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

Iron deprivation induces apoptosis in some sensitive cultured tumour cells, while other cells are resistant. In order to elucidate the mechanisms involved in apoptosis induction by iron deprivation, we studied the expression of p53 and the expression of selected p53-regulated genes. To discriminate between changes coupled only with iron deprivation and changes involved in apoptosis induction by iron deprivation, we compared the expression of the genes in sensitive (human Raji, mouse 38C13) versus resistant (human HeLa, mouse EL4) cells under iron deprivation. Iron deprivation was achieved by incubation in a defined iron-free medium. The level of p53 mRNA decreased significantly under iron deprivation in sensitive cells, but it did not change in resistant cells. On the contrary, the level of the p53 protein under iron deprivation was slightly increased in sensitive cells while it was not changed in resistant cells. The activity of p53 was assessed by the expression of selected p53-regulated targets, i.e. p21(WAF1/CIP1) gene, mdm2, bcl-2 and bax. We did not detect any relevant change in mRNA levels as well as in protein levels of these genes under iron deprivation with the exception of p21(WAF1/CIP1). We detected a significant increase in the level of p21 mRNA in both (sensitive and resistant) mouse cell lines tested, however, we did not find any change in both (sensitive and resistant) human cell lines. Moreover, the p21(WAF1/CIP1) protein was accumulated in mouse-sensitive 38C13 cells under iron deprivation while all other cell lines tested, including human-sensitive cell line Raji, did not show any accumulation of p21(WAF1/CIP1) protein. It seems that the p21(WAF1/CIP1) mRNA, as well as protein accumulation, is not specifically coupled with apoptosis induction by iron deprivation and that it is rather cell-line specific. Taken together, we suggest that iron deprivation induces apoptosis at least in some cell types independently of the p53 pathway.

• MeSH
• Apoptosis * MeSH
• Humans MeSH
• Mice MeSH
• Tumor Cells, Cultured MeSH
• Tumor Suppressor Protein p53 physiology   MeSH
• bcl-2-Associated X Protein MeSH
• Proto-Oncogene Proteins c-bcl-2 genetics   MeSH
• Proto-Oncogene Proteins genetics   MeSH
• Gene Expression Regulation MeSH
• Iron physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• BAX protein, human MeSH Browser
• Bax protein, mouse MeSH Browser
• Tumor Suppressor Protein p53 MeSH
• bcl-2-Associated X Protein MeSH
• Proto-Oncogene Proteins c-bcl-2 MeSH
• Proto-Oncogene Proteins MeSH
• Iron MeSH

We studied the sensitivity of tumor cells to the induction of apoptosis by iron deprivation. Iron deprivation was achieved by the employment of a defined iron-deficient culture medium. Mouse 38C13 cells and human Raji cells die within 48 and 96 h of incubation in iron-deficient medium, respectively. On the contrary, mouse EL4 cells and human HeLa cells are completely resistant to the induction of death under the same experimental arrangement. Deoxyribonucleic acid fragmentation analysis by agarose gel electrophoresis as well as flow cytometric analysis after propidium iodide staining detected in 38C13 and Raji cells, but not in EL4 and HeLa cells, changes characteristic to apoptosis. The 38C13 cells, sensitive to iron deprivation, also displayed a similar degree of sensitivity to apoptosis induction by thiol deprivation (achieved by 2-mercaptoethanol withdrawal from the culture medium) as well as by rotenone (50 nM), hydroxyurea (50 microM), methotrexate (20 nM), and doxorubicin (100 nM). Raji cells shared with 38C13 cells a sensitivity to rotenone, methotrexate, doxorubicin, and, to a certain degree, to hydroxyurea. However, Raji cells were completely resistant to thiol deprivation. EI4 and HeLa cells, resistant to iron deprivation, also displayed a greater degree of resistance to most of the other apoptotic stimuli than did their sensitive counterparts. We conclude that some tumor cells in vitro are sensitive to apoptosis induction by iron deprivation, while other tumor cells are resistant. All the tumors found to be sensitive to iron deprivation in this study (four cell lines) are of hematopoietic origin. The mechanism of resistance to apoptosis induction by iron deprivation differs from the mechanism of resistance to thiol deprivation.

• MeSH
• Apoptosis * drug effects   MeSH
• Lymphoma, B-Cell MeSH
• Coloring Agents MeSH
• Cell Division MeSH
• Burkitt Lymphoma MeSH
• Doxorubicin pharmacology   MeSH
• Electrophoresis, Agar Gel MeSH
• DNA Fragmentation MeSH
• HeLa Cells MeSH
• Hydroxyurea pharmacology   MeSH
• Culture Media MeSH
• Humans MeSH
• Lymphoma, T-Cell MeSH
• Methotrexate pharmacology   MeSH
• Mice MeSH
• Tumor Cells, Cultured MeSH
• Neoplasms pathology   MeSH
• Propidium MeSH
• Flow Cytometry MeSH
• Rotenone pharmacology   MeSH
• Transferrin MeSH
• Cell Survival MeSH
• Iron administration & dosage   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Coloring Agents MeSH
• Doxorubicin MeSH
• Hydroxyurea MeSH
• Culture Media MeSH
• Methotrexate MeSH
• Propidium MeSH
• Rotenone MeSH
• Transferrin MeSH
• Iron MeSH

We studied the factors that determine the differing growth requirements of low-iron-tolerant (LIT) versus high-iron-dependent (HID) cells for extracellular nontransferrin iron. The growth of LIT cells HeLa and THP-1, when transferred from transferrin (5 micrograms/ml) medium into low-iron (5 microM ferric citrate) medium, was not significantly affected while HID cells Jiyoye and K562 showed nearly no growth. HeLa and THP-1 cells, as well as Jiyoye and K562 cells, do not produce transferrin in sufficient amounts to support their growth in low-iron medium. Surprisingly, similar rates of iron uptake in low-iron medium (0.033 and 0.032 nmol Fe/min and 10(6) cells) were found for LIT cells HeLa and HID cells K562. Furthermore, the intracellular iron level (4.64 nmol/10(6) cells) of HeLa cells grown in low-iron medium was much higher than iron levels (0.15 or 0.20 nmol/10(6) cells) of HeLa or K562 cells grown in transferrin medium. We demonstrated that the activity (ratio activated/total) of the iron regulatory protein (IRP) in HID cells Jiyoye and K562 increased more than twofold (from 0.32 to 0.79 and from 0.47 to 1.12, respectively) within 48 h after their transfer into low-iron medium. In the case of LIT cells HeLa and THP-1, IRP activity stayed at similar or slightly decreased levels (0.86-0.73 and 0.58-0.55, respectively). Addition of iron chelator deferoxamine (50 microM, i.e., about half-maximal growth-inhibitory dose) resulted in significantly increased activity of IRP also in HeLa and THP-1 cells. We hypothesize that the relatively higher bioavailability of nontransferrin iron in LIT cells, over that in HID cells, determines the differing responses observed under low-iron conditions.

• MeSH
• Leukemia, Erythroblastic, Acute MeSH
• Leukemia, Monocytic, Acute MeSH
• Biological Transport MeSH
• Cell Division * MeSH
• Burkitt Lymphoma MeSH
• Deferoxamine pharmacology   MeSH
• HeLa Cells MeSH
• Kinetics MeSH
• Culture Media MeSH
• Humans MeSH
• Tumor Cells, Cultured MeSH
• Iron-Sulfur Proteins metabolism   MeSH
• Iron-Regulatory Proteins MeSH
• RNA-Binding Proteins metabolism   MeSH
• Transferrin pharmacology   MeSH
• Iron metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Deferoxamine MeSH
• Culture Media MeSH
• Iron-Sulfur Proteins MeSH
• Iron-Regulatory Proteins MeSH
• RNA-Binding Proteins MeSH
• Transferrin MeSH
• Iron MeSH