Retinoid-regulated gene expression in normal and leukemic myeloid cells

Physiological concentrations of retinoic acid can induce acute alterations in the expression of the enzyme tissue transglutaminase in cultured macrophages. The induction of this enzyme offers a probe to study the mechanism of retinoid action in both normal and leukemic cells.     

Production of a colony-stimulating factor following differentiation of leukemic myeloblasts to macrophages

Leukemic cells in the myeloblastic stage from a murine myeloid leukemia cell line (M1) were induced to differentiate to macrophages by lipopolysaccharide (LPS) from Gram-negative bacteria. A granulocyte-macrophage colony-stimulating factor (CSF) was produced only during differentiation. After induction of differentiation, the continued presence of LPS was necessary to stimulate the macrophages to release CSF. In contrast, a macrophage cell line (Mm-1) derived from the M1 line produced CSF without LPS-stimulation, but CSF release was stimulated by the presence of LPS.     

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.     

Inducers of leukemic cell differentiation cause down-regulation of RB gene expression.

Expression of the retinoblastoma (RB) tumor suppressor gene during cell differentiation induced by dimethyl sulfoxide or sodium butyrate was studied in HL-60 human promyelocytic leukemia cells. As cells progressed through the cell cycle, the amount of RB protein per cell increased with homeostasis maintained, so that the amount of RB protein relative to the total cell mass remained almost constant. Dimethyl sulfoxide was used to induce these promyelocytic leukemia cells to undergo terminal differentiation into mature myeloid cells. There was an early reduction in the RB protein expressed per cell. The reduction in expression was similar for cells in all cell cycle phases. There was also progressively reduced expression at later times as cells terminally differentiated. This was compared to the case in which sodium butyrate was used to induce the differentiation of HL-60 cells into mature monocytic cells. An early reduction in RB protein expression per cell also occurred. It occurred for cells in all cell cycle phases as well. Thus, the induced differentiation of HL-60 cells along either the myeloid or the monocytic differentiation lineage involves an early reduction in RB expression, which is common to both pathways. The reduction anteceded proliferative arrest or differentiation. In both cases, the final, resulting G0-differentiated cells had less RB protein per cell than the proliferating, immature, leukemic precursor cells.     

Terminal erythroid differentiation in normal and leukemic mouse cells.

Friend virus-transformed murine erythroleukemic cells (FL cells) have been used as an in vitro model for the study of the expression of the genetic program involved in the final stages of erythroid differentiation. Treatment of the FL cells with chemical inducers such as dimethylsulfoxide results in their differentiation from 'pro-erythroblasts' to orthochromatic normoblasts and the appearance of several erythroid markers including hemoglobin, enzymes of the heme pathway, heme, glycophorin, and spectrin. These markers appear in an ordered sequence, suggesting that two genetic programs are involved in the erythroid differentiation of the cells. Preliminary studies with erythropoietin-stimulated fetal liver cultures in vitro suggest that the same is true for normal erythroid differentiation.     

T cell receptor delta gene organization and expression in normal and leukemic natural killer cells

In peripheral blood most NK activity is mediated by CD3- cells with large granular lymphocyte morphology which cannot be assigned to a specific hemopoietic lineage. In accordance with previous studies we have analyzed the organization of the TCR delta gene, which rearranges early in thymic ontogeny, in normal NK cells, and in granular lymphocytes proliferative disorders (GLPD), in an effort to further define their relationship to the T cell differentiation pathway and to identify a possible marker of clonality for CD3- GLPD. The alpha/delta locus was rearranged in five cases of CD3+ GLPD with a biallelic deletion of the C delta region, suggesting V-J alpha rearrangement, whereas CD3- GLPD and normal CD3- NK cells had the delta gene in germ-line configuration, but surprisingly expressed high levels of TCR delta-related mRNA. On the basis of this finding and of the presence of truncated TCR-beta and CD3-epsilon mRNA, we are led to speculate on a possible ontogenic relationship of NK cells to the T cell differentiation pathway at stages preceding TCR gene rearrangement.     

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.     

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.     

Molecular control of cell differentiation and programmed cell death during digit development

During the hand plate development, the processes of cell differentiation and control of cell death are relevant to ensure a correct shape of the limb. The progenitor cell pool that later will differentiate into cartilage to form the digits arises from undifferentiated mesenchymal cells beneath the apical ectodermal ridge (AER). Once these cells abandon the area of influence of signals from AER and ectoderm, some cells are committed to chondrocyte lineage forming the digital rays. However, if the cells are not committed to chondrocyte lineage, they will form the prospective interdigits that in species with free digits will subsequently die. In this work, we provide the overview of the molecular interactions between different signaling pathways responsible for the formation of digit and interdigit regions. In addition, we briefly describe some experiments concerning the most important signals responsible for promoting cell death. Finally, on the basis that the interdigital tissue has chondrogenic potential, we discuss the hypothesis that apoptotic-promoting signals might also act as antichondrogenic factors and chondrogenic factors might operate as anti-apoptotic factors.     

Molecular dissection of gp130-dependent pathways in hepatocytes during liver regeneration

Interleukin-6 (IL-6) via its signal transducer gp130 is an important mediator of liver regeneration involved in protecting from lipopolysaccharide (LPS)-induced liver injury after partial hepatectomy (PH). Here we generated mice either defective (Delta) in hepatocyte-specific gp130-dependent Ras or STAT activation to define their role during liver regeneration. Deletion of gp130-dependent signaling had major impact on acute phase gene (APG) regulation after PH. APG expression was blocked in gp130-DeltaSTAT animals, whereas gp130-DeltaRas mice showed an enhanced APG response and stronger SOCS3 regulation correlating with delayed hepatocyte proliferation. To define the role of SOCS3 during hepatocyte proliferation, primary hepatocytes were co-stimulated with IL-6 and hepatocyte growth factor. Higher SOCS3 expression in gp130-DeltaRas hepatocytes correlated with delayed hepatocyte proliferation. Next, we tested the impact of LPS, mimicking bacterial infection, on liver regeneration. LPS and PH induced SOCS3 and APG in all animal strains and delayed cell cycle progression. Additionally, IL-6/gp130-dependent STAT3 activation in hepatocytes was essential in mediating protection and thus required for maximal proliferation. Unexpectedly, oncostatin M was most strongly induced in gp130-DeltaSTAT animals after PH/LPS-induced stress and was associated with hepatocyte proliferation in this strain. In summary, gp130-dependent STAT3 activation and concomitant SOCS3 during liver regeneration is involved in timing of DNA synthesis and protects hepatocyte proliferation during stress conditions.     

Molecular dissection of regulated secretory pathways in human gastric enterochromaffin-like cells: an immunohistochemical analysis

Enterochromaffin-like (ECL) cells regulate gastric acid secretion through vesicular release of histamine. Until now, the molecular machinery of human ECL cells involved in the formation and release of vesicles is largely unknown. We analyzed tissue samples obtained from normal human gastric mucosa (n=4) and ECLomas (n=5) immunohistochemically using the APAAP method or double immunofluorescence confocal laser microscopy. Human pheochromocytomas (n=5) were investigated in parallel and compared to ECL cells. Secretory pathways were characterized using antibodies specific for marker proteins of large dense-core vesicles (LDCVs; islet cell antigen 512, chromogranin A, pancreastatin, and vesicular monoamine transporter 2) and small synaptic vesicle (SSV) analogues (synaptophysin). Tissues were also analyzed for expression of the peptide hormone processing enzymes, carboxypeptidase E and prohormone convertase 1, as well as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, 25-kDa synaptosome-associated protein (SNAP25), syntaxin, and synaptobrevin. Immunoreactivity for markers of LDCVs and SSV analogues were detected in normal ECL cells and ECLomas. Both tissues also showed expression of carboxypeptidase E and prohormone convertase 1. Analysis of vesicular SNARE (v-SNARE) and target membrane SNARE (t-SNARE) proteins revealed the presence of SNAP25, syntaxin, and synaptobrevin in normal and neoplastic ECL cells. Our data suggest that ECL cells possess the two vesicle types of regulated neuroendocrine secretory pathways, LDCVs and SSV analogues. Since ECL cells also contain typical SNARE proteins, the molecular machinery underlying secretory processes in this cell type appears to be identical to the secretory apparatus of neuroendocrine cells and neurons. In addition, our findings suggest that the secretory apparatus of ECL cells is maintained during neoplastic transformation. Accepted: 10 June 1999     

Molecular properties of a galaptin and its activity in normal and leukemic erythroid tissue

Recently, the generic term “galaptins” was proposed for the group of low molecular weight, acidic, β-galactoside-specific protein lectins that have been isolated from a wide variety of animal tissues and are thought to have a role in cell-cell recognition and adhesion. A molecule of this type, called erythroid developmental agglutinin (EDA), has been isolated from rabbit bone marow where it seems to mediate the intererythroblast adhesion seen in erythroblastic islands during erythropoiesis in vivo. Here, we show that after purification, EDA shows 95%-100% Coomassie blue staining as a single component on electrophoresis in native, urea, and SDS polyacrylamide gels and electrofocuses as a single band at pH 5.6. EDA has a subunit molecular weight of 13,000 in SDS gels and, unlike the majority of other galaptins, which arc dimeric, native EDA is monomeric in solution. Another monomeric galaptin, chicken lactose lectin II, has been described recently, and it therefore seems that there may be two classes of galaptin distinguishable by their aggregation state in solution. We have previously reported that EDA agglutinates rabbit erythroblasts in vitro and that this reaction is inhibited by β-galactoside-containing sugars and by anti-EDA Fab fragments suggesting that EDA bridges directly between cell surface glycoproteins. The insensitivity of this reaction to cooling, or to the disruption of cellular metabolism or the cytoskeleton demonstrated here further supports this hypothesis. EDA-mediated erythroblast agglutination was also shown to be independent of divalent cations. Since galaptins are thought to be important in cohesion between normal cells, the possibility that EDA is not active in leukemic erythroid tissue was examined. The murine erythroleukemia cell line (MELC) provided an excellent system for this study since MELC are thought to be derived from an erythroid committed cell transformed at an early stage of development and can be induced by a number of chemical agents to differentiate terminally along the erythroid developmental pathway in culture. EDA of rabbit origin was found to agglutinate mouse erythroblasts in vitro and was used to investigate the response of MELC to EDA. It was found that the transformed cells were not readily agglutinated by EDA but on induction, and the concomitant loss of many of their transformed characteristics, MELC gained aggregation competence for EDA. The possible causes of these differences are discussed.     

Induced differentiation of avian myeloblastosis virus-transformed myeloblasts: phenotypic alteration without altered expression of the viral oncogene.

Cells of a clone of avian myeloblastosis virus-transformed myeloblasts were induced to differentiate to adherent myelomonocytic cells by treatment with lipopolysaccharide. These adherent cells were subcultured and maintained as a line for more than 6 months with lipopolysaccharide present. Cells of this line were induced to differentiate to nondividing macrophage-like cells by the addition of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. In this way, the following homogeneous cell populations representing three distinct stages of myeloid differentiation were obtained: I, actively dividing myeloblasts that grew in suspension: II, actively dividing adherent cells; and III, fully differentiated nondividing cells resembling macrophages. When the expression of v-myb (the oncogene of avian myeloblastosis virus) was examined in cells of these three differentiation stages, it was found that the protein encoded by v-myb (p45v-myb) continued to be synthesized in similar quantities and showed no obvious alteration (assessed by partial proteolytic digestion and two-dimensional gel electrophoresis) during differentiation. These results show that cells transformed by v-myb can be induced to differentiate without affecting the expression of v-myb and imply that, during differentiation, the effect of v-myb is suppressed by a mechanism other than altered expression of the oncogene.     

Separation of leukemic myeloblasts and splenic lymphocytes based upon differences in light scatter profile.

Cell sorting based upon differences in light scatter profile was carried out to sort cells which differ in size. This technique permitted the acquisition of subpopulations of murine spleen cells enriched for leukemic myeloblasts or lymphocytes. Morphologic assessment of the sorted populations was confirmed by malignancy assay $\text{in}$$\text{vivo}$. The same technique has been applied to human leukemic cells and significant enrichment for proliferating and quiescent cells was accomplished.