We have found that phagocytic leukocytes exposed to the tumor-promoting agent, 12-O-tetradecanoyl phorbol-13-acetate, efficiently release carbon-i of 2-deoxyglucose in the form of CO2 with concurrent intracellular accumulation of a phosphorylated 5-carbon intermediate. In the absence of 12-atetradecanoyl phorbol-13-acetate, these cells release barely detectable amounts of CO2 from 2-deoxyglucose. 12-0-Tetradecanoyl phorbol-13-acetate, at a concentration of 1 ng/ml, has an immedate effect on CO2 release, which is temperature-dependent and linear with time and cell number. The ability of a number of phorbol ester-like compounds to enhance this catabolic pathway for 2-deoxyglucose correlates with their ability to act as tumor promotors and inflammatory agents. Although this effect of phorbol esters appears to be restricted to granulocytes, monocytes, and macrop a es, the possibility arises that other mammalian cells are capable of catabolizing or can be induced to catabolize 2-deoxyglucose. Thus, 2-deoxyglucose decarboxylation should be considered whenever this analog of mannose and glucose is used as an indicator for sugar transport, especially when pharmacodynamic agents are present.12-O-Tetradecanoyl phorbol-1S-acetate (TPA), the most active tumor-promoting agent isolated from croton oil (1, 2), acts as an inflammatory agent (3), increases the level of proteases in mouse skin (4), and enhances the carcinogenicity of a number of agents applied to the skin (5). TPA is capable of evoking a multiplicity of changes in cultured cells which are generally considered to be characteristic of the transformed phenotype. Responses include an increase in plasminogen activator production (6-8), an increase in the rate of sugar transport (9), a decrease in LETS protein (10), altered morphology (11), and inhibition of terminal differentiation (12)(13)(14)(15)(16)(17).TPA also increases the rate of decarboxylation of glucose in human granulocytes via the hexose monophosphate shunt (18,19 fetal bovine serum. The human myeloid line HL-60 (22) was maintained in the same medium with a supplement of 20% heat-inactivated fetal bovine serum. For preparation of cell-free extracts, washed cells were suspended in 3 mM EDTA/30 ,uM NADP+/20 mM Hepes, pH 7.5, at a concentration of 107/ml. After three cycles of rapid freezing and thawing, particulate matter was removed by centrifugation at 15,000 rpm for 5 min. The supernatant contained all activity for NADP+-dependent CO2 release from glucose and 2-dGlc.Assay for Release of CO2. CO2 released by cells incubated under conditions described in figure and table captions was trapped in a Hyamine-soaked filter paper in a glass well suspended above the incubation mixture. The incubation was terminated by injection of concentrated sulfuric acid (100 ,ul/ml) and incubation was continued for 30 min at 37°C to ensure that all dissolved CO2 was released from the acidified medium. Radioactivity in the filter paper was determined in a scintillation counter with toluene-based fluor.Chromatographic Me...

A protein factor contained in a 1 M KCI extract of L-cell ribosomes and partially purified by chromatography on DEAE-cellulose forms a specific ternary complex with rat-liver Met-tRNAf and GTP. The complex is measured by its quantitative retention on nitrocellulose membranes. Complex assembly is optimal at 100 mM KC1 and 0.2 mM MgCl2, and is independent of mRNA and of ribosomes. The GTP requirement can be replaced over 65% by its methylene analogue GDPCH2P, indicating that GTP hydrolysis is not involved. Complex formation is inhibited by 10 MM aurintricarboxylic acid, but is unaffected by 100 MM pactamycin, 100 MM fusidic acid, or by excess uncharged methionine tRNAf. The ternary complex is relatively stable and appears at the void volume during filtration on Sephadex G-100. At 1-3 mM MgCl2 and in the presence of other factors, the ternary complex is implicated in protein initiation by (i) its capacity to bind to the 40S ribosomal subunit to form a 48S complex; and (ii) the subsequent association of the 48S complex with a 60S subunit to form a functional "80S complex."There is considerable evidence to suggest that in eukaryotes, the rate-determining step in protein synthesis occurs during the initiation phase of translation. Such a rate-limiting step has been observed in such diverse tissues as cells in mitosis (1), muscles of diabetic animals (2), livers of fasted animals (3, 4), as well as in cell cultures exposed to aminoacid-deficient medium (5, 6), temperature changes (7), and alterations in sodium-potassium ratios (8). Other studies in both prokaryotes and eukaryotes indicate that the sequence of events leading to the formation of an 80S initiation complex is mediated by the stepwise participation of specific ribosome factors (9)(10)(11)(12)(13)(14). Consequently, the first steps in the initiation sequence are of particular importance if it is assumed that they represent logical sites for the operation of regulatory controls.Recent studies inI prokaryotic systems have rel)orted that one of the earliest steps in the initiation sequence is the formation of a ternary complex containing the ribosome initiation factor and GTP (15,16).In this report, we describe the in vitro formation of a eukaryotic ternary complex with similar properties, composed of a partially purified ribosome factor from L-929 mouse fibroblasts (L-cells), rat-liver Met-tRNAf, and GTP. Preliminary data were presented in a previous communication (17

Butyrate, in relatively low concentrations, has been shown to induce synthesis of enzymes, cause changes in cell morphology, and inhibit growth of a variety of mammalian cells in tissue culture (reviewed in [1]).In this communication, we report our observations on the effect of butyrate on lymphocyte activation. Butyrate completely and reversibly inhibits mitogeninduced blast formation. We present evidence that it does not interfere with the binding of mitogens, that it does not inhibit a number of the "early" reactions involved in activation, and that it does not affect ongoing DNA synthesis for an extended period of time. However, butyrate rapidly inhibits any increase in the rate of DNA synthesis. Materials and MethodsMouse spleen lymphocytes were isolated from 18-25 g (6-9-wk-old) male or female Swiss Webster mice, by the method of Bcyum (2). Freshly isolated lymphocytes, from the spleen of a single mouse, were incubated either in RPMI-1640 or minimum essential medium. All media were supplemented with 10% heat-inactivated fetal calf serum and 10 -5 M 2-mercaptoethanol. Two separate duplicate cultures were prepared for each experimental point. Each culture consisted of 1.0-1.5 × 106 cells in 1 ml medium in a 12 × 75-mm plastic tissue culture tube maintained for the indicated time at 37°C, with an atmosphere of 5% CO2. Butyric acid (neutralized with NaOH) was used in 2 mM final concentration or as indicated in Table I.To measure the rate of DNA synthesis, cells were labeled with 1 or 2.5 ~Ci/ml [3H]thymidine (sp act: 60 Ci/mmol) for 2 h, filtered onto Millipore membrane filters (pore size: 0.45 ~m), washed with 2 × 1 ml Dulbecco's phosphate-buffered saline minus Ca ++ and Mg +÷ (PBS), and with 3 × 5 ml 5% TCA. Acid insoluble radioactivity on dried filters was determined in a liquid scintillation counter. All experiments were repeated a minimum of five times. The data presented are from typical experiments. Results 2 mM butyrate inhibits the induction of DNA synthesis in mouse spleen lymphocytes by PHA, Concanavalin A, or endotoxin (Table I A). Although incorporation of [3H]thymidine is inhibited by at least 90%, cell viability, as determined by trypan blue exclusion, is not affected. With lower concentrations

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