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Alterations in protein tyrosine phosphate (PTP), lactate, and fructose 2,6-bisphosphate (F-2,6-P2) levels have been associated with induced MEL cell differentiation and commitment to terminal cell division (TCD). The possible relationships of perturbations in PTP metabolism and reduction in lactate formation during differentiation were investigated utilizing sodium orthovanadate, Na3VO4, primarily an inhibitor of PTP phosphatases, and levamisole, considered an alkaline phosphatase inhibitor. Both of these compounds were found to effectively inhibit the TCD-associated differentiation induced by DMSO, HMBA, and Na butyrate and to abrogate the differentiation-associated reduction in lactate accumulation due to these agents. However, they were found not to inhibit hemin-induced hemoglobin synthesis which is independent of TCD and does not alter lactate metabolism. Two brominated levamisole analogs, L-p-bromotetramisole and D-p-bromotetramisole, were also found to be inhibitors of TCD-associated differentiation and to be effective at even lower concentrations than levamisole. The changes in TCD-associated differentiation and lactate production exhibited the same concentration-dependence with respect to the inhibitors. These findings strengthened the theory that TCD-associated differentiation, decreased lactate production, and sensitivity to phosphatase inhibitors are all associated. Since the induction of MEL cell differentiation has been shown to be associated with, and is thought to be due to, the induction of PTP phosphatase activity and Na3VO4 is thought to inhibit the differentiation by inhibiting PTP phosphatase activity, the effect of levamisole on PTP levels was determined. Levamisole, like Na3VO4, was found to increase the tyrosine phosphate levels of proteins of similar molecular weights in intact cells in both the presence and absence of a differentiation inducer. Several phosphotyrosine-containing, similarly sized proteins were particularly affected by differentiation induction and by Na3VO4 and levamisole treatment. Changes in the levels of tyrosine phosphate-containing proteins of approximately 92-96, 60, and 38 kd were particularly noticeable. The induction of differentiation reduced PTP levels and inhibition of differentiation due to treatment with either Na3VO4 or levamisole increased their levels. These data suggest relationships between signal transduction pathways involved in differentiation and TCD, the regulation of lactate and F-2,6-P2 metabolism, and PTP levels.

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Mouse erythroleukemia (MEL) cell erythroid differentiation induced by dimethyl sulfoxide or hexamethylene bisacetamide (HMBA) is accompanied by the production of hemoglobin, terminal cell division and decreases in lactate production and fructose 2,6-bisphosphate levels. A number of studies have suggested that decreases in the cellular level of protein phosphotyrosine content may play a role in MEL cell differentiation. In particular, it was shown that the expression of several protein tyrosine phosphatase genes accompany this process and that the transfection of one of these genes into MEL cells followed by its subsequent expression induced eythroid differentiation. However, none of the physiological substrates for these protein tyrosine phosphatases have been identified. It is shown here that MEL cell differentiation is accompanied by decreases in tyrosine phosphorylation of Vav and possibly of the erythropoietin receptor (EpoR). Immunoprecipitation of the EpoR and analysis of co-precipitated proteins, indicates that the EpoR associates with Vav, STAT5 and an unidentified 60 Kd protein, . HMBA-induced erythroid differentiation abrogates these associations. The phosphatase inhibitors, Na3VO4 and levamisole, inhibit HMBA-induced differentiation as well as the association of the EpoR with Vav, STAT5 and the 60 Kd protein. This is of interest since Na3VO4, at the concentrations used here, has been shown to be a potent inhibitor of the activity of protein tyrosine phosphatases. These results suggest that levamisole, at least indirectly, acts by a molecular mechanism similar to that of Na3VO4 and that the loss of the association of the EpoR with Vav, STAT5, and and/or the reduction in the level of tyrosine phosphorylation of these proteins may play a role in MEL cell differentiation.

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The fraction of glucose metabolized to lactate is dramatically reduced during erythroid differentiation of mouse erythroleukemia (MEL) cells induced by dimethyl sulfoxide (DMSO), hexamethylene bisacetamide (HMBA), or sodium butyrate treatment. In order to determine the mechanism of the reduction in lactate production, several enzymatic steps in glucose catabolism were investigated. No changes in glycolytic enzyme levels were found during differentiation that could account for the alteration in lactate production and alterations in pyruvate kinase activity are known not to occur during MEL cell differentiation. Further, utilizing D-mannoheptulose, a specific inhibitor of hepatic-/tumor-specific glucokinase, no dependence on the activity of this enzyme for growth or differentiation was observed. Therefore, the possibility was entertained that the decrease in lactate production reflected a decrease in fructose 2,6-bisphosphate (F-2,6-P-2) which is a major regulator of the lactate production due to its ability to allosterically stimulate phosphofructokinase-1 (PFK-1) activity. PFK-1 cannot function in the absence of F-2,6-P-2 when only a suboptimal concentration of one of its substrates, fructose-6-phosphate (F-6-P), is present. When assayed under limiting F-6-P concentrations, it was found that following DMSO- or HMBA-induced differentiation, PFK-1 activity was decreased 7-20-fold. This finding suggested that F-2,6-P-2 levels might be controlling lactate production in this system. In keeping with this idea, marked decreases in F-2,6-P-2 levels were found to occur during DMSO- or HMBA-induced differentiation. These data suggest that decreasing F-2,6-P-2 levels account for the decrease in lactate accumulation that occurs during MEL cell differentiation.

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Patients with acute promyelocytic leukemia (APL) associated with the t(15;17) translocation and fusion of the promyelocytic leukemia (PML) and retinoic acid receptor-alpha (RAR-alpha) genes achieve complete remission but not cure with all-trans retinoic acid (RA), NB4, a cell line derived from a patient with t(15;17) APL that undergoes granulocytic differentiation when treated with pharmacologic doses of RA, was used as a model for differentiation therapy of APL. We found that NB4 cells are resistant to differentiation by nonretinoid inducers such as hexamethylene bisacetamide (HMBA), butyrates, vitamin D3, or hypoxanthine, all of which can induce differentiation in the commonly used HL60 leukemia cell line. Preexposure of NB4 cells to low concentrations of RA for a period as short as 30 minutes abolished resistance to nonretinoids and potentiated differentiation. Sequential RA and HMBA treatment yielded maximal differentiation by 3 days of drug exposure, whereas the effect of RA alone peaked after 6 days and yielded a smaller percentage of differentiated cells. RA also reversed NB4 cell resistance to butyrates and allowed for synergistic differentiation by these agents. Pretreatment with HMBA before exposure to RA failed to stimulate differentiation. Sequential RA/HMBA treatment also markedly increased the extent of differentiation of primary cultures of bone marrow and peripheral blood mononuclear cells from three APL patients. In one case RA/HMBA treatment overcame resistance to RA in vitro. Together, these results suggest that intermittent low doses of RA followed by either HMBA or butyrates may be a useful combination in the treatment of APL. This clinical strategy may help prevent or overcome RA resistance in APL.

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Treatment of mouse erythroleukemia (MEL) cells with hexamethylene bisacetamide induces a program of erythrodifferentiation, as judged by an increase in the synthesis of globins and other erythroid-specific products. This induction can be inhibited by glucocorticoids, e.g. dexamethasone. All globin and other erythroid-specific genes tested contain GATA response elements (GATA-RE) and can be transactivated by GATA-1, a transcription factor. GATA-1 is highly expressed in erythroid cells, including MEL cells. We noted a glucocorticoid receptor (GR) response element motif near a GATA-RE motif in the promoter region of the mouse beta-major and beta-minor globin genes and about 130 bases away from a GATA-RE in the alpha 1-globin gene promoter and, therefore, investigated the possibility that the dexamethasone-induced inhibition of induced MEL cell differentiation may involve effects of the GR on GATA-1 activity. Evidence obtained from transfection assays and DNA electrophoretic mobility shift assays indicates that the GR binds GATA-1 and interferes with its function before any interaction with DNA, but that the presence of a glucocorticoid response element near a GATA-RE augments the GR effect. The N-terminal 106-amino acid domain of the GR was found to be essential for the effect, possibly by binding to GATA-1. Since GATA-1 is autoregulatory, i.e. it has been shown by others to bind to its own promoter and up-regulate its own transcription, the finding that activated GR can interfere with GATA-1 function may provide an explanation for the inhibition by glucocorticoids of the entire program of erythroid differentiation in MEL cells. That is, by interfering with GATA-1 function, the GR inhibits not only the expression of erythroid structural genes, but may also inhibit the expression of a primary erythroid regulatory gene, GATA-1. It was also shown that the GATA-RE in each of the beta-globin promoters responds to mouse GATA-1 in a functional transfection assay.

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Endogenous glutamate is thought to be a major neurotransmitter. After binding to a cell membrane receptor there can be a stimulation of what can be called the nitric oxide (NO)-mediated neurotransmission pathway (NO-MNP). The activity of the enzyme that produces NO from arginine, NO synthase, and the level of NO become elevated. NO has little activity within the cell in which it is produced, but it rapidly leaks out of that cell and produces effects in neighboring cells. The NO-MNP can be activated to release NO in endothelial cells which in turn acts on neighboring vascular smooth muscle cells to induce vasodilation. Therefore, we suggest that exogenous, ingested glutamate, like endogenous glutamate, can lead to the same stimulation of the NO-MNP in sensitive individuals which would then cause the symptoms of the Chinese restaurant syndrome and/or glutamate-induced asthma. Further, since ingested nitrite and related compounds can be metabolized to NO, NO may more directly cause the symptoms of 'hot dog headache'. In addition, it has been suggested that NO production can also be controlled in endothelial cells by fluid forces that stimulate pressure receptors. Therefore, elevations of NO and stimulation of the NO-MNP may occur due to sudden, local, alterations of blood pressure during pugilistic activities and play a role in the symptoms of pugilistic Alzheimer's disease. If these ideas are correct, then inhibitors of the NO-MNP and/or temporary reduction of the plasma level of arginine may be useful in preventing at least some of the symptoms of these disorders.

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Lactate accumulation in the medium and glucose utilization decreased during the induction of in vitro differentiation of mouse erythroleukemia (MEL) and human myeloid leukemia (HL-60) cells. The decrease in lactate accumulation occurred as early as 24 h after inducer treatment was initiated and occurred prior to the decrease in glucose utilization. The decrease in lactate accumulation was greater than that predicted by the decrease in glucose utilization, i.e., the ratio of glucose used glycolytically, as measured by lactate accumulation, to glucose used in other pathways ('glycolytic ratio') markedly decreased during differentiation in these cell lines. Differentiation correlated with the abrogation of the high levels of lactate accumulation first described by Warburg as characteristic of some transformed and neoplastic cells. Studies on both parental and differentiation-resistant variant MEL cell lines indicated that the changes in lactate accumulation were not dependent on the changes in glucose utilization and could be dissociated from them. Moreover, the changes in lactate accumulation only occurred in cells able to undergo differentiation-induced terminal cell division. This regulatable expression of lactate accumulation in MEL and HL-60 cells in vitro may make them useful model systems for the elucidation of the molecular mechanisms controlling lactate formation in malignant cells.

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Upon treatment with the phorbol ester, tetradecanoylphorbol 13-acetate (PMA), peripheral mononuclear blood cells from patients with acute myeloid leukemia secrete into serum-free cell-conditioned media (PMA-CCM) at least three distinct nondialysable 'hematopoietic' factors: granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage-colony-stimulating factor (GM-CSF) and erythroid differentiation factor (EDF, activin A). G-CSF was identified by its stimulation of [3H]thymidine incorporation into a G-CSF-responsive cell line, NSF-60, and the inhibition of its stimulation by a G-CSF-specific monoclonal antibody (MAB). GM-CSF was identified by its stimulation of [3H]thymidine incorporation into a GM-CSF-responsive line, TALL-101, and the inhibition of its stimulation by a GM-CSF-specific MAB. EDF was identified by its ability to stimulate erythroid differentiation in mouse erythroleukemia cell lines, its identical retention times to those of authentic EDF on three successive reverse-phase HPLC columns and characterization of its penultimate N-terminal residue as leucine which is the same as that of authentic EDF. Both authentic EDF and the erythroid-stimulating activity in PMA-CCM were found to act synergistically with a suboptimal inducing concentration of a well-studied inducing agent, dimethyl sulfoxide, in inducing erythroid differentiation. In addition, a fourth activity was observed in PMA-CCM: normal human fetal bone marrow cell-proliferation stimulating activity (FBMC-PSA). FBMC-PSA was identified by its ability to stimulate the growth of granulocytes and macrophages in FBMC suspension cultures, which neither recombinant G-CSF or GM-CSF were found to do.

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The effects of 5-fluorouracil (5-FUra), in combination with various differentiation inducers on the growth and differentiation of mouse erythroleukemia (MEL) cells were investigated. The cells were first treated with 5-FUra, washed, and then treated with various concentrations of differentiation inducers: hexamethylene bisacetamide (HMBA), dimethyl sulfoxide (DMSO), and N-methylformamide. Pretreatment with 5-FUra, shown here to be a weak inducer of MEL cell differentiation, enhanced the subsequent HMBA induction of differentiation. The three inducers of differentiation markedly inhibited cell growth and increased cell death in a dose- and time-dependent manner if given immediately after cells were exposed to 5-FUra. In contrast, 5-FUra at similar concentrations inhibited cell growth, but only slightly increased cell death, while inducers without 5-FUra had little effect on cell growth or viability. When placed in fresh drug-free medium for 6 days following drug treatments, the cells completely recovered from the growth inhibition of 5-FUra as a single agent, whereas in cells previously treated with only HMBA there was a inhibition of cell growth without loss of viability. In contrast, a profound and prolonged growth inhibition with 98% cell death occurred in cells previously treated with 5-FUra followed by HMBA. The enhancement of 5-FUra cytotoxicity appeared to be directly related to the degree of differentiation and to biochemical events that occur during the commitment to terminal cell division induced by N-methylformamide, DMSO, or HMBA. An increase in Okazaki fragments was found in MEL cells treated with HMBA or DMSO when committed to terminal cell division. DNA breaks also follow 5-FUra treatment (A. Yoshioka et al., J. Biol. Chem., 262: 8235-8241, 1987) and may be the events that lead to cell death. The marked increase in cell death resulting from 5-FUra/HMBA treatment may be, at least partly, a consequence of increased DNA breaks due to 5-FUra followed by inhibition of DNA repair which is known to occur following the HMBA or DMSO induction of differentiation and commitment to terminal cell division. This combined sequential cytotoxic-differentiation therapy resulting in synergistic cytotoxicity and differentiation may be the basis of a new approach to cancer therapy and may aid in reducing the amounts of chemotherapeutic agents required for effective treatment, while maintaining or even increasing their therapeutic effects.

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The hemoglobin minor/hemoglobin major ratio expressed in mouse erythroleukemia (MEL) cells grown in vitro varies according to the differentiation inducer utilized. For example, butyrate and hemin induce higher hemoglobin minor/hemoglobin major ratios than do dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA). Benzyl alcohol in non-toxic concentrations was found to markedly reduce the hemoglobin minor/hemoglobin major ratio and to moderately reduce the total hemoglobin induced by DMSO or HMBA in MEL cells, while only slightly decreasing the ratio induced by hemin or butyrate. The addition of dexamethasone (another and more potent inhibitor of the induction of hemoglobin synthesis than benzyl alcohol) to the media during HMBA induction of differentiation increased the hemoglobin minor/hemoglobin major ratio. This is similar to other "inhibitory" treatments (i.e., treatments that result in sub-optimal hemoglobin production) that have been previously reported. Therefore, although benzyl alcohol and dexamethasone both partly inhibit the induction of total hemoglobin production, they have opposite effects on the induced hemoglobin phenotype: benzyl alcohol decreases the hemoglobin minor/hemoglobin major ratio while dexamethasone increases it. The mechanism(s) of the alteration in the hemoglobin phenotype is unknown as is the mechanism of induction by any of the various inducing agents or of the inhibition of induction by any treatment. However, it appears that if the signal for the induction of hemoglobin minor is sufficiently potent (as it is during butyrate or hemin induction), it cannot be overcome by benzyl alcohol at a "non-toxic" concentration.

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When mouse erythroleukemia (MEL) cells were induced to differentiate by growth in the presence of dimethyl sulfoxide, hexamethylene bisacetamide (HMBA), or hemin, the apparent activity of DNA ligase extractable from inducer-treated cells decreased 70 to 80% when compared to untreated cells. Earlier work had indicated that these changes did not occur in a differentiation-resistant MEL cell variant and suggested that the decrease in the level of DNA ligase activity might be related to the differentiation process. Since the MEL cells accumulate high levels of both hemoglobin-bound and non-hemoglobin-bound heme, the effect of both hemoglobin and hemin on DNA ligase activity of MEL cell extracts was tested. When cell-free extracts containing DNA ligase activity were preincubated with hemin at concentrations up to 150 microM, an 80% or greater inhibition of the DNA ligase activity resulted. The ATP-dependent DNA ligase from bacteriophage T4 was also inhibited by hemin, but the NAD-dependent DNA ligase from Escherichia coli was not sensitive to this treatment. Preincubation of these same extracts with hemoglobin at levels comparable to those present in differentiating cells did not result in inhibition of any of the ATP-dependent DNA ligases tested. Culturing the cells with dimethyl sulfoxide in the presence of imidazole resulted in a marked decrease in globin chain accumulation but did not reverse the dimethyl sulfoxide-related decrease in DNA ligase activity. These data suggest the possibility that heme or its metabolites, but not globin or hemoglobin, could serve to modify the process of DNA replication and/or repair in differentiating MEL cells via inhibition of DNA ligase activity. These data are consistent with the findings of Lo et al. (S.C. Lo, R. Aft, and G.C. Mueller, Cancer Res., 41: 864-870, 1981) which correlated the onset of differentiation-related terminal cell division in MEL cells with the levels of nonhemoglobin heme present in these cells.

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Proteases have been shown to be capable of inducing two of the most fundamental biologic processes: mitogenesis and cellular differentiation. Their proteolytic activity has been the most widely studied enzymatic activity implicated in inducing these processes. Protease induction of mitogenesis is a major system for studying the control of this process and studies of possible transmembrane signals have been initiated. Proteases have only recently begun to be used as tools for probing the induction of differentiation, but at least three cell line systems have been studied. Extracellular proteases such as thrombin may play physiologic roles in inducing mitogenesis in vivo as suggested by several laboratories. Although the amount of data bearing on the similar possibility of the induction of differentiation by extracellular proteases is negligible, it remains a possibility. For example, macrophages not only have surface-bound proteases (6), but also release proteases (261, 308) as well as cytokines. Any of these agents, individually or in combination, may play a role in inducing erythroid differentiation in vivo and may provide a raison d'etre for the "blood islands" consisting of erythroblasts surrounding a "nurse" macrophage which are so frequently seen in bone marrow.

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The addition of one of several proteases to cultures of mouse erythroleukaemia (MEL) or human K-562 leukaemia cells can induce a substantial portion of the cells to undergo erythroid differentiation. This effect is due, at least in part, to the proteolytic action of these enzymes. The critical substrate(s) for this proteolytic action is not a component of the medium or a long-lived substance(s) released from the cells. In order to determine if the substrate(s) is located on the cell surface or intracellularly, a comparison of the ability of non-immobilized papain and immobilized papain (i.e. covalently linked to Sepharose beads which were larger than the cells) to induce MEL cell differentiation was undertaken. Both papain preparations induced the same level of differentiation. The proteolytic activity of the bead-linked papain remained associated with the beads. Therefore, proteases induce erythroid differentiation in these cells by acting proteolytically on a substrate(s) that is exterior to the cell.

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The induction of differentiation in several tumor lines serves as a basis for a new approach to cancer treatment. In vitro studies in the mouse erythroleukemia (MEL) cell system have identified about 300 agents capable of inducing differentiation by mechanisms that remain to be elucidated. The design of differentiation therapy will depend on the specific tumor cell type, an effective time course, and the synergistic interaction among combinations of two or more inducers. The induction of differentiation may be followed by terminal cell division (TCD) or programmed cell death in several tumor cell systems. This mechanism for the destruction of tumor cells is one goal of differentiation therapy and differs from nonspecific cytotoxic therapy. To evaluate the effect of differentiation therapy, a clear distinction must be made between nonspecific cytotoxicity and the programmed TCD of induced cytodifferentiation. One possible parameter for assessing the commitment to TCD in the MEL cell system is a selective decrease in DNA ligase activity, which does not appear to occur following treatment with nonspecific cytotoxic agents. These biological and biochemical parameters should be helpful in designing agents capable of inducing TCD in vivo.

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The addition of certain proteases to cultures of Friend virus-infected mouse erythroleukemia cells can induce up to 90% of the cells in culture to become hemoglobin-containing, as assessed by positive staining for benzidine (B+). Because the mechanism of this protease action is unknown, media components were studied as possible targets for protease activity. Aliquots of medium plus serum were incubated for various times with levels of protease sufficient to induce approximately 50% of the cells to the B+ state. Cells were added to protease-pretreated serum either before or after inactivation of the protease. In all cases, enzymatically active protease had to be present with the cells to induce B+ cells to form. Serum and other components of the medium pretreated with protease were inactive. Mouse erythroleukemia cells grown in the absence of serum were also induced by proteases to form B+ cells. These data imply that the inducing action of proteases cannot be passively transferred by protease-pretreated serum or medium nor is serum required for protease-mediated induction of B+ cells. Taken together, these conclusions suggest that the protease action is on the cells or on cellular products intimately associated with cells.

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The relative amounts of hemoglobin (Hb) major and Hb minor accumulated during induction of erythrodifferentiation in mouse erythroleukemia (MEL) cells were studied. The ratio of major to minor was found to depend not only upon the inducer tested (as reported previously by others), but also upon the concentration of the inducer and the time of exposure to the inducer as well as the specific cell line of MEL cells studied. At concentrations required for optimal induction of differentiation, certain agents led to the accumulation of predominantly Hb major, but suboptimal concentrations of the same inducers led to predominantly Hb minor accumulation. After a relatively short induction time (2 da) utilizing a given inducer either the level of Hb minor was higher than that of Hb major or the levels of the two Hb's were approximately equal, but after longer induction periods (3-7 da) Hb major was more abundant than Hb minor. In addition, it was found that the three proteases tested induced predominantly Hb minor. The addition of suboptimal concentrations of low molecular weight inducers acted synergistically with a given protease to produce a high yield of Hb-containing cells. When these agents were added singly they induced relatively low Hb major/Hb minor ratios, but when a low molecular weight inducer was added together with a protease in a "synergistic" combination, elevated ratios were induced. The proportions of hemoglobin types induced in MEL cells may be related in part to the intensity of the induction response. In view of these data, classifications of inducers based solely on the ratios of Hb types produced must be guarded.

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Several protease preparations of varied specificity increased hemoglobin levels in K562 cells. These are the first enzymes shown to stimulate this process in these cells. Hemin, at a concentration at which it did not act as a potent inducer of hemoglobin production, was found to synergistically stimulate induction by proteases. As seen in some other cell types, six different protease preparations also stimulated K562 cell yield. Hemin did not enhance the protease stimulation of cell yield, but was, instead, slightly inhibitory. Trypsin was one of the most potent inducers of the proteases tested. A combination of trypsin with a "synergistic" concentration of hemin did not decrease the size of K562 cells during induction of hemoglobin production, suggesting that these cells were not irreversibly differentiated nor induced to terminal cell division by this treatment. This was supported, although not proven, by an assay that demonstrated no progressive decrease in the rate of cell multiplication associated with the induction of hemoglobin synthesis.

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Monomeric (G-) actin and filamentous (F-) actin levels were determined in Triton X-100 extracts prepared from mouse erythroid cells at various stages of differentiation. G-actin and F-actin were found in the Triton-soluble fraction and in the Triton-insoluble fraction, respectively. G-actin levels in untreated and dimethyl sulfoxide-treated (differentiated) erythroleukemia cells, reticulocytes, and erythrocytes were 48, 33, 2.8, and 0.37 microgram/mg protein, respectively, and F-actin levels were 17, 35, 45, and 59 micrograms/mg protein, respectively. G-actin/F-actin ratios were successively lower in cells representing the more mature stages of development.

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Proteases stimulate mouse erythroleukemia (MEL) cell differentiation and multiplication. The stimulation of differentiation is synergistically increased by low concentrations of dimethyl sulfoxide. Synergism of other low molecular weight inducers with representative proteases, alpha-chymotrypsin and protease V8, was tested. Hemin. hypoxanthine, actinomycin D, aminonucleoside of puromycin, hexamethylene bisacetamide, and 5-azacytidine were also found to act synergistically with this protease to augment MEL cell hemoglobin production, but not cell multiplication. Fatty acids (acetate, propionate, butyrate, isobutyrate, and valerate), prostaglandins A1 and E1, amino acids, and amino acid analogs and metabolites did not act synergistically with chymotrypsin. Some physiologic amino acids were found to be weak inducers. Several of the low molecular weight inducers also acted synergistically with protease V8 in inducing differentiation, and, as with chymotrypsin, did not act synergistically in stimulating cell multiplication. Like chymotrypsin, protease V8 did not act synergistically with butyrate. The earlier finding that proteases, but not low molecular weight inducers, stimulate cell multiplication during the induction of differentiation was confirmed. Carboxypeptidase A also was found to be an inducer.

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