Leukemia-associated Inhibitory Activity Research Articles

Ferritin: An expanded role in metabolic regulation

Synthesis of ferritin, a constitutive protein, is increased by iron. This protein is well recognized as a protein which detoxifies, stores and transports iron. The 24 subunits of ferritin assemble to form a protomer of M r 480 000. This protein shell can sequester up to 4500g atoms of iron as ferrichydroxyphosphate. Ferritin in vitro and in vivo binds other metal ions such as Cu, Zn, Cd, Pb, Be and Al. Next to Fe it binds large quantities of Be. Therefore, in vitro ferritin protects against and reverses the inhibition by Be of enzymes susceptible to this metal ion. Also, rats pretreated with Fe survive otherwise toxic levels of either pulmonary or intravenous exposure of Be. Liver ferritin from rats injected with Zn contains some of the injected metal ion. Incubation of such ferritin-zinc complex with zinc-requiring apoenzymes restores their activity. Fe(III) of ferritin is released only after its reduction to Fe(II) by a reductant. Incubation of phosphoglucomutase, a phosphoserine containing enzyme with ferritin and a reductant causes irreversible inactivation of the enzyme and removes 70% of its phosphate. Some other phosphoproteins are similarly inactivated but without the loss of the bound phosphate. Thus, uncontrolled release of iron from ferritin, in the presence of a reductant and oxygen can modify several biomolecules and can affect metabolic processes. A subclass of ferritin, acidic isoferritins, have been implicated in leukemia-associated inhibitory activity and has been suggested to inhibit production of Ia + macrophage progenitors. These observations suggest that ferritin, in addition to its role in iron storage and transport, may participate in other metabolic processes. An expanded role for ferritin in metabolic regulation is suggested.

Leukemia-associated inhibitory activity in acute leukemia developed from myelodysplastic syndrome.

Clinical responses of patients with acute leukemia (AL) following the myelodysplastic syndrome (MDS) to the standard therapy are poor. It is considered that the greatly decreased hemopoiesis in these cases is responsible for their clinical picture. We studied leukemia-associated inhibitory activity (LIA), which inhibited human granulocyte-macrophage progenitors, in these patients. Peripheral blood (PB) mononuclear cells (MNC) suppressive to granulocyte-macrophage colony formation were present in 3 of 5 cases of de novo AL and in 3 of 4 cases of AL following MDS. The PB MNC-cultured media from these cases also suppressed colony formation. The elution patterns of LIA of these cases were almost identical in gel chromatography. These results suggest that LIA may be responsible for the suppression of normal granulopoiesis in some patients with AL developed from MDS, and that the profound derangement of normal hemopoietic capability in these cases may be due to multiple complex factors.

Monocyte-Macrophage-Derived Acidic Isoferritins: Normal Feedback Regulators of Granulocyte-Macrophage Progenitor Cells In Vitro

Abstract 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.

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.

Acidic Isoferritins (Leukemia-Associated Inhibitory Activity) Fail to Inhibit Blast Proliferation in Acute Myelogenous Leukemia

AbstractCell-free extracts of bone marrow and blood cells from patients with leukemia contain an inhibitor of normal granulocyte/macrophage progenitor (CFU-GM) proliferation (leukemia-associated inhibitory activity, LIA) identified as acidic isoferritins. A comparison was made of the action of crude LIA prepared from frozen-thawed leukemic blood cells and purified spleen ferritin from a patient with chronic myelogenous leukemia, on the proliferation of blast progenitors from patients with acute myelogenous leukemia (AML), and on the promyelocyte leukemia cell line, HL-60. Crude LIA showed no inhibition of blast progenitor or HL-60 proliferation at low concentrations, but inhibited the proliferation of CFU-GM. At higher concentrations, crude LIA inhibited both blast cells and CFU-GM. Purified spleen ferritin failed to inhibit blast progenitors or HL-60 cells at any concentration tested, but inhibited both 7-day and 14-day CFU-GM. Using the thymidine “suicide” technique, the action of LIA was confirmed as being on CFU-GM in S-phase, but it failed to affect the proliferation of blast cells in S-phase. It is concluded that acidic isoferritins inhibit normal CFU-GM but not blast cells from patients with AML. Acidic isoferritins could confer a proliferative advantage of the leukemic clone over its normal counterparts.

Acidic isoferritins (leukemia-associated inhibitory activity) fail to inhibit blast proliferation in acute myelogenous leukemia

Cell-free extracts of bone marrow and blood cells from patients with leukemia contain an inhibitor of normal granulocyte/macrophage progenitor (CFU-GM) proliferation (leukemia-associated inhibitory activity, LIA) identified as acidic isoferritins. A comparison was made of the action of crude LIA prepared from frozen-thawed leukemic blood cells and purified spleen ferritin from a patient with chronic myelogenous leukemia, on the proliferation of blast progenitors from patients with acute myelogenous leukemia (AML), and on the promyelocytic leukemia cell line, HL-60. Crude LIA showed no inhibition of blast progenitor or HL-60 proliferation at low concentrations, but inhibited the proliferation of CFU-GM. At higher concentrations, crude LIA inhibited both blast cells and CFU-GM. Purified spleen ferritin failed to inhibit blast progenitors or HL-60 cells at any concentration tested, but inhibited both 70-day and 14-day CFU-GM. Using the thymidine “suicide„ technique, the action of LIA was confirmed as being on CFU-GM in S-phase, but it failed to affect the proliferation of blast cell in S-phase. It is concluded that acidic isoferritins inhibit normal CFU-GM but not blast cells from patients with AML. Acidic isoferritins could confer a proliferative advantage of the leukemic clone over its normal counterparts.

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.

Acidic isoferritins as feedback regulators in normal and leukemic myelopoiesis.

The absence of normal hematopoiesis during acute leukemia not in remission and the recovery of apparently normal blood cells during chemotherapy-induced remission suggest that suppressive cell interactions may be involved in the pathogenesis of acute leukemia. Others have shown inhibition of normal progenitor cell proliferation and differentiation by cells from patients with leukemia, but little or no information was provided regarding the actual characterization of the inhibitory cells or the mechanisms of action (Broxmeyer and Moore 1978). We have demonstrated the existence of an S-phase specific inhibitory activity [leukemia-associated inhibitory activity (LIA)] against normal granulocyte-macrophage progenitor cells (CFU-GM) which was produced by bone marrow, spleen, and blood cells from patients with acute and chronic myeloid and lymphoid leukemia and “preleukemia” (Broxmeyer et al. 1978a,b, 1979a,b). Greater concentrations of LIA were found during acute leukemia (newly diagnosed and untreated, or on therapy but not in remission) than during chronic leukemia (Broxmeyer et al. 1978a,b). Remission of acute leukemia was associated with low levels of LIA (Broxmeyer et al. 1978b, 1979a), and LIA at that time was not found in hemopoietic cells from normal donors (Broxmeyer et al. 1978a,b, 1979a,b). In contrast to its action on normal CFU-GM, LIA was not effective in suppressing the growth of normal CFU-GM from patients with acute leukemia who were not in remission and from many patients with acute leukemia during remission and with chronic leukemia (Broxmeyer et al. 1978b, 1979a,b). We postulated that LIA may thus confer a proliferative advantage to abnormally responsive cells (Broxmeyer et al. 1978b).

Isolation and biochemical characterization of leukemia-associated inhibitory activity that suppresses colony and cluster formation of cells

Leukimia-associated inhibitory activity suppresses colony and cluster formation in vitro of cells derived from granulocyte-macrophage progenitor cells of normal donors. It does not inhibit these same progenitor cells from patients with leukemia and it may contribute to the proliferative advantage leukemia cells appear to possess over normal hematopoietic cells during acute leukemia. The inhibitory activity was isolated by a combination of procedures including: ultracentrifugation, Sephadex G-200, carboxymethylcellulose, SDS-polyacrylamide gel electrophoresis, thin-layer and preparative isoelectric focusing and concanavalin A-Sepharose. Leukemia-associated inhibitory activity was characterized as a glycoprotein. It was inactivated by trypsin, chymotrypsin, pronase and periodate treatment. It bound to and was eluted by α-methylmannose from concanavalin A-Sepharose columns and had an apparent M r range of 450–550 000 and an isoelectric focus value between pH 4.6 and 4.9. Crude leukemia associated inhibitory activity was temperature sensitive but the more purified preparations were heat stable.

A murine model system for the study of the human leukemia-associated inhibitory activity

Mice inoculated with the RFV strain of Friend virus develop an erythroleukemia that spontaneously regresses. Bone marrow cells from leukemic mice produce a factor that inhibits colony formation in agar by normal CFU-Cs. CFU-Cs from leukemic animals are resistant to the factor. Bone marrow cells from some mice whose leukemia has regressed continue to produce the factor, and CFU-Cs from some regressed mice retain resistance to the factor. These observations dupllicate findings reported for patients with acute leukemias and those with leukemia in remission. The RFV system thus serves as an accurate experimental model for the study of hematopoietic cell interactions and the proliferative advantage of neoplastic cells in human leukemias.

A Murine Model System for the Study of the Human Leukemia-Associated Inhibitory Activity

AbstractMice inoculated with the RFV strain of Friend virus develop an erythroleukemia that spontaneously regresses. Bone marrow cells from leukemic mice produce a factor that inhibits colony formation in agar by normal CFU-Cs. CFU-Cs from leukemic animals are resistant to the factor. Bone marrow cells from some mice whose leukemia has regressed continue to produce the factor, and CFU-Cs from some regressed mice retain resistance to the factor. These observations duplicate findings reported for patients with acute leukemias and those with leukemia in remission. The RFV system thus serves as an accurate experimental model for the study of hematopoietic cell interactions and the proliferative advantage of neoplastic cells in human leukemias.

Persistence of Inhibitory Activity against Normal Bone-Marrow Cells during Remission of Acute Leukemia

Bone-marrow cells from patients with acute leukemia in remission were tested for their capacity to produce a substance (leukemia-associated inhibitory activity, LIA) that inhibits the formation of granulocyte and macrophage colonies in cultures of normal, but not of leukemic, bone marrow. LIA was detected in extracts of whole marrow in only eight of 83 patients in remission. However, extracts of slowly sedimenting cells, separated by velocity sedimentation from the marrows of eight patients in remission whose whole marrow had produced no LIA, produced inhibitory material in all cases. Extracts of the more rapidly sedimenting cells from these marrows contained an inactivator of LIA. Three of six patients in remission whose unfractionated marrow was unresponsive to LIA had a subpopulation of colony-forming cells that was sensitive to the inhibitor. These observations suggest that certain cellular functions dot not completely return to normal during remission of acute leukemia.