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Regulation, mechanisms and proposed function of ferritin translocation to cell nuclei

¹ Department of Neuroscience and Anatomy, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, PA 17033, USA
² Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, PA 17033, USA
³ Department of Pathology, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, PA 17033, USA

View larger version (160K): [in a new window]   Fig. 1. H-ferritin immunostaining in a human astrocytoma tumors. A total of six different tumors were analyzed. The sections in A-D represent contiguous sections from the same tumor. Note the blood vessel containing red blood cells in the upper left-hand corner for orientation. (A) H-ferritin was detected with the HS-59 monoclonal mouse anti-human rH-ferritin antibody (red immunoreaction product). The arrows indicate examples of positive cell nuclei. (B) A contiguous section was reacted for H-ferritin (red) and a blue counterstain was applied to show the presence of cell nuclei. An example of a ferritin positive nucleus is indicated by the arrow. A non-ferritin positive nucleus is indicated by the arrowhead and appears blue. (C) This section was stained for Mouse IgG as a nonspecific control for the secondary antibody. No staining is visible, although nuclei can be seen in the field (arrow). (D) This section was counterstained to reveal the cell nuclei (blue, arrow) after incubation with mouse IgG. (E) This section represents GFAP staining in the human astrocytoma. Intermediate (GFAP-positive) filaments are seen coursing throughout the cytoplasm (red). The section was counterstained to visualize the nuclei that appear blue (arrow). The staining with GFAP represents a control for nuclear ferritin staining with a different human monoclonal antibody that would stain cellular features of the astrocytoma. (F) An immunoreaction for neurofilament was performed on the astrocytoma as an irrelevant control monoclonal antibody. There is no red reaction product for neurofilaments. The section was counterstained blue to reveal nuclei (arrow). Bar, 10.8 µm.

View larger version (63K): [in a new window]   Fig. 2. H-ferritin immunostaining in SW1088 human astrocytoma cells is not regulated by DNA synthesis but does change in response to iron chelation. H-ferritin was detected with the HO2 monoclonal mouse anti-human rH-ferritin antibody (A,C,E,G,I). The sections were colocalized with DAPI in order to visualize the nucleus (B,F,H,J) or BrdU for DNA synthesis (D). (A-D) Cells that have been triple-stained for H-ferritin (A,C), DAPI (B) and BrdU incorporation (D). (A,B) H-ferritin is detected in the nucleus of astrocytoma cells grown in standard media. The arrow indicates an example of a single cell nucleus. The cell nuclei from A are shown in B using DAPI. The arrow indicates the same cell in A and B. (C,D) A is reproduced as C in order to compare H-ferritin staining with that of BrdU (D). H-ferritin is found in the nuclei of both BrdU and non-BrdU positive cells. (D) The arrows in C,D indicate the nucleus of the same cell that is both H-ferritin and BrdU positive. The asterisk near the cell in C indicates a cell that has H-ferritin in the nucleus but is not BrdU positive. (E,F) H-ferritin is detected in the nucleus and cytoplasm of astrocytoma cells (E) but is not localized to the nucleus in dividing cells (F). The DAPI stain in F reveals two cells (arrows) that are dividing and the area containing the chromosomes is not visible in those cells in E. (G,H) H-ferritin is only cytoplasmic in most (>85%) DFO-treated cells. The nuclei of the cells in G are demonstrated using DAPI (H) and the arrows depict a cell in which nuclear ferritin is not detected. (I,J) H-ferritin is detected in the nucleus after being returned to standard media after DFO treatment (I). The arrows indicate the nucleus of a cell that is stained for H-ferritin (I) and DAPI (J). Bar, 10 µm.

View larger version (24K): [in a new window]   Fig. 3. Ferric ammonium citrate, cytokines and hydrogen peroxide affect ferritin nuclear concentrations in SW 1088 human astrocytoma cells. SW 1088 astrocytoma cells were treated with deferoxamine and then placed in standard media (control) or standard media, including varying concentrations of ferric ammonium citrate (FAC), tumor necrosis factor (TNF), interleukin 1ß (IL-1ß) or hydrogen peroxide (H2O2). Nuclear and cytosolic extracts were isolated and ferritin levels were determined using the HO2 monoclonal mouse anti-human rH-ferritin antibody. Ferritin levels are expressed as a percentage of control values. The concentration of the experimental factors is shown at the bottom of each graph. (A) Nuclear and cytosolic ferritin protein levels both increase in response to FAC exposure, but the increase is not concentration dependent. (B) Nuclear ferritin levels are increased in response to TNF, but cytosolic levels are decreased when compared with control at each concentration. (C) Nuclear ferritin levels increase at the lowest dose of IL 1ß, but decrease at the higher doses. The cytosolic levels of ferritin are not different from control. (D) Nuclear ferritin levels increase in response to H2O2 only at 100 µM H2O2, while cytosolic levels remain constant. The asterisks indicate a statistically significant difference from control values. **P<0.001, *P<0.05.

View larger version (75K): [in a new window]   Fig. 4. Ferritin nuclear translocation in SW1088 human astrocytoma cells responds to iron chelation treatment and is ferritin-subunit specific. These are a series of confocal images that are shown for each condition as a representative of the entire cell population. Each experiment was repeated three times and random cells chosen. The arrow in each panel points to the cell nucleus. Fluorophore-labeled proteins (5 µM) were added to permeabilized cells for 60 minutes prior to visualization. (A) TRITC-dextran (70,000 kDa) enters the cells but does not translocate to the nucleus, indicating that the nuclear membrane remained intact following digitonin treatment. (B) FITC-rH ferritin enters the cells but is not imported into the nucleus of cells plated in standard culture conditions. (C) If cells are pretreated with deferoxamine and then returned to standard medium containing FITC rH-ferritin, the labeled ferritin translocates to the nucleus. (D) The combination of DFO and ferric ammonium citrate (FAC) results in the formation of ferrioxamine B that limits the chelating ability of DFO. Under this condition, FITC rH-ferritin does not translocate to the nucleus but remains cytoplasmic. (E) FITC-labeled BSA was added to the standard media following DFO pretreatment (same condition as in B) as a control for the selectivity of the uptake mechanism and to show the nuclear membrane is still intact following permeabilization. FITC-BSA enters the cell but does not enter the nucleus. (F) The 222 mutant, an H-ferritin that lacks a ferroxidase center enters the cell nuclei (two cells are shown in this micrograph, one is indicated by the arrow) of DFO-treated cells indicating the iron status of ferritin is not a factor in the nuclear uptake. (G) To determine if there is a preferred subunit type of ferritin that is translocated to the nucleus, FITC rL-ferritin was added to the medium after pretreatment with DFO. This subunit of ferritin does not translocate to the nucleus under these conditions, indicating there is a preference for H-rich ferritin and also demonstrating the integrity of the nuclear membrane after digitonin permeabilization. (A-G) Exogenously applied ferritin. In H, an example of a cell transfected with Myc-tagged ferritin is shown. The epitope tagged ferritin is found in the nucleus of SW1088 cells. These data illustrate that endogenously expressed tagged H-ferritin can translocate to the nucleus. Bars, 7 µm.

View larger version (124K): [in a new window]   Fig. 5. Mechanism of nuclear translocation for FITC labeled rH-ferritin. SW1088 cells were pretreated with deferoxamine for 72 hours followed by digitonin treatment. The cells were then exposed to treatments that affect nuclear translocation of proteins in the presence of FITC-conjugated H-ferritin. The cells in the micrographs are representative of the general population. The arrows indicate the nuclei of individual cells. Each experiment was performed in triplicate. (A) As previously established, FITC-labeled rH-ferritin translocates to the nucleus. (B) Treatment of the cells with wheat germ agglutinin (WGA), to block the nuclear pore complex, blocks nuclear translocation of FITC-labeled recombinant H-ferritin, which collects at the perinuclear membrane. (C) Incubation of cells with FITC-labeled recombinant H-ferritin at 4°C decreased the appearance of the ferritin in cell nuclei. FITC-labeled rH-ferritin collects at the perinuclear membrane. (D) Depletion of ATP in the cells with apyrase results in a decreased appearance of FITC-labeled recombinant H-ferritin in cell nuclei. FITC-labeled rH-ferritin collects at the perinuclear membrane. (E) Regeneration of ATP following apyrase treatment results in FITC-labeled rH-ferritin nuclear translocation. (F) Exposure of the cells to NEM does not inhibit ferritin nuclear translocation. Two cells are shown in this micrograph with immunolabeled nuclei (arrows). These studies demonstrate that ferritin uptake into the nucleus is via the nuclear pore complex; it requires energy but does not require an NLS bearing cytosolic protein. Bars, 7 µm (A-E); 4 µm (F).

View larger version (15K): [in a new window]   Fig. 6. 125I-rH-ferritin crosslinks to DNA in whole cells. 125I-rH ferritin was incubated with SW1088 astrocytoma cells grown in standard, untreated medium as a control (CN) or in the presence of 100 µM deferoxamine (DFO). (A) One group of cells from both CN and DFO conditions were placed in 1% formaldehyde to crosslink DNA-protein complexes (lanes 3 and 4). The other group was not subjected to crosslinking (lanes 1 and 2). (B) The membranes containing the crosslinked complexes were incubated with a rH-ferritin-specific antibody to demonstrate that the crosslinked protein was ferritin. Lane 1, CN; lane 2, DFO; lane 3, CN crosslinked; lane 4, DFO crosslinked. (C) The numerical values obtained from the densitometric analysis of each lane in A are shown. The data shown in the figure are representative of three different experiments. 125I-rH ferritin-DNA complexes were identified in CN and DFO crosslinked conditions with the greatest level found in the DFO group.

View larger version (25K): [in a new window]   Fig. 7. Ferritin protects DNA from iron-induced oxidative damage, in vitro. (A) Electrophoretic supercoil relaxation assay. The positions to which supercoiled (I), relaxed circular (II) and linear (III) monomeric DNAs migrated are indicated in the on the left. Reaction mixtures contained supercoiled pUC19 DNA in the following conditions: lane 1, DNA alone; lanes 2-7, DNA plus 50 µM FeCl3 and 10 mM H2O2; lanes 3-7, recombinant H-ferritin at increasing concentrations (95 nM, 240 nM, 475 nM, 5 µM and 14.9 µM) in addition to the 50 µM FeCl3 and 10 mM H2O2. (B) Graph of the mole fraction of form I DNA remaining at the end of the reaction as a function of the concentrations of ferritin and non-ferritin proteins. Reactions were performed as described in A, with the following ferritin samples: rH-ferritin (black square); apoferritin ferritin (black circle); recombinant 222 mutant ferritin (white square); and transferrin (black triangle). The CAP protein (white circle) was used in place of ferritin as a control for DNA-binding proteins. As controls for nonspecific protein effects, ferritin was replaced by ovalbumin (crossed white square), bovine serum albumin (white triangle) or chymotrypsin (black diamond) in duplicate reactions. All experiments were repeated three times. These data demonstrate that ferritin will protect DNA from iron-induced oxidative damage.

View larger version (71K): [in a new window]   Fig. 8. Cell culture model to study ferritin and DNA damage. To demonstrate that SW1088 astrocytoma cells in culture are susceptible to DNA damage when nuclear ferritin is not present, we used the following model. Intracellular ferritin was depleted by deferoxamine and then the cells were reincubated with standard media, which results in a reappearance of ferritin in the nucleus. However, one group of cells was exposed to wheat germ agglutinin (WGA), to block the translocation of ferritin to the nucleus, and the other group was not exposed to WGA. Both sets of SW1088 cells were treated with digitonin, which is necessary to allow WGA to enter the cells. DNA strand breaks are detected using the TUNEL assay (green; Boehringer Mannheim). DAPI staining (blue) was used to locate cell nuclei. The same microscopic field is represented in both right and left panels. (A) In this experiment, 100 µM H2O2 was added to the reincubation media after DFO pretreatment. If WGA was included (+WGA) in the reincubation media (to block ferritin translocation to the nucleus), most of the cells became TUNEL positive (green). If WGA was excluded from the reincubation media (-WGA) (permitting ferritin translocation to the nucleus), no TUNEL-positive cells were present. (B) In this experiment, ferric ammonium citrate (100 µM) was added to the reincubation media. There were no TUNEL-positive cells regardless of whether or not WGA was absent (-WGA) or present (+WGA) in the reincubation media. The micrographs have been chosen as representative. Experiments were repeated three times each.

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