Activation of normal genes in malignant cells: activation of chemotaxis in relation to other stages of normal differentiation in myeloid leukemia.

Genetically differerent clones of myeloid leukemic cells have been used to study the activation of normal genes in these malignant cells by the normal physiological inducer of myeloid cell differentiation, the protein MGI. In appropriate clones, MGI induced the normal differentiation-associated property of chemotaxis to a variety of compounds including the steroid hormone dexamethasone. The induced cells could also distinguish among different steroids by chemotaxis, suggesting that there are specific membrane interaction sites for steroids. The sequence of differentiation in these cells was the formation of C3 and Fc rosettes leads to phagocytosis of these rosettes and chemotaxis leads to synthesis and secretion of lysozyme leads to mature macrophages or granulocytes. The use of appropriate mutants and the comparison of induction by MGI and dexamethasone has shown that chemotaxis to casein can be dissociated from: chemotaxis to dexamethasone, ATP, and bacterial factor; formation of C3 or Fc rosettes; phagocytosis of these rosettes; synthesis of lysozyme; and the formation of mature cells. It is suggested from this dissection of normal differentiation that there are different membrane changes for specific chemotaxis, formation of these rosettes, and their phagocytosis, and that induction of each of these properties requires activation of different genes.     

Control of normal differentiation of myeloid leukemic cells. XIII. Inducibility for some stages of differentiation by dimethylsulfoxide and its disassociation from inducibility by MGI.

There are clones of myeloid leukemic cells that can be induced to differentiate by the normal differentiation-inducing protein MGI to form Fc and C3 rosettes, mature macrophages and granulocytes. One of these clones (MGI+DMSO+) was also inducible by dimethylsulfoxide (DMSO) for C3 but not Fc rosettes, and for mature macrophages but not for mature granulocytes. Other clones (MGI+DMSO-) were inducible by MGI but not DMSO and a third type of clone (MGI-DMSO-) was not inducible by either compound. Clones that differed in their inducibility by DMSO showed a similar inhibition of cell multiplication by DMSO. The results indicate, that some stages of differentiation can be induced by DMSO in an appropriate clone of myeloid leukemic cells and that there are different cellular sites for induction by DMSO and MGI.     

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

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 ³⁵S-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 of normal differentiation of myeloid leukemic cells. XII. Isolation of normal myeloid colony-forming cells from bone marrow and the sequence of differentiation to mature granulocytes in normal and D+ myeloid leukemic cells

An enriched population of early myeloid cells has been obtained from normal mouse bone marrow by injection of mice with sodium caseinate and the removal of cells with C3 (EAC) rosettes by Ficoll-Hypaque density centrifugation. This enriched population had no EAC or Fc (EA) rosettes and contained 87% early myeloid cells stained for myeloperoxidase and/or AS-D-chloroacetate esterase, 7% cells in later stages (ring forms) of myeloid differentiation and 6% unstained cells, 2% of which were small lymphocytes. After seeding in agar with the macrophage and granulocyte inducer MGI, the enriched population showed a cloning efficiency of 14% when removed from the animal and of 24% after one day in mass culture. Both the enriched and the unfractionated bone marrow cells gave the same proportion of macrophage and granulocyte colonies. The normal early myeloid cells were induced to differentiate by MGI in mass culture in liquid medium to mature granulocytes and macrophages. The sequence of granulocyte differentiation was the formation of EA and EAC rosettes followed by the synthesis and secretion of lysozyme and morphological differentiation to mature cells. D⁺ myeloid leukemic cells with no EA or EAC rosettes had a similar morphology to normal early myeloid cells and showed the same sequence of differentiation. The induction of EA and EAC rosettes occurred at the same time in both the normal and D⁺ leukemic cells, but lysozyme synthesis and the formation of mature granulocytes was induced later in the leukemic than in the normal cells. The results indicate that selection for non-rosette-forming normal early myeloid cells also selected for myeloid colony forming cells, that these normal early myeloid cells can form colonies with differentiation to macrophages and granulocytes, that normal and D⁺ myeloid leukemic cells have a similar sequence of differentiation and that the normal cells had a greater sensitivity for the formation of mature cells by MGI.     

Control of normal differentiation of myeloid leukemic cells. XI. Induction of a specific requirement for cell viability and growth during the differentiation of myeloid leukemic cells

Normal hematopoietic cells require the presence of a protein (MGI) in the appropriate conditioned medium (CM) for cell viability and growth and for differentiation to mature macrophages and granulocytes. Clones of myeloid leukemic cells have been established in culture (D⁺ clones) which require CM with this protein for differentiation, but not for cell viability and growth. It has been shown that these leukemic cells can be induced by CM to again require, like normal cells, the presence of CM for cell viability and growth. Induction of this requirement, which will be referred to as RVG, occurred before the D⁺ cells differentiated to mature granulocytes. Clones of myeloid leukemic cells (D^? clones) that could not be induced to differentiate to mature cells, did not show the induction of RVG. The steroid hormones prednisolone and dexamethasone can induce some, but not all the changes associated with differentiation of D⁺ cells. Incubation with these steroids did not result in the induction of a requirement for these steroids for cell growth and viability. Studies with CM from different sources have shown, that all batches that induced RVG also induced differentiation of D⁺ cells and that both activities were inhibited after treating the CM with trypsin. It is suggested that the same protein (MGI) may be involved in both activities. Incubation of D⁺ cells with CM resulted in an increase in agglutinability by concanavalin A and this increase was maintained even in the absence of CM. This suggests, that the induction of RVG in D⁺ myeloid leukemic cells is associated with a change in the cell surface membrane.     

Differences in surface membrane ecto-ATPase and ecto-AMPase in normal and malignant cells. I. Decrease in ecto-ATPase in myeloid leukemic cells and the independent regulation of ecto-ATPase and ecto-AMPase.

The hydrolysis of ATP and AMP by enzymes located on the external side of the plasma membrane (ecto-ATPase and ecto-AMPase) was studied in mouse myeloid leukemic cells, normal early myeloid cells, and normal mature granulocytes and macrophages. Nine clones of myeloid leukemic cells were used belonging to three groups that differ in their ability to be induced to differentiate by the differentiation-inducing protein MGI. These three groups consisted of MGI+D+ that can be induced to undergo complete differentiation, MGI+D- that can be induced to partially differentiate and MGI-D- with no induction of differentiation. The ecto-ATPase activity of normal early myeloid cells was similar to that of normal mature granulocytes and macrophages and higher than that of any of the leukemic cells. Among the leukemic cells, the MGI-D- cells had the highest level of ecto-ATPase activity. The behaviour of ecto-AMPase differed from that of ecto-ATPase. Some MGI-D- clones had a higher ecto-AMPase activity than normal cells and MGI+D- and MGI+D+ cells showed no detectable activity. Neither the ecto-ATP-ase nor ecto-AMPase activities changed after induction of differentiation in normal early myeloid or MGI+D+ leukemic cells. The results indicate that the myeloid leukemic cells had a decreased ability to hydrolyse external ATP, that there can be an independent regulation of ecto-ATPase and ecto-AMPase and that neither of these enzyme activities changed during differentiation.     

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.

Growth and differentiation of normal myeloid haematopoietic cells are regulated by a family of macrophage- and granulocyte-inducing (MGI) proteins. Some of these proteins (MGI-1) induce cell growth and others (MGI-2) induce cell differentiation. Addition of MGI-1 to normal myeloid cells induces growth and also induces the endogenous production of MGI-2. This induction of differentiation-inducing protein by growth-inducing protein then ensures the coupling between growth and differentiation found in normal cells. There are myeloid leukemic cells that constitutively produce their own MGI-1, but the cells do not differentiate in culture medium containing horse or calf serum. By removing serum from the medium, or in medium with mouse or rat serum, these leukemic cells are induced to differentiate to mature cells, which like normal mature cells, then no longer multiply. Leukemic cells with constitutive production of MGI-1 continuously cultured in serum-free medium with transferrin were also induced to differentiate by removing transferrin. This induction of differentiation was in all these cases associated with the endogenous production of MGI-2 by the cells. The results indicate that changes in specific constituents of the culture medium can result in autoinduction of differentiation in these leukemic cells due to restoration of the induction of MGI-2 by MGI-1, which then restores the normal coupling of growth and differentiation.     

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

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.     

Induction of lysozyme activity by adenosine 3':5' cyclic monophosphate in cultured mouse myeloid leukemic cells.

Mouse myeloid leukemic cells(Ml) could be induced by glucocorticoids to form Fc receptors, phagocytize, migrate in agar, induce lysosomal enzyme activities, and change into forms that were morphologically similar to macrophages and granulocytes. Adenosine 3′:5′ cyclic monophosphate also induced lysosomal enzyme activities, but not the other differentiation-associated properties. The induction of lysozyme activity was marked, the activity reaching about 400 times the initial activity at 5 days after treatment. This suggests that adenosine 3′:5′ cyclic monophosphate may be important in induction of lysozyme activity during differentiation of the cells.     

Differences in membrane unsaturated fatty acids and electron spin resonance in different types of myeloid leukemia cells.

The fatty acid composition and some physical properties of intact cells and isolated plasma membranes of two types of mouse myeloid leukemia cell clone grown in culture have been examined. One clone type, MGI+D+, can be induced by the macrophage and granulocyte-inducing protein (MGI) to differentiate into mature macrophages and granulocytes. The other clone type, MGI+D-, could not be induced to differentiate into mature cells. A two-fold increase in the ratio of saturated fatty acid to unsaturated fatty acid was found in the MGI+D- compared to the MGI+D+ clones. The MGI+D- clones produced an unusual polyunsaturated C20:5 fatty acid at 28 degrees C, whereas the MGI+D+ clones did not grow at this temperature. The cells and their isolated plasma membranes were studied by electron spin resonance. The motion of the 5-nitroxide stearate spin label was found to be higher in the intact cells and in the membranes of MGI+D- clones than of the MGI+D+ clones. The cells of MGI+D+ clones showed a similar freedom of motion to normal myeloblasts from the bone marrow. The results indicate that myeloid leukemia cells which differ in their competence to be induced to differentiate into mature cells have different physical properties of their plasma membranes and that this is correlated with their fatty acid acyl chain composition.     

The network of hemopoietic regulatory proteins in myeloid cell differentiation.

There are clones of myeloid leukemic cells that can be induced to undergo terminal cell differentiation to macrophages by normal hemopoietic regulatory proteins. Induction of differentiation in two different clones of myeloid leukemic cells with interleukin 6 (IL-6) or granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in induction of mRNA for the hemopoietic regulatory proteins IL-6, GM-CSF, interleukin 1 alpha and interleukin 1 beta, tumor necrosis factor, and transforming growth factor beta 1. In one of these clones, induction of differentiation with GM-CSF was also associated with induction of mRNA for macrophage colony-stimulating factor (M-CSF) but not for the receptor for M-CSF (c-fms), whereas in the other clone, induction of differentiation with IL-6 was associated with induction of mRNA for both c-fms and M-CSF. The clones also differed in their responsiveness to these regulators. There was no induction of mRNA for granulocyte colony-stimulating factor or interleukin 3 during differentiation of either clone. The results indicate that the genes for a nearly normal network of positive and negative hemopoietic regulatory proteins are induced during differentiation of these myeloid leukemic cells and that there are leukemic clones with specific defects in this network.     

Enhancement of hormone action by a phorbol ester and anti-tubulin alkaloids involves different mechanisms

The tumor-promoting phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) enhanced 1-isoproterenol and prostaglandin E1 stimulated cyclic AMP formation in clones of mouse myeloid leukemic cells. The enhancement was found up to 3h after TPA treatment and had disappeared after 24h, indicating its reversibility. The effect of TPA was not inhibited by removal of extracellular Ca2+ or pre-treatment with the calcium ionophore A23187. This enhancement by TPA seems to involve a different pathway than enhancement of response to the same hormones after treatment with the anti-tubulin alkaloids colchicine or vinblastine, since a myeloid leukemic cell mutant clone that was non-responsive to the anti-tubulin alkaloids responded to TPA. Furthermore, combined treatment of colchicine-sensitive cells with TPA and colchicine showed an additive stimulating effect. The enhancement of cell response to hormones by TPA was found in myeloid leukemic cell clones that either were or were not induced to differentiate after treatment with TPA. This suggests that enhancement of the effect of these and possibly other hormones by TPA may be an initial step of TPA action, but that this enhancement is not sufficient to induce the wide repertoire of TPA effects including induction of differentiation.     

Regulation of cell surface receptors for different hematopoietic growth factors on myeloid leukemic cells.

There are clones of myeloid leukemic cells which are different from normal myeloid cells in that they have become independent of hematopoietic growth factor for cell viability and growth. The ability of these clones to bind three types of hematopoietic growth factors (MGI-1GM = GM-CSF, IL-3 = multi-CSF and MGI-1M = M-CSF = CSF-1) was measured using the method of quantitative absorption at 1 degree C and low pH elution of cell-bound biological activity. Results of binding to normal myeloid and lymphoid cells were similar to those obtained by radioreceptor assays. The results indicate that the number of receptors on different clones of these leukemic cells varied from 0 to 1,300 per cell. The receptors have a high binding affinity. Receptors for different growth factors can be independently expressed in different clones. There was no relationship between expression of receptors for these growth factors and the phenotype of the leukemic cells regarding their ability to be induced to differentiate. The number of receptors on the leukemic cells was lower than on normal mature macrophages. Myeloid leukemic cells induced to differentiate by normal myeloid cell differentiation factor MGI-2 (= DF), or by low doses of actinomycin D or cytosine arabinoside, showed an up-regulation of the number of MGI-1GM and IL-3 receptors. Induction of differentiation of leukemic cells by MGI-2 also induced production and secretion of the growth factor MGI-1GM, and this induced MGI-1GM saturated the up-regulated MGI-1GM receptors. It is suggested that up-regulation of these receptors during differentiation is required for the functioning of differentiated cells.     

Suppression of the in vivo malignancy and in vitro cell multiplication of myeloid leukemic cells by hybridization with normal macrophages.

Mouse myeloid leukemic cells that multiplied in vitro and were malignant in vivo were hybridized with non-malignant non-multiplying normal mouse or human macrophages. Both the hybrids with mouse and with human macrophages were non-malignant and non-multiplying. The suppression of malignancy and cell multiplication in these hybrids was associated with the expression of eight other properties expressed in normal macrophages but not in the myeloid leukemic cells. These properties were C3 and Fc rosettes and immune phagocytosis, rosettes and phagocytosis of uncoated erythrocytes, synthesis and secretion of lysozyme and a high frequency of cap formation by concanavalin A (ConA). The hybrids also expressed two properties of the myeloid leukemic cells, a lack of cell attachment to the surface of a Petri dish and staining for myeloperoxidase. The use of specific antibodies has shown that the lysozyme produced by hybrids between human macrophages and mouse myeloid leukemic cells was human lysozyme. The results indicate that the in vivo malignancy and in vitro cell multiplication of myeloid leukemic cells was suppressed in hybrids with normal macrophages, and that his suppression can be dissociated from the normal macrophage property of cell attachment.     

Control of normal differentiation of myeloid leukemic cells. IV. Induction of differentiation by serum from endotoxin treated mice

Sera from different strains of mice injected with endotoxin induced clones (D⁺) from a cultured line of myeloid leukemic cells to undergo normal differentiation to mature granulocytes and macrophages. Other clones (D^?) derived from the same cell line were not inducible by these sera to undergo normal cell differentiation. Sera from the same strains of mice that had not been injected with endotoxin, increased the cloning efficiency of D⁺ and D ^? clones but did not induce differentiation. Endotoxin serum induced differentiation in D⁺ cells at dilutions up to 1:64, but increased the cloning efficiency of these cells at dilutions up to 1:2048. The end point of the dilution of endotoxin serum that induced differentiation in D⁺ cells, was also the end point that induced the formation of colonies with differentiation from normal bone marrow cells. The results indicate that serum from endotoxin treated animals can serve as a good in vivo source to induce normal differentiation in D⁺ myeloid leukemic cells; that the progeny of a single leukemic cell was induced to undergo differentiation to both macrophages and granulocytes; that endotoxin serum contained two activities, one that increased cloning efficiency and the other that induced cell differentiation; and that the same material in endotoxin serum induced cell differentiation in normal and leukemic cells.     

K562 leukemia cells transfected with the human c-fes gene acquire the ability to undergo myeloid differentiation

Expression of the proto-oncogene p93c-fes and its associated tyrosine kinase activity is marked in mature granulocytes, monocytes, differentiated HL-60 leukemia cells, and leukemia cell lines KG-1, THP-1, HEL, and U-937, which can be induced to differentiate along the granulocyte/monocyte pathway. Conversely, p93-c-fes expression is absent in the K562 cell line, which is resistant to myeloid differentiation. Upon transfection and clonal selection of K562 cells using a mammalian expression vector containing the 13-kilobase pair c-fes gene, c-fes mRNA was transcribed and p93-c-fes tyrosine activity kinase was expressed. Clones expressing c-fes underwent myeloid differentiation as assessed by the appearance of phagocytic activity, Fc receptors, nitro blue tetrazolium reduction, Mac-1 immunofluorescence, and lysozyme production. These results indicate that the expression of the c-fes protooncogene and its associated tyrosine kinase activity plays a major role in the initiation of myeloid differentiation.     

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

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.     

Induction by recombinant human granulocyte colony-stimulating factor of differentiation of mouse myeloid leukemic M1 cells

The effect of recombinant human granulocyte colony-stimulating factor (G-CSF) on induction of differentiation of mouse myeloid leukemic M1 cells was examined. Purified G-CSF caused dose-dependent induction of phagocytic activity and lysozyme activity in M1 cells. Its half-maximally effective concentration was 10 ng/ml. On treatment of M1 cells with G-CSF (100 ng/ml) for 4 days, 30-50% of the cells differentiated morphologically into macrophage cells; 30-40% of the cells were blast cells and 20-30% of the cells were forms intermediate between blastic cells and mature macrophages.     

Synchrony of gene expression and the differentiation of myeloid leukemic cells: reversion from constitutive to inducible protein synthesis

There are mutant myeloid leukemic cells that cannot be induced to differentiate in serum-free culture medium, or medium with calf serum by the macrophage and granulocyte differentiation-inducing protein (MGI-2) that induces differentiation in normal myeloid cells. These mutants can be induced to differentiate by MGI-2 in medium with mouse serum. The mechanism of this induction of differentiation has been analysed by using two-dimensional gel electrophoresis to study changes in the synthesis of cytoplasmic proteins. In calf serum, 46 of the protein changes that were induced by MGI-2 in normally differentiating cells were constitutive in the differentiation-defective mutant cells. Treatment with mouse serum reverted 13 of these proteins from the constitutive to the non-constitutive state. This reversion was associated with a gain of inducibility for various differentiation-associated properties, so that 23 proteins were induced by MGI-2 for the same type of change as in normal differentiation. A normal developmental program requires synchrony of gene expression. The existence of constitutive instead of inducible gene expression can produce asynchrony in this program and thus produce blocks in differentiation. The results indicate that it is possible to treat these mutant cells so as to induce the reversion of specific proteins from the constitutive to the non-constitutive state, and that this can then restore the synchrony required for induction of differentiation. It is suggested that this mechanism may also allow induction of differentiation in other types of differentiation-defective cells.