Biomedical subjects
Publications and source records attributed to K Marushige.
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Rat liver chromatin has been fractionated into two fractions on the basis of precipitability in standard saline after mild treatment with DNase II. The major portion of liver chromatin contains little nonhistone protein and is enriched in histones, while a minor portion of such chromatin, with which RNA polymerase is associated, is highly enriched in proteins other than histone and impoverished in histones. Although the relative concentrations of the several histone species present in two chromatin fractions are identical, binding of histones to DNA in the minor fractions appears to be modified, presumably by the presence of proteins other than histones.
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Trigeminal neurinoma cells were used to characterize the involvement of ERK, JNK, p38 and phosphatidylinositol 3-kinase (PI3-K)/Akt signaling pathways in the induction of apoptosis. Activation of JNK by anisomycin, the inhibition of ERK activation by PD098059 or a blockage of the PI3-K/Akt pathway by wortmannin or LY294002 alone, was not sufficient for the induction of apoptosis. Apoptosis was rapidly induced when the activation of JNK was coupled with the inhibition of PI3-K/Akt, and the induction was further enhanced by a concurrent inhibition of ERK activation. The p38 inhibitor, PD169316, reduced the activities of ERK and Akt. Rapid induction of apoptosis occurred when the inhibition of p38 was coupled with JNK activation, and a concurrent inhibition of PI3-K/Akt potentiated the induction. Apoptosis was also induced without JNK activation, though at a slower rate, by a combined treatment with PD169316 and LY294002. A concomitant inhibition of ERK and Akt activation induced apoptosis without JNK activation, although with a considerable delay of its onset. These results suggest that ERK, JNK, p38 and PI3-K/Akt signaling pathways interact to form an integrated network, and the induction of apoptosis requires coordinated changes in these signaling pathways.
Nerve growth factor (NGF) inhibited cellular DNA synthesis of rat T9 anaplastic glioma cells in a dose-dependent manner in the range of 0.5-5 micrograms/ml. Oxidation of 2 to 3 tryptophan residues of NGF, which had been known to destroy biological and immunological activity, greatly diminished its inhibitory effect on DNA synthesis. The inhibition was also abolished by anti-NGF IgG. Flow cytometric analyses and immunocytochemical assays of DNA synthesis using bromodeoxyuridine incorporation at various times during cell exposure to NGF revealed that the growth inhibition was attributable to gradual accumulation of growth-arrested cells at the G1 phase. Synthesis of nuclear regulatory proteins JUN and p53 was inhibited preferentially and progressively by NGF as inhibition of DNA synthesis increased.
Nerve growth factor (NGF) and glia maturation factor (GMF) reverse some of the transformed characteristics of T9 glioma cells (Marushige et al., Cancer Res 47: 4109-4115, 1987). As an attempt to define the mechanisms of such actions, various chemical agents which modulate second messenger systems were examined for their effects on growth and morphological characteristics of these cells. Administration of bromo-cAMP, forskolin and methylisobutylxanthine retarded cell growth and induced formation of long, branching processes which were similar to those induced by GMF. Perturbation of Ca2(+)-mediated processes by a Ca2+ ionophore, ionomycin, and by calmodulin antagonists, chlorpromazine and W-7, on the other hand, arrested cell growth, and caused clustering of cells, spreading of the cytoplasm and development of lamellipodium-like protrusions which were reminiscent of the effects of NGF. Administration of bromo-cAMP in combination with chlorpromazine, W-7 or ionomycin prevented spreading of the cytoplasm and produced compact cell bodies with well-developed processes. The results of this study demonstrate that modulation of specific second messenger systems by chemical agents are capable of simulating selective morphological changes inducible by NGF and GMF.
Changes in cytoskeletal organization in T9 anaplastic glioma cells have been examined during morphological changes induced by nerve growth factor (NGF) and by glia maturation factor (GMF). Indirect immunofluorescent labeling of cytoskeletal proteins has revealed that while neither GMF nor NGF induces expression of glial fibrillary acidic protein in this cell line, changes in cytoskeletal organization induced by these factors show some features similar to those observable during maturation of normal glial cells. Changing cell shapes induced by these factors are clearly outlined by the prominent distribution of microfilaments along cellular margins. Microtubules and intermediate filaments gradually extend during morphological changes and fill the characteristic cytoplasmic processes induced by NGF and GMF.
Chromatin condensation during apoptosis induced by TGF beta 1 in T24 glioma and 476-16 trigeminal neurinoma (Schwannoma) cells was examined and compared with that occurring during mitosis. Apoptotic (round-up) cells were selectively detached from the culture surface by a mechanical shock. Their histones were analysed in comparison with those obtained from TGF beta 1-treated cells remaining attached to the culture surface, from control cells not treated with TGF beta 1 and from metaphase cells. While mitosis-specific hyperphosphorylation of histones H1 and phosphorylation of histone H3 was not observed in apoptotic cells, apoptotic chromatin lacked ubiquitinated histone H2A (histone uH2A) as did metaphase chromosomes. The cellular level of free ubiquitin and the overall pattern of ubiquitin-conjugated proteins were, however, found to remain unaltered in apoptotic cells, suggesting that the ubiquitin conjugating machinery for histone H2A may be specifically perturbed during the chromatin condensation occurring in apoptosis.
Transforming growth factor beta 1 (TGF beta 1) inhibits cell proliferation in T24 glioma and 476-16 trigeminal neurinoma (Schwannoma) cells. In both cell types, the inhibition of cell proliferation is followed by cell rounding and detachment as well as internucleosomal DNA fragmentation, nuclear condensation and cell shrinkage, cellular changes that are characteristic for apoptosis. While the induction of apoptosis is closely coupled with the inhibition of cell proliferation in these tumor cells, the mode of apoptosis appears to differ between the two cell types. In 476-16 cells whose proliferation is highly susceptible to TGF beta 1, apoptosis occurs primarily after growth arrest at the G1 phase. Apoptotic cell death of 476-16 cells pretreated with TGF beta 1 is stimulated by serum deprivation, and it is inhibited by mitogenic growth factors such as insulin and platelet-derived growth factors. In T24 cells whose DNA replication is inhibited only moderately by TGF beta 1, apoptosis occurs in the presence of TGF beta 1 during the final cell division cycle when cells undergo density-induced growth arrest. While staurosporine accelerates TGF beta 1-induced apoptosis in both 476-16 and T24 cells, 12-O-tetradecanoylphorbol 13-acetate inhibits TGF beta 1-induced apoptosis of 476-16 cells but stimulates that of T24 cells. The present results suggest that TGF beta 1 may potentially be utilized for the management of neurogenic tumors of glial and Schwann cell origin.
Nerve growth factor (NGF) reduces the development of trigeminal neurinomas in vivo. To characterize the action of NGF on these tumors, clonal cells (476-16) isolated from a rat trigeminal neurinoma cell line were synchronized at early S phase by aphidicolin, a reversible inhibitor of DNA polymerase alpha, and effects of NGF on DNA replication were examined in vitro. While NGF did not inhibit DNA replication in the ongoing S phase, it reduced the level of DNA synthesis in the succeeding S phase without altering its timing and duration. The inhibitory action was elicited by a brief exposure to NGF during progression through the preceding S to G1 phases with decreasing effectiveness in the later stage of G1.
Exposure of trigeminal neurinoma 476-16 cells to C2-ceramide in a serum-deprived medium induces cell rounding followed by cell death characterized by cytoplasmic shrinkage and nuclear condensation. The induction of cell rounding and death occurs in proliferating cells but not in essentially quiescent cells of confluent cultures. Trypan blue-unpermeable round cells formed as a result of ceramide treatment undergo apoptosis without ceramide in a serum-containing medium, suggesting that they are irreversibly committed to cell death. The induction of cell rounding by ceramide is inhibited by low doses of the protein-tyrosine phosphatase inhibitors, orthovanadate and pervandate, and stimulated by high doses of pervanadate. The inhibition of protein-tyrosine phosphatases interfers the induction of cell death by ceramide. The protein-serine/threonine phosphatase inhibitor calyculin A induces cell rounding in proliferating cells. This cell rounding does not lead to cell death, thus calyculin A inhibits the induction of cell death by ceramide. While orthovanadate inhibits the induction of cell rounding by calyculin A, the latter is potentiated by ceramide. A combination of ceramide and calyculin A induces cell rounding and cell death in confluent cultures. These results demonstrate that modulation of phosphorylation of the serine/threonine and tyrosine of cellular proteins is intimately involved in the process of cell rounding and apoptosis in anchorage-dependent cells.