Progress 07/01/03 to 06/30/06
Outputs
Iron is an essential trace element for all dividing cells. To examine the consequences of depriving cells of iron, Jurkat T-lymphocytes were made iron-deficient by exposing them to the iron chelator, deferoxamine (DFO). DFO caused morphological changes characteristic of apoptosis, including cell shrinkage, plasma membrane blebbing, chromatin condensation and fragmentation, and also formation of apoptotic bodies. Additionally, proteolytic cleavage of poly(ADP-ribose)polymerase was detected, suggesting the involvement of caspases in initiating the apoptosis. In support of this possibility, pre-incubating the cells with a selective caspase-3 inhibitor prevented the apoptotic effects of DFO. These data suggest that DFO caused apoptosis via a caspase-3-dependent cell death pathway. During the early induction period of apoptosis induced by DFO, no effect on GRP78 and HSP70 mRNA expression was found. In contrast, there was increased mRNA expression of Growth Arrest and DNA
Damage-inducible gene 153 (GADD153), which seemed to be at the level of transcription rather than mRNA stability. Moreover, fortifying the cells with antioxidants did not prevent the increased GADD153 mRNA expression, and no evidence of single-strand breaks in DNA was found, suggesting that neither free radicals nor DNA damage were involved in triggering GADD153 gene activation. Because GADD153 is recognized as a pro-apoptotic gene, these findings suggest that GADD153 may play a role in mediating apoptosis in the iron-deficient cells. Other research has focused on the importance of iron deficiency in killing cancer cells. As a cofactor for ribonucleotide reductase involved in DNA synthesis, iron is necessary for cell proliferation. On the other hand, a potential role of iron in facilitating DNA repair has not been examined particularly in cancer cells made iron-deficient with DFO, which can also be used as a cancer chemotherapeutic drug. In preliminary experiments that will expand in
the future, it has been found that exposing HCT-116 (expressing wild-type p53 gene) and HCT-15 (expressing mutant p53 gene) human colon cancer cells to DFO induced DNA damage that led eventually to apoptosis. However, treating HCT-116 cells with iron (ferrous sulfate) not only reversed the DNA damage, but also rescued them from committing apoptosis. The effect of iron was not as noticeable in HCT-15 cells. These data suggest that iron is important in the repair of DNA damaged by DFO and that p53 could play a role in mediating the DNA repair following iron replenishment of the iron-deficient cells. Also, the new findings reveal that the induction of DNA damage by DFO does not automatically sentence the cancer cells to apoptosis. Evidently, cancer cells expressing wild type p53 can undesirably recover from DFO treatment, whereas cancer cells expressing mutant p53 are not able to recover as well making them more prone to killing by creating a state of iron deficiency with DFO.
Impacts
The present and anticipated findings are expected to help ascertain whether severely iron-deficient cells are automatically committed to death or alternatively can be revitalized by iron replenishment so as to result in activation of certain genes to promote DNA structural integrity needed for cell viability.
Publications
- HOPKINS RG, EDWARDS VL, LOO G. 2003. Cellular iron deficiency upregulates GADD153 stress-response gene expression. FASEB J 17, A1159 (abstract #712.12).
- PAN YJ, HOPKINS RG, LOO G. 2004. Increased GADD153 gene expression during iron chelation-induced apoptosis in Jurkat T-lymphocytes. Biochim Biophys Acta 1691, 41-50.
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Progress 10/01/04 to 09/30/05
Outputs
Cells are highly dependent on iron as a vital nutrient for their proliferation. Iron is essential for the biosynthesis of DNA, but the significance of iron in the repair of damaged DNA is unknown. Using two distinct iron chelators, namely, deferoxamine (DFO) and dipyridyl (DPD), a state of iron deficiency was induced in HCT116 human colon adenocarcinoma cells. Within several hours of treatment with DFO or DPD, the cells showed morphological changes consistent with the notion that the cells were a few steps from actually dying. Further analysis of such iron-deficient but viable cells revealed single strand breaks in DNA. Eventually, such cells would be expected to commit apoptosis. On the other hand, we asked the question if replenishing these iron-deficient cells with iron could rejuvenate them, thus aborting the initiation of apoptosis. In preliminary experiments, replenishing the iron-deficient cells with iron at the appropriate time diminished the DNA single strand
breaks and averted the cells from committing apoptosis. These findings suggest that iron is required to facilitate repair of the DNA damage caused by iron deficiency. The mechanism needs to be investigated. In studying gene expression, GADD45 and GADD153 were upregulated in the iron-deficient cells. The potential role of GADD45 and GADD153, as well as p53, in the repair of the DNA damage caused by DFO and DPD is under investigation.
Impacts
The present and anticipated findings are expected to help ascertain whether severely iron-deficient cells are automatically committed to death or alternatively can be revitalized by iron replenishment so as to result in activation of certain genes to promote DNA structural integrity needed for cell viability.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
At the cellular level, proper levels of the micronutrient, iron, are needed by cells to help ensure their viability and proliferative capacity. Thus, depriving cells of iron stresses them and can lead to cell death. To determine if the expression of certain stress-response genes is affected by celluar iron deficiency with the aim of establishing a potential association between stress-response gene expression and occurrence of cell death, Jurkat T-lymphocytes were made iron-deficient by exposing them to the iron chelator, deferoxamine (DFO). Such treatment produced evidence of cell death by the process of apoptosis, that is, cell shrinkage, membrane blebbing, chromatin condensation and fragmentation, and also formation of apoptotic bodies. Additionally, proteolytic cleavage of poly(ADP-ribose)polymerase was detected, suggesting involvement of caspases in initiating apoptosis. Indeed, a selective caspase-3 inhibitor prevented the effects of DFO. During the early
induction period of apoptosis, GRP78 and HSP70 mRNA expression were not affected. In contrast, there was mainly increased mRNA expression of Growth Arrest and DNA Damage-inducible gene 153 (GADD153), which seemed to be at the level of transcription rather than mRNA stability. Furthermore, fortifying cells with antioxidants did not prevent the increased GADD153 mRNA expression, and no evidence of single-strand breaks in DNA was found, suggesting that neither reactive oxygen species nor DNA damage were involved in triggering GADD153 gene activation. DFO also caused GADD153 protein to be expressed. Because GADD153 is recognized as a pro-apoptotic gene, these findings generate the notion that GADD153 might help mediate apoptosis in iron-deficient cells.
Impacts
These new basic research findings permit a greater mechanistic understanding of why iron deficiency adversely affects lymphocytes, which are cells known to normally function in the immune system in defending against infectious diseases. Additionally, the findings suggest that GADD153 could serve as a novel molecular marker for the diagnosis of severe iron deficiency.
Publications
- HOPKINS RG, EDWARDS VL, LOO G. 2003. Cellular iron deficiency upregulates GADD153 stress-response gene expression. FASEB J 17, A1159 (abstract #712.12).
- PAN YJ, HOPKINS RG, LOO G. 2004. Increased GADD153 Gene Expression During Iron Chelation-Induced Apoptosis in Jurkat T-Lymphocytes. Biochim Biophys Acta 1691, 41-50.
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