Regulation of choline kinase activity and phosphatidylcholine biosynthesis by mitogenic growth factors in 3T3 fibroblasts.

The regulation of choline kinase activity by fetal bovine serum and the regulation of phosphatidylcholine biosynthesis by choline kinase have been investigated in 3T3 fibroblasts. Treatment of quiescent 3T3 fibroblasts with serum was shown in previous work to increase phosphocholine pool size and phosphatidylcholine biosynthesis. We now report that treatment of 3T3 cells with serum increased intracellular choline kinase activity by 2-3-fold with a concomitant 2-3-fold decrease of intracellular free choline concentrations.

Cystamine augments the stimulation of DNA synthesis by peptide growth factors and microtubule-disrupting agents in cultures of 3T3 mouse fibroblasts.

Cystamine together with colchicine markedly enhanced the uptake of [3H]-thymidine into DNA of quiescent cultures of insulin-stimulated Swiss 3T3 mouse fibroblasts. Flow cytofluorometric analyses showed an increased rate of transition of cells from G0/G1----S + G2 in response to combinations of insulin, colchicine, and cystamine. Cystamine, the most effective of several thiol compounds, gave maximal augmentation at 200 microM and was toxic at 300-500 microM.

Regulation of phosphatidylcholine biosynthesis by mitogenic growth factors.

Phosphatidylcholine (PC) biosynthesis in cultured 3T3 fibroblasts was increased in varying degrees by these mitogenic growth factors: fetal bovine serum, insulin, 12-O-tetradecanoylphorbol-13-acetate, epidermal growth factor, vasopressin, fibroblast growth factor and insulin-like growth factors I and II. PC synthesis was increased 2-4-fold by 10% serum, up to 4-fold by growth factors alone, and up to 8-fold by combinations of two or more growth factors. Single growth factors had no effect on the incorporation of [3H]choline into the acid-soluble precursors of PC, while serum or combinations of two or more mitogens could increase the incorporation of [3H]choline into acid-soluble material by up to 2-fold.

The relationship between the disassembly of microtubules during G1 and the enhancement of DNA synthesis by colchicine in mouse fibroblasts stimulated with peptide growth hormones.

By immunofluorescent staining to visualize the cytoplasmic microtubular cytoskeleton in mouse fibroblasts we have ascertained that after a relatively short exposure of cells to colchicine, microtubules remain disassembled for a prolonged period of time after cells are transferred to a colchicine-free medium. In contrast to the persisting effects of colchicine, a brief exposure of cells to nocodazole first induces the expected disruption of microtubules followed by regeneration of the cytoskeleton within a few hours after removal of extracellular drug. These results shed light on our previous finding that quiescent mouse fibroblasts first treated with colchicine and then transferred to colchicine-free medium exhibit an enhanced proliferative response to EGF and insulin, whereas cells treated in a similar manner with nocodazole show no enhancement of DNA synthesis stimulated by peptide growth hormones.

18F-5-Fluorouridine, a new probe for measuring the proliferation of tissue in vivo.

(1) Increased metabolic trapping of labeled fluorouridine reflects the interaction of three parameters in rapidly proliferating tissues: increased rates of intracellular phosphorylation, increased rates of transport, and increased rates of synthesis of RNA. (2) We have taken advantage of these metabolic phenomena, demonstrating in this paper that the uptake of 18F-5-fluorouridine, a positron-emitting radiopharmaceutical, can provide a very practical means for measuring changes in proliferative states of tissues in vivo. (3) Two major changes in proliferative states have been examined: one involves changes in growth of normal mouse tissues induced by pharmacological agents; the other involves tumor growth and neoplastic infiltration in mice and rabbits.

Colchicine inhibits epidermal growth factor degradation in 3T3 cells.

Colchicine (2 microM) did not affect the initial rate of association of 125I-labeled epidermal growth factor (125I-EGF) to Swiss 3T3 cells but continued incubation (up to 24 hr) led to an increase in cell-associated radioactivity. The effect is also produced by Colcemid, vinblastine, and podophyllotoxin but not by lumicolchicine. Disruption of microtubules with colchicine does not alter the rate of "down regulation" of EGF receptors, suggesting the binding and internalization of the factor proceed unchanged.

Antitubulin agents enhance the stimulation of DNA synthesis by polypeptide growth factors in 3T3 mouse fibroblasts.

Colchicine and other antitubulin agents markedly enhanced the stimulation of DNA synthesis by combinations of various growth factors such as epidermal growth factor, insulin, fibroblast-derived growth factor, and vasopressin in serum-free cultures of several quiescent 3T3 mouse fibroblast cell lines. Enhancing effects were observed based on continuous incorporation of [3H]thymidine into DNA as well as by autoradiographic labeling of cell nuclei. The concentration of colchicine and podophyllotoxin required to produce half-maximal enhancement of DNA synthesis stimulated by epidermal growth factor and insulin was 25-50 nM.

Mid-G1 marker protein(s) in 3T3 mouse fibroblast cells.

Quiescent 3T3 mouse fibroblast cells in a state of growth arrest due to serum deprivation were exposed to [14C]isoleucine. The cell cultures were then stimulated by the addition of 10% fetal calf serum. At various times after stimulation, the 14C-labeled cells were exposed to [3H]isoleucine.

Polyglutamyl and polylysyl derivatives of the lysine analogues of folic acid and homofolic acid.

A series of Nepsilon-poly-alpha-glutamyl and Nepsilon-polylysyl derivatives of Nalpha-pteroyllysine and Nalpha-homopteroyllysine, analogues of the naturally occurring gamma-polyglutamyl forms of folate, was prepared and tested as substrates for dihydrofolate reductase and as substrates and inhibitors of thymidylate synthetase. Nalpha-Dihydropteroyl-Nepsilon-(tri-alpha-glutamyl)lysine was 1.8 times as active as Nalpha-dihydropteroyl glutamate (dihydrofolate) as a substrate for L1210 murine leukemia dihydrofolate reductase.

Inhibition of thymidylate synthetase and dihydrofolate reductase by naturally occurring oligoglutamate derivatives of folic acid.

Naturally occurring oligoglutamate derivatives of folic acid in extracts of Escherichia coli have been isolated on the basis of their inhibitory actions toward thymidylate synthetase and dihydrofolate reductase. The inhibitor of thymidylate synthetase has been identified as N-5-formyl-H4pteroyloligoglutamate (approximately 5 amino acid residues). It is 150-fold more inhibitory than the monoglutamate.

Genetic and immunological studies of bacteriophage T4 thymidylate synthetase.

Thymidylate synthetase, which appears after infection of Escherichia coli with bacteriophage T4, has been partially purified. The phage enzyme is immunologically distinct from the host enzyme and has a molecular weight of 50,000 in comparison to 68,000 for the host enzyme. A system has been developed to characterize T4 td mutants previously known to have impaired expression of phage thymidylate synthetase.

Detection of DNA synthesis in intact organisms with positron-emitting (methyl- 11 C)thymidine.

(11)CO(2) produced in the Brookhaven 152-cm cyclotron was converted to formaldehyde, which in turn was used for the enzymatic conversion of deoxyuridine-5'-phosphate to [(11)C]thymidylate. Enzymatic treatment of the nucleotide with alkaline phosphatase gave [(11)C]thymidine.The preparation of [(11)C]thymidine from cyclotron-generated (11)CO(2) required 110 min (about 5 half-lives): 35 min for the synthesis of H(11)CHO, 25 min for the enzymatic conversion to [(11)C]thymidylate, 20 min for column chromatography, 5 min for phosphatase treatment, 10 min for evaporation, 2 min for filtration through an anion-exchange resin, and 13 min for miscellaneous manipulations.