We studied the effect of iron deficiency, i.e., 24-h preincubation in iron-free medium, and the effect of high level of non-transferrin iron, i.e., the preincubation in ferric citrate medium containing 500 microM ferric citrate, on the expression of DMT1, Dcytb, ferroportin, hephaestin, and ceruloplasmin in various functional types of human cells. The expression of these proteins potentially involved in non-transferrin iron transport across cell membranes was tested on mRNA level by quantitative real-time PCR as well as on protein level by western blot analysis in Caco-2 (colorectal carcinoma), K562 (erythroleukemia), and HEP-G2 (hepatocellular carcinoma) cells. We found that changes in non-transferrin iron availability, i.e., iron deficiency and high level of non-transferrin iron, affect the expression of tested proteins in a cell type-specific manner. We also demonstrated that changes in the expression on mRNA level do not often correlate with relevant changes on protein level.

• MeSH
• Cell Membrane metabolism   MeSH
• Cell Line MeSH
• Ceruloplasmin genetics   metabolism   MeSH
• Cytochrome b Group genetics   metabolism   MeSH
• Gene Expression * MeSH
• Ferroportin MeSH
• Culture Media chemistry   MeSH
• Humans MeSH
• Membrane Proteins * genetics   metabolism   MeSH
• Oxidoreductases genetics   metabolism   MeSH
• Cation Transport Proteins genetics   metabolism   MeSH
• Transferrin metabolism   MeSH
• Iron metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Ceruloplasmin MeSH
• CYBRD1 protein, human MeSH Browser
• Cytochrome b Group MeSH
• Ferroportin MeSH
• HEPH protein, human MeSH Browser
• Culture Media MeSH
• Membrane Proteins * MeSH
• Oxidoreductases MeSH
• Cation Transport Proteins MeSH
• solute carrier family 11- (proton-coupled divalent metal ion transporters), member 2 MeSH Browser
• Transferrin MeSH
• Iron MeSH

We have shown previously that iron deprivation significantly stimulates the uptake of non-transferrin ferric iron from ferric citrate by erythroleukemia K562 cells and that this stimulation depends on protein synthesis. However, we have not detected increased expression of any known iron transport protein (Kovar J. et al. (2006) Blood Cells Mol Dis 37:95-99). Therefore, in order to identify membrane proteins of K562 cells with increased expression under iron deprivation, we employed the isolation of membrane proteins by two-phase partitioning system, protein separation by high-resolution 2D electrophoresis, computer differential analysis, and tandem mass spectrometry. Employing these techniques we identified two proteins with statistically significant upregulation, i.e., aldolase A (ALDA) and voltage-dependent anion channel 2 (VDAC2). The upregulation of aldolase A and VDAC2 in K562 cells under iron deprivation was also confirmed by western blot analysis. This is the first time when the control of aldolase A and VDAC2 levels by iron status of the cell is demonstrated.

• MeSH
• Fructose-Bisphosphate Aldolase genetics   metabolism   MeSH
• K562 Cells * MeSH
• Iron Deficiencies * MeSH
• Humans MeSH
• Voltage-Dependent Anion Channel 2 genetics   metabolism   MeSH
• Gene Expression Regulation * MeSH
• Up-Regulation MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fructose-Bisphosphate Aldolase MeSH
• Voltage-Dependent Anion Channel 2 MeSH
• VDAC2 protein, human MeSH Browser

We tested the effect of iron deprivation on cell death induction in human Raji cells pre-adapted to differing availability of extracellular iron. Iron deprivation was achieved by incubation in a defined iron-free medium. Original Raji cells have previously been adapted to long-term culture in a defined medium with 5 microg/ml of iron-saturated human transferrin as a source of iron. Raji/lowFe cells were derived from original Raji cells by subsequent adaptation to culture in the medium with 50 microm ferric citrate as a source of iron. Raji/lowFe-re cells were derived from Raji/lowFe cells by re-adaptation to the transferrin-containing (5 microg/ml) medium. Iron deprivation induced cell death in both Raji cells and Raji/lowFe-re cells; that is, cells pre-adapted to a near optimum source of extracellular iron (5 microg/ml of transferrin). However, Raji/lowFe cells preadapted to a limited source of extracellular iron (50 microm ferric citrate) became resistant to the induction of cell death by iron deprivation. We demonstrated that cell death induction by iron deprivation in Raji cells correlates with the activation of executioner caspase-3 and the cleavage of caspase-3 substrate, poly-ADP ribose polymerase. Two other executioner caspases, caspase-7 and caspase-6, were not activated. Taken together, we suggest that in human Raji cells, iron deprivation induces apoptotic cell death related to caspase-3 activation. However, the sensitivity of the cells to death induction by iron deprivation can be reversibly changed by extracellular iron availability. The cells pre-adapted to a limited source of extracellular iron became resistant.

• MeSH
• Enzyme Activation MeSH
• Apoptosis drug effects   physiology   MeSH
• Cell Division drug effects   physiology   MeSH
• Burkitt Lymphoma MeSH
• Iron Deficiencies * MeSH
• Caspase 3 metabolism   MeSH
• Caspase 6 metabolism   MeSH
• Caspase 7 metabolism   MeSH
• Culture Media MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Cell Survival drug effects   physiology   MeSH
• Iron pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CASP6 protein, human MeSH Browser
• Caspase 3 MeSH
• Caspase 6 MeSH
• Caspase 7 MeSH
• Culture Media MeSH
• Iron MeSH