Polymorphisms and mutations of human TMPRSS6 in iron deficiency anemia.

Male subjects with iron deficiency from the general population were examined for polymorphisms or sporadic mutations in TMPRSS6 to identify genetic risk factors for iron deficiency anemia. Three uncommon non-synonymous polymorphisms were identified, G228D, R446W, and V795I (allele frequencies 0.0074, 0.023 and 0.0074 respectively), of which the R446W polymorphism appeared to be overrepresented in the anemic population. In addition, three children with iron refractory iron deficiency anemia, and one sibling with iron responsive iron deficiency anemia were also examined for polymorphisms or sporadic mutations in TMPRSS6. Two children (family 1) were compound heterozygotes for a L674F mutation and a previously described splicing defect predicted to cause skipping of exon 13 (IVS13+1 G>A). One child from the second family was homozygous for a deletion (497T) causing a frameshift (L166X+36) and premature termination. The sibling and mother from the second family were compound heterozygotes for the L166X mutation and the uncommon R446W polymorphism. Although in vitro expression studies demonstrated that the R446W isoform was biologically similar to wildtype Tmprss6, clinical data indicate that the R446W produces a milder disease when carried in trans with severe mutation in Tmprss6. The four children carrying mutations in TMPRSS6 all exhibited inappropriately high urinary hepcidin levels for the degree of iron deficiency.

Iron deprivation induces apoptosis via mitochondrial changes related to Bax translocation.

In order to elucidate the mechanisms involved in apoptosis induction by iron deprivation, we compared cells sensitive (38C13) and resistant (EL4) to apoptosis induced by iron deprivation. Iron deprivation was achieved by incubation in a defined iron-free medium. We detected the activation of caspase-3 as well as the activation of caspase-9 in sensitive cells but not in resistant cells under iron deprivation. Iron deprivation led to the release of cytochrome c from mitochondria into the cytosol only in sensitive cells but it did not affect the cytosolic localization of Apaf-1 in both sensitive and resistant cells. The mitochondrial membrane potential (Deltapsi(m)) was dissipated within 24 h in sensitive cells due to iron deprivation. The antiapoptotic Bcl-2 protein was found to be associated with mitochondria in both sensitive and resistant cells and the association did not change under iron deprivation. On the other hand, under iron deprivation we detected translocation of the proapoptotic Bax protein from the cytosol to mitochondria in sensitive cells but not in resistant cells. Taken together, we suggest that iron deprivation induces apoptosis via mitochondrial changes concerning proapoptotic Bax translocation to mitochondria, collapse of the mitochondrial membrane potential, release of cytochrome c from mitochondria, and activation of caspase-9 and caspase-3.

Iron deprivation induces apoptosis independently of p53 in human and murine tumour cells.

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.

Apoptosis induction in lymphoma cells: thiol deprivation versus thiol excess.

We studied the effects of thiol availability on apoptosis induction in B-cell lymphoma 38C13, T-cell lymphoma EL4, and also other cells. Compounds with a free SH group are required for survival and growth of 38C13 cells but not of EL4 cells. Thiol deprivation (2-mercaptoethanol concentrations about 0.3 microM and lower) induced apoptosis in 38C13 cells. On the other hand, thiol excess (2-mercaptoethanol concentrations higher than 300 microM) induced apoptosis in 38C13 cells and EL4 cells as well as in other cells (e.g. Raji, HeLa). L-cystine and non-thiol antioxidant ascorbic acid were unable to support survival of 38C13 cells. Ascorbic acid induced cell death at concentrations higher than 600 microM. Thiol cross-linking compound diamide (100 microM and higher) abrogated the survival-supporting effect of 2-mercaptoethanol (50 microM). Apoptosis induction by thiol deprivation and by thiol excess was not directly related to a specific significant change in the p53 level or p53 activation. Apoptosis induction by thiol excess was associated with a certain decrease in the Bcl-2 level while the Bax level did not change. We conclude that both thiol deprivation and thiol excess can induce apoptosis in lymphoma cells. Apoptosis induction by thiol deprivation is specifically related to the presence of a free SH group. However, apoptosis induction by thiol excess does not seem to be specifically related to the presence of a free SH group. It probably results from the excess of a reductant. Apoptotic control protein p53 does not seem to play a significant role in apoptosis induction either by thiol deprivation or by thiol excess.