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.     

Effects of murine erythroleukemia inducers on mouse erythroid colony formation in culture.

The effect of various agents which are known to increase the differentiation of Friend erythroleukemia cells was investigated in cultures of mouse bone marrow cells. N,N-dimethylacetamide (5 and 15 mM) and acetamide (60 mM) significantly increased the number of erythroid colonies observed. Tetramethylurea, dimethylformamide, pyridine N-oxide, and butyric acid were ineffective. Dimethylsulfoxide at a concentration of 1% significantly increased colony number in cultures of marrow cells obtained from male mice, but had no effect in cultures of female bone marrow cells.     

Effects of brefeldin A on the processing of viral envelope glycoproteins in murine erythroleukemia cells.

This paper documents the effects of brefeldin A (BFA) on the processing and transport of viral envelope glycoproteins in a retrovirus-transformed murine erythroleukemia (MEL) cell line. BFA is a fungal metabolite that disrupts intracellular membrane traffic at the endoplasmic reticulum (ER)-Golgi complex junction. In MEL cells, BFA inhibited the processing of the newly synthesized precursor, gPr90env, of the murine leukemia virus envelope protein, gp70, and curtailed the budding of virions into the culture medium by blocking the transport of this protein out of the ER. The block resulted in the intracellular accumulation of gPr90env and two putative products of its processing (78 and 66 kDa). The results of endoglycosidase (endo) H and D digestion of the viral glycoproteins in the presence and absence of BFA indicated that (i) there was no glycoprotein processing during the first approximately 2 h of the BFA block; (ii) active Golgi enzymes relocated to the ER in approximately 2 h during BFA treatment, resulting in the production of partially endo H-resistant forms of the spleen focus-forming virus glycoprotein, gp55 (in controls, this glycoprotein was generally retained in the ER as an endo H-sensitive entity); and (iii) proteolytic processing of gPr90env to gp70 occurred prior to the acquisition of endo H resistance and at approximately the same time as endo D sensitivity (i.e. in a cis Golgi compartment). In control cells, the spleen focus-forming virus glycoprotein, gp55, underwent turnover with a half-life of approximately 5 h. In contrast, its turnover was considerably slower during BFA treatment (t 1/2 = approximately 20 h), suggesting that transport of gp55 out of the ER was required for its degradation or that BFA afforded it protection from proteolysis within the ER.     

Effects of inhibitors of ion-motive ATPases on the plasma membrane potential of murine erythroleukemia cells

The membrane electric effects of N,N'-dicyclohexyl-carbodiimide (DCCD) and vanadate were studied in murine erythroleukemia cells (MELC), comparing the patch-clamp technique and the accumulation ratio (ARexp) of [3H]-tetraphenylphosphonium (TPP+). Electrophysiological measurements showed that both these inhibitors produce, at micromolar concentrations, a 20-30 mV hyperpolarization of resting potential (delta psi p) of MELC, which is abolished when the electrochemical equilibrium potential of K+ (EK) is brought close to zero. DCCD and vanadate turned out to have distinct targets on the plasma membrane of MELC (an H+ pump and the Na+,K(+)-ATPase, respectively). Measurements of ARexp showed that: (i) patch-clamp measurements of delta psi p were equivalent to those based on ARexp of antimycin-pretreated cells (ARANT); (ii) DCCD produced a strong increase in ARANT, that was antagonized by carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) and diethylstilbestrol (DES); (iii) vanadate determined a marked increase in ARANT that was insensitive to FCCP, but antagonized by ouabain; (iv) incubation in high K+ medium (HK) brought ARANT to 1.0 in the controls, but did not lower this ratio below 3.0 in the presence of DCCD or vanadate; (v) the total amount of TPP+ taken up by the cells was in any case water extractable by a freezing and thawing procedure. On the whole, our data indicate that DCCD and vanadate hyperpolarize the MELC by increasing the K+ conductance and, at the same time, enhance the TPP+ binding, probably by changing the electrostatic potential profile of the plasma membrane. These effects seem to involve functional modifications of the target pumps, apparently related to the ion-occluding state of these enzymes.     

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.     

Nucleosome-associated protein kinases in murine erythroleukemia cells.

Chromatin subunits from murine erythroleukemia cells were prepared by a method which releases actively transcribing genes. Two casein kinase activities (CK₁ and CK₂) were isolated from these nucleosomes by gel nitration in 0.5 m NaCl. CK₁ (M_r ~ 200,000) and CK₂ (M_r ~ 35,000) were further purified by phosphocellulose chromatography and characterized with regard to several parameters which may regulate their activity in vivo. CK₁ has an NaCl optimum of 0.14 m, utilizes GTP as phosphate donor ~25% as efficiently as ATP, and phosphorylates a discrete group of high molecular weight nonhistone proteins in the unfractionated chromatin starting material. CK₂ has an NaCl optimum of 0.24 m, cannot utilize GTP, and modifies a different group of nonhistones. Both kinases are inhibited by concentrations of hemin (<50 μm) which efficiently induce globin gene expression in erythroleukemia cells. A histone kinase resolved during the gel filtration step is unaffected by hemin. An investigation of the mode of hemin inhibition reveals that CK₁ and CK₂ interact in different fashions with the inhibitor.     

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.     

Stochastic model for mast cell proliferation in culture of murine peritoneal cells

We recently identified two types of mast cell colonies derived from murine peritoneal cells: type 1 and type 2. Type 1 mast cell colonies consisted of berberine sulfate(+)- safranin(+) connective tissue-type mast cells (CTMC) and were derived from mature CTMC in the heaviest fraction obtained by Percoll density gradient centrifugation. In contrast, type 2 mast cell colonies consisted of alcian blue(+)- berberine sulfate(-)- safranin(-) mucosal mast cells (MMC) and were derived from immature progenitors in low density fractions. We replated a total of 60 type 1 and 60 type 2 mast cell colonies and examined their capability for producing secondary colonies. Although all of the primary colonies yielded secondary colonies, the replating efficiencies of individual colonies varied over a wide range. Cumulative distributions of secondary colonies from both type 1 and type 2 primary colonies could be fitted well by gamma distributions obtained by computer simulation. These findings are in agreement with the stochastic model for CTMC- and MMC proliferation. Cytological analyses of secondary colonies from primary type 1 colonies revealed heterogeneous distributions of alcian blue(+)- safranin(-)- berberine sulfate(-) mast cells, suggesting that transdifferentiation from mature CTMC to safranin(-)- berberine sulfate(-) mast cells is also governed by stochastic mechanisms.     

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.     

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.     

Induction of glycophorin gene expression in cultured murine erythroleukemia cells

In order to identify the mRNA for mouse glycophorin, mRNA was isolated from immature erythroid cells obtained from the spleens of anemic mice, translated in vitro in mRNA-dependent rabbit reticulocyte lysate, and then immunoprecipitated with a specific antiserum. Glycophorin mRNA was shown to be present only in erythroid cells. In immunofluorescent and in vitro translation studies, it was shown that glycophorin mRNA is absent in uninduced murine erythroleukemia (MEL) cells, but is induced in dimethylsulfoxide-treated differentiating cells.     

Stability of globin mRNA in terminally differentiating murine erythroleukemia cells

The stability of globin mRNA in terminally differentiating MEL cells has been reevaluated. Previously, it had been reported that globin mRNA has a half-life of approximately 17 hr in terminally differentiating MEL cells. We show that the previous measurements of this parameter were confounded by physical instability of differentiating MEL cells. By using culture conditions that physically stabilize end-stage cells we show that the stability of globin mRNA in terminally differentiating MEL cells is equal to the value observed for ribosomal RNA, a half-life greater than 60 hr.     

Effects of Brefeldin A on the Golgi complex, endoplasmic reticulum and viral envelope glycoproteins in murine erythroleukemia cells

This report concerns the effects of Brefeldin A (BFA): i) on the Golgi complex and the ER of retrovirus-transformed murine erythroleukemia (MEL) cells and, ii) on the viral proteins these cells express. Golgi complexes were extensively disorganized by BFA. Within 5 min, most stacked cisternae were converted to vesicles scattered throughout the centrosphere region. By 30 min, the Golgi complexes were completely disassembled. Only clusters of small vesicles ("Golgi remnants") persisted in the vicinity of the centrioles and microtubule-organizing centers. Some of these small vesicles had a simple coat structure on their membranes. Over the next 1 to 2 h of BFA treatment, the number of vesicles in the Golgi area decreased concomitantly with the expansion of a predominantly smooth membrane portion of the ER, consisting of a network of dilated tubules in continuity with regular RER cisternae, annulate lamellae and the nuclear envelope. By electron microscopy, viral glycoproteins appeared to accumulate on the membranes of this network, and immature virions were found to bud preferentially into its cisternal space. Viral accumulations increased with time under BFA. The rest of the RER appeared normal, apparently unaffected by the drug. Preferential virion budding suggests that this expanding network is a chemically differentiated part of the ER. By immunofluorescence, antibodies to viral envelope proteins gave a punctate staining at the surface of control cells, presumably in the areas of virion budding, whereas relatively large intracellular masses of antigens were found in BFA-treated cells. We assume that these masses represent the differentiated parts of the ER. Taken together, these findings suggest that BFA blocks intracellular transport of newly synthesized cellular and viral proteins immediately distal to the distinct compartment of the ER in which virion budding preferentially occurs. BFA effects are rapidly and fully reversible. Within 1 min of the removal of the drug, stacks of Golgi cisternae began to reappear in the vicinity of the centrioles, and by 30 min, Golgi complexes regained their normal structural appearance.     

Characterization of spermine uptake by Ehrlich tumour cells in culture

Summary. Spermine is taken up by Ehrlich ascites tumour cells through a specific, saturable, temperature and energy-dependent transport system with a remarkably low affinity constant for spermine (around 1 μM). In the absence of a potassium ion gradient through the plasma membrane, spermine uptake remains saturable but the value of the K_m for spermine is much higher (153 μM). Difluormethylornithine treatment (3 mM for 48 h) induces significant increases in V_max values (up to 9-fold) and changes in the K_m values with scarce statistical significance. Among the biogenic amines tested, only spermidine and, partly, agmatine seem to share the same transport system with spermine. No difference is observed in the rate of spermine transport when assays are carried out in the presence of 50-fold excess of ornithine or calcium, or 100-fold excess of glutamine. Received April 25, 2000 Accepted November 1, 2000     

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.     

TFAR19 gene changes the biophysical properties of murine erythroleukemia cells

TFAR19 is a novel apoptosis-related gene and can accelerate cell apoptosis in the presence of apoptosis inducements. Here, we studied the effects of TFAR19 on some biophysical properties of mouse erythroleukemia (MEL) cells and their molecular and structural basis. After transfected with TFAR19 and apoptosis inducement, MEL revealed a high cell membrane fluidity, a decrease in resynthesis of phospholipids, an increase in the proteins/nucleic acids ratio, a relatively orderly cytoskeleton network, an impaired deformability, a low integrin aM expression, and a decrease in adhesion to endothelial cells. These findings suggest the potential of TFAR19 for antitumor cell migration, and thus for antitumor gene therapy.