H-ferritin subunit overexpression in erythroid cells reduces the oxidative stress response and induces multidrug resistance properties.

The labile iron pool (LIP) of animal cells has been implicated in cell iron regulation and as a key component of the oxidative-stress response. A major mechanism commonly implied in the downregulation of LIP has been the induced expression of ferritin (FT), particularly the heavy subunits (H-FT) that display ferroxidase activity. The effects of H-FT on LIP and other physiological parameters were studied in murine erythroleukemia (MEL) cells stably transfected with H-FT subunits. Clones expressing different levels of H-FT displayed similar concentrations of total cell iron (0.3 +/- 0.1 mmol/L) and of reduced/total glutathione. However, with increasing H-FT levels the cells expressed lower levels of LIP and reactive oxygen species (ROS) and ensuing cell death after iron loads and oxidative challenges. These results provide direct experimental support for the alleged roles of H-FT as a regulator of labile cell iron and as a possible attenuator of the oxidative cell response. H-FT overexpression was of no apparent consequence to the cellular proliferative capacity. However, concomitant with the acquisition of iron and redox regulatory capacities, the H-FT-transfectant cells commensurately acquired multidrug resistance (MDR) properties. These properties were identified as increased expression of MDR1 mRNA (by reverse transcription polymerase chain reaction [RT-PCR]), P-glycoprotein (Western immunoblotting), drug transport activity (verapamil-sensitive drug efflux), and drug cytotoxicity associated with increased MDR1 or PgP. Although enhanced MDR expression per se evoked no significant changes in either LIP levels or ROS production, it might be essential for the survival of H-FT transfectants, possibly by expediting the export of cell-generated metabolites.

Role of ferritin in the control of the labile iron pool in murine erythroleukemia cells.

In vitro studies have shown that ferritin iron incorporation is mediated by a ferroxidase activity associated with ferritin H subunits (H-Ft) and a nucleation center associated with ferritin L subunits (L-Ft). To assess the role played by the ferritin subunits in regulating intracellular iron distribution, we transfected mouse erythroleukemia cells with the H-Ft subunit gene mutated in the iron-responsive element. Stable transfectants displayed high H-Ft levels and reduced endogenous L-Ft levels, resulting in a marked change in the H:L subunit ratio from 1:1 in control cells to as high as 20:1 in some transfected clones. The effects of H-Ft overexpression on the labile iron pool were determined in intact cells by a novel method based on the fluorescent metallosensor calcein. H-Ft overexpression resulted in a significant reduction in the iron pool, from 1.3 microM in control cells to 0.56 microM in H-Ft transfectants, and in higher buffering capacity following iron loads. A fraction of the H-Ft-associated iron was labile, available to cell-permeant, but not cell-impermeant, chelators. The results of this study provide the first in vivo direct demonstration of the capacity of H-Ft to sequester cell iron and to regulate the levels of the labile iron pool.

Fluorescence analysis of the labile iron pool of mammalian cells.

The labile iron pool (LIP) of cells constitutes a cytosolic fraction of iron which is accessible to permeant chelators and contains the cells' metabolically and catalytically reactive iron. LIP is maintained by a balanced movement of iron from extra- and intracellular sources. We describe here an approach for tracing LIP levels in living cells based on the fluorescent probe calcein (CA). This probe binds Fe(II) rapidly, stoichiometrically, and reversibly while forming fluorescence-quenched CA-Fe complexes. Cells are loaded with CA via its acetomethoxy precursor CA-AM, attaining 1-10 microM intracellular concentrations and retaining full viability. LIP is defined here operationally as the sum of "free" and CA-bound iron of the cell. The method for assessing LIP is based on the measurement of: (a) the total intracellular concentration of CA in CA-loaded cells ([CA]1), which is estimated from fluorimetric measurements of CA in a given suspension of cells, the number of cells, and the cell volume; (b) the intracellular [CA-Fe], the concentration of [CA] bound to metals (> 95% iron), which is assessed from the relative rise in fluorescence (delta F) elicited by addition of highly permeant and high-affinity binding chelators such as salicyladehyde-isonicotinoyl-hydrazone (SIH) and the value of [CA]1; and (c) the "free" cell iron concentration [Fe(II)], which is computed from the experimentally determined values of CA-Fe(II)'s dissociation constant (Kd) in various cell lines grown in suspension (Kd = 0.22 +/- 0.01 microM). The value of cellular LIP is defined as the sum of [CA-Fe] and [Fe]. It is derived from the experimental determination of [CA]1 and [CA-Fe] and from calculation of [Fe] by application of the mass law equation using the Kd value of [CA-Fe]. The estimated values of LIP for resting erythroid and myeloid cells are in the range of 0.2-1.5 microM. The values varied commensurately with cell iron loads and iron chelator treatment. The method provides a simple, noninvasive tool for on-line monitoring of cytosolic iron under normal and abnormal conditions of cell iron supply and for assessing the dynamics of intracellular iron in living cells.

Dynamics of the cytosolic chelatable iron pool of K562 cells.

The labile iron pool of cells (LIP) constitutes the primary source of metabolic and catalytically reactive iron in the cytosol. We studied LIP homeostasis in K562 cells using the fluorescent metal-sensitive probe calcein. Following brief exposure to iron(II) salts or to oxidative or reductive stress, LIP rose by up to 120% relative to the normal level of 350nM. However, the rate of recovery to normal LIP level differed markedly with each treatment (respective t1/2s of 27, 65-88 and < or = 17 min). We show that the capacity of K562 cells to adjust LIP levels is highly dependent on the origin of the LIP increase and on the pre-existing cellular iron status.

Iron acquired from transferrin by K562 cells is delivered into a cytoplasmic pool of chelatable iron(II).

The release of iron from transferrin (Tf) in the acidic milieu of endosomes and its translocation into the cytosol are integral steps in the process of iron acquisition via receptor-mediated endocytosis (RME). The translocated metal is thought to enter a low molecular weight cytoplasmic pool, presumed to contain the form of iron which is apparently sensed by iron responsive proteins and is the direct target of iron chelators. The process of iron delivery into the cytoplasmic chelatable pool of K562 cells was studied in situ by continuous monitoring of the fluorescence of cells loaded with the metal-sensitive probe calcein. Upon exposure to Tf at 37 degrees C, intracellular fluorescence decayed, corresponding to an initial iron uptake of 40 nM/min. The Tf-mediated iron uptake was profoundly inhibited by weak bases, the protonophore monensin, energy depletion, or low temperatures (< 25 degrees C), all properties characteristic of RME. Cell iron levels were affected by the slowly permeating chelator desferrioxamine only after prolonged incubations. Conversely, rapidly penetrating, lipophilic iron-(II) chelators such as 2,2'-bipyridyl, evoked swift increases in cell calcein fluorescence, equivalent to sequestration of 0.2-0.5 microM cytosolic iron, depending on the degree of pre-exposure to Tf. Addition of iron(III) chelators to permeabilized 2,2'-bipyridyl-treated cells, failed to reveal significant levels of chelatable iron(III). The finding that the bulk of the in situ cell chelatable pool is comprised of iron(II) was corroborated by pulsing K562 cells with Tf-55Fe, followed by addition of iron(II) and/or iron(III) chelators and extraction of chelator-55Fe complexes into organic solvent. Virtually all of the accumulated 55Fe in the chelatable pool could be complexed by iron(II) chelators. The cytoplasmic concentration of iron(II) fluctuated between 0.3 and 0.5 microM, and its mean transit time through the chelatable pool was 1-2 h. We conclude that after iron is translocated from the endosomes, it is maintained in the cytosol as a transit pool of chelatable iron(II). The ostensible absence of chelatable iron(III) implicates the intracellular operation of vigorous reductive mechanisms.

Transport of iron and other transition metals into cells as revealed by a fluorescent probe.

Transport of nontransferrin-bound iron into cells is thought to be mediated by a facilitated mechanism involving either the trivalent form Fe(III) or the divalent form Fe(II) following reduction of Fe(III) at the cell surface. We have made use of the probe calcein, whose fluorescence is rapidly and stoichiometrically quenched by divalent metals such as Fe(II), Cu(II), Co(II), and Ni(II) and is minimally affected by variations in ionic strength, Ca(II) and Mg(II). Addition of Fe(II) salts to calcein-loaded human erythroleukemia K-562 cells elicited a slow quenching response that was markedly accelerated by the ionophore A-23187 and was reversed by membrane-permeant but not by impermeant chelators. These observations were confirmed by fluorescence imaging of cells. Other divalent metals such as Co(II), Ni(II), and Mn(II) permeated into cells at roughly similar rates, and their uptake, like that of Fe(II), was blocked by trifluoperazine, bepridil, and impermeant sulfhydryl-reactive organomercurials, indicating the operation of a common transport mechanism. This method could provide a versatile tool for studying the transport of iron and other transition metals into cells.