- Publikační typ
- abstrakt z konference MeSH
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
- buněčná membrána metabolismus MeSH
- buněčné linie MeSH
- ceruloplasmin genetika metabolismus MeSH
- cytochromy typu b genetika metabolismus MeSH
- exprese genu MeSH
- kultivační média chemie MeSH
- lidé MeSH
- membránové proteiny genetika metabolismus MeSH
- oxidoreduktasy genetika metabolismus MeSH
- proteiny přenášející kationty genetika metabolismus MeSH
- transferin metabolismus MeSH
- železo metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem 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.
We tested the effect of iron deprivation on the uptake of iron from ferric citrate by human erythroleukemia K562 cells. The iron uptake after 24-h preincubation in defined iron-free medium was approximately 2-3x higher than after the preincubation in control transferrin-containing medium. The preincubation of K562 cells in iron-free medium together with the inhibitor of protein synthesis cycloheximide completely abrogated the stimulation of the iron uptake. The preincubation in iron-free medium resulted in a slight decrease (20%) of DMT1 mRNA level. The level of Dcytb, ferroportin and hephaestin mRNA did not exert any significant change. We also did not find any significant effect on the protein level of DMT1, Dcytb, ferroportin and hephaestin. We conclude that iron deprivation stimulates the uptake of non-transferrin iron in K562 cells and that this stimulation depends on protein synthesis. It seems that the expression of an unknown or seemingly unrelated protein(s) is involved.
- MeSH
- buňky K562 MeSH
- časové faktory MeSH
- cykloheximid farmakologie MeSH
- financování organizované MeSH
- lidé MeSH
- nádorové buňky kultivované MeSH
- proteiny regulující obsah železa antagonisté a inhibitory biosyntéza MeSH
- transferin metabolismus MeSH
- vztahy mezi strukturou a aktivitou MeSH
- železo farmakokinetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- srovnávací studie MeSH
