This report identifies a group of compounds, polymethylene bisacetamides (acetylated diamines), which are potent inducers of erythroid differentiation in murine erythroleukemia cells. A known inducing agent, N-methylacetamide, was dimerized through varying numbers of methylenes in an attempt to increase the local effective concentration at adjacent target sites. The simple dimer was no more effective than N-methylacetamide alone; introduction of five to eight methylenes between acetamide groups substantially increased the effectiveness of these compounds. The hexamethylene bisacetamide was active between 0.5 mM and 5 mM; the percentage of cells induced and the rate at which they were recruited to differentiation was dependent upon the concentration of inducer within this range. At 5 mM hexamethylene bisacetamide essentially the entire population (>99%) was induced to a pathway of erythroid differentiation which was greater differentiation of the cultured cells than with any inducer yet tested.The murine erythroleukemia cell line established by Friend (1) shows a low percentage (<1%) of spontaneous erythroid differentiation; inclusion of 280 mM dimethylsulfoxide (Me2SO) in the culture medium results in an increased number of cells synthesizing hemoglobin (2). The program of erythroid differentiation induced by Me2SO includes characteristic changes in morphology (2), the appearance of globin mRNA (3, 4), an increase in iron uptake and heme synthesis (2), and the appearance of erythroid membrane antigens (5).Dimethylsulfoxide is not unique in this action in that several other compounds, having in common a hydrophilic or polar group covalently joined to a hydrophobic group, also induce differentiation in these cells (6, 7). With the knowledge that several types of organic compounds are active as inducing agents, we attempted to find a compound that would be better than those already investigated in terms of both lower effective concentrations and the percentage of cells that differentiate. One approach to this problem was to test dimers of a known inducing agent in order to increase the local effective concentration of inducer at adjacent target sites. The inducing agent N-methylacetamide was dimerized by linkage at nitrogen through a varying number of methylene groups. We have identified a group of compounds, polymethylene bisacetamides (acetylated diamines), that are potent inducers, in that, at concentrations well below the optimal for Me2SO, essentially the entire culture (>99%) of murine erythroleukemia cells differentiate. These compounds are structurally related to certain naturally occurring diamines, which are shown to be weak inducers. MATERIALS AND METHODSStrain 745A, a murine erythroleukemia cell line, was provided by Dr. Charlotte Friend, and maintained in continuous culture in our laboratory for 3 years as previously described (8). The extent of differentiation in a culture was determined by either of two methods. The percentage of hemoglobin-containing cells was determined by the benzidine rea...

Complementary DNA coding for human monocyte interleukin 1 (IL-1), pl 7 form, was expressed in Escherichia coli. During purification, IL-1 activity on murine T cells was associated with the recombinant protein. Homogeneous human recombinant IL-1 (hrIL-1) was tested in several assays to demonstrate the immunological and inflammatory properties attributed to this molecule. hrIL-1 induced proliferative responses in a cloned murine T cell in the presence of suboptimal concentrations of mitogen, whereas no effect was observed with hrIL-1 alone. At concentrations of 0.05 ng/ml, hrIL-1 doubled the response to mitogen (5 X 106 half maximal units/mg). Human peripheral blood T cells depleted of adherent cells underwent a blastogenic response and released interleukin 2 in the presence of hrIL-1 and mitogen. hrIL-1 was a potent inflammatory agent by its ability to induce human dermal fibroblast prostaglandin E2 production in vitro and to produce monophasic (endogenous pyrogen) fever when injected into rabbits or endotoxin-resistant mice. These studies establish that the dominant pl 7 form of recombinant human IL-1 possesses immunological and inflammatory properties and acts on the central nervous system to produce fever.

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Previous studies demonstrated that 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a tumor promoter, is a potent inhibitor of inducer-mediated differentiation of murine erythroleukemia cells. Inhibition of cell differentiation was associated with inhibition of cell growth. The present studies, employing a cell line adapted for growth in TPA, demonstrate that inhibition of differentiation is not dependent upon inhibition of cell growth or a change in the cellfdivision cycle; neither is inhibition of differentiation accompanied by detectable effect on cell uptake of [3H]hexamethylene bisacetamide, the inducer used in these studies. TPA causes an inhibition of expression of all hexamethylene bisacetamide-inducible erythroid characteristics measured, including commitment to terminal cell division, accumulation of globin mRNA, and synthesis of globins, spectrin, heme synthetic enzymes (5-aminolevulinic acid dehydratase and uroporphyrinogen-I synthase) and heme. A hypothetical model for the inhibitory action of tumor promoters on terminal cell differentiation is discussed. 12-0-Tetradecanoyl-phorbol-13-acetate (TPA) is one of a group of plant diterpenes that are tumor promoters in the two-stage mouse skin carcinogenesis system (1-4). TPA is a reversible inhibitor of cell differentiation in several in vitro systems, including murine erythroleukemia cells (MELC) (5, 6), murine neuroblastoma cells (7), chicken myoblasts and chondroblasts (8,9), and the fibroblast-adipocyte conversion (10). It has been postulated that tumor promotion itself may involve interference in the process of differentiation (1, 6, 11). Of the several systems examined to date, MELC is the best characterized with respect to the molecular events during expression of differentiation. Upon exposure to various agents such as dimethyl sulfoxide (12), hexamethylene bisacetamide (HMBA) (13), or butyric acid (14), these cells initiate erythroid differentiation, including characteristic morphological changes (12), synthesis and accumulation of globin mRNA (15-18), synthesis of a and ( globins (19,20), increase in spectrin content (21,22), increase in the activity of heme synthetic enzymes and heme content (23,24), and loss of the capacity for cell division (12,25,26).We have previously found that TPA-mediated inhibition of differentiation is accompanied by an early and pronounced inhibition of cell growth (6,27). In the present studies we demonstrate that an effect on cell growth is not required for the effect on differentiation. Furthermore, we show that, under conditions in which TPA has no detectable effect on cell growth or uptake of inducer, it inhibits the expression of many characteristics of erythroid differentiation, including commitment to terminal cell division and accumulation of globin mRNA, globin, spectrin, heme synthetic enzymes, and heme. MATERIALS AND METHODSMELC, subclone DS19-1OTS, a TPA-sensitive cell line isolated from strain 745A-DS19, was maintained in culture and characterized as described (27). DS19-1OTS(TPA) cells were obtain...

A DNA-binding protein has been purified from Escherichia coli infected with bacteriophage T7 by DNA-cellulose chromatography. The protein is absent in uninfected cells. The purified protein has a molecular weight of 31,000 and binds strongly and preferentially to single-stranded DNA. In vitro studies show that this protein can stimulate the rate of polymerization catalyzed by the T7-induced DNA polymerase 10-15 times under conditions where the polymerase is unable to effectively use a single-stranded template. The degree of stimulation is dependent upon the ratio of binding protein to DNA template and is independent of polymerase concentration. The observed stimulation is specific for the T7 DNA polymerase in that addition of the protein to reactions catalyzed by E. coli DNA polymerases I, II, or III or T4 DNA polymerase is without effect.