The expression of eIF3a, p27^kip1, NDRG1 and TfR1 mRNA and protein is regulated in response to cellular iron depletion using iron chelators and also hypoxia.

<p>(A) Following incubation of MCF7 cells with DFO (250 µM), 311 (25 µM) or hypoxia (1% O₂) for 24 h/37°C, total mRNA was extracted and RT-PCR was conducted. (B) MCF7 cells were incubated for 24 h/37°C with 311 or the pre-formed 311-iron complex (311-Fe; 2∶1 ligand-metal complex; 25 µM 311∶ 12.5 µM Fe) and total mRNA was extracted and RT-PCR conducted. (C) MCF7 cells incubated for 24 h/37°C with iron as ferric ammonium citrate (FAC; 100 µg/mL) showed an opposite expression pattern when compared to cells incubated with DFO (250 µM) for 311 (25 µM). (D) Wild-type (<i>HIF-1α</i>^+/+) and <i>HIF-1α</i>–knockout (KO; <i>HIF-1α</i>^-/-) murine embryo fibroblasts (MEFs) were incubated with either control medium, DFO (250 µM), 311 (25 µM), or hypoxia (1% O₂) and their mRNA extracted for analysis using RT-PCR. (E) Following incubation of MCF7 cells for 24 h/37°C with the same conditions as in (A) above, total protein was extracted and western blot analysis was conducted. (F) MCF7 cells were incubated under the same conditions as in (C) and western analysis performed as in (E). The gel photographs and blots are representative of 3 independent experiments and the densitometric analysis is expressed as mean ± SD. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 relative to control cells.</p

Schematic overview of the down-stream genes regulated by eIF3a and the resultant functional effects. (A)

<p>A working model that describes eIF3a’s role in regulating NDRG1 and p27^kip1 expression. <b>(i)</b> When eIF3a is over-expressed, iron depletion up-regulates <i>NDRG1</i> transcription and eIF3a stimulates translation of nascent <i>NDRG1</i> transcripts due to its pro-translation role as an initiation factor subunit. This facilitates <i>de novo</i> NDRG1 synthesis, while translation of non-essential transcripts is suppressed during stress <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Anderson1" target="_blank">[15]</a>. Conversely, our observation that p27^kip1 protein, but not mRNA, is down-regulated by eIF3a over-expression suggests p27^kip1 transcripts may be instead recruited to stress granules, the production of which is dynamically regulated by eIF3a, thereby suppressing p27^kip1 synthesis. (<b>ii)</b> In contrast, when eIF3a is ablated, eIF3a-containing stress granules do not form and <i>p27^kip1</i> transcripts are, by default, recruited by the translational apparatus, thereby increasing p27^kip1 protein expression during iron depletion. In the absence of eIF3a, <i>NDRG1</i> transcripts continue to be directed to the translational apparatus, but are translated at a slower rate due to the loss of eIF3a. This model is consistent with the data presented in this study and with the known ability of eIF3a to negatively regulate p27^kip1 expression by a translational mechanism <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Dong2" target="_blank">[7]</a>. <b>(B)</b> Schematic summarizing some of the functions of eIF3a, including those demonstrated in this study. First, when eIF3a is over-expressed such as in early stages of cancer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Pincheira1" target="_blank">[80]</a> there is: <b>(i)</b> up-regulation of the metastasis suppressor, NDRG1, leading to both increased differentiation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Kovacevic1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-vanBelzen1" target="_blank">[71]</a> and decreased metastasis/invasion (shown herein); <b>(ii)</b> down-regulation of the cyclin-dependent kinase inhibitor, p27^kip1, resulting in activation of the cell cycle and proliferation (shown herein and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Dong2" target="_blank">[7]</a>); and <b>(iii)</b> increased expression of the ribonucleotide reductase M2 subunit (RRM2) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Dong1" target="_blank">[6]</a> allowing DNA synthesis and growth. Second, when eIF3a expression is abrogated, such as occurs in hypoxic tissues that are typical of advanced tumors <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Chen2" target="_blank">[77]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Dellas1" target="_blank">[78]</a>, there is: <b>(i)</b> down-regulation of NDRG1, leading to a loss of differentiation and increased metastasis/invasion (shown herein); <b>(ii)</b> up-regulation of p27^kip1, resulting in the inactivation of the cell cycle and inhibited proliferation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Dong2" target="_blank">[7]</a>; and <b>(iii)</b> decreased expression of RRM2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057273#pone.0057273-Dong1" target="_blank">[6]</a>, preventing DNA synthesis and growth.</p

Role of eIF3a in the regulation of <i>p27^kip1</i> and <i>NDRG1</i> mRNA and protein expression

<p>. Tetracycline (TET)-off MCF7 cells stably transfected with the eIF3a-sense plasmid (eIF3a-S; <b>A, C</b>) or the eIF3a-antisense (eIF3a-AS; <b>B, D</b>) plasmid were incubated with or without TET in either control medium, or this medium containing DFO (250 µM) or 311 (25 µM) for 24 h/37°C and their mRNA or protein extracted for analysis using RT-PCR or western blot. The gel photographs are representative of 3 independent experiments and the densitometric analysis is expressed as mean ± SD. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 relative to the respective control.</p

Primers for amplification of genes used in this study.

<p>Primers for amplification of genes used in this study.</p

Correction: N-myc Downstream Regulated 1 (NDRG1) Is Regulated by Eukaryotic Initiation Factor 3a (eIF3a) during Cellular Stress Caused by Iron Depletion

<p>Correction: N-myc Downstream Regulated 1 (NDRG1) Is Regulated by Eukaryotic Initiation Factor 3a (eIF3a) during Cellular Stress Caused by Iron Depletion</p

Altered cell migration, invasion and proliferation implementing TET-regulated eIF3a-S and eIF3a-AS transfected MCF7 cells.

<p>(A) The images show the result of a migration assay and were taken after a 24 h/37°C incubation of eIF3a-S and eIF3a-AS transfected MCF7 cells in the presence or absence of TET. Cell density indicates the number of cells that were able to pass through a polycarbonate membrane with a pore size of 8 µm. Scale bar: 200 µm. (B) The graph quantifies the result of the migration assays performed in (A) and is expressed as the fold change of the number of cells relative to the control. (C) The graph illustrates the result of invasion assays as the fold change in the number of cells that were able to pass through a basement membrane relative to the control. The western blots in (D) and (E) represent the expression of eIF3a, p27^kip1 and NDRG1 under the conditions shown using eIF3a-S and eIF3a-AS cells in the presence and absence of TET. (F, G) The graphs show the proliferation of eIF3a-S and eIF3a-AS transfected MCF7 cells in the presence and absence of TET over a 24, 48, 72 and 96 h period. The photographs in (A) and western blots in (D, E) are representative of 3 experiments. Migration (B), invasion (C) and densitometry (D, E) are expressed as mean ± SD (3 experiments), while proliferation results (F, G) are expressed as mean ± SD (8 experiments). **<i>p</i><0.01, ***<i>p</i><0.001.</p

Siderocalin/Lcn2/NGAL/24p3 Does Not Drive Apoptosis Through Gentisic Acid Mediated Iron Withdrawal in Hematopoietic Cell Lines

<div><p>Siderocalin (also lipocalin 2, NGAL or 24p3) binds iron as complexes with specific siderophores, which are low molecular weight, ferric ion-specific chelators. In innate immunity, siderocalin slows the growth of infecting bacteria by sequestering bacterial ferric siderophores. Siderocalin also binds simple catechols, which can serve as siderophores in the damaged urinary tract. Siderocalin has also been proposed to alter cellular iron trafficking, for instance, driving apoptosis through iron efflux via BOCT. An endogenous siderophore composed of gentisic acid (2,5-dihydroxybenzoic acid) substituents was proposed to mediate cellular efflux. However, binding studies reported herein contradict the proposal that gentisic acid forms high-affinity ternary complexes with siderocalin and iron, or that gentisic acid can serve as an endogenous siderophore at neutral pH. We also demonstrate that siderocalin does not induce cellular iron efflux or stimulate apoptosis, questioning the role siderocalin plays in modulating iron metabolism.</p> </div

Stably-induced expression of Scn does not drive apoptosis in FL5.12 cells.

<p>(A) FL5.12 cells were transduced with the pCVL-SFFV-muScn-IRES-GFP lentivirus and GFP mean fluorescence intensity was determined one-week post-transduction by cytometry, confirming transgene functionality. (B) A Western blot of supernatants, concentrated from 32 µL, from FL5.12 cells shows that the transduced cells constitutively express Scn, while parental cells in the presence or absence of IL-3 do not secrete detectable amounts of Scn after 72 h in culture. (C) Transduced FL5.12 were incubated with a variety of siderophores in order to assess the role of exogenous siderophores on cell viability (NT: no treatment). The hexadentate chelators DFO and Ent at 100 µM produce robust apoptosis, while the bidentate chelators at 300 µM do not affect viability.</p

Scn has no effect on iron release or iron uptake from HeLa cells.

<p>Control HeLa/X7 (transfected with empty vector) or HeLa/24p3R-L cells were labeled with either (A) 2.5 µM ⁵⁹FeCl₃ or (B) 0.75 µM ⁵⁹FeTf and re-incubated with 2 µM murine Scn or control medium for 5 h (dotted columns) or 24 h (checked columns); 100 µM DFO was used as a positive control. Expression of <i>BOCT</i> in transfected HeLa/24p3R-L cells was confirmed by RT-PCR (C). In (C), a typical result from three experiments is shown. In (D), control HeLa/X7 (white columns) and HeLa/24p3R-L cells (black columns) were incubated for 4 h in the presence of 2 µM ⁵⁹FeCl₃, 2 µM ⁵⁹FeEnt, 2 µM murine Scn with bound ⁵⁹FeEnt (⁵⁹FeEnt+Scn) or in the presence of 2 µM ⁵⁹FeEnt plus 2 µM human albumin (⁵⁹FeEnt+Alb). Internalized ⁵⁹Fe was determined by γ-counting. Albumin was added in (D) as an additional control for non-specific binding. Error was calculated as the standard deviation among three experiments.</p

Added Scn does not affect the expression of iron responsive genes.

<p>Expression of <i>H-ferritin</i> (<i>FTH-1</i>) and <i>NDRG1</i> in HeLa/X7 and HeLa/24p3R-L cells was assayed by RT-PCR (A) and Western blot (B). Cells were untreated or treated with 2 µM murine Scn or DFO (100 µM or 250 µM) for 24 h. Densitometry results (right) were calculated relative to β-actin; error was calculated from the standard deviation among three experiments; a typical result from three experiments is shown in (A) and (B).</p