gene expression in vivo during I/R of rat liver were investigated by molecular biology procedures. We also analyzed transferrin-bound iron uptake into surviving liver slices. Increased amounts of TfR protein and messenger RNA (mRNA) were found 2 to 6 hours after reestablishment of blood supply. RNA bandshift analysis showed that iron regulatory protein (IRP) activity was decreased in the first hours of reperfusion, thus indicating that IRP-mediated mRNA stabilization was not involved in early TfR upregulation. On the contrary, increased transcription of the TfR gene in isolated nuclei was observed during reperfusion; during the ischemic phase this was preceded by enhanced binding of hypoxia inducible factor (HIF-1) to a DNA sequence derived from the TfR promoter. TfR2 mRNA levels were also enhanced after reperfusion. The increased expression of TfR at the cell surface resulted in increased uptake of transferrin-bound-iron into surviving liver slices; however, iron was not incorporated into ferritin. In conclusion, HIF-1 mediated activation of TfR gene transcription and IRP-mediated increase of TfR mRNA stability ensure a steady induction of TfR, and hence higher iron uptake in reperfused rat liver. TfR-mediated entry of the metal into liver cells may represent a source of catalitically active iron, which may play a role in reperfusion damage. (H EPATOLOGY 2002;36:103-111.)

Human Mitochondrial Ferritin Expressed in HeLa Cells Incorporates Iron and Affects Cellular Iron Metabolism

Mitochondrial ferritin (MtF) is a newly identified ferritin encoded by an intronless gene on chromosome 5q23.1. The mature recombinant MtF has a ferroxidase center and binds iron in vitro similarly to H-ferritin. To explore the structural and functional aspects of MtF, we expressed the following forms in HeLa cells: the MtF precursor (approximately 28 kDa), a mutant MtF precursor with a mutated ferroxidase center, a truncated MtF lacking the approximately 6-kDa mitochondrial leader sequence, and a chimeric H-ferritin with this leader sequence. The experiments show that all constructs with the leader sequence were processed into approximately 22-kDa subunits that assembled into multimeric shells electrophoretically distinct from the cytosolic ferritins. Mature MtF was found in the matrix of mitochondria, where it is a homopolymer. The wild type MtF and the mitochondrially targeted H-ferritin both incorporated the (55)Fe label in vivo. The mutant MtF with an inactivated ferroxidase center did not take up iron, nor did the truncated MtF expressed transiently in cytoplasm. Increased levels of MtF both in transient and in stable transfectants resulted in a greater retention of iron as MtF in mitochondria, a decrease in the levels of cytosolic ferritins, and up-regulation of transferrin receptor. Neither effect occurred with the mutant MtF with the inactivated ferroxidase center. Our results indicate that exogenous iron is as available to mitochondrial ferritin as it is to cytosolic ferritins and that the level of MtF expression may have profound consequences for cellular iron homeostasis.

Desferioxamine increases iron depletion and apoptosis induced by ara-C of human myeloid leukaemic cells.

We investigated whether changes in iron metabolism and the transferrin receptor (TRF-R) expression were involved in the antileukaemic effects of arabinoside cytosine (ara-C). Treatment with 100 nM ara-C for 48h reduced thymidine uptake and increased the surface expression of the TRF-R on leukaemic blasts derived from 13/16 (81%) patients and on the HL-60 and U-937 cell lines. Whereas intracellular non-haem iron was strongly depleted 24 h after ara-C addition, TRF-R up-regulation and recovery of intracellular non-haem iron concentration occurred together after a longer exposure of the cultured cells to the drug. Since iron is an essential regulator of cell proliferation we have evaluated the effects of the combination between ara-C and the iron chelator desferioxamine (DSF) on the growth of HL-60 and U-937 cells. We found that desferioxamine strongly potentiated the effects of ara-C on leukaemic cell growth inhibition and apoptosis. This is the first report of a positive interaction between ara-C and an iron chelator in terms of antileukaemic effects.

Correlation between the Expression of Ki67 and HLA-DR Antigens in Fote-mustine-Treated Melanoma Cell Lines

We studied the effect of fotemustine (FM) on the expression of several melanoma-associated antigens (MAA) and HLA antigens defined by a panel of seven monoclonal antibodies (mabs). We also looked for the effect of the culture conditions on the antigen expression. We used eight established melanoma cell lines, grown as monolayers in (a) modified Leibowitz L15 medium for 0, 48, 96 h; (b) with 12.8 μg/ml fotemustine for 48 h; (c) with 12.8 μg/ml fotemustine for 48 h followed by incubation with modified Leibowitz L15 medium for another 48 h. Incubation of melanoma cell lines for 48 or 96 h in growth medium (a) resulted in a modest upregulation of the trans-ferrin receptor (TFR), the very late antigen 2 (VLA-2), and the intercellular adhesion molecule 1 (ICAM-1) in the majority of our cell lines. Following the addition of FM (b) no relevant change in the antigen expression was observed during continuous incubation for 48 h. However, an additional incubation of the FM-treated melanoma cells for another 48 h in FM-free medium (c) showed a striking upregulation of TFR, VLA-2 andICAM-1. Furthermore, the nuclear proliferation-associated antigen detected by Ki67 was upregulated markedly only in the presence of at least 80% HLA-DR-positive cells.

Iron requirements of human lymphocytes: relative contributions of intra- and extra-cellular iron.

A supply of iron is of vital importance if lymphocyte proliferation is to proceed successfully and two major sources of iron are available, intracellular stores and serum transferrin. We have investigated the relative importance to the human T lymphocyte of these two sources of iron by depleting them of intracellular iron with the chelator desferrioxamine and by culturing them in medium completely depleted of transferrin iron. The chelator decreased mitogen-stimulated proliferation of human peripheral blood T cells, in a dose-dependent manner, in the absence of extracellular transferrin-iron. By culturing the cells in iron-depleted medium, we found that normal lymphocytes proliferated, to a degree, in the absence of extracellular transferrin-iron. We also observed that transferrin receptor mRNA expression was sustained in mitogen-stimulated, iron-deprived lymphocytes, compared with untreated cells suggesting that up-regulation of transferrin receptor may occur in these cells through stabilization of the mRNA. We propose that intra- and extra-cellular iron may contribute to early and late activation processes and that a low level of intracellular iron in lymphocytes, chronically activated in the iron-deficient environment associated with chronic inflammatory diseases such as rheumatoid arthritis, may be a factor in the abnormal cell-mediated immunity associated with such diseases.

α-Interferon Induces Depletion of Intracellular Iron Content and Up Regulation of Functional Transferrin Receptors on Human Epidermoid Cancer kb Cells

We have demonstrated that interferon-α (IFNα) upregulated the epidermal growth factor receptor (EGF-R) on human epidermoid carcinoma cells. Here we report that IFNα induces growth inhibition and upregulation of transferrin receptor (TRF-R) on epidermoid cancer KB cells. IFNα does not alter TRF-R affinity for its ligand and induces a two-fold increase of TRF binding sites. IFNα does not modify receptor internalization and cycling. Intracellular iron levels are known to regulate TRF-R expression: we have, therefore, evaluated whether changes in the iron content could be determined by IFNα. Iron levels are transiently increased after addition of fresh growth medium in untreated controls but not in KB cells exposed for 48 h to IFNα. Iron depletion is however completely reversed 24 h later when maximal TRF-R upregulation occurs in IFN α-treated cells. We suggest that IFNα-induced iron depletion elicits a homeostatic cellular response through upregulation of TRF-R.