Morris Friedkin

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.

Enhancement of DNA synthesis by colchicine in 3T3 mouse fibroblasts stimulated with growth factors.

Abstract When quiescent cultures of Swiss 3T3 mouse fibroblasts were stimulated to enter the S phase by peptide growth hormones added in serum-free medium, DNA synthesis was markedly enhanced by treatment of the cells with colchicine. The enhancement could be shown by increased incorporation of [3H]thymidine ([3H]TdR) into DNA as well as by increased transition from G0/G1→S in DNA histograms obtained by flow cytofluorometric analyses. The enhancement induced by colchicine occurs at concentrations known to cause depolymerization of cytoplasmic microtubules in mouse fibroblasts. The onset of action of colchicine was shown to be a function of unmediated transport. One hour of exposure to 2 μM colchicine produced enhancements that could be related to persistent intracellular binding of the antitubulin agent. Disruption of the cytoplasmic microtubules could be delayed 3–4 h after stimulation of quiescent cells by EGF, insulin and FDGF, yet significant enhancement of DNA synthesis was observed. Once DNA synthesis was initiated in the S phase, enhancement was not obtained. When cells were pretreated with either FDGF or fetal bovine serum for 3 h, washed and then reincubated with insulin, colchicine added after the initial exposure enhanced the stimulation of DNA synthesis. These results show that the enhancing action of colchicine is exerted in G1 at least 3 h after addition of peptide growth factors. These findings raise the possibility that the microtubular network regulates the action of growth-promoting factors subsequently to their initial interaction with the cell.

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.

The role of cytoplasmic microtubules in the regulation of the activity of peptide growth factors

The enhancement of DNA stimulation by peptide growth factors in colchicine-treated cultures of Swiss 3T3 mouse fibroblasts appears to be associated with disassembly of cytoplasmic microtubules. Other antitubulin agents (colcemid, vinblastine, podophyllotoxin, nocodazole, and maytansine) also potentiate the effects of growth promoting factors such as insulin and EGF. The cytoplasmic microtubules are disrupted by antitubulin agents at concentrations very similar to those required for depolymerization of mitotic spindle microtubules. In the presence of highly radioactive [3H]thymidine, DNA synthesis can be stimulated in quiescent mouse fibroblasts without completion of the cell cycle. This makes possible studies of the potentiating effects of colchicine during the prereplicative period of the cycle uncomplicated by effects of the antitubulin agent on the mitotic spindle. The enhancing effects of colchicine can be demonstrated with sparse cultures of 3T3 fibroblasts as well as with 3T6 cells. Maytansine and colchicine need not be present during the initial period of interaction of peptides with the cell to produce enhancement of DNA synthesis. The enhancement can be obtained after transient exposure of cells to fetal bovine serum. The potentiating effects of antitubulin agents such as nocodazole, podophyllotoxin, and maytansine can be easily reversed whereas the enhancement with colchicine persists despite washing of cells. The stimulatory effects of insulin and EGF persist in cultures treated with antitubulin agents. The findings support the premise that after internalization the receptor-mitogen complex may remain active within endocytic vesicles when cells are exposed to colchicine.

EMPIRICAL VS. RATIONAL APPROACHES IN CANCER CHEMOTHERAPY

According to Webster’s Dictionary, empiricism relies solely on experiments and observation without benefit of principles, whereas rationalism is in itself a source of insight independent of experience. I think most of us would agree that pure rationalism is extremely rare in the life sciences. Although molecular biology has opened up new vistas of rationalism, most of what we know, what we have learned, and what we might predict is the result of hard work at the laboratory bench. A well-known example of empiricism in chemotherapy is the mundane screening of compounds without regard to their structures. A less empirical approach is to determine the structure of a drug discovered by mere chance and then to modify it in a systematic way with the hope of developing a better derivative. A semirational approach is the design of chemotherapeutic agents based on a knowledge of metabolic events in normal and neoplastic cells. This concept leads to more selective screening of only those compounds that show some structural similarity to important cell metabolities, coenzymes, hormones, and allosteric regulators. This seems most appropriate and satisfying, since it guides our research in an apparently meaningful way. We no longer act like blind men groping for a way out. Such a strategy seems to hold out hope for those of us who try to design more effective and more specific inhibitors. There is always the sense of anticipation that we are finally on the right track. Yet, insight alone, although seemingly superior to the empirical approach, is not enough. We do not have a precise picture of everything we need to know. Our knowledge is too fragmentary. We may be in the position to make a good guess about potential efficacy but we cannot predict toxicity. Let us reverse the history of penicillin. Suppose the whole beautiful story of bacterial cell wall synthesis had been worked out before the discovery of antibiotics and as a result of this knowledge it became clear that the bacterial cell wall was chemically quite different from animal cell walls. It is conceivable that some bright biochemist might have reasoned that an inhibitor of the transpeptidation reaction in the last stage of murein synthesis should cause irreparable harm to bacteria but not to animal cells. An effective synthetic penicillin-like compound could have come out of such a study. However, no prediction could have been made about its toxicity. This is where empiricism enters the picture. After we are all through inhibiting our favorite enzymes,

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.

Reversible inactivation and reactivation of thymidylate synthetase

Abstract The reversible inactivation and reactivation of E. coli thymidylate synthetase has been studied in a number of ways. The enzyme was rapidly inactivated at 0 °0 in the presence of 7 m urea. Maximal reactivation occurred when dithiothreitol was present at a concentration of 10 m m during inactivation and reactivation. Mercaptocthanol at 0.1 m was also found to be an effective thiol. The kinetics of reactivation were studied after dilution of the ureatreated enzyme and incubation either at 0 °0 or at room temperature. The reversible inactivation based on kinetic studies can be described as a denaturation process leading to unfolding and refolding of a polypeptide chain. Disruption into subunits and reassembly appears to be an unlikely possibility under the conditions used in the present study. It was possible to carry out gel filtration of the inactivated enzyme with Sephadex G-100 equilibrated with 5 m uread and to restore activity upon dilution of column fractions. These experiments also supported the concept that a reversible conformational transition had occurred. Further studies using the methods of optical rotatory dispersion and circular dichoism seem most appropriate.