Differentiation Of Normal Myeloid Cells Research Articles

Abstract TP130: Transcriptome Analysis of Mononuclear Cell Responses After Ischemic Stroke in Type 2 Diabetic Mice

Introduction: Type 2 diabetes mellitus (T2DM) is a major risk factor of ischemic stroke and is associated with poor outcome after stroke. However, the mechanisms underlying the detrimental effects of diabetes are largely unclear. We used single-cell RNA sequencing (scRNA seq) to determine the transcriptome profiles of blood peripheral mononuclear cells (BPMNs) under diabetic and ischemic conditions using T2DM mouse model. Hypothesis: The altered gene expression under diabetic condition affects the normal differentiation of BPMNs, resulting in the aggravation of ischemic brain injury. Methods: We performed scRNA seq for BPMNs from four groups of mice, consisting of db/db mice with or without stroke and db/+ mice with or without stroke. Stroke was induced by distal middle cerebral artery occlusion. We specified the cell types of BPMNs and compared the transcriptome among the groups of mice. We assessed the biological role of identified differentially expressed genes (DEGs) by enrichment analysis. Results: In the comparison between db/db and db/+ groups, 168 up-regulated and 245 down-regulated DEGs were identified. The db/db group was composed of a larger population of macrophages and a smaller population of lymphocytes compared to db/+ group. The gene Cebpa , known to be associated with myeloid maturation, was significantly down-regulated in monocytes/macrophages, indicating the adverse effect of diabetes on the normal differentiation of myeloid cells. In addition, enrichment analysis revealed that up-regulated DEGs were related to the gene ontology term translation in B cells and innate immune response in monocytes. On the other hand, the down-regulated DEGs were related to antigen processing and presentation in macrophages. In the comparison between stroke and non-stroke groups, 21 up-regulated and 223 down-regulated DEGs were identified, in which down-regulated DEGs were related to cellular oxidant detoxification in T cells. Conclusions: The present study demonstrated altered gene expression profile and molecular network across various cell types in PBMCs in response to diabetic and ischemic conditions by scRNAseq, providing a clue to the underlying mechanism of the adverse effect of T2DM on native immunity and response to stroke.

Cell Intrinsic and Extrinsic Mechanisms Mediate the Role of c-Myc Haploinsufficiency in Controlling HSC Fate

It has been shown that loss of c-Myc leads to accumulation of hematopoietic stem cells (HSCs) and severe cytopenia as a consequence of a blockage of HSC differentiation. Here we report a role of c-Myc haploinsufficiency in regulating HSC quiescence and self-renewal. We showed that c-Myc haploinsufficient mice displayed decreased white blood count and number of lymphocytes with normal myeloid cell differentiation. The number of HSCs and hematopoietic progenitor cells (HPCs) were all decreased significantly in c-Myc haploinsufficient mice as compared with control mice. We found that c-Myc haploinsufficiency inhibited HSC self-renewal capacity, increased proliferation and decreased quiescence of HSCs in vivo. By transplantation assays, we showed that c-Myc haploinsufficiency has extrinsic and intrinsic effects on the maintenance of HSCs in vivo. Our study suggests that loss of c-Myc activity and reduced dosage of c-Myc have distinct effects on HSC functions.c-Myc is a critical downstream mediator of the Wnt/b-catenin pathway. We showed that c-Myc haploinsufficiency is sufficient to prevent severe anemia in Apc heterozygous mice, and to significantly prolong the survival of Apc heterozygous mice. In addition, treatment of Apc haploinsufficient mice by a c-Myc inhibitor significantly reversed anemia in Apc-deficient mice.By transplantation assay, we further demonstrated that reduced expression of c-Myc in the bone marrow niche is responsible for prevention of severe anemia in Apc-deficient mice. However, we found that reduction of c-Myc by loss of a single allele of c-Myc did not rescue defective self-renewal capacity of Apc haploinsufficient HSCs. Taken together, our studies indicate that c-Myc mediates the function of the Wnt/b-catenin signaling pathway in bone marrow niche but not in HSCs. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of the Enzyme Dihydroorotate Dehydrogenase Overcomes Differentiation Blockade in Acute Myeloid Leukemia

BackgroundThe prognosis for adults diagnosed with acute myeloid leukemia (AML) remains poor, with a five-year survival of 25%. This prognosis is even more dismal in older patients, and those who are not well enough to receive standard induction chemotherapy. Speaking to the need for new therapies is the fact that our therapeutic backbone - a combination of cytarabine and an anthracycline - remains unchanged for 40 years.Differentiation therapy (e.g. ATRA and Arsenic trioxide) revolutionized care for the small subset of patients diagnosed with acute promyelocytic leukemia (APL). Where APL was once the worst form of myeloid leukemia, it now carries the best prognosis, with a five-year survival exceeding 85%. Replicating the success of differentiation therapy in the remaining 90% of patients with AML has remained elusive, and was the motivation behind our studies.HypothesisAML comprises disparate genetic sub-types, but one shared hallmark is that the leukemic blasts are frozen at an immature and self-renewing stage of development. Despite AML's heterogeneity, 70% of cases overexpress HoxA9, a gene that must be downregulated in the process of normal myeloid cell differentiation. We hypothesized that the process through which HoxA9 maintains the undifferentiated state represented a commonly dysregulated node, and that therapies that bypass this node would be useful in the treatment of AML. Thus, we asked the question: 'can we identify small molecules that overcome myeloid differentiation arrest?'A Phenotypic Differentiation ScreenWe established a HoxA9-based AML screening system that permitted an unbiased flow cytometry phenotypic differentiation screen. We screened 330,000 small molecules for those with myeloid differentiation activity. Target identification of our lead compounds led to the unexpected discovery that inhibitors of dihydroorotate dehydrogenase (DHODH) can overcome myeloid differentiation arrest. DHODH is a mitochondrial enzyme involved in the endogenous synthesis of pyrimidines. The modulation of DHODH had not previously been shown to affect myeloid differentiation.DHODH Inhibitors in the Treatment of LeukemiaThe small molecule brequinar is a potent DHODH inhibitor (Panel A). Brequinar triggered myeloid differentiation in vitro (Panel B) and in vivo. Brequinar was highly active in vivo, as demonstrated in syngeneic murine AML models (HoxA9+Meis1 and MLL/AF9) as well as xenotransplant AML models (THP1, HL60, MOLM13, OCI/AML3).In an aggressive MLL/AF9 model of AML, treatment of mice with brequinar caused myeloid differentiation (Panel D), reduced leukemic cell burden, and improved overall survival (Panel C). Furthermore, treatment with brequinar reduced the number of leukemia stem cells, reduced colony-formation activity, and depleted the number of leukemia initiating cell activity (Panel E). These findings support the hypothesis that the myeloid differentiation resulting from DHODH inhibition is both phenotypic and functional, such that the matured leukemia cells lose their self-renewing capability.DHODH Inhibitors and Normal HematopoiesisTreatment with brequinar was better-tolerated and more effective than treatment with cytotoxic chemotherapy. Unlike cytarabine and doxorubicin, brequinar could be given for many weeks without cumulative toxicity. To assay the effect of DHODH inhibition on normal cells, we performed competitive bone marrow transplantation assays. Mice were treated with brequinar, 5-fluorouracil or induction chemotherapy. Their bone marrow was transplanted in competition (1:1) with normal (untreated) bone marrow, to gauge the effect of therapy on hematopoietic stem cell (HSC) function. Treatment with 5-FU and induction chemotherapy led to a dramatic loss of HSC fitness. In contrast, HSCs from mice treated with brequinar were functionally equivalent to those of untreated mice (Panel F) supporting the notion that DHODH inhibition does not lead to detrimental differentiation of the HSC compartment.DiscussionThe mechanism for the selective vulnerability of leukemia cells to DHODH inhibition remains under investigation. Our studies point towards DHODH as a new metabolic target; DHODH inhibitors may provide differentiation therapy for patients with AML. [Display omitted] DisclosuresSykes:Bayer Pharma AG: Research Funding. Meyer:Bayer Pharma AG: Employment. Stoeckigt:Bayer Pharma AG: Employment. Janzer:Bayer Pharma AG: Employment. Scadden:GlaxoSmithKline: Research Funding; Dr. Reddy’s: Consultancy; Fate Therapeutics: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Teva: Consultancy; Apotex: Consultancy; Bone Therapeutics: Consultancy; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

Stat3 and C/EBPβ synergize to induce miR-21 and miR-181b expression during sepsis.

Myeloid-derived suppressor cells (MDSCs) increase late sepsis immunosuppression and mortality in mice. We reported that microRNA (miR) 21 and miR-181b expression in Gr1+CD11b+ myeloid progenitors increase septic MDSCs in mice by arresting macrophage and dendritic cell differentiation. Here, we report how sepsis regulates miR-21 and miR-181b transcription. In vivo and in vitro binding studies have shown that C/EBPα transcription factor, which promotes normal myeloid cell differentiation, binds both miRNA promoters in Gr1+CD11b+ cells from sham mice. In contrast, in sepsis Gr1+CD11b+ MDSCs miR-21 and miR-181b promoters bind both transcription factors Stat3 and C/EBPβ, which co-imunoprecipitate as a single complex. Mechanistically, transcription factor Rb phosphorylation supports Stat3 and C/EBPβ accumulation at both miRNA promoters, and C/EBPβ or Stat3 depletion by siRNA in sepsis Gr1+CD11b+ MDSCs inhibits miR-21 and miR-181b expression. To further support this molecular path for MDSC accumulation, we found that Stat3 and C/EBP binding at miR-21 or miR-181b promoter was induced by IL-6, using a luciferase reporter gene transfection into naive Gr1+CD11b+ cells. Identifying how sepsis MDSCs are generated may inform new treatments to reverse sepsis immunosuppression.

Tumor-induced myeloid dysfunction and its implications for cancer immunotherapy.

Immune function relies on an appropriate balance of the lymphoid and myeloid responses. In the case of neoplasia, this balance is readily perturbed by the dramatic expansion of immature or dysfunctional myeloid cells accompanied by a reciprocal decline in the quantity/quality of the lymphoid response. In this review, we seek to: (1) define the nature of the atypical myelopoiesis observed in cancer patients and the impact of this perturbation on clinical outcomes; (2) examine the potential mechanisms underlying these clinical manifestations; and (3) explore potential strategies to restore normal myeloid cell differentiation to improve activation of the host antitumor immune response. We posit that fundamental alterations in myeloid homeostasis triggered by the neoplastic process represent critical checkpoints that govern therapeutic efficacy, as well as offer novel cellular-based biomarkers for tracking changes in disease status or relapse.

Expression of c-Fes Protein Isoforms Correlates with Differentiation in Myeloid Leukemias

The cellular fes gene encodes a 93-kilodalton protein-tyrosine kinase (p93) that is expressed in both normal and neoplastic myeloid cells. Increased c-Fes expression is associated with differentiation in normal myeloid cells and cell lines. Our hypothesis was that primary leukemia cells would show a similar pattern of increased expression in more differentiated cells. Therefore, we compared c-Fes expression in cells with an undifferentiated, blast phenotype (acute myelogenous leukemia--AML) to cells with a differentiated phenotype (chronic myelogenous leukemia--CML). Instead of differences in p93 expression levels, we found complex patterns of c-Fes immunoreactive proteins that corresponded with differentiation in normal and leukemic myeloid cells. The "blast" pattern consisted of c-Fes immunoreactive proteins p93, p74, and p70; the "differentiated" pattern showed two additional c-Fes immunoreactive proteins, p67 and p62. Using mRNA from mouse and human cell lines, we found deletion of one or more exons in the c-fes mRNA. Those deletions predicted truncation of conserved domains (CDC15/FCH and SH2) involved in protein-protein interactions. No deletions were found, however, within the kinase domain. We infer that alternative splicing generates a family of c-Fes proteins. This may be a mechanism to direct the c-Fes kinase domain to different subcellular locations and/or substrates at specific stages of myeloid cell differentiation.

Targeting Hypomethylation of DNA to Achieve Cellular Differentiation in Myelodysplastic Syndromes (MDS)

Considerable progress has been made recently in defining and understanding the diverse members of the group of hematologic disorders now known as the myelodysplastic syndrome (MDS). New systems of classification, based on the latest cytogenetic methodologies, have generated better prognostic data, and basic research has more closely associated molecular mechanisms with clinical subgroups. The mechanisms underlying most cases of myelodysplasia appear to be an array of chromosomal abnormalities leading to suppression of normal myeloid cell differentiation and dominance of abnormal, immature cells. The process is progressive and is mediated by a variety of cytokines, potential loss of tumor suppressor genes, aberrations in signal transduction pathways, and perhaps immune mechanisms. Hypermethylation of specific DNA sequences has been implicated in the pathogenesis of MDS. Until recently, treatment options have been few, high risk, and mostly ineffective. New discoveries, particularly in the area of stimulating remaining normal myeloid cells to resume growth and differentiation, hold promise for safer treatment regimens and improved outcomes. Among the promising new agents are nucleoside analogues, such as 5-azacytidine and decitabine, which reactivate tumor suppressor gene transcription through effects on DNA methylation.

Expression of myeloid differentiation antigens in acute nonlymphocytic leukemia: increased concentration of CD33 antigen predicts poor outcome--a report from the Childrens Cancer Study Group.

Ninety-eight cryopreserved specimens of acute nonlymphocytic leukemia (ANLL) cells obtained at initial diagnosis of children enrolled on the Childrens Cancer Study Group 251 protocol (CCG 251) were examined by indirect immunofluorescence using four monoclonal antibodies to myeloid differentiation antigens. The relationship between the level of differentiation of ANLL cells as determined by their antigen phenotype and the clinical outcome of treatment, including complete remission (CR) rate, survival, and event-free survival, was evaluated. Most leukemic specimens were determined to express the CD33 antigen (L4F3), a 67-kD protein. Because the level of differentiation of normal myeloid cells is reflected by the concentration of the CD33 antigen expressed, samples were categorized as CD33-bright (immature) versus CD33-dull (mature). Patients with CD33-bright leukemic blasts had a marginally inferior CR rate to those with CD33-dull blasts (P = 0.08). With respect to survival and event-free survival, there was a significantly inferior outcome in the CD33-bright patients (P = 0.04 and P = 0.06, respectively). Reactions of ANLL with anti-CD15 antibody (1G10), anti-CD36 antibody (5F1), or anti-CD17 antibody (T5A7) did not predict clinical outcome. This study indicates that patients whose ANLL blasts displayed the CD33 antigen in an amount associated with immature myeloid cells experienced a worse outcome than patients with ANLL blasts that expressed a phenotype associated with more mature cells.

Evidences that fibroblasts and epithelial cells produce a specific type of macrophage and granulocyte inducer, also known as colony-stimulating factor, and that monocyte-macrophages can produce another factor with proliferative inducing activity on myeloid cells and differentiative activity on macrophages.

Molecules with the property to induce proliferation of bone marrow cells in liquid cultures, and with colony-stimulating activity, were found on media conditioned (MC) by lung fibroblasts and kidney epithelial cells. These factors presented an apparent mol wt of 70,000 and 22,000 d respectively. Also when MC by epithelial cells from lungs was tested for the induction of proliferation of bone marrow cells a molecule with 22,000 d was detected. These molecules are thought to be CSF because they induce colony formation, and they are also similar in mol wt to two of the already known CSF. In fact the GM-CSF obtained from endotoxic lungs with a large epithelial cell content has a mot wt of 22,000 d, and the CSF-1 produced by a fibroblast cell line had 70,000. When the MC by fibroblast was used to induce bone marrow cells to proliferate, three new molecules with colony-stimulating activity were secreted. These molecules with apparent mol wts of 45,000, 30,000 and 17,000 d were also found in the MC by bone marrow cells when induced to proliferate with MC by epithelial cells. When the 45,000-d molecules was used in induced bone marrow cells to proliferate, once again the 30,000- and the 17,000-d molecules were secreted. Evidence is also provided that the 45,000-d molecule is produced by the monocyte-macrophage cells, and that it can induce Fc receptors or resident and elicited peritoneal macrophages. The possibility that the production of CSF is cell specific is discussed together with two models to explain the way in which these molecules can participate as proliferative (MGI-1) and differentiative (MGI-2) function in normal myeloid cell differentiation. Finally, a new terminology is proposed to classify this family of molecules.

In Vivo Control of Differentiation of Myeloid Leukemic Cells by Cyclosporine A and Recombinant Interleukin-1α

There are different types of hematopoietic regulatory proteins that regulate the multiplication and differentiation of normal myeloid cells. These different types include four growth-inducing proteins called colony-stimulating factors (CSF), including interleukin-3 (IL-3), or macrophage and granulocyte inducers, type 1 (MGI-1); another type (called MGI-2) that induces myeloid differentiation of normal myeloid cells without inducing myeloid cell multiplication; and interleukin-1 (IL-1), which can act on myeloid precursor cells. Different clones of myeloid leukemic cells can differ in their ability to be induced to undergo terminal cell differentiation by different hematopoietic regulatory proteins. We have now studied the ability of cyclosporine A and recombinant IL-1 alpha to regulate in vivo differentiation of different clones of myeloid leukemic cells that are either susceptible or resistant to induction of differentiation by IL-1 in vitro. The results show that (a) cyclosporine A, like other immune-suppressing compounds such as cyclophosphamide, inhibited in vivo differentiation of myeloid leukemic cells and differentiation was restored by injecting recombinant GM-CSF; (b) recombinant IL-1 alpha induced in vivo terminal differentiation of IL-1-sensitive but not IL-1-resistant clones of myeloid leukemic cells; (c) IL-1 alpha and GM-CSF synergistically induced differentiation in vivo in a GM-CSF-responsive and IL-1-nonresponsive clone of leukemic cells; and (d) IL-1 alpha induced in vivo the rapid production and release into serum of the differentiation-inducing protein MGI-2 as well as the growth-inducing proteins M-CSF and G-CSF.

In vivo control of differentiation of myeloid leukemic cells by cyclosporine A and recombinant interleukin-1 alpha

There are different types of hematopoietic regulatory proteins that regulate the multiplication and differentiation of normal myeloid cells. These different types include four growth-inducing proteins called colony-stimulating factors (CSF), including interleukin-3 (IL- 3), or macrophage and granulocyte inducers, type 1 (MGI-1); another type (called MGI-2) that induces myeloid differentiation of normal myeloid cells without inducing myeloid cell multiplication; and interleukin-1 (IL-1), which can act on myeloid precursor cells. Different clones of myeloid leukemic cells can differ in their ability to be induced to undergo terminal cell differentiation by different hematopoietic regulatory proteins. We have now studied the ability of cyclosporine A and recombinant IL-1 alpha to regulate in vivo differentiation of different clones of myeloid leukemic cells that are either susceptible or resistant to induction of differentiation by IL-1 in vitro. The results show that (a) cyclosporine A, like other immune- suppressing compounds such as cyclophosphamide, inhibited in vivo differentiation of myeloid leukemic cells and differentiation was restored by injecting recombinant GM-CSF; (b) recombinant IL-1 alpha induced in vivo terminal differentiation of IL-1-sensitive but not IL-1- resistant clones of myeloid leukemic cells; (c) IL-1 alpha and GM-CSF synergistically induced differentiation in vivo in a GM-CSF-responsive and IL-1-nonresponsive clone of leukemic cells; and (d) IL-1 alpha induced in vivo the rapid production and release into serum of the differentiation-inducing protein MGI-2 as well as the growth-inducing proteins M-CSF and G-CSF.

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.

The Pattern of HLA-DR and HLA-DQ Antigen Expression on Clonable Subpopulations of Human Myeloid Progenitor Cells

Three subpopulations of human myeloid progenitor cells (CFU-GM) can be distinguished by differences in their kinetics of development; the liquid phase pre-CFU-GM, the day 14 CFU-GM, and the day 7 CFU-GM. The relative cell membrane densities of the HLA-DR and HLA-DQ antigens expressed by the three subpopulations was investigated by comparing the amount of antibody required to deplete bone marrow cell preparations of each cell type. Three separate approaches were used--complement (C') cytotoxicity, antiglobulin/C'-cytotoxicity and immune rosette depletion. Similar results were obtained for all three procedures, although the latter two gave a tenfold greater sensitivity over the standard C'-cytotoxicity method. At saturating anti-HLA-DR antibody concentrations, 85% to 95% of cells within the three myeloid subpopulations were found to express HLA-DR antigens. However, the relative amount of HLA-DR expressed by these subpopulations increased from the pre-CFU-GM to the day 7 CFU-GM. The expression of HLA-DQ antigens was considerably lower and could only be detected by using the more sensitive procedures. Only 50% of day 7 and 14 CFU-GM progenitor cells expressed detectable HLA-DQ antigens, whereas a greater proportion (80%) of the pre-CFU-GM were HLA-DQ positive. The pattern of HLA-DQ expression on these clonable precursors was quite distinct and opposite to the cell membrane density of the HLA-DR antigens. Because these three progenitor cell populations are thought to be linked in differentiation sequence, these results provide indirect support for the hypothesis that HLA class II antigens are implicated in regulatory mechanisms during normal myeloid cell differentiation.

The pattern of HLA-DR and HLA-DQ antigen expression on clonable subpopulations of human myeloid progenitor cells

Three subpopulations of human myeloid progenitor cells (CFU-GM) can be distinguished by differences in their kinetics of development; the liquid phase pre-CFU-GM, the day 14 CFU-GM, and the day 7 CFU-GM. The relative cell membrane densities of the HLA-DR and HLA-DQ antigens expressed by the three subpopulations was investigated by comparing the amount of antibody required to deplete bone marrow cell preparations of each cell type. Three separate approaches were used--complement (C') cytotoxicity, antiglobulin/C'-cytotoxicity and immune rosette depletion. Similar results were obtained for all three procedures, although the latter two gave a tenfold greater sensitivity over the standard C'-cytotoxicity method. At saturating anti-HLA-DR antibody concentrations, 85% to 95% of cells within the three myeloid subpopulations were found to express HLA-DR antigens. However, the relative amount of HLA-DR expressed by these subpopulations increased from the pre-CFU-GM to the day 7 CFU-GM. The expression of HLA-DQ antigens was considerably lower and could only be detected by using the more sensitive procedures. Only 50% of day 7 and 14 CFU-GM progenitor cells expressed detectable HLA-DQ antigens, whereas a greater proportion (80%) of the pre-CFU-GM were HLA-DQ positive. The pattern of HLA-DQ expression on these clonable precursors was quite distinct and opposite to the cell membrane density of the HLA-DR antigens. Because these three progenitor cell populations are thought to be linked in differentiation sequence, these results provide indirect support for the hypothesis that HLA class II antigens are implicated in regulatory mechanisms during normal myeloid cell differentiation.

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.

Expression of myeloid differentiation antigens on normal and malignant myeloid cells.

A series of monoclonal antibodies have been characterized that define four surface antigens (MY3, MY4, MY7, and MY8) of human myeloid cells. They were derived from a fusion of the NS-1 plasmacytoma cell line with splenocytes from a mouse immunized with human acute myelomonocytic leukemia cells. MY3 and MY4 are expressed by normal monocytes and by greater than 90% of patients with acute monocytic leukemia or acute myelomonocytic leukemia, but are detected much less often on other types of myeloid leukemia. MY7 is expressed by granulocytes, monocytes, and 5% of normal bone marrow cells. 79% of all acute myeloblastic leukemia (AML) patients tested (72 patients) express MY7 without preferential expression by any AML subtype. MY8 is expressed by normal monocytes, granulocytes, all peroxidase-positive bone marrow cells, and 50% of AML patients. MY3, MY4, and MY8 define myeloid differentiation antigens in that they are not detected on myeloid precursor cells and appear at discrete stages of differentiation. These antigens are not expressed by lymphocytes, erythrocytes, platelets, or lymphoid malignancies. The monoclonal antisera defining these antigens have been used to study differentiation of normal myeloid cells and malignant cell lines.

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

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.

Indirect induction of differentiation in myeloid leukemic cells by lipid A

Normal myeloid and MGI(+)D(+) clones of myeloid leukemic cells can be induced for Fc and complement component 3 rosettes, lysozme, and mature macrophages and granulocytes by a protein with macrophage- and granulocyte-inducing (MGI) activity, whereas MGI(+)D(-) clones can be induced by this protein for rosettes and lysozme but not mature cells. Lipopolysaccharides (LPS) from different bacteria induced the appearance of rosettes, lysozyme, and macrophages in some MGI(+)D(+) clones but did not induce any of these changes in MGI(+)D(-) clones. Lipid A gave the same results as LPS. Incubation of MGI(+)D(+) cells with LPS also induced an MGI activity detectable in the culture medium. This activity behaved like MGI in inducing (i) rosettes, lysozyme, and mature cells in MGI(+)D(+) leukemic cells including a clone resistant to LPS, (ii) rosettes and lysozyme in MGI(+)D(-) leukemic cells, and (iii) differentiation of normal myeloid cells to mature macrophages and granulocytes. This activity was induced in MGI(+)D(+) cells by LPS before the induction of rosettes or lysozyme. The results indicate that the lipid A portion of LPS indirectly induces differentiation of MGI(+)D(+) myeloid leukemic cells by inducing MGI protein. It is suggested that induction of specific regulatory proteins may be a more general mechanism for the induction of differentiation by surface-acting compounds.