Pure Ferritin Research Articles

In vitro and in vivo comparison of DTPA- and DOTA-conjugated antiferritin monoclonal antibody for imaging and therapy of pancreatic cancer

Pancreatic cancer has a very poor prognosis with a less than 5% survival rate at 5 years. Neither external beam radiation nor chemotherapy, alone or in combination, have given encouraging results so far. A possible solution might come from the use of targeted therapy such as radioimmunotherapy. We present here the results obtained from the preclinical development of a new monoclonal antiferritin antibody (Ab), AMB8LK. Ferritin is overexpressed in pancreatic cancer and could thus be used as a target for the delivery of radioactivity at the tumour sites. The AMB8LK Ab was conjugated to three chelating agents: the 2-(4-isothiocyanatobenzyl)-diethylenetriamine pentaacetic acid (PSCN-Bz-DTPA), the (R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid (p5CN-Bz-CHX-A"-DTPA) and the 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (pSCN-Bz-DOTA). Radiolabelling of the three immunoconjugates with indium 111 and yttrium 90 as well as in vitro stability and immunoreactivity against pure ferritin and cells expressing ferritin were analysed. In vivo biodistribution studies were conducted on normal and on human pancreatic adenocarcinoma CAPAN-1 tumour bearing mice. These experiments demonstrated good radiolabelling (>95%), stability and immunoreactivity of the three compounds. In the biodistribution studies, differences between the three immunoconjugates were apparent in the rate of blood clearance and in tumour, liver and bone uptake. A very good pancreatic adenocarcinoma tumour targeting was observed especially with the Bz-DTPA-AMB8LK: 20% of the injected dose of the indium-labelled compound 3 days after injection; 15% of the injected dose 5 days after that of the yttrium-labelled Ab. Altogether, these results in animal models suggest that (90)Y-Bz-DTPA-AMB8LK is a good candidate for further therapeutic efficacy studies.

Ferritin and hemocyanin: 210Po molecular traps in marine fish, oyster and lobster

er , BP 800, 98012 Monaco Cedex ABSTRACT: The relative degree of binding of 210 Po with fish, mollusc and crustacean ferritins was investigated. Comparison of 210 Po concentrations in the purified ferritins from liver of the Atlantic mackerel Scomber scombrus and from the visceral mass of oysters Crassostrea gigas confirmed the high affinity of polonium for these iron-containing proteins. The ferritin fraction in lobster Homarus gammarus hepatopancreas contained an order of magnitude more 210 Po than pure ferritin from fish and oyster; however, the hepatopancreatic ferritin fraction was not pure and it also contained the res- piratory protein hemocyanin. A high performance size-exclusion chromatography analysis further revealed the important contribution of hemocyanin to 210 Po fixation in lobster. The combined 210 Po binding capacity of ferritin and hemocyanin in lobster hepatopancreas most probably accounts for the very high 210 Po concentrations found in the hepatopancreas of many higher crustaceans.

Rapid and efficient isolation of transferrin and ferritin from Manduca sexta

We report methods for the rapid purification of two iron-binding proteins from larval hemolymph of Manduca sexta. Ferritin was purified in two steps by density gradient ultracentrifugation. To accomplish this, we utilized the relatively high level of ferritin present in the hemolymph of this animal and augmented the density of the protein in vivo by injection of iron sulfate. Nitrocellulose blots analyzed by laser densitometry showed hemolymph from iron-injected insects contained about 0.4 mg of ferritin per ml (approximately 0.7% of total hemolymph protein); of this, 62% was found as pure ferritin in the pellet formed during ultracentrifugation. Following the density ultracentrifugation, we purified transferrin from the hemolymph subphase by immobilized metal ion affinity chromatography using a new gel, Novarose-SE1000/40 coupled to dipicolylamine (DPA) chelated with nickel. Higher capacity Ni 2+DPA-gel permitted good resolution of transferrin in the first chromatography; a lower capacity of the same gel allowed purification of transferrin in a second step. Overall transferrin recovery was 52%. Larval hemolymph contained 0.770 mg transferrin/ml, representing about 1.3% of the total protein.

Mouse hepatoma and liver ferritins: Comparative structural studies

Pure ferritin from male mouse liver produces a single band of monomers ( R F = 0.199) with electrophoresis in polyacrylamide gels at pH 9.0. The five sub-bands within this monomeric band appear to represent charge isomers having the same molecular size. Ferritin from BH3 transplantable mouse hepatoma shows two overlapping bands of monomers ( R F A = 0.208 and R F B = 0.240); further electrophoretic studies show that these bands represent two subpopulations of molecules differing both in charge and size. Sub-bands are not found in this hepatoma ferritin. The larger tumor ferritin reaches the same end migration position as all liver isoferritins on gradient gels, signifying a very similar or identical molecular size; however, the absence of sub-bands indicates that this hepatoma ferritin differs in charge from the homologous liver proteins. Liver and hepatoma ferritins both produces a single prominent subunit band corresponding to nominal molecular weights of 22 250 and 21 700, with polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. With electrophoresis on polyacrylamide gradient slabs containing sodium dodecyl sulfate and dithiothreitol, both liver and hepatoma ferritins now reveal two subunits bands situated at identical positions. The polypeptides of these two closely spaced bands have a nominal molecular weight difference of less than 1000. Neither the hepatoma nor the liver seems to produce the ferritins found in the other tissue. Nevertheless, all these ferritins are composed of the same two types of subunits, albeit in different relative amounts. Observed distinctions in the ferritins from these normal or neoplastic cells must reflect differences in assembly and processing, as well as in the regulated expression of the same ferritin genes.

Multiple isoferritins in mouse liver: Demonstration by polyacrylamide gel electrophoresis

Unfractionated pure ferritin isolated from the livers of female or male mice forms five narrow protein-positive bands on standard 5% polyacrylamide gel disc electrophoresis (pH 9.0). Since all of these sub-bands also contain iron, they are interpreted as being isoferritins. The multiple sub-bands are very unlikely to be artifactually generated by the analytical procedure used since they are not found in horse spleen ferritin when this is coelectrophoresed with female mouse liver ferritin. The present results provide an independent indication that many isoferritins indeed can be found within a single organ.

Immuno-Electronmicroscopic Studies of Surface Antigens of Blood Elements: I. Autoantibody on Erythrocytes in Acute Hemolytic Anemia

Abstract Tagging, by means of the immunoferritin technic, of autoantibody on the erythrocytes of a patient with autoimmune hemolytic anemia, and on cells from a panel of blood of normal individuals of blood group O which have been incubated either with the patient’s serum or eluate from his cells, is described. It was found that: 1) Ferritin-labeled antibody to human IgG was localized on the surface of the patient’s erythrocytes at fairly even intervals. 2) Ferritin-labeled antibody to human IgM, β1C, rat globulin or pure ferritin alone was not bound to the patient’s cells. 3) None of the ferritin-conjugates mentioned in 1) or 2) or pure ferritin was bound to red cells from normal individuals represented in the panel. 4) Only ferritin-conjugated antibody to human IgG was localized, in a similar pattern, on the surface of the normal red cells which had been incubated either with the patient’s serum or the eluate from his cells, whereas none of the conjugates in 2) or pure ferritin was bound to these treated cells.

SMALL-SCALE ISOLATION OF FERRITIN FOR THE ASSAY OF THE INCORPORATION OF 14C-LABELLED AMINO ACIDS.

1. A new procedure is described for the isolation of pure ferritin from small amounts of tissue. After the removal of most of the tissue proteins by heat coagulation, the ferritin fraction was chromatographed successively on CM-cellulose and Sephadex G-200. 2. The isolated ferritin appeared to be free from other proteins, as judged by its sedimentation pattern in the ultracentrifuge, by electrophoresis on polyacrylamide gel and by immunoelectrophoresis. 3. After the injection of [(14)C]leucine into a series of rats, the specific activity of liver ferritin isolated by the new procedure bore a constant relationship to that of liver ferritin separated by antigen-antibody precipitation. The procedure can thus be used to obtain ferritin of suitable purity for studies of amino acid incorporation. 4. The new procedure can be used to measure the total ferritin protein content of a tissue. It is not possible to use ultraviolet absorption for the measurement of ferritin protein because of considerable interference from the iron that it contains.

Iron Metabolism in the Bone Marrow as Seen by Electron Microscopy: A Critical Review

Abstract High resolution electron microscopy has made possible the visualization of transport and storage iron in the form of ferritin, both in dispersed form and in aggregates and in the form of "iron micelles" in mitochondria. Hemosiderin was found to consist either of pure ferritin in crystalline clusters or, more frequently, of ferritin associated with other substances, including a lipid component in the form of myelinic figures and PAS positive material. In the following paragraphs we have summarized the new morphologic findings and what appears to us the most likely interpretation in the light of known biochemical and isotopic studies. Alternative interpretations have been discussed in the body of the paper. Electron microscopy has established the erythroblastic island as a morphologic and functional unit of the bone marrow. A central reticular "nurse cell" appears to impart nutrients to surrounding rows of erythroblasts by the process of rhopheocytosis. Transfer of ferritin by this process is probably a passive phenomenon, since the amount transferred parallels the amount of iron present in the central reticular cell. Ferritin is increased both in the reticular cell and in erythroblasts in hemochromatosis. It is absent in iron deficiency, although rhopheocytosis remains prominent. Normally all erythroblasts (proerythroblasts and normoblasts) and reticulocytes contain ferritin. Only the larger aggregates can be visualized by the Prussian blue reaction in sideroblasts and siderocytes. Ferritin generally disappears when reticulocytes mature, even in hemochromatosis and infections, two conditions in which there is an excess of ferritin in erythroblasts. Interestingly, the increase in infections is entirely in form of dispersed ferritin and cannot be visualized by the Prussian blue reaction; i.e., sideroblasts are absent, in contrast to hemochromatosis where they are normal or increased. It appears most likely that ferritin disappears from normal maturing reticulocytes because it is utilized for hemoglobin formation. It persists in mature red cells in Cooley’s anemia, hypersideremia, hypochromic anemia and lead poisoning where hemoglobin formation is disturbed. The origin of the ferritin in the nurse cells and the extent to which ferritin rather than siderophilin contributes to hemoglobin synthesis are unsolved problems. Isotopic studies indicate that almost all of the iron used for hemoglobin synthesis is derived from siderophilin and hemoglobin synthesis can proceed without any visible ferritin, as in iron deficiency anemia. These facts must be reconciled with the electron microscopic observations which suggest that normally some iron reutilization within the marrow proceeds by way of erythrophagocytosis, fragmentation, intracellular hemolysis of red cells, formation of ferritin and ropheocytosis. Iron derived from erythrophagocytosis elsewhere in the body probably reaches the marrow bound to siderophilin. Such iron can be incorporated into ferritin of reticular cells as may be seen in hyperferremia and following injection of iron compounds. The process of rhopheocytosis would then lead to utilization of at least part of this ferritin iron for hemoglobin synthesis. In certain pathologic states, accumulation of ferritin and related visible dispersed or conglomerated iron micelles may point to the sites where hemoglobin synthesis or iron transport is blocked. In Cooley’s anemia and the hypersideremic hypochromic (non-thalassemic) anemias, iron accumulates in the mitochondria, which are known to be involved in hemoglobin synthesis. In lead poisoning, the mitochondria are markedly abnormal, and probably correspond to the areas of punctate basophilia. However, the iron accumulates in other areas of the cell, suggesting a different type of block.

The nature of storage iron in idiopathic hemochromatosis and in hemosiderosis. Electron optical, chemical, and serologic studies on isolated hemosiderin granules.

Using three different methods of cells fractionation, hemosiderin granules were isolated from tissues (liver and/or spleen) of three patients. The samples were obtained from a case of idiopathic hemochromatosis, a case of thalassemia major with secondary (transfusional?) hemosiderosis, and a case of transfusional hemosiderosis associated with an unclassified anemia. Iron, nitrogen, and protein content of the hemosiderin granules varied over a wide range. Electron microscopy of sectioned granules revealed aggregates of dense particles of different shapes, with diameters ranging from 10 A to about 75 A. In some of the granules dense particles corresponding to the iron hydroxide micelles of ferritin molecules were abundant. But many of the granules contained very few of these molecules. The presence of ferritin and apoferritin in the samples of hemosiderin granules was demonstrated by means of precipitin tests in agar-gel, using rabbit antiferritin sera with known antibody nitrogen concentrations. At least three antigenic components were detected in highly purified crystalline ferritin prepared from tissues of the three patients; the hemosiderin granules contained the same antigens, but probably in much smaller quantities. Both ferritin and apoferritin molecules were extracted from hemosiderin granules, and were demonstrated in the electron microscope after suitable preparation. The solubility curve of human ferritin in solutions of (NH(4))(2)SO(4) was investigated. The results indicate that substantial quantities of ferritin or apoferritin can be lost in saline, aqueous media during isolation of hemosiderin granules from cells. It was shown by means of electron microdiffraction on selected hemosiderin granules that the dense particles represent forms of partly hydrated alpha-Fe(2)O(3). The conditions necessary for electron microdiffraction in an electron microscope precluded an exact determination of the state of hydration of the alpha-Fe(2)O(3) or of its structural relation to (FeOOH) micelles of pure ferritin in its undenatured state. The findings were considered in the light of evidence on the structure and disposition of hemosiderin in situ in cells, and on the structure of ferritin. Differences between endogenous hemosiderin and hemosiderin derived from injections of colloidal iron compounds were pointed out. The evidence indicates that in hemochromatosis and in secondary hemosiderosis much of the inorganic storage iron in liver and spleen is derived from degraded ferritin. The findings suggest that an abnormal cellular metabolic pathway of ferritin is implicated in the pathogenesis of hemochromatosis and transfusional hemosiderosis.