DNA synthesis is not required for the commitment of murine erythroleukemia cells

We have assessed the relationship between DNA synthesis and the differentiation of MEL cells induced by DMSO. Under conditions where the rate of incorporation of ³H-deoxyadenosine into DNA was inhibited by 99%, the rate at which MEL cells become committed to terminal erythroid differentiation was identical to that of a culture treated with inducer alone. We conclude that commitment of MEL cells does not require concomitant DNA synthesis.     

Ribavirin induced differentiation of murine erythroleukemia cells

Summary The synthetic nucleoside, ribavirin (1--D-ribofuranosyl-1,2,4-triazole-3-carboxamide), a broad spectrum antiviral agent currently being tested in clinical studies with AIDS patients; and mycophenolic acid, a non-nucleoside inhibitor of inosinate (IMP) dehydrogenase, are effective inducers of terminal differentiation of Friend virus transformed murine erythroleukemia cells. The inhibition of cell division and the induced maturation produced by these agents appears to be a consequence of inhibition of IMP dehydrogenase, since growth inhibition is reversed and differentiation is prevented by the simultaneous exposure of cells treated with the agents to exogenous guanine or guanosine, which circumvents the effects of blockage of IMP dehydrogenase. However, while the effects mycophenolic acid, a pure IMP dehydrogenase inhibitor with no other biochemical effects, were completely reversed by guanine salvage supplies, cells exposed to ribavirin responded in a different manner. At levels of guanine salvage supplies below 50 M, growth inhibition and cell differentiation were partially reversed. At salvage supply concentrations greater than 50 M, while differentiation was completely blocked, the toxicity of ribavirin was increased and cell division was greatly diminished. These results indicate additional biochemical effects for ribavirin unrelated to the inhibition of IMP dehydrogenase, which may be related to its antiviral properties.     

Messenger RNA changes during differentiation of murine erythroleukemia cells

During differentiation of murine erythroleukemia cells, the levels of certain mRNA were observed to change. To characterize the various patterns of changes that occur during differentiation, cDNA libraries made from RNA isolated from uninduced and differentiating cells were screened with labeled cDNA or RNA labeled in vivo for different periods of time. cDNA clones that corresponded to individual mRNAs whose level remained constant, increased, or decreased during differentiation were identified. These clones were used to analyze Northern blots containing RNA from uninduced and differentiated cells. A number of characteristic changes in individual mRNAs in differentiating murine erythroleukemia cells could be identified, such as no change, increase in concentration, increase in concentration and slight change in size, decrease in concentration, decrease in concentration and change in size, appearance of new band(s) of entirely different size, and change in relative concentrations of two related mRNAs. Measurements of rates of mRNA synthesis and degradation suggest that both parameters change during differentiation and that these changes are instrumental in establishing cellular concentration of specific mRNAs. It seems that the changes in mRNA stability observed in differentiating murine erythroleukemia cells may be associated with changes in the primary structure of the transcribed portion of mRNA. The observation that specific mRNA synthesized before and after induction may have very different stabilities at the same point in differentiation supports this hypothesis.     

Nuclear translocation of active AKT is required for erythroid differentiation in erythropoietin treated K562 erythroleukemia cells

Erythroid differentiation of human erythroleukemia cell line K562 induced by erythropoietin is a complex process that involves modifications at nuclear level, including nuclear translocation of phosphatidyl-inositol 3-kinase. In this work we show that erythropoietin stimulation of K562 cells can induce nuclear translocation of active Akt, a downstream molecule of the phosphatidyl-inositol 3-kinase signaling pathway. Akt shows a peak of activity in whole cell homogenates at earlier stage when compared to the nucleus, which shows a peak delayed of 10 min. Akt increases its intranuclear amount and activity rapidly and transiently in response to EPO. Almost all Akt kinase that translocates to the nucleus shows a marked phosphorylation on serine 473. Nuclear enzyme translocation is blocked by the phosphatidyl-inositol 3-kinase inhibitor Ly294002 or Wortmannin. The specific Akt pharmacological inhibitor VI, VII and VIII that act as blocking enzyme activation inhibited translocation as well, whereas Akt inhibitor IX, that inhibits Akt activity, did not block Akt nuclear translocation. When cells were treated by means of siRNA sequences or with the Akt inhibitors the differentiation process was arrested, thus showing the requirement of the nuclear translocation of the active enzyme to differentiate. These findings strongly suggest that the intranuclear translocation of active Akt kinase represents an important step in the signaling pathway that mediates erythropoietin-induced erythroid differentiation.     

Hedgehog signaling is required for the differentiation of ES cells into neurectoderm

Mouse embryonic stem cells can differentiate in vitro into cells of the nervous system, neurons and glia. This differentiation mimics stages observed in vivo, including the generation of primitive ectoderm and neurectoderm in embryoid body culture. We demonstrate here that embryonic stem cell lines mutant for components of the Hedgehog signaling cascade are deficient at generating neurectoderm-containing embryoid bodies. The embryoid bodies derived from mutant cells are also unable to respond to retinoic acid treatment by producing nestin-positive neural stem cells, a response observed in cultures of heterozygous cells, and contain cores apparently arrested at the primitive ectoderm stage. The mutant cultures are also deficient in their capacity to differentiate into mature neurons and glia. These data are consistent with a role for Hedgehog signaling in generating neurectoderm capable of producing the appropriate neuronal and glial progenitors in ES cell culture.     

Sulfhydryl groups and the differentiation of murine erythroleukemia cells.

The importance of cysteine and sulfhydryl groups has been demonstrated in relation to the differentiation and respiration of Friend erythroleukemia cells (FLC). The respiratory rate of undifferentiated FLC was higher basally (5.06 ± 0.16 vs. 3.10 ± 0.09 nmoles 02/min/10⁶ cells) and was further 70% stimulated by addition of cysteine, whereas DMSO-induced differentiated cells were insensitive. A sulfhydryl blocking agent (PCMS) was capable of maintaining the differentiated state of FLC cultured in the absence of DMSO and this effect appeared to be reversible upon removal of the PCMS.     

Characterization of cellular receptors for erythroid differentiation factor on murine erythroleukemia cells

Erythroid differentiation factor (EDF), which is structurally related to transforming growth factor-beta family and induces differentiation of murine erythroleukemia cell clone F5-5, has been labeled with 125I to characterize its interaction with cellular receptors. Binding of 125I-EDF to F5-5 cells is time- and temperature-dependent, specific, saturable, and reversible. Transforming growth factor-beta 1 has no significant effects on growth of F5-5 cells and binding of 125I-EDF to F5-5 cells. Scatchard analysis of the binding data indicated that F5-5 cells have a single class of binding sites (3,200/cell) with an apparent Kd of 3.1 X 10(-10) M. Affinity cross-linking experiments demonstrated three radiolabeled components of 140,000, 76,000, and 67,000 daltons under both reducing and nonreducing conditions. Labeling of these three components has been inhibited by incubation of the cells with excess unlabeled EDF. These results imply molecular weights of 115,000, 51,000, and 42,000 for the EDF receptors on this cell line.     

A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly

Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III1-7). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III1 (FNDeltaIII1), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III9-10 to a position close to the NH2-terminal assembly domain, as well as a large deletion spanning repeats III4-7, had no effect on assembly. In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site. Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.     

Extracellular matrix is required for the survival and differentiation of transplanted hepatic progenitor cells

Engelbreth-Holm-Swarm (EHS) gel has been reported to maintain the mature hepatocyte phenotypes in primary cultured hepatocytes. We investigated the effect of EHS gel on the differentiation of fetal liver cells, which contain stem/progenitor cells. The isolated fetal liver cells cultured on EHS gel formed a spherical shape and increased liver-specific gene expressions compared with cells cultured on collagen. The hepatic progenitor cells that were transplanted subcutaneously to BALB/c nude mice could survive and express hepatocyte marker alpha-fetoprotein when the cells were suspended with EHS gel. These findings demonstrate that EHS gel supports cytodifferentiation from immature progenitor cells to hepatocytes and maintain its differentiated phenotypes in vitro and in vivo.     

Distribution of the inducer of differentiation bis-acetyl-diaminopentane in murine erythroleukemia cells

Exposure of murine leukemia cells in culture to bis-acetyl-diaminopentane (BADP) caused erythroid maturation as measured by the accumulation of hemoglobin in treated cells. The appearance of differentiated cells in cultures exposed to BADP occurred 18 to 20 hours earlier than in those treated with dimethylsulfoxide (DMSO), a standard inducer of differentiation in this system. Studies with [³H]BADP indicated the occurrence of relatively rapid association of the inducer with cells, and subsequent linear accumulation. Fractionation of cellular components and measurement of radioactivity from BADP therein demonstrated that this agent preferentially associates with a fraction enriched for plasma membrane. In addition, [³H]BADP was capable of binding to the plasma membrane-enriched fraction isolated from murine erythroleukemia cells as measured by gel filtration. These findings support the concept that interaction of inducers of murine erythroleukemia differentiation such as BADP with components of the surface membrane may be important in the cascade of events that lead to the erythroid maturation of these leukemic cells.     

Changes in proteinase activities during the differentiation of murine erythroleukemia cells

Changes in intracellular proteinase activities were examined during DMSO-induced differentiation of murine erythroleukemia cells. Suc-APA-MCA hydrolytic activity was significantly decreased, and apparent ATP-dependent multicatalytic proteinase activity was also decreased with MEL cell differentiation. Cathepsin B and L activity was mainly present in the microsomal fraction of control cells, but a part of this activity had shifted to the lysosomal fraction of differentiated cells. With the translocation of cathepsin B from the microsomal to the lysosomal fraction, the pro-enzyme form of cathepsin B was converted into the mature enzyme. These results suggest that the lysosomal pathway contributes to the degradation of specific proteins with cell differentiation.     

Notch-1 inhibits apoptosis in murine erythroleukemia cells and is necessary for differentiation induced by hybrid polar compounds

Strikingly increased expression of notch-1 has been demonstrated in several human malignancies and pre-neoplastic lesions. However, the functional consequences of notch-1 overexpression in transformed cells remain unclear. We investigated whether endogenously expressed notch-1 controls cell fate determination in mouse erythroleukemia (MEL) cells during pharmacologically induced differentiation. We found that notch-1 expression is modulated during MEL cell differentiation. Premature downregulation of notch-1 during differentiation, by antisense S-oligonucleotides or by enforced expression of antisense notch-1 mRNA, causes MEL cells to abort the differentiation program and undergo apoptosis. Downregulation of notch-1 expression in the absence of differentiation inducer increases the likelihood of spontaneous apoptosis. We conclude that in MEL cells, endogenous notch-1 expression controls the apoptotic threshold during differentiation and growth. In these cells, notch-1 allows differentiation by preventing apoptosis of pre-committed cells. This novel function of notch-1 may play a role in regulating apoptosis susceptibility in notch-1 expressing tumor cells.     

Terminal differentiation of murine erythroleukemia cells: physical stabilization of end- stage cells

An important limitation in the use of the murine erythroleukenia (MEL) cell system as an in vitro system for the study of terminal erythroid differentiation has been the inability to produce significant numbers of cells which represent the end-point of the pathway in vitro. We show here that a major reason for the failure to observe end-stage cells in vitro is that such cells are physically unstable under the standard culture conditions used for MEL cell differentiation. Modification of these culture conditions by the addition of either bovine serum albumin or Ficoll leads to physical stabilization of end-stage cells. Under such culture conditions, uniform cultures of terminally differentiated MEL cells with morphological characteristics similar to those of normal mouse orthochromatophilic erythroblasts and reticulocytes are observed. Examination of physical and biochemical parameters of these cell populations give values which are similar to values characteristic of mouse reticulocytes. A physically stabilized MEL cell shows a narrow cell volume distribution with an average value of approximately 100 mum(3), similar to the cell volume distribution observed for mouse reticulocytes, while a typical MEL cell culture treated with DMSO but without a stabilizing agent exhibits a broader, more heterogeneous cell volume distribution with an average value of approximately 500 mum(3). Globin mRNA levels and levels of globin synthesis reach values almost equal to those in mouse reticulocytes in cultures of physically stabilized MEL cells while differentiating cultures not treated with a stabilizing agent reach substantially lower values for these parameters. We suggest that the ability to produce populations of MEL cells which undergo complete terminal erythroid differentiation in vitro will allow the analysis of the molecular mechanisms which control the terminal stages of the erythroid differentiation process.     

Serum-free, defined medium for the growth and differentiation of murine erythroleukemia cells.

Murine erythroleukemia (MEL) cells are frequently employed to study both cell growth and erythroid differentiation. Although these cells are easily cultured and induced to differentiate, they are routinely maintained in a medium that contains 10%-15% fetal bovine serum. Because of the variability between different lots and the cost of serum, it was desirable to define a serum-free medium in which to culture MEL cells. In the present work, a totally serum-free, defined medium is described that supports both normal cell growth and dimethyl sulfoxide induced differentiation in the two MEL cell lines examined (DS-19 and 270). A variety of hormones and biological compounds are examined in this medium to determine their effects on growth and differentiation. This medium does not support the growth of the mouse hepatoma cell line.     

Erythroid differentiation of cultured murine erythroleukemia cells by the spermine analogue canavalmine

Canavalmine, an analogue of spermine, induced erythroid differentiation of murine erythroleukemia cells 745A, as evidenced by benzidine staining and heme content of cultured cells. Benzidine-positive cells synthesizing hemoglobin appeared on day 4 after addition of 250 μM canavalmine. The canavalmine-induced cell differentiation was inhibited by the addition of agents which alter the structure of the cell membrane, such as local anesthetics (procainamide and lidocaine) or Ca²⁺ antagonists (nifedipine and verapamil) at dosages not toxic for the cell growth. Canavalmine did not significantly affect the levels of conjugated polyamines in the acid-insoluble fraction of the cells. In contrast, the level of free spermidine in the acid-soluble fraction greatly decreased during the 18 h after canavalmine treatment. Putrescine and spermidine, when added externally to the growth medium, showed dose-dependent inhibition of canavalmine-induced cell differentiation. Neither cadaverine nor spermine had any significant effect. These results suggest that not only structural change of cell membrane but alteration of the polyamine metabolism, especially a regulation of the cellular level of free spermidine, might have a key importance in erythroid differentiation of murine erythroleukemia cells induced by canavalmine.