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Biomedical subjects

M Friedkin

Publications and source records attributed to M Friedkin.

At least 19 recordsLinked to original sources
PubMed1985

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. Initial rates of choline transport were the same in quiescent and serum-treated cells, whereas choline kinase activity was 2-3-fold higher in serum-treated cells. As a consequence, free choline concentrations were 2-3-fold lower in serum-stimulated cells than in control quiescent cells. Phosphocholine turnover rates were increased 2-fold by serum treatment both as a consequence of a serum-dependent increase of phosphocholine pools and as a result of a serum-dependent lowering of the phosphocholine half-life. Thus, the overall response of 3T3 cells to serum stimulation included decreased choline pools and increased choline kinase activity, phosphocholine pool size, phosphocholine turnover, and phosphatidylcholine biosynthesis.

Animals↗
PubMed1984

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. Serum was shown to increase choline phosphorylation, choline kinase activity and the size of the phosphocholine pool. These data were utilized to calculate the radioactive specific activity of phosphocholine. Serum did not increase phosphocholine specific activity above control values; thus the increased incorporation of labelled choline into PC after serum stimulation resulted from increased PC synthesis and not from a simple change in specific activity of precursor phosphocholine.

Animals↗
PubMed1984

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. Amplification of DNA synthesis by cystamine was also obtained with epidermal growth factor, vasopressin, and 0.5% fetal bovine serum. Combinations of cystamine and other microtubule-disrupting agents such as nocodazole, maytansine, and podophyllotoxin enhanced DNA synthesis in insulin-stimulated cells. In experiments involving sequential addition of agents, significant enhancement of DNA synthesis was observed when the addition of colchicine to cystamine-treated cells was delayed or conversely when the addition of cystamine to colchicine-treated cultures was delayed. This reciprocal interaction between cystamine and colchicine suggests that a prereplicative intermediate accumulates in response to the action of these dissimilar compounds. We consider the possibility that cystamine may act by forming mixed disulfides with thiol groups of unknown protein(s) that regulate DNA replication.

Animals↗
PubMed1983

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. We conclude that cytoplasmic microtubules must remain disaggregated during the prereplicative G1 period in order for cells to exhibit the enhancing effects of the microtubule-disrupting drugs on DNA synthesis.

Animals↗
PubMed1982

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. (4) We describe tracer experiments with 18F-5-fluorouridylate, prepared by enzymatic means, and with 18F-5-fluorouridine, prepared by both enzymatic means and direct radiochemical procedures. (5) Uptakes of 18F after a pulse of 18F-5-fluorouridine were increased in mouse spleen following phenylhydrazine treatment to induce increased splenic erythropoiesis. (6) Uptakes of 18F in various mouse tissues were decreased following pretreatment with actinomycin D. This finding is consistent with the known inhibitory action of actinomycin on RNA synthesis. (7) Intracerebral Zimmerman ependymoblastoma tumors showed extraordinarily high uptakes of fluorine-18 in mice injected intravenously with 18F-5-fluorouridylate or with 18F-5-fluorouridine in contrast to very low uptakes by normal brain tissue. (8) After intracerebral injection of mice with suspensions of L1210 leukemia cells, distant organs such as lung, liver, and spleen became involved. These tissues showed significant increases of radioactivity after pulse labeling with 18F-5-fluorouridylate consistent with histological evidence for infiltration of these tissues by neoplastic cells. (9) Intramuscular VX2 carcinoma tumors in rabbits showed localized uptakes of 18F significantly higher than surrounding normal muscle tissue. (10) The most important clinical implication of the present work is the promise that 18F-5-fluorouridine uptakes can be followed in humans by positron emission tomography. This would provide a direct means of measuring different rates of in vivo proliferation in neoplasms, hematologic tissues and other organs undergoing rapid growth changes.

Animals↗
PubMed1980

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. However, colchicine markedly decreases the rate of appearance of acid-soluble radioactivity from cells either incubated continuously with 125I-EGF for 24 hr or exposed to the radioactive peptide for only 1 or 3 hr. The results indicate that colchicine decreases the rate of degradation of internalized 125I-EGF. Because antitubulin agents enhance the mitogenic effect of EGF our results suggest that peptide degradation can be dissociated from the long-term biological effect.

Ammonium Chloride↗
PubMed1979

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. Lumicolchicine did not produce enhancing effects. The disassembly of microtubules resulting from the action of colchicine, Colcemid, and vinblastine did not inhibit the stimulation of DNA synthesis in quiescent Swiss 3T3 fibroblasts by fetal bovine serum. We conclude that the cytoplasmic microtubule network in 3T3 mouse fibroblasts does not exert a positive regulatory function in the initiation of DNA synthesis but rather can produce a constraint on the initial action of the peptide growth factors in serum-free media.

Animals↗
PubMed1978

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. Cytoplasmic extracts from the double-labeled cells were subjected to sodium dodecyl sulfate/slab gel electrophoresis. By these procedures it was found that the relative rate of synthesis of a protein species of Mr 50,000 increased after stimulation of quiescent cells, reached a maximum at 5 hr, and then decreased before the 3T3 cells began to enter the S phase. The characteristic peaking profile of mid-G1 protein synthesis exhibited by the Mr 50,000 polypeptide can serve as a useful marker for the progression of events in G1 prior to exit into S.

Cell Cycle↗
PubMed1976

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. N-alpha-Dihydropteroyl-Nepsilon-(di-alpha-lysyl)lysine was 1.2 times as active as dihydrofolate in spite of its strong positive charge. The most active compound tested, Nepsilon-(tert-butyloxycarbonyl)lysine, was 3.5 times as active as dihydrofolate. None of the enzymatically prepared Nalpha-tetrahydropteroyllysine derivatives tested was as active as Nalpha-tetrahydropteroyl glutamate (tetrahydrofolate) as a substrate for E. coli thymidylate synthetase. However, there was a progressive increase in activity with the addition of each alpha-glutamyl residue, the Nepsilon-(penta-alpha-glutamyl)lysine being 88% as active as tetrahydrofolate. Nalpha-Tetrahydropteroyl-Nepsilon-(di-alpha-lysyl)lysine was the most active thymidylate synthetase substrate of the polylysine derivatives, being 67% as active as tetrahydrofolate. Addition or deletion of lysyl residues resulted in diminished activity. It is noteworthy that substrate activity is retained in spite of the positively charged poly(amino acid) side chain. None of the enzymatically prepared tetrahydrohomopteroyl derivatives tested was as active as Nalpha-tetrahydrohomopteroyl glutamate (tetrahydrohomofolate) as an inhibitor of E. coli thymidylate synthetase.

Animals↗
PubMed1975

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. Synthetic N-5-formyl derivatives containing 3 to 6 glutamyl residues were prepared and found to be 67- to 200-fold more inhibitory than the monoglutamate. N-5-Formimino-H4pteroyltriglutamate is one-twentieth as inhibitory as the corresponding N-5-formyl derivative. The inhibitor of mouse leukemia dihydrofolate reductase has been identified as N-10-formylpteropentaglutamate. It is approximately 7 times as inhibitory as N-10-formylpteroylmonoglutamate. It is 4,400 times as inhibitory toward mouse leukemia dihydrofolate reductase compared with the enzyme from E. coli. Lysine analogs of N-5-formyl-H4folate containing alpha0glutamyl groups in peptide linkage to the epsilon-amino group of lysine were relatively poor inhibitors of thymidylate synthetase. The inhibitory action of folic acid oligoglutamates on E. coli thymidylate synthetase was subject to reversal with 0.4 M NaCl, an effect that was more marked with various pteroyloligoglutamates than with H4homopteroylmonoglutamate and N-5, N-8-deaza-N-10-methylpteroylmonoglutamate.

Chromatography, DEAE-Cellulose↗