Modification of ferritin during iron loading.

Recombinant human ferritin loaded with iron via its own ferroxidase activity did not sediment through a sucrose-density gradient as a function of iron content. Analysis of the recombinant ferritin by native PAGE demonstrated an increase in altered migration pattern of the ferritins with increasing sedimentation, indicating an alteration of the overall charge of ferritin. Additionally, analysis of the ferritin by SDS-PAGE under nonreducing conditions demonstrated that the ferritin had formed large aggregates, which suggests disulfide bonds are involved in the aggregation. The hydroxyl radical was detected by electron spin resonance spectroscopy during iron loading into recombinant ferritin by its own ferroxidase activity. However, recombinant human ferritin loaded with iron in the presence of ceruloplasmin sedimented through a sucrose-density gradient similar to native ferritin. This ferritin was shown to sediment as a function of iron content. The addition of ceruloplasmin to the iron loading assay eliminated the detection of the DMPO-*OH adduct observed during loading using the ferroxidase activity of ferritin. The elimination of the DMPO-*OH adduct was determined to be due to the ability of ceruloplasmin to completely reduce oxygen to water during the oxidation of the ferrous iron. The implications of these data for the present models for iron uptake into ferritin are discussed.

The consequences of hydroxyl radical formation on the stoichiometry and kinetics of ferrous iron oxidation by human apoferritin.

Despite previous detection of hydroxyl radical formation during iron deposition into ferritin, no reports exist in the literature concerning how it might affect ferritin function. In the present study, hydroxyl radical formation during Fe(II) oxidation by apoferritin was found to be contingent on the "ferroxidase" activity (i.e., H subunit composition) exhibited by apoferritin. Hydroxyl radical formation was found to affect both the stoichiometry and kinetics of Fe(II) oxidation by apoferritin. The stoichiometry of Fe(II) oxidation by apoferritin in an unbuffered solution of 50 mM NaCl, pH 7.0, was approximately 3.1 Fe(II)/O(2) at all iron-to-protein ratios tested. The addition of HEPES as an alternate reactant for the hydroxyl radical resulted in a stoichiometry of about 2 Fe(II)/O(2) at all iron-to-protein ratios. HEPES functioned to protect apoferritin from oxidative modification, for its omission from reaction mixtures containing Fe(II) and apoferritin resulted in alterations to the ferritin consistent with oxidative damage. The kinetic parameters for the reaction of recombinant human H apoferritin with Fe(II) in HEPES buffer (100 mM) were: K(m) = 60 microM, k(cat) = 10 s(-1), and k(cat)/K(m) = 1.7 x 10(5) M(-1) x (-1). Collectively, these results contradict the "crystal growth model" for iron deposition into ferritin and, while our data would seem to imply that the ferroxidase activity of ferritin is adequate in facilitating Fe(II) oxidation at all stages of iron deposition into ferritin, it is important to note that these data were obtained in vitro using nonphysiologic conditions. The possibility that these findings may have physiological significance is discussed.

Intact human ceruloplasmin is required for the incorporation of iron into human ferritin.

We have previously reported several studies on the loading of iron into ferritin by ceruloplasmin using proteins from rats. Loading iron into human ferritin using human serum ceruloplasmin is complicated by the fact that human ceruloplasmin is very susceptible to proteolysis (T. P. Ryan, T. A. Grover, and S. D. Aust, 1992, Arch. Biochem Biophys. 293, 1-8). The present study investigated the effect of proteolysis on the ability of human ceruloplasmin to load iron into human ferritin. SDS-PAGE revealed one major band with an apparent molecular weight of 116 kDa for a proteolytically degraded form of ceruloplasmin versus a 132-kDa band for an intact form of the enzyme. Both forms of the enzyme possessed ferroxidase activity, although that of the proteolytically degraded enzyme was approximately twofold less than that of the intact enzyme (4.9 nmol (min)-1 vs 8.3 nmol (min)-1). Only the intact form of ceruloplasmin was able to catalyze iron loading into ferritin without altering the physical characteristics of the ferritin protein during the process. Abnormal migration in nondenaturing PAGE gels, as well as a decrease in the amount of detectable ferritin protein, was observed when ferritin was incubated with iron alone or with proteolytically degraded ceruloplasmin and iron. It was concluded that the structural integrity of ceruloplasmin is required for the enzyme to effectively catalyze iron loading into ferritin.

Production of recombinant human apoferritin heteromers.

We are interested in learning how iron is safely inserted and stored in ferritin. Recombinant DNA technology has considerable potential in determining the functional roles of the two ferritin subunits (H and L). In previous studies, we have observed that recombinant rat H ferritin was repressive to cell growth in both prokaryotic and eukaryotic expression systems (Guo et al., Biochem. Biophys. Res. Commun. 242, 39-45 (1998)). This results in the protein being expressed at very low levels. This problem was partially bypassed by the use of an inducible expression system, which utilizes T7 RNA polymerase dependent expression of the gene, induced by isopropyl beta-D-thiogalactopyranoside (IPTG). Simultaneously expressing the H and L ferritin genes in this system resulted in only a narrow range of ferritin heteromers, which predominantly consisted of the L subunit. Addition of rifampicin to cultures, 1 h following the induction of protein synthesis by IPTG, increased the production of the H subunit and thus increased the range of ferritin H:L subunit ratios. Simultaneous expression of the H and L ferritin genes in Escherichia coli grown in a deficient medium with minimal iron and with the addition of rifampicin resulted in the production of a range of recombinant human apoferritin heteromers that could be separated based on their subunit composition.

Spontaneous release of interleukin-6 by primary cultures of lymphoid and tumor cell populations purified from human ovarian carcinoma.

Interleukin-6 (IL-6) is a cytokine that has been implicated as a growth factor in human ovarian carcinoma, yet the in vivo source of IL-6 in patients remains undefined. We measured IL-6 by ELISA in cell-free ascites (CFA) of 19 patients with ovarian carcinoma. IL-6 was detectable in all samples (mean level 3.3 ng/ml). To identify the cellular source of IL-6, we measured this cytokine by ELISA in 24-48 h supernatants of cultured lymphocyte-, macrophage-, and tumor cell-enriched populations purified from three solid ovarian carcinomas by centrifugal elutriation. All cell populations spontaneously released IL-6; however, tumor cells and tumor-associated macrophage released levels of IL-6 that greatly exceeded those released by tumor-associated lymphocytes. Kinetic studies revealed that IL-6 was detectable at 6 h and that levels increased in all cultures examined over a 48 h time course. These data suggest that both tumor and infiltrating host cells may be the source of the high levels of IL-6 found in carcinomatous ascites. Furthermore, although all three cell types examined may contribute to IL-6 production in patients with ovarian carcinoma, tumor cells are perhaps the most clinically significant source.

Host-tumor interaction in ovarian cancer. Spontaneous release of tumor necrosis factor and interleukin-1 inhibitors by purified cell populations from human ovarian carcinoma in vitro.

The biological activity of tumor necrosis factor (TNF alpha/beta) and interleukin-1 beta (IL-1 beta) can be blocked by soluble, naturally occurring molecules--TNF alpha/beta binding proteins (BP-55 and BP-75), derived from the extracellular portion of the 55- and 75-kDa TNF alpha/beta membrane receptors, and IL-1 receptor antagonist (IL-1ra), respectively. We examined the levels of these cytokines and their inhibitors in cell-free ascites of 18 patients with advanced ovarian carcinoma by ELISA. Levels of both TNF BP and IL-1ra dramatically exceeded those of TNF and IL-1; thus, it is unlikely that these cytokines are active in ascites from patients with this disease. We then elutriated solid tumor samples from three additional patients, yielding pure populations of tumor cells, macrophages, and lymphocytes. Cells were cultured for up to 48 hr and the spontaneous production of TNF, IL-1, and their inhibitors was measured by ELISA. Tumor cells and macrophages both released inhibitors for TNF and IL-1. Tumor cells released IL-1ra and BP-55, while macrophages released IL-1ra and BP-75. Kinetic studies showed that both tumor cells and macrophages produced an initial burst of TNF alpha and IL-1 beta which was overtaken within 48 hr by a sustained production of TNF BP and IL-1ra. Lymphocytes released no TNF alpha or TNF beta, which alone suggests that tumor associated lymphocytes are locally quiescent in vivo. TNF and IL-1 inhibitors originate from tumor cells and tumor associated macrophages and probably block TNF and IL-1 activity locally and regionally in ovarian carcinoma patients. Whether this phenomenon contributes to the pathogenesis of this disease remains to be determined.