Separation of different molecular forms of macrophage‐ and granulocyte‐inducing proteins for normal and leukemic myeloid cells

Abstract

It is shown that serum of mice treated with endotoxin (ES) contains three separable and functionally distinct forms of macrophage- and granulocyte-inducing (MGI) proteins. One form (MGI-1M) induced the formation of macrophage colonies from normal bone-marrow cells and showed on gel filtration an apparent molecular weight of 300,000; a second form (MGI-1G) induced the formation of granulocyte colonies from normal bone-marrow cells and had an apparent molecular weight of 45-100,000; and the third form (MGI-2) induced the normal differentiation of MGI+D+ myeloid leukemic cells to macrophages and granulocytes and had an apparent molecular weight of 28,000. Studies on the time course of the decrease of these three activities in ES have indicated that MGI-2 was more readily inactivated in vivo than MGI-1M and MGI-1G. The MGI-1M in ES isolated after gel filtration was completely neutralized by an antiserum to MGI-1 from mouse L-cells, whereas the isolated MGI-1G and MGI-2 were not affected by this antiserum. Gel filtration under dissociating conditions (6 M guanidinium chloride) resulted in a reduction of the apparent molecular weights of MGI-1M from 300,000 to 42,000, and of MGI-1G from 45-100,000 to 28,000, while it produced no change in the 28,000 apparent molecular weight of MGI-2. Similar studies with conditioned medium produced in vitro from mouse lung and peritoneal macrophages showed that in these conditioned media, MGI-1 (both G and M) in the native form had an apparent molecular weight of 41,000 and MGI-2 of 24,000, and that both MGI-1 and 2 had an apparent molecular weight of 24,000 under dissociating conditions. The results indicate that MGI-1 exists in serum in vivo and in these conditioned media as aggregated proteins, whereas MGI-2 does not, and that macrophages and lung tissue are not the only source of the MGI proteins found in ES. It is suggested that all three forms of MGI activity are derived from one precursor protein; that only the MGI-2 form assayed on leukemic cells should be used for treatment based on the induction of normal cell differentiation in myeloid leukemia; and that MGI-2 may serve as a survey mechanism for inducing differentiation in myeloid leukemic cells that have lost their responsiveness to the MGI-1 molecules that control the viability, proliferation and differentiation of normal myeloblasts.

Mechanisms that uncouple growth and differentiation in myeloid leukemia cells: restoration of requirement for normal growth-inducing protein without restoring induction of differentiation-inducing protein.

“…induce formation of macrophage colonies (MGI-iM) (4,6,7), granulocyte colonies (MGI-IG). (4,6), or both types of colonies (MGI-IGM) (8)(9)(10).…”

Hemopoietic Growth Factors and Oncogenes in Myeloid Leukemia Development

“…Extensive studies have documented the presence in various conditioned media of differentiation-inducing factors (DF, DIF or MGI-2) that do not appear to be proliferative agents but are able to induce similar suppressive effects to G-CSF on a variety of murine and human leukemic cell lines, the most extensively studied being the murine M1 model [31][32][33]. In vivo evidence indicates that production of the murine factor can be T -cell-dependent [34], unlike the situation with G-CSF, and biochemical purification studies have indicated that DF is separable and distinct from G-CSF [33,35].…”

Autoinduction of differentiation in myeloid leukemic cells: restoration of normal coupling between growth and differentiation in leukemic cells that constitutively produce their own growth-inducing protein.

“…Assays for MGI-I and MGI-2 activity, rosettes, lysozyme, and cell morphology MGI-l activity was assayed by seeding 5 x 104 nucleated bone marrow cells in soft agar as described (Pluznik and Sachs, 1965;Lotem et al, 1980) in EM with 2007o horse serum and the number of macrophage and granulocyte colonies with >50 cells was counted 7 days after seeding. One unit of MGI-l activity is defined as the amount inducing the formation of one colony per 5 x 104 nucleated bone marrow cells.…”