Identification of leukemia-associated inhibitory activity as acidic isoferritins. A regulatory role for acidic isoferritins in the production of granulocytes and macrophages.

Acidic isoferritins have been identified as leukemia-associated inhibitory activity (LIA), which suppresses colony and cluster formation of colony-forming unit-granulocyte macrophages from normal donors but not from patients with leukemia. LIA was detected in all ferritin preparations tested, including ferritin isolated from normal heart, spleen, liver, and placental tissues, and from the spleens of patients with chronic myelogenous leukemia and Hodgkin's disease. Purified preparations of LIA were composed almost entirely of acidic isoferritins, as determined by immunoassay, radioimmunoassay, and isoelectric focusing. The inhibitory activity in the LIA and ferritin samples was inactivated by a battery of antisera specific for ferritin, including those prepared against acidic isoferritins from normal heart and spleen tissues from patients with Hodgkin's disease, and those previously absorbed with basic isoferritins. Antisera absorbed with acidic isoferritins did not inactivate the inhibitory activity. Separation of LIA and chronic myelogenous leukemia and normal spleen ferritin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing confirmed that the regions of peak inhibitory activity corresponded in each to an apparent molecular weight of approximately 550,000 and to a pI value of 4.7. Similar physicochemical characteristics included inactivation by methods that dissociate ferritin molecules into subunits and by treatment with trypsin, chymotrypsin, pronase, and periodate. The purified preparations were extremely stable to heat treatment. The glycoprotein nature of the inhibitory activity was substantiated because it bound to concanavalin A-Sepharose and was eluted off by alpha-methyl mannose. Inhibitory activity of the activity of the acidic isoferritins was detected at concentrations as low as 10(-17)-10(-19) M and iron saturation did not appear to be necessary for its action. These results implicate acidic isoferritins in the regulation of normal myelopoiesis and suggest a role for them in the progression of leukemia.

Ferritin synthesis by human T lymphocytes.

Mononuclear cells from peripheral blood of normal humans, unselected spleen cells from patients with Hodgkin's disease, and selected T and non-T lymphoid cells from normal peripheral blood and from the spleens of Hodgkin's disease patients were examined for de novo synthesis and secretion of ferritin. After precipitation of labeled lysates and supernatants from unseparated and selected T cells with antiserum to human liver ferritin, two bands were visible on sodium dodecyl sulfate-polyacrylimide gel analysis. The two bands were detected in molecular weight regions 19,000 and 21,000, which are thought to represent the L and H subunits of the ferritin molecule, respectively. The slower band (subunit H) was more radioactive than the faster band (subunit L). The H subunit is found in greater amounts in the serum of some tumor patients, but its cellular origin has not been established. The present findings indicate that cells of the immune system contribute to the synthesis and secretion of a ferritin molecule with a high proportion of H subunits.

Relative subunit composition of the ferritin synthesized by selected human lymphomyeloid cell populations.

Biosynthesis of ferritin subunits by cell sets isolated from normal human peripheral blood, spleens of Hodgkin's disease patients, and tumor cell lines were investigated. Normal mature hematopoietic cells made a ferritin with more H (21K) than L (19K) subunits. The reverse was found for a promyelocytic tumor cell line and tumor cell lines derived from other tissues. Two dimensional electrophoresis indicated H has a lower pI than L. Therefore relative proportions of the two subunits contribute to the electrophoretically distinct forms of the isoferritins. In response to increasing concentrations of iron in vitro, a selected monocyte population synthesized more H than L; L biosynthesis however increased more than H. Some possible regulatory implications of these observations are discussed.

Expression and structural and functional properties of human ferritin L-chain from Escherichia coli.

The human ferritin L-chain cDNA was cloned into a vector for overproduction in Escherichia coli, under the regulation of a lambda promoter. The plasmid obtained contains the full L-chain coding region modified at the first two codons. It is able to direct the synthesis of the L-chain which can constitute up to 15% of the total soluble protein of bacterial extract. The L-chains assemble to form a ferritin homopolymer with electrophoretic mobility, molecular weight, thermal stability, spectroscopic, and immunological properties analogous to natural ferritin from human liver (95% L-chain). This recombinant L-ferritin is able to incorporate and retain iron in solution at physiological pH values. At variance with the H-ferritin, the L form does not uptake iron at acidic pH values and does not show detectable ferroxidase activity. It is concluded that ferritin L-chain lacks the ferroxidase site present in the H-chain and that the two chains may have specialized functions in intracellular iron metabolism.

Model for intermediate steps in monocytic differentiation: c-myc, c-fms, and ferritin as markers.

A mature hematopoietic cell represents the end product of a stepwise differentiation process. As a model system for studying differentiation, the human promyelocytic leukemia cell line HL-60 undergoes terminal monocytic/macrophagic differentiation following exposure to either phorbol 12-myristate 13-acetate or 1,25-dihydroxyvitamin D3. We have derived and analyzed a variant HL-60 cell line, 1F10, that permits the study of several intermediate steps in the myeloid differentiation process. These intermediate steps are documented by cell cycle data and phenotype analysis as well as markers such as c-myc, c-fms, and both subunits of ferritin.

Biosynthesis of ferritin subunits from different cell lines of HL-60 human promyelocytic leukaemia cells and the release of acidic isoferritin-inhibitory activity against normal granulocyte-macrophage progenitor cells.

Biosynthesis of acidic isoferritins was investigated in human promyelocytic HL-60 cells, characterized by diploid (2C), tetraploid (4C) and mixed diploid--tetraploid (2C-4C) DNA cell lines. The three cell lines were studied for the biosynthesis of ferritin and its subunits and for the release of acidic isoferritin-inhibitory activity against normal CFU-GM before and after addition of DMSO. While the tetraploid and mixed diploid--tetraploid cell lines synthesized more H-(Mr = 21) than L-subunits (Mr = 19) after induction, the tetraploid line synthesized more H-subunit before and after induction, compared to the diploid line. The release of acidic isoferritin-inhibitory activity was greater before than after induction in both cell lines, but the tetraploid cell line released more acidic isoferritin-inhibitory activity consistent with its greater production of Mr = 21 subunit. However, after induction no inhibitory activity could be detected from the diploid cells and much less activity was detected with the tetraploid cells, suggesting that differentiation caused a decrease in production of acidic isoferritin-inhibitory activity.

Serum ferritin in relation to the course of Hodgkin's disease.

Serum levels of patients with Hodgkin's disease were evaluated during the course of the disease. Significant correlations were seen in relation to the stage of the disease, to sex and to various hematological data. An increase of Fer levels during progression and a decrease during remission was observed. Possible pathogenetic mechanisms are discussed.

Structure of human ferritin light subunit messenger RNA: comparison with heavy subunit message and functional implications.

Ferritin has a protein shell of 5 X 10(6) Da consisting of 24 subunits of two types, a heavier (H) chain of 21,000 Da and a lighter (L) chain of 19,000 Da. A cDNA clone of the messenger for the L subunit has been isolated from a human monocyte-like leukemia cell line. The clone contains an open reading frame of 522 nucleotides coding for an amino acid sequence matching 97% of the published sequence of human liver ferritin L subunit determined by sequenator, but it corresponds to only 55% of the reported amino acid sequence of a human liver H-subunit clone. Nevertheless, computer analysis of the subunit conformations predicted from the open reading frames of the L and H clones shows that most of the amino acid differences are conservative and would allow both subunits to form the five alpha-helices and beta-turns established by x-ray crystallography for horse spleen ferritin subunits. This suggests that L and H subunits are structurally interchangeable in forming an apoferritin shell. The 5' untranslated region of our human ferritin L clone has considerable homology with that of the rat liver ferritin L clone in the region immediately upstream from the initiator codon, notably showing an identical sequence of 10 nucleotides at the same position in both subunit clones that may participate in regulating the known activation of ferritin mRNA after iron administration. Extensive homology, including several blocks of nucleotides, was identified between the 3' untranslated regions of the human and rat L clones. The common structural features of the H and L subunits lead us to conclude that they have diverged from a single ancestral gene.

Monocyte-macrophage-derived acidic isoferritins: normal feedback regulators of granulocyte-macrophage progenitor cells in vitro.

The recent identification of a leukemia-associated inhibitory activity (LIA) against granulocyte-macrophage progenitor cells (CFU-GM) as acidic isoferritins has now led to detection of this activity in normal bone marrow and blood cells. Detection of this activity depends on stimulation of CFU-GM by granulocyte-macrophage colony stimulatory factors (GM-CSF), and some conditioned media (CM) sources of GM-CSF (human placental and monocyte, mouse macrophage and WEHI-3) contained low levels of acidic isoferritin that lowered colony formation. Inactivation or removal of this activity increased the stimulatory capacity of the CM. CM depleted of acidic isoferritins or CM originally devoid of this activity (human GCT, 5637, Mo, lymphocytes: mouse L cells or pokeweed-mitogen-stimulated spleen cells) increased the sensitivity of the assay to detect acidic isoferritin inhibitory activity. This activity was selectively contained and released from normal monocytes and macrophages. Restriction of this activity to mononuclear phagocytes was substantiated, as only continuous cell lines of monocytes and macrophages or lines capable of induction to this lineage contained and released acidic isoferritin inhibitory activity. The cells of origin and target cells of action suggest that acidic isoferritin-inhibitory activity can be considered as a negative feedback regulator, at least in vitro.