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.     

ADP-ribosyltransferase activity during the friend virus-induced murine erythroleukemia cell differentiation

A variety of chemical agents that are known to induce erythrodifferentiation in the Friend virus-induced murine erythroleukemia (MEL) cell have been suggested to mediate DNA cleavage in cultured cells prior to differentiation. The activation of the nuclear enzyme, ADP-ribosyltransferase, depends upon the presence of single strand breaks in DNA. If dimethyl sulfoxide (Me2SO) causes DNA breakage, it would be expected that the activity of ADP-ribosyltransferase would increase. A study of ADP-ribosyltransferase activity during cell growth indicates that both Me2SO-treated and untreated MEL cells exhibit a similar increase in the enzyme activity but the increase in Me2SO-treated cells is delayed by a few hours. When examined at comparable stages of growth, both treated and untreated cells show almost identical levels of enzyme activity. The present data thus do not support the contention that Me2SO induces DNA breakage in the MEL cells.     

DNA strand scission and ADP-ribosyltransferase activity during murine erythroleukemia cell differentiation

The accumulation of DNA strand breaks and activation of ADP-ribosyltransferase (ADPRT) have recently been associated with cellular differentiation. Murine erythroleukemia (MEL) cells undergo erythropoietic differentiation when exposed to dimethyl sulfoxide (Me2SO) and several studies have suggested that DNA strand scission induced by this agent is a prerequisite for expression of the differentiated phenotype. Me2SO induction of MEL cells has also been associated with increases in ADPRT activity in one study, but not in another. We have monitored the effects of Me2SO on DNA strand breaks in preformed and replicating MEL cell DNA. The results clearly demonstrate that DNA fragmentation is not detectable during Me2SO induction of MEL differentiation, even in the presence of 3-aminobenzamide, an inhibitor of ADPRT. Further, these results are consistent with an absence of detectable changes in both endogenous and total potential ADPRT activity during Me2SO-induced MEL differentiation. These findings would argue against Me2SO induction of DNA strand scission and ADPRT in MEL cells undergoing differentiation.     

Changes in purine nucleoside phosphorylase activity during thymosin-induced human null cell differentiation

Purine nucleoside phosphorylase (PNP) is a purine salvage pathway enzyme which we have found to be 8-10 times more active (per cell) in human peripheral blood null lymphocytes than in T lymphocytes. To test the hypothesis that null cells are, in part, pre-T lymphocytes we have defined an in vitro system for null cell differentiation into T cells and examined PNP activity during this differentiation process. We found that about 10% of human null cells could be driven to differentiate into T cells using thymosin fraction 5 (TF5) an extract of bovine thymus glands. The response to TF5 was dose related to up to 250 micrograms/ml with a maximum response occurring by 42-46 hr incubation. Exposure to TF5 was necessary for more than 4 hr but no more than 8 hr in order to obtain a maximum response. Both OKT4 and OKT8 positive cells were present in the newly differentiated T cell population but OKT8 positive cells appeared to predominate (OKT4/OKT8 = 0.698 +/- 0.30, mean +/- 1 SD). The differentiation process did not involve DNA synthesis but was inhibited at 4 degrees C. In the newly differentiated T cells PNP activity per cell was 8- to 10-fold lower (36 +/- 23 nm/hr/106 cells) than in null cells (311 +/- 136), and was at a level similar to mature T cells (56 +/- 7). Thus, human peripheral blood null cells can be induced to differentiate into T lymphocytes which can be characterized by both surface markers and biochemical parameters. Future studies will look at the function of TF5-induced T cells and the regulation of PNP activity during the differentiation process.     

Changes in surface architecture during murine erythroleukemia cell differentiation as detected by lectin binding and agglutination

Cell surface alterations occurred during murine erythroleukemia cell (clone 745) differentiation that were detected by both agglutination and lectin binding. Agglutination of erythroleukemia cells was produced by wheat germ agglutinin; whereas, concanavalin A, Ricin, soybean agglutinin and fucose-binding protein were either ineffective or much less efficacious. Treatment of leukemia cells with the inducer of erythroid differentiation dimethylsulfoxide (DMSO) caused a progressive accumulation of hemoglobin-containing cells in culture and a decrease in the rate of agglutination by wheat germ agglutinin, which began at 24 h after exposure to the polar solvent, reached a nadir at 48 h, and remained essentially constant thereafter. The binding of radioactive wheat germ agglutinin by untreated control erythroleukemia cells increased with time in culture, reaching a maximum value at 48 h, and decreased progressively thereafter. Although an increase in ³H-labeled wheat germ agglutinin binding also occurred in DMSO-treated cells, the level bound was significantly lower than that observed in control cells at 24–96 h. The treatment of erythroleukemia cells with various concentrations of DMSO resulted in a decrease in the number of wheat germ agglutinin receptor sites. Other inducers of differentiation (i.e., dimethylformamide, bis(acetyl)diaminopentane) also inhibited the rate of wheat germ agglutinin-induced agglutination of erythroleukemia cells while, in contrast, the inducer tetramethylurea did not. These studies indicate that membrane changes occur during differentiation and suggest that there may be more than one mechanism involved in the initiation of maturation which ultimately leads to the common pathway of erythroid development.     

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.     

Changes in adenyl cyclase specific activity during differentiation on an established myogenic cell line

The variation of specific activity of adenyl cyclase has been studied during differentiation of an established line of myoblast, strain L₆D and of a temperature sensitive developmental variant strain, H₆, derived from it. The specific activities of both basal and NaF stimulated adenyl cyclase were found to decrease 2 to 3 folds after fusion of myoblasts into myotubes in cultures of L₆D. Cultures of strain H₆ displayed the same decrease in specific activity of adenyl cyclase when grown at temperature which allows differentiation, while no decrease was observed at the temperature which does not allow cell fusion. These results indicare that the decrease in specific activity of adenyl cyclase is associated with cell fusion and reflects membrane changes ocurring during differentiation of myogenic cells.     

Suppressor T cell growth and differentiation: production of suppressor T cell differentiation factor by the murine thymoma BW5147

Previous studies have identified a lymphokine, termed Ts differentiation factor (TsDF), in primary MLR supernatants that induces effector function of alloantigen-primed MLR-Ts. The present report describes constitutive production of TsDF by the murine thymoma BW5147, and its use to analyze alloantigen and TsDF requirements for MLR-Ts activation to TsF production. Serum-free supernatants of BW5147 restored the capacity of MLR-TsF production to alloantigen-primed MLR-Ts cultured with glutaraldehyde-fixed allogeneic stimulator cells, and were not themselves directly suppressive in the MLR assay. BW5147 supernatant induced MLR-TsF production from primed L3T4-Ly2+ MLR-Ts in the absence of concomitant proliferation, suggesting that the function of BW5147 supernatant, like that of MLR-derived TsDF, is a differentiative rather than a proliferative one, and is required for the synthesis or release of TsF. The differentiative activity of BW5147 supernatant was associated with a molecular species of approximately 14,500 m.w. by HPLC fractionation and was expressed independently of detectable IL 2, IL 3, IFN-gamma, and IL 1. The functional activity of BW5147 supernatant has therefore been provisionally designated BW5147-derived Ts differentiative factor, or BW-TsDF. By using BW-TsDF, it was demonstrated that MLR-Ts fail to respond to TsDF in the absence of, or preceding, reexposure to priming alloantigen. Instead, alloantigen binding by primed MLR-Ts appears to create a transient state of TsDF responsiveness. Primed MLR-Ts were fully sensitive to delayed addition of TsDF for approximately 12 hr after reexposure to alloantigen, but became TsDF-unresponsive within 24 to 36 hr. MLR-Ts cultured alone for 36 hr were fully responsive to the combined addition of TsDF and alloantigen. Thus, MLR-Ts activation to TsF release requires the sequential events of specific alloantigen binding, which induces a TsDF-responsive state, followed by interaction with TsDF. The transience of induced TsDF responsiveness suggests a precise mechanism for control of antigen-initiated Ts activation to effector function.     

Changes in p34cdc2 kinase activity and cyclin A during induced differentiation of murine erythroleukemia cells.

Hexamethylene bisacetamide (HMBA)-induced murine erythroleukemia (MELC) differentiation is characterized by a prolongation of the initial G1 which follows passage through S phase in the presence of inducer. Commitment to terminal cell division is first detected in a portion of the cell population during this prolonged G1. HMBA-induced commitment is stochastic. This study has examined changes in two known cell cycle regulators, p34cdc2 and cyclin A, in cycle-synchronized MELC in the absence and presence of HMBA. Histone H1 kinase activity of p34cdc2, and the levels of CDC2Mm mRNA, 1.8-kilobase mRNA of cyclin A, and cyclin A protein changed during cell cycle progression in MELC, and all of them were suppressed during G1. The suppression of the H1 kinase activity and cyclin A expression continued through the prolonged G1 in MELC cultured with HMBA, whereas p34cdc2 protein level did not vary through the cell cycle in MELC cultured without or with inducer. Phosphorylation of p34cdc2 in uninduced MELC gradually increased as cells progressed from G1 to S. In induced MELC, an increase in phosphorylation of p34cdc2 occurred during the prolonged G1, and prior to the exit of the bulk of the cells from G1 to S. These results suggest that in HMBA-induced MELC, p34cdc2 phosphorylation per se is not a limiting factor in determining G1 to S progression. The persistent suppression of cyclin A expression and histone H1 kinase activity may play a role in HMBA-induced commitment to terminal differentiation.     

Changes in cell migration of mesenchymal cells during osteogenic differentiation

We showed that the migration, morphology and adhesiveness of undifferentiated mesenchymal cells dramatically changed during osteogenic differentiation. The migration of these cells was transiently upregulated early in osteogenic differentiation. At a later stage, migration was decreased but adhesiveness was increased. Furthermore, Cdc42 and Rac1 Rho-family small GTPases were activated at early stages of differentiation and the phosphorylation level of FAK decreased as differentiation progressed. We also showed cell migration was promoted by inhibition of the Rho-ROCK-myosin signaling. Finally, using a mouse model of ectopic bone formation, we confirmed that treatment with ROCK inhibitor, Y-27632 increased cell movement into bone formation sites, resulting in enhanced osteogenesis. These results provide a new insight into the link between cell migration and osteogenic differentiation.     

Alteration in sialyltransferase and sialic acid expression accompanies cell differentiation in rat intestine

The subcellular distribution of sialyltransferase and its product of action, sialic acid, was investigated in the undifferentiated cells of the rat intestinal crypts and compared with the pattern observed in the differentiated cells present in the surface epithelium. Sialyltransferase was immunocytochemically detected with an antibody, affinity-purified on a beta-galactosidase/sialyltransferase fusion protein, which recognizes only protein epitopes of the enzyme. A similar pattern and intensity of immunolabeling were observed in the Golgi apparatus, apical and basolateral plasma membranes of both undifferentiated and differentiated absorptive cells. However, in the goblet cells, the mucus was only weakly labeled in cells present in the basal portion of the crypts but increased in intensity through the zone of migration to the surface epithelium. Sialic acid as detected with the Limax flavus lectin was observed in the Golgi apparatus and post-Golgi apparatus structures of both absorptive and goblet cells regardless of their position along the crypt-to-surface epithelium axis. However, a striking difference in the plasma membrane distribution of sialic acid existed between undifferentiated cells of the lower half of the crypts and those of the upper half and the surface epithelium: in the former, label was present in both the apical and basolateral domain, whereas in the latter it became restricted to the apical domain. These results suggest that the presence of sialyltransferase immunoreactivity in the goblet cell mucus and the polarization of sialic acid to the apical plasma membrane of both goblet and absorptive cells may be markers for the differentiated state.     

Changes in enzyme activity during differentiation in Chlamydomonas reinhardtii

The patterns of alanine dehydrogenase, glutamate dehydrogenase and malate dehydrogenase activity were studied during the normal vegetative cell cycle and during the process of gametic differentiation and dedifferentiation in synchronized cultures of Chlamydomonas reinhardtii. During all three phases of growth and differentiation the synthesis of DNA was also measured. During gametic differentiation all three enzyme levels were suppressed compared to vegetative cells although DNA and cell number were comparable. During gametic dedifferentiation no DNA synthesis occurred during the first 24 h cycle and only a doubling during the second. It was not until the third cycle that a normal 4-fold increase in DNA was observed. Cell number followed a similar pattern. Athough the levels of alanine dehydrogenase and malate dehydrogenase were uniformly low during the first cycle when glutamate dehydrogenase increased 4-fold, during the second cycle the patterns of these enzymes changed markedly. The enzymes did not attain levels characteristic of vegetative cells until the third cycle.     

Changes in β-Galactosidase activity during differentiation and dedifferentiation inDictyostelium discoideum

β-Galactosidase (EC 3.2.1.23) ofDictyostelium discoideum was investigated for its properties and activity during differentiation and dedifferentiation. β-Galactosidase of this organism had a pH optimum at 3.5. The specific activity of this enzyme was increased gradually from the time of initiation of differentiation and reached a peak at the aggregation stage. Then the activity of the enzyme showed a slight decrease followed by a further increase and reached a maximum at early culmination. During dedifferentiation of cells disaggregated from a slug, the activity of the enzyme was increased, reached a maximum after 3 hr of incubation and then decreased nearly to the original level of activity after completion of dedifferentiation. This increase in the enzyme activity coincided with decomposition of acid mucopolysaccharide contained in the prespore specific vacuoles, and both processes were sensitive to cycloheximide. No increase in the activity of acetylglucosaminidase (EC 3.2.1.30), another lysosomal enzyme, was observed during the process. Possible roles of β-galactosidase in cell type conversion as well as in dedifferentiation of the prespore cell were discussed.     

Changes in DNA composition during cell differentiation in a green alga

In the coenobia-forming green alga, Hydrodictyon reticulatum, changes in DNA composition during growth and differentiation of reproductive cells were followed. A “loss” of DNA per nucleus could be attributed to underreplication of an amplified DNA fraction being part of a (GC)-rich satellite band in CsCl gradients of DNA from young coenobia. This (GC)-rich satellite DNA contains up to 3 % unique DNA sequences intermingled with repetitive DNA. It does not (or nearly not) contain ribosomal DNA. A possible role of this DNA fraction in differentiation is suggested.