DMSO increases tyrosine residue phosphorylation in membranes from murine erythroleukemia cells

Phosphorylation of membranes from murine erythroleukemia cells was performed in the presence and absence of the polar solvent dimethyl sulfoxide. Quantitation of the phosphoamino acid content revealed that DMSO stimulated phosphotyrosine accumulation by three-fold; serine and threonine phosphorylation decreased significantly. We had previously shown that DMSO stimulated tyrosine residue phosphorylation of the hepatic epidermal growth factor receptor. EGF had little effect in MEL membranes; therefore, DMSO results in accumulation of phosphotyrosine in cell membranes that do not exhibit significant EGF-dependent phosphorylation.     

Growth inhibition and differentiation induction in murine erythroleukemia cells by 4-hydroxynonenal

4-Hydroxynonenal (HNE) is one of the major end products of lipid peroxidation. Here we show that the exposure of murine erythroleukemia (MEL) cells to 1 μM HNE, for 10.5 h over 2 days, induces a differentiation comparable with that observed in cells exposed to DMSO for the whole experiment (7 days). The exposure of MEL cells for the same length of time demonstrates a higher degree of differentiation in HNE-treated than in DMSO-treated MEL cells. The protooncogene c-myc is down-modulated early, in HNE-induced MEL cells as well as in DMSO-treated cells. However, ornithine decarboxylase gene expression first increases and then decreases, during the lowering of the proliferation rate. These findings indicate that HNE, at a concentration physiologically found in many normal tissues and in the plasma, induces MEL cell differentiation by modulation of specific gene expression.     

The synthesis and accumulation of membrane protein 4.1 in Friend erythroleukemia cells

The effect of extensive differentiation on the synthesis and accumulation of protein 4.1 were studied on Friend erythroleukemia cells grown in suspension and on fibronectin coated dishes. Whole membranes of Friend erythroleukemia cells (FELC) contained a protein 4.1a and 4.1b doublet of Mr 76 and 74 kDa and two minor bands of Mr 105 and 43 kDa that cross-reacted with anti-human protein 4.1 IgG. These proteins were present even in uninduced cells. The synthesis of protein 4.1 was maximal after 4 days of induction in both suspension culture and in fibronectin-coated dishes whereas the protein 4.1 continued to accumulate until the seventh day. More protein 4.1 accumulated in cells grown on fibronectin-coated dishes, at each stage of differentiation, than in cells grown in suspension. The protein 4.1a/4.1b ratio changed during differentiation. The amounts of protein 4.1b increased progressively after induction until the protein 4.1a/4.1b ratio was similar to that of mouse mature erythrocyte. The protein 4.1a/4.1b ratio appears to be an internal marker of erythroid differentiation.     

Induction of differentiation in mouse neuroblastoma cells by hexamethylene bisacetamide

Hexamethylene bisacetamide (HMBA), a potent inducer of erythroid differentiation in murine erythroleukemia cells (1), induces differentiation in mouse neuroblastoma cells, as indicated by the extension of neurites and the development of an excitable membrane. HMBA is effective at concentrations 50-fold lower than dimethylsulfoxide (2), another inducer of differentiation in both mouse neuroblastoma and murine erythroleukemia cells.     

Specific activation of murine B cells by low molecular weight polyamino-polycarboxylic acids (ampholytes).

Commercial preparations of ampholytes (AM), which consist of mixtures of large numbers of polyamino-polycarboxylic acids of molecular weight less than 1000 and whose chemical composition is otherwise not specified by the manufacturer, were found to induce blastogenesis in spleen cell cultures from various strains of mice. The blastogenic response of the spleen cells to the ampholytes was 2 to 12 times greater than that of the unstimulated cultures, and peak stimulatory activity occurred 2–4 days after stimulation. Preparations consisting of either acidic, neutral, or alkaline ampholytes were all found to be mitogenic, although the alkaline ampholytes generally induced the highest stimulation and were active over the widest concentration range (0.08–80 μg per culture). Studies using spleen cells from nu/nu mice, CBA/N mice, organ distribution studies, and the use of cytotoxic antisera and complement for the depletion of thymus-derived (T) cells or bone marrow-derived (B) cells suggested that ampholytes are mitogenic for murine B cells. These cells arise early in ontogeny, because the ampholytes were found to be as mitogenic for spleen cells from newborn as for adult mice. Further, in concordance with the characteristics of other B-cell mitogens, injection of mice with ampholytes induced polyclonal antibody synthesis. The possibility that blastogenic stimulation was due to a contaminant was ruled out by demonstrating that anti-lipid A-treated, ultrafiltered, and isoelectric-focused ampholytes retained stimulatory activity. The results of these investigations suggest that commercial ampholyte preparations contain an undetermined number of low molecular weight (< 1000) acidic, neutral, and alkaline polyamino-polycarboxylic acids which are specific mitogens for a primitive population of murine B cells.     

P2X7 receptor activation induces cell death and microparticle release in murine erythroleukemia cells

Extracellular ATP induces cation fluxes in and impairs the growth of murine erythroleukemia (MEL) cells in a manner characteristic of the purinergic P2X7 receptor, however the presence of P2X7 in these cells is unknown. This study investigated whether MEL cells express functional P2X7. RT-PCR, immunoblotting and immunofluorescence staining demonstrated the presence of P2X7 in MEL cells. Cytofluorometric measurements demonstrated that ATP induced ethidium⁺ uptake into MEL cells in a concentration-dependent fashion and with an EC₅₀ of ∼ 154 μM. The most potent P2X7 agonist 2′- and 3′-0(4-benzoylbenzoyl) ATP, but not ADP or UTP, induced ethidium⁺ uptake. ATP-induced ethidium⁺ and YO-PRO-1²⁺ uptake were impaired by the P2X7 antagonist, A-438079. A colourmetric assay demonstrated that ATP impaired MEL cell growth. A cytofluorometric assay showed that ATP induced MEL cell death and that this process was impaired by A-438079. Finally, cytofluorometric measurements of Annexin-V binding and bio-maleimide staining demonstrated that ATP could induce rapid phosphatidylserine exposure and microparticle release in MEL cells respectively, both of which were impaired by A-438079. These results demonstrate that MEL cells express functional P2X7, and indicate that activation of this receptor may be important in the death and release of microparticles from red blood cells in vivo.     

Relationship of Friend murine leukemia virus production to growth and hemoglobin synthesis in cultured erythroleukemia cells.

The factors that control oncornavirus formation were analyzed in Friend leukemia cells that undergo hematopoiesis when treated with dimethyl sulfoxide. Suspension cultures of Ostertag FSD-1 cell line were found to enter a G or resting state at the end of their proliferative phase and to simultaneously cease producing helper and dependent components of Friend virus. Whereas the decline in virus production is at least 100-fold, rates of cellular RNA and protein synthesis are only slightly lower in resting than in growing cells. Both resting and growing cells contain similarly large concentrations of the viral proteins P(30) and P(12). Dimethyl sulfoxide induces hemoglobin synthesis in growing cells, but its effects on virus production appear to be indirect results of its action to inhibit cell growth and thus to delay entry of cells into the G resting state. Furthermore, variant cell lines were obtained with differing abilities to synthesize virus or hemoglobin. Some lines no longer produce infectious virus, although they all harbor murine leukemia virus genes which are expressed to varying extents. The major internal protein of these oncornaviruses, P(30), is synthesized in large amounts by all of the cell lines. These results suggest that Friend virus production is not coinduced with erythroid differentiation, as had been proposed, but rather is controlled by a cellular growth cycle.     

Inhibition of respiration by a phorbol ester tumor promoter in murine cultured cells

The tumor promoter 12-O-tetradecanoyl phorbol-13-acetate is a potent inhibitor of mitochondrial respiration in both normal and methylcholanthrene-transformed C3H $\text{10T1}\text{2}$ mouse fibroblasts. This inhibition is seen at concentrations of tumor promoter in the range of 10^?8M, occurs within a few minutes after exposure of the intact cells, and is not seen with a biologically inactive analog. The effect appears to be exerted through inhibition of the function of an oligomycin-sensitive ATPase. It is possible, therefore, that alterations in mitochondrial function are associated with the process of tumor promotion.     

Inhibition of protein synthesis and induction of helical polysomes in rat liver by N-hydroxy-2-fluorenylacetamide

After a single intraperitoneal injection of the hepatocarcinogen N-hydroxy-2-fluorenylacetamide (OH-FAA), numerous helical polysomes were found in the hepatocytic cytoplasm at 2 and 6 but not 24 h after treatment. Electron microscopy also demonstrated nucleolar segregation, disarray of endoplasmic reticuium (ER), and disaggregation of polyribosomes at the times when helical polysomes were present. Polyribosome disaggregation was confirmed and quantified by determining size distribution of polyribosomes at 2 h after OH-FAA treatment. Protein synthesis was inhibited at the time of helical polysome induction but the degree of inhibition did not noticeably alter the number of helical polysomes found electron microscopically.     

Increased synthesis of a low molecular weight protein in vincristine-resistant cells

A 19,000-dalton peptide (pI = 5.7) that is synthesized in increased amounts in vincristine-resistant Chinese hamster cells ($\text{DC-3F}\text{VCRd-5}$) has been identified by two-dimensional gel electrophoresis. Reduced amounts of the protein were present in a revertant line of $\text{DC-3F}\text{VCRd-5}$, and only trace amounts were detected in control DC-3F cells. A similar protein (M_r = 19,000; pI = 5.7) was also found in a vincristine-resistant mouse line. Two vincristine-resistant human neuroblastoma cell lines likewise contained elevated levels of a low molecular weight acidic protein. Increased biosynthesis of the 19,000-dalton polypeptide in $\text{DC-3F}\text{VCRd-5}$ cells coincides with the presence of a homogeneously staining region, HSR, on a metaphase chromosome.     

Inhibition by 1 alpha,25-dihydroxyvitamin D3 of dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells

Regulation of erythroid differentiation by vitamin D3 derivatives was examined in Friend erythroleukemia cells. After Friend cells were cultured for 5 days with 1.5% dimethyl sulfoxide (DMSO), as much as 70% of the cells became benzidine-positive and the hemoglobin content increased in parallel with the increase of benzidine-positive cells. The DMSO-induced erythroid differentiation was markedly inhibited by concurrent addition of the active form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]. Of the vitamin D3 derivatives tested, 1 alpha,25(OH)2D3 was the most potent in inhibiting DMSO-induced erythroid differentiation. 1 alpha,25(OH)2D3 alone was totally ineffective in both cell growth and erythroid differentiation. These results together with our previous reports indicate that 1 alpha,25(OH)2D3 is somehow involved not only in myeloid differentiation, but also in erythroid differentiation.     

Inhibitory studies of DNA, RNA and protein synthesis in Escherichia coli by platinum containing complexes

Previous studies have shown various platinum containing compounds to be effective anti-tumor agents in man and animals. Many of these compounds have also been shown to be effective inhibitors of bacterial DNA, RNA and protein synthesis. Data is presented here which compares the inhibitory effectiveness of a number of recently synthesized platinum compounds toward the inhibition of the synthesis of DNA, RNA and protein in Escherichia coli. We also compared the effectiveness of these compounds toward the inhibition of bacterial growth. Some of these new derivatives appear to be nearly 3-fold more potent than the more thoroughly studied cis-diamminedichloroplatinum(II) (cis-PDD) and trans-diamminedichloroplatinum(II) (trans-PDD).