Barbara Hoffman-Liebermann

Molecular dissection of differentiation in normal and leukemic myeloblasts: separately programmed pathways of gene expression.

Abstract The regulation of gene expression in leukemic and normal myeloblasts induced to differentiate by the normal macrophage and granulocyte inducing protein MGI was studied by analysis of protein changes using two-dimensional polyacrylamide gel electrophoresis. During the 6-day period of differentiation from myeloblasts to mature cells, there was a programmed sequential change in the rate of synthesis of 217 of the 450 proteins detected in a MGI+D+ leukemic clone. The developmental program was initiated with a decrease in the synthesis of many proteins within the first hour, whereas the synthesis of new proteins occurred later, mostly between the second and fourth days. The mature cells showed a specialization associated with a changeover and increased synthesis of the major protein species. Both the MGI+D+ leukemic and the normal myeloblasts showed a similar sequence of protein changes during differentiation. The normal developmental program was thus maintained in the MGI+D+ leukemic cells. Cell mutants which differ in their competence to be induced to differentiate by MGI were used to dissect the developmental program of differentiation. Sixty-six protein changes were induced by MGI in partially differentiatable MGI+D− clones, whereas only 12 or 16 protein changes were induced in different MGI−D− clones which had not been induced for any previously known differentiation-associated property. In these mutant clone types, the induced protein changes were subsets of those induced by MGI in the MGI+D+ leukemic and normal myeloblasts. These subsets spanned the whole 6-day period of differentiation and had the same developmental sequence as in the fully differentiatable MGI+D+ cells. These data indicate that the protein changes during differentiation are not organized as one sequence, but rather as multiple, parallel sequences which can be separately induced. MGI induced some, but not all, of these sequences in the mutant clones. It is concluded that differentiation consists of multiple, parallel, separately programmed pathways of gene expression. Analysis of the initial differences between the proteins synthesized in untreated normal and leukemic myeloblasts has shown that all the leukemic clones, when compared to normal myeloblasts, constitutively expressed the MGI-induced state for a common subset of 14 proteins. In addition, the MGI+D− and MGI−D− clones, compared to MGI+D+, constitutively expressed the differentiated state for other subsets of proteins. The size of these constitutively expressed subsets was larger in MGI−D− than in MGI+D− clones. It is, therefore, suggested that the constitutive expression of some pathways of gene expression results in leukemia, whereas the constitutive expression of other pathways results in a decreased competence for the induction of differentiation.

Regulation of actin and other proteins in the differentiation of myeloid leukemic cells

Abstract The regulation of cytoplasmic proteins in mutants of mouse myeloid leukemic cells, differing in their competence to be induced to differentiate by the normal macrophage- and granulocyte-inducing protein (MGI) and the steroid inducer dexamethasone, was analyzed using SDS-polyacrylamide gel electrophoresis of 35 S-methionine-labeled proteins. Before induction, no consistent differences in the pattern of cytoplasmic proteins were found between clones with different capabilities to differentiate. Four MGI + D + clones, which are induced by MGI for Fc and C3 rosettes, the synthesis and secretion of lysozyme, and the formation of mature macrophages and granulocytes, all showed the same nine prominent changes in cytoplasmic proteins after induction. Five of these changes were either an increase or a decrease in proteins present in uninduced cells; four proteins appeared to be newly synthesized. One of the proteins that increased after induction was identified as actin. The pattern of cytoplasmic proteins from MGI-induced MGI + D + clones more closely resembled that of normal peritoneal macrophages and granulocytes than the pattern of the uninduced clones. The relationship of these protein changes to cell differentiation was further substantiated by the finding that MGI + D − cells, which can be induced by MGI for Fc and C3 rosettes and lysozyme, but not for mature cells, showed only four cytoplasmic protein changes which were quantitatively less than those found for MGI + D + clones. An MGI − D − clone which was not inducible for any differentiation-associated properties by MGI showed no alteration in protein synthesis. Thus in all the clones studied, there was a correlation between the number and extent of protein changes and the degree of MGI-induced differentiation. In MGI + D + clones, some of the differentiation-associated properties induced by MGI can be induced by the steroid hormone dexamethasone. Of the nine protein changes induced by MGI, six were also induced by dexamethasone, and no changes were induced by dexamethasone which were not also induced by MGI. These results, which were also shown by two-dimensional polyacrylamide gel electrophoresis, indicate that in cells which can respond to both MGI and dexamethasone, the proteins induced by dexamethasone were a subset of those induced by MGI.

Control mechanisms regulating gene expression during normal differentiation of myeloid leukemic cells: Differentiation defective mutants blocked in mRNA production and mRNA translation

Abstract Regulation of gene expression during myeloid cell differentiation has been analyzed using clones of myeloid leukemic cells that differ in their competence to be induced to differentiate by the normal macrophage- and granulocyte-inducing protein MGI. Changes in the relative rate of synthesis for specific proteins were compared to changes in the relative amounts of corresponding translatable poly(A)+ mRNAs, assayed in the reticulocyte cell-free translation system, using two-dimensional gel electrophoresis. Of the 217 proteins which changed during MGI-induced differentiation of normally differentiating MGI+D+ leukemic cells, 136 could be identified as products of cell-free translation. Eighty-four percent of the 70 decreases in synthesis, most of which occurred early during differentiation, were not accompanied by a parallel decrease in the amount of translatable mRNA, but were accompanied by a parallel shift of the corresponding mRNAs from the polysomal to the monosomal and free mRNA fractions. These results indicate that most of the early decreases in the synthesis of proteins were translationally regulated. In contrast, 81% of the proteins which increased in synthesis and 71% of the proteins that were induced de novo were regulated at the level of mRNA production. Experiments with differentiation defective mutants have shown that they were blocked both at the level of mRNA production and mRNA translation. The data with these mutants have suggested that there were different subsets of translationally regulated proteins which were separately regulated. The translational blocks for several proteins in these mutant clones have also made it possible to identify additional translational sites of regulation for protein changes that were controlled at the level of mRNA production during normal differentiation. The results indicate that translational regulation may predominantly have a different function in cell differentiation than regulation by mRNA production, and that differentiation-defective mutants can be blocked at either level.

Regulation of endogenous type C virus expression during normal myeloid cell differentiation Evidence for a role in promoting myeloid cell proliferation and differentiation

Abstract A specific regulatory pattern for the expression of endogenous type C virus was found during differentiation of normal myeloblasts from different strains of mice. Endogenous virus was expressed in the myeloblasts. Upon induction of differentiation by the normal macrophage- and granulocyte-inducing protein MGI, there was a rapid enhancement of virus production followed by an inhibition of virus secretion and an intracellular accumulation of viral proteins. There was finally a complete shutoff of viral protein synthesis at the terminal stage of differentiation, so that the mature cells no longer expressed viral proteins. The comparison of normal and fully differentiatable MGI + D + leukemic myeloblasts has shown that the regulatory pattern for virus production during differentiation in the normal myeloblasts was similar to that during differentiation in the leukemic myeloblasts. Ecotropic and xenotropic viruses were detected in the normal myeloblasts, whereas only ecotropic virus was detected in the leukemic myeloblasts. Only the production of ecotropic virus was enhanced upon induction of differentiation of the normal myeloblasts by MGI, whereas there was a shutoff of both xeno- and ecotropic viruses at the terminal stage of differentiation. Dexamethasone, lipopolysaccharide, dimethylsulfoxide, and low concentrations of actinomycin D increased the MGI-induced enhancement of ecotropic virus in both the normal and the leukemic myeloblasts, but did not affect the production of the xenotropic virus. Superinfection of the normal myeloblasts with high titers of their own endogenous ecotropic virus, as in the case of infection with virus from MGI + D + leukemic cells, increased the proliferation of myeloblasts, and in the presence of MGI this then resulted in an increased number of mature cells. It is concluded that the observed pattern for endogenous ecotropic type C virus expression is part of the developmental program of mouse myeloid cell differentiation and that endogenous type C viruses may serve as normal physiological agents for the promotion of myeloblast cell proliferation, which in the presence of the normal inducer of differentiation then results in an increased number of differentiated cells.