Induction of erythroid differentiation in Friend murine erythroleukemic cells by inorganic selenium compounds.

Inorganic selenium compounds are shown to be inducers of hemoglobin synthesis in malignant murine erythroleukemia (MEL) cells. SeO₂ can induce hemoglobin synthesis at $\text{1}\text{20}$ the concentration of butyric acid and $\text{1}\text{5000}$ the concentration of dimethylsulfoxide (DMSO), two potent inducers of erythroid differentiation in MEL cells. SeO₂ and H₂SeO₃ showed an equivalent capacity to stimulate hemoglobin synthesis in three different MEL cell lines. The incorporation of ³H-glycine into hemoglobin was demonstrated in lysates of SeO₂-induced MEL cells.     

Changes in acid proteolytic activity during differentiation of murine erythroleukemic cells

Proteolytic activity was measured in murine erythroleukemic 745 cell line grown in culture, before and after the addition of agents which promote differentiation. The 36,000 × g soluble fraction of the cells degraded [¹⁴C]globin with maximal activity at pH 3.6, while the insoluble fraction failed to degrade [¹⁴C]globin within a pH range of 2.5–9.0.The acid protease activity in the soluble fraction of the undifferentiated murine erythroleukemic cells increased during the first 2 days in culture and remained constant during the following 4 days. We suggest that this activity resides in the lysosomes since it migrates together with the lysosomal marker α-mannosidase on colloidal silica gradients, shows maximum activity at acid pH and is sensitive towards inhibition by pepstatin. Induced differentiation of the cells by dimethyl sulfoxide, butyric acid or hexamethylene bisacetamide was concomitantly associated with a marked reduction in protease activity and the accumulation of hemoglobin within the cells. In contrast, in a non-inducible variant of 745 cell line DMSO failed to affect proteolysis. It is suggested that in murine erythroleukemic cells changes in acid protease activity are associated with the cellular triggered by chemical inducers.     

Membrane protein redistribution during differentiation of cultured human erythroleukemic cells.

Human erythroleukemic (K562) cells differentiate along the erythroid differentiation pathway in vitro when 0.05 mM hemin is included in the growth medium. In the presence of the inducer the cells continue to proliferate and, after a delay of 24 to 48 h, start to synthesize hemoglobin. However, during differentiation, no changes in the major cell surface proteins were detected using lactoperoxidase-catalyzed iodination, and no change in the synthesis of spectrin, the major cytoskeletal protein of the mature erythrocyte, was detected by specific immune precipitation. Despite this absence of major changes in cell surface proteins, profound changes take place in the organization of the cell membranes. A process similar but not identical to the enucleation observed in erythroid differentiation in vivo occurs in which a smooth-surfaced cell, about 10 micrometers in diameter, is divided from the nucleus-containing part of the cell. With the exception of ribosomes, these reticulocyte-like cells contain no organelles when examined by transmission electron microscopy, but contain much of the parent cell's hemoglobin, spectrin, and glycophorin.     

Poly(A) polymerase activity during cell cycle and erythropoietic differentiation in erythroleukemic mouse spleen cells.

Poly(A) polymerase activity was studied in lysates of cultured murine erythroleukemic cells (Friend cells). Incorporation of ATP into acid-precipitable products is dependendent on the presence of Mn2+ or Mg2+ and of an RNA primer. The reaction is specific for ATP as the substrate (KM=290 290 micron, it is not inhibited by actinomycin D and only slightly interferred with by ethidium bromide. Cordycepin 5'-triphosphate and sodium pyrophosphate inhibit the enzyme activity. The chain length of the products of the reaction is dependent on the primer concentration and reaches up to 30 nucleotides. Poly(A) polymerase activity is low in resting (G1 phase) cells 75 nmol ATP incorporated/h per 10(6) cells) and increases to a level about twice as high in early S phase of the cell cycle. A possible model for regulation of enzyme activity is discussed. Polymerase activity in the early phase of erythropoietic differentiation of the cells induced by butyric acid does not show any difference in comparison to untreated controls. A decrease in enzyme activity to levels characteristic for cells in G1 phase accompanies shutdown of cell growth in the course of the ongoing differentiation. Analysis of the DNA content of the cells revealed that erythropoietic differentiation of Friend cells induced by butyric acid is characterized by arrest of the cells in G1 phase of the cell cycle. Poly(A) polymerase activity in erythroleukemic cells is thus controlled only by the phase of the cell cycle; it is not affected by changes in gene expression during erythroid differentiation.     

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.     

Relationship of the decreases in protein synthesis and intracellular Na+ during friend murine erythroleukemic cell differentiation

The earliest known ionic event during Friend murine erythroleukemic (MEL) cell differentiation along the erythroid pathway is a 45% drop in intracellular sodium concentration ([Na+]i) due to a decrease in Na+ influx (Lannigan, D. A., and Knauf, P. A. (1985) J. Biol. Chem. 260, 7322-7324). We have analyzed the mechanism of the decreased Na+ influx. The Na+ influx in uninduced cells was insensitive to dimethylamiloride, bumetanide, and diisothiocyanostilbene disulfonate. The intracellular pH (pHi) did not change up to 15 h after dimethyl sulfoxide induction, at which time Na+ influx has decreased by approximately 40%; thus, the decrease in Na+ influx is not coupled to a change in pHi. A substantial amount of the decrease in Na+ influx seems to result from a drop in amino acid-dependent Na+ transport. This reduction in amino acid-dependent Na+ influx reflects a decrease in net Na+ influx rather than solely in Na+/Na+ exchange and can account for an appreciable portion of the reduction in [Na+]i seen during differentiation. The drop in amino acid-dependent Na+ influx could not be explained by membrane depolarization but was correlated with a decrease in protein synthesis. Inhibition of protein synthesis in uninduced cells by cycloheximide also caused a decrease in Na+ influx. We conclude that during differentiation the reduction in protein synthesis decreases amino acid-dependent Na+ influx which in turn causes a drop in [Na+]i leading to a reduction in the Na+/K+ pump rate.     

Differential expression of alpha- and beta-globin genes during differentiation of cultured erythroleukemic cells.

Murine erythroleukemic cells induced to differentiate in vitro with dimethylsulfoxide provide a model for events involved in the regulated expression of the globin genes. Here we examine alpha- and beta-globin gene expression in such cells which contain no detectable globin RNA prior to induction. To quantitate alpha- and beta-globin RNAs in cellular RNA samples by molecular hybridization techniques, highly radioactive complementary DNAs were synthesized using mouse alpha- and beta-globin RNAs purified by formamide gel electrophoresis. Maximally induced erythroleukemic cells and mouse reticulocytes contain nearly equal relative amounts of alpha- and beta-globin RNA. During the period in which globin RNA accumulates in differentiating erythroleukemic cells, however, alpha- and beta-globin RNAs are not present in equivalent amounts. alphaRNA is present in substantial excess (alpha/beta ratio 3.7) early in induction, and the alpha/beta RNA ratio progressively approaches 1 as differentiation proceeds further. These observations directly suggest that the alpha- and beta-globin genes are differentially expressed during cellular differentiation and raise questions as to how relative expression of globin genes is controlled during normal development.     

Relationship of 25-hydroxyvitamin D3 side chain structure to biological activity.

27-nor-25-Hydroxyvitamin D3, 26,27-bisnor-25-hydroxyvitamin D3, and 22-27-hexanor-20-hydroxyvitamin D3 and the corresponding 5,6-trans isomers have been synthesized. All compounds were tested for their ability to induce intestinal calcium transport and bone calcium mobilization in normal and anephric rats. The 27-nor- and 26,27-bisnor-25-hydroxyvitamin D3 analog are capable of stimulating intestinal calcium transport and bone calcium mobilization in normal rats but are 10 to 100 times less active than 25-hydroxyvitamin D3. Although these analogs are inactive in anephric rats, their corresponding 5,6-trans isomer are capable of stimulating both intestine and bone activity in these animals. The 22-27-hexanor-20-hydroxyvitamin D3 and its corresponding 5,6-trans isomer are incapable of stimulating either intestinal calcium transport or bone calcium mobilization. These results suggest that minor alterations in the side chain significantly decrease the biopotency of 25-hydroxyvitamin D3. Since these analogs are biologically active in normal but not in anephric animals, it appears that the kidney 1alpha-hydroxylation is necessary for activity. Since 22-27-hexanor=20-hydroxyvitamin D3 and its corresponding 5,6-trans analog are biologically inactive, it is likely that at least part of the side chain is necessary for 25-hydroxyvitamin D3 to stimulate intestinal calcium transport and bone calcium mobilization.     

Modulation of murine erythroleukemic cell differentiation by inhibitors of polyamine biosynthesis

The differentiation of murine erythroleukemic cells induced by hexamethylene bisacetamide is shown to be differently affected by two inhibitors of polyamine biosynthesis. Methyl glyoxal bis(guanyl hydrazone) (inhibitor or S-adenosyl methionine decarboxylase) inhibited this differentiation process. By using a novel experiment protocol the inhibitory effect of this drug on the induced differentiation was dissociated from pleiotropic effects on cell growth. Methyl glyoxal bis(guanyl hydrazone) only inhibited the induced differentiation if present during the first 6 h of culture of the cells with the inducer. No effect on the induced differentiation was observed if the drug was added to the culture medium 6 h after the inducer. alpha-Difluoro methylornithine (inhibitor of ornithine decarboxylase) stimulated the differentiation of these cells. Polyamine analysis demonstrated that alpha-difluoro methylornithine increased the rapidity and the amplitude of the changes in intracellular polyamines associated with this induced differentiation. The presence of methyl glyoxal bis(guanyl hydrazone) during the first 3 h with the inducer was sufficient to produce opposing changes in the intracellular polyamines. These results suggest that changes in either intracellular polyamines or the activities of polyamine biosynthetic enzymes play a regulatory role in the differentiation process induced in murine erythroleukemic cells by hexamethylene bisacetamide.     

Erythroid and megakaryocytic differentiation of K562 erythroleukemic cells by monochloramine

Abstract

The induction of leukemic cell differentiation is a hopeful therapeutic modality. We studied the effects of monochloramine (NH₂Cl) on erythroleukemic K562 cell differentiation, and compared the effects observed with those of U0126 and staurosporine, which are known inducers of erythroid and megakaryocytic differentiation, respectively. CD235 (glycophorin) expression, a marker of erythroid differentiation, was significantly increased by NH₂Cl and U0126, along with an increase in cd235 mRNA levels. Other erythroid markers such as γ-globin and CD71 (transferrin receptor) were also increased by NH₂Cl and U0126. In contrast, CD61 (integrin β3) and CD42b (GP1bα) expression, markers of megakaryocytic differentiation, was increased by staurosporine, but did not change significantly by NH₂Cl and U0126. NH₂Cl retarded cell proliferation without a marked loss of viability. When ERK phosphorylation (T202/Y204) and CD235 expression were compared using various chemicals, a strong negative correlation was observed (r = ?0.76). Paradoxically, NH₂Cl and staurosporine, but not U0126, induced large cells with multiple or lobulated nuclei, which was characteristic to megakaryocytes. NH₂Cl increased the mRNA levels of gata1 and scl, decreased that of gata2, and did not change those of pu.1 and klf1. The changes observed in mRNA expression were different from those of U0126 or staurosporine. These results suggest that NH₂Cl induces the bidirectional differentiation of K562. Oxidative stress may be effective in inducing leukemic cell differentiation.     

四氢蒽醌类化合物结构及其生物活性研究进展

四氢蒽醌类化合物是一类比较少见的天然结构,以微生物次生代谢产物居多,少量来源于植物,具有细胞毒活性、抗菌活性、抗疟原虫等生物活性。本文主要从四氢蒽醌类化合物及其衍生物的结构和生物活性两方面来对天然四氢蒽醌化合物进行综述,共综述了54个四氢蒽醌类化合物,45个来源于微生物,9个来源于植物南山花的根中,其中altersolanol A具有很好的抗肿瘤活性,是一个有很大吸引力的抗癌先导化合物。通过对四氢蒽醌类化合物的综述,为四氢蒽醌类化合物的进一步研究和开发提供依据。     

Transfer of human globin genes to erythroleukemic mouse cells.

Thymidine kinase negative (TK-) Friend cells were transformed with recombinant molecules carrying human globin genes and the thymidine kinase gene of herpes simplex virus type 1 DNA. Transformation frequencies of 1 transformant/microgram donor DNA/1 x 10(6) cells were obtained by standard procedures and this was increased 20- to 30-fold by treating recipient cells with dimethyl sulfoxide or glycerol. Transformed cell lines expressed thymidine kinase activity of viral origin as determined by its insensitivity to 0.2 mM dTTP and electrophoretic mobility in polyacrylamide gels. The physical status of donor DNA in the transformed cells was examined in Hirt precipitates and supernatants by Southern blot hybridization and spot hybridization techniques. This analysis showed that most donor sequences were present in a circular or concatenate configuration, but also was suggestive of some donor sequences being integrated into high molecular weight DNA. Expression of human globin genes and particularly the epsilon-globin gene in the transformed Friend cells was studied by Northern blot hybridization analysis.