Changes in the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt signaling associated with the induction of apoptosis.

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.

Modulation of cell rounding and apoptosis in trigeminal neurinoma cells by protein phosphatase inhibitors.

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.

Alterations in focal adhesion and cytoskeletal proteins during apoptosis.

Ceramide induces cell rounding and subsequent apoptotic cell death in trigeminal neurinoma 476-16 cells. A protein tyrosine phosphatase inhibitor, orthovanadate, inhibits cell rounding and subsequent apoptotic death, while a serine/threonine phosphatase inhibitor, calyculin A, stimulates cell rounding but inhibits apoptosis (reference 11). In an attempt to determine critical cellular changes associated with cell rounding during the induction of apoptosis, focal adhesion and cytoskeletal proteins in apoptotic round cells induced by ceramide were examined by immunoblotting and compared with those of non-apoptotic round cells and adherent cells. As compared with adherent cells, tyrosine phosphorylation of a group of proteins between 110-125 KDa, including p125 focal adhesion kinase (FAK) is reduced in the apoptotic round cells as well as in non-apoptotic round cells induced by calyculin A and metaphase cells in mitosis. However, a concerted decrease of vinculin, paxillin and FAK, preceding the changes of whole cellular proteins, is seen in the apoptotic round cells but not in the non-apoptotic round cells. The inhibition of ceramide-induced apoptosis by orthovanadate is accompanied by a prevention of such a decrease in focal adhesion proteins. It thus appears that these focal adhesion proteins are degraded during the cell rounding occurring during apoptosis. Proteolysis of focal adhesion components may not only irreversibly disrupt cell adhesion but also impede transduction of growth and survival signals, and may play a critical role in the initiation and execution of apoptosis.

Disappearance of ubiquitinated histone H2A during chromatin condensation in TGF beta 1-induced apoptosis.

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.

Induction of apoptosis by transforming growth factor beta 1 in glioma and trigeminal neurinoma cells.

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.

Characterization of single-stranded DNA binding proteins in rat glial-enriched nuclei.

Single-stranded DNA binding proteins (SSBs) are those proteins which preferentially bind single-stranded DNA as opposed to double-stranded DNA and are known to be involved in recombination, amplification, and repair of DNA. To characterize single-stranded DNA binding proteins of glial cells and to examine their potential involvement in induction of neurogenic tumors in rats, nuclei were isolated from target glia and non-target liver of carcinogenically sensitive Sprague-Dawley (SD) and resistant Berlin-Druckrey-IV (BD-IV) rats of various ages and rapidly proliferating glioma cells. Nuclei were fractionated into chromatin, a preribosomal RNA protein complex, heterogeneous nuclear ribonucleoprotein complex (hnRNP), and nucleoplasm. SSBs were isolated, quantitated, and characterized by electrophoresis. A comparison of the contents of SSBs relative to RNA and their electrophoretic profiles between chromatin and hnRNP revealed that SSBs of liver chromatin were mainly associated with RNA. However, it was found that glial chromatin, particularly that of juvenile rats, was enriched with a heterogeneous series of SSBs which were not found in liver chromatin and presumably associated with chromosomal DNA. Some of these SSBs were enriched in glial chromatin of sensitive SD rats compared with that of resistant BD-IV rats. High mobility group proteins (HMG) 1 and 2 constituted major SSB components in the nucleoplasm and a greater amount of these HMGs were found in juvenile glia, compared to adult glia and juvenile and adult liver. Fractionation of glial SSBs and determination of their biological functions may contribute to the further understanding of the role these proteins may play in the processes of carcinogenesis.

Growth inhibition of synchronized trigeminal neurinoma cells by nerve growth factor.

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.

Growth inhibition of anaplastic glioma cells by nerve growth factor.

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.

The use of nerve growth factor as a reverse transforming agent for the treatment of neurogenic tumors: in vivo results.

The rationale behind the evaluation of natural differentiating agents, such as nerve growth factor (NGF), for reverse transforming potential is based on the theory that such compounds may represent a nontoxic means of controlling tumor growth. Previous in vitro experiments have shown that NGF is capable of retarding growth and of inducing persistent differentiation of neurogenic tumor cell lines. In vivo, NGF is capable of causing a persistent reduction in the number of ethylnitrosourea-induced neurinomas and of increasing survival time following intracerebral implantation of F98 anaplastic glioma cells. In this study, anaplastic glioma and neurinoma implants were treated with NGF to evaluate the reverse transforming potential of NGF in vivo. Results indicate that NGF is capable of causing a significant decrease in the growth rate of subcutaneous T9 (anaplastic glioma) and clone 16 (anaplastic neurinoma) implants. Significantly, NGF treatment was accompanied by adverse effects that were minimal and transient. Continued tumor growth (although greatly retarded) following NGF treatment is an aspect that requires further investigation. However, the results of this study suggest that NGF may prove useful, alone or in combination with other types of therapy, for the treatment of tumors of neurogenic origin.

The reverse transforming effects of nerve growth factor on five human neurogenic tumor cell lines: in vitro results.

The role of nerve growth factor (NGF) in the development, maintenance and regeneration of the mammalian sensory and sympathetic nervous systems has been well characterized, as has the ability of NGF to induce a variety of neoplastic cell lines of neuroecto-dermal (neurogenic) origin to differentiate. The ability to stimulate neoplastic cells of neurogenic origin to differentiate suggests that NGF may prove useful as a reverse transforming agent for the treatment of neurogenic tumors. Five human neurogenic tumor cell lines were evaluated for their response to NGF in vitro to determine whether the NGF is capable of inducing changes consistent with a reverse transforming response. Results indicate that NGF was able to reverse some of the transformed properties of these tumor cell lines, as NGF treatment stimulated neoplastic cells to develop a more differentiated phenotype, diminished or arrested growth, and induced changes that were persistent.

Effect of nerve growth factor on the transplacental induction of neurinomas by ethylnitrosourea in Sprague-Dawley rats.

Administration of nerve growth factor (NGF) to the offspring of Sprague-Dawley rats transplacentally exposed to 50 mg/kg ethylnitrosourea on the 20th day of gestation resulted in a significant reduction of trigeminal and peripheral nerve neurinomas. Forty, 60, and 80 micrograms of NGF was administered in five s.c. doses, one dose on each of days 12-16, 90-94, and 210-214 postnatally. Of the 34 rats in the NGF-treated group, 11 animals were affected with trigeminal nerve neurinomas as compared to 18/34 in the NGF-untreated group (P less than 0.05). In the peripheral nerves (spinal cord nerve roots) there were five and 11 neurinomas, respectively, in each group of 34 rats. When the total numbers of neurinomas (trigeminal and peripheral nerves) between these groups were compared (16/34 versus 29/34), the significance of neurinoma reduction was P less than 0.01. Five trigeminal and two peripheral neurinomas in the NGF-untreated group were shown by immunohistochemical staining to contain nerve growth factor receptor protein, whereas none of the neurinomas in the NGF-treated group were positive for the receptor protein. The results obtained from this experiment lend support to the hypothesis that NGF has the capability to reduce the oncogenic consequences of ethylnitrosourea exposure perhaps by the process of maturation and/or differentiation of the transformed cells, and that this effect may depend upon the presence of receptor binding sites.

Chemical control of growth and morphological characteristics of anaplastic glioma cells.

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.

Cytoskeletal reorganization induced by nerve growth factor and glia maturation factor in anaplastic glioma cells.

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.

Modulation of growth and of morphological characteristics in glioma cells by nerve growth factor and glia maturation factor.

Anaplastic glioma T9 cells were treated with either nerve growth factor (NGF) or glia maturation factor (GMF) or both. It was found that, when T9 cells were treated with these factors in a chemically defined medium, both NGF and GMF induced characteristic changes of cell morphology and growth pattern. Several differences in the effects of NGF and GMF were noted. NGF retarded growth rate, whereas GMF did not. The cells treated with NGF were characterized by a flattened extended cytoplasm with numerous protruding processes. The cell masses were somatically connected by cell bridges. GMF, on the other hand, produced slender cells with long, branching processes forming an interconnecting cell net. Concomitant administration of NGF and GMF retarded cell growth as was demonstrated with NGF alone and induced morphological changes predominantly attributable to GMF. The maximal effect of either NGF or GMF or both was attained after 4 days of treatment. A withdrawal of the factors from the medium following various periods of treatment revealed that the effects of GMF were readily reversible while morphological changes induced by NGF persisted in its absence.

Proteolysis of somatic type histones in transforming rat spermatid chromatin.

Elongated rat spermatid nuclei have been isolated on the basis of their resistance to sonication in 0.32 M sucrose containing 1.5 mM CaCl2. Chemical analyses indicate that approx. 35% of the DNA in these nuclei is associated with somatic type histones, while the remainder represents sperm histone-DNA complex. In contrast to nuclei of somatic cells, when elongated spermatid nuclei are incubated under appropriate conditions, somatic type histones but not sperm histone are rapidly degraded. Differential extraction of elongated spermatid nuclei with 5 mM HCl and then with various concentrations of NaCl followed by 0.2 M HCl has revealed that they contain two kinds of proteases. The protease in the 5 mM HCl extract is acrosin (EC 4.3.21.10). Rapid degradation of somatic type histones is, however, observable upon incubation of elongated spermatid nuclei which have been treated with 5 mM HCl and are therefore free of acrosin or upon incubation of elongated spermatid chromatin where the majority of acrosin is removed, suggesting that the observed proteolysis of somatic type histones is not due to acrosin. Proteases which may represent the enzymes responsible for the histone degradation are extractable from acrosin-free spermatid nuclei with NaCl (0.9 M) and by subsequent treatment of the salt-extracted nuclei with 0.2 M HCl. The proteases in the NaCl and the 0.2 M HCl extract possess identical properties and appear to be the same enzyme which may exist in spermatid chromatin in two different forms.

Alkylation of isolated chromatin with N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea.

When isolated chromatin is incubated with the carcinogens N-methyl-N-nitrosourea (MeNU) and N-ethyl-N-nitrosourea (EtNU), DNA and chromosomal proteins become alkylated to increasingly greater extents as the carcinogen concentrations increase. With either MeNU or EtNU, the core and linker DNA of chromatin are alkylated to essentially identical extents. Alkylation of chromatin DNA as well as free DNA is drastically reduced at physiological ionic strengths (e.g. 0.15 M NaCl). The presence of 0.15 M NaCl, on the other hand, enhances alkylation of chromosomal proteins. While EtNU is much less reactive to DNA than MeNU, alkylation of chromosomal proteins relative to that of chromatin DNA has been found to be markedly greater with EtNU than with MeNU. Such a difference in their relative reactivities toward DNA and proteins may be related to the known difference of carcinogenic potency between these N-nitroso compounds.

Template properties of DNA alkylated with N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea.

Alkylation of DNA with N-methyl-N-nitrosourea (MeNU) and N-ethyl-N-nitrosourea (EtNU) reduces its ability to support RNA synthesis catalyzed by exogenously added RNA polymerase. It is likely that 7-alkylguanine and alkyl phosphotriester in DNA are mainly responsible for the inhibition of RNA synthesis. The inhibitory effect of alkyl groups varies depending upon divalent metal ions and the type of RNA polymerase used as well as upon the presence of chromosomal proteins on DNA templates. Analyses of RNA products indicate that inhibition occurs primarily at the initiation step.

Dispersion of mammalian sperm chromatin during fertilization: an in vitro study.

When "denuded spermatozoa" (spermatozoa stripped of the greater part of their acrosomes and resembling in may respects spermatozoa after acrosomal reaction) of the bull are incubated with 0.1 M 2-mercaptoethanol (pH 8), sperm chromatin is degraded extensively by a protease in the sperm head. The morphological pattern of sperm nuclear dispersion upon in vitro incubation is similar to that observed in the newly fertilized egg. Following disintegration of the outer layers of the sperm nucleus, chromatin dispersion commences from the periphery of the posterior half and proceeds to the anterior end and to the core of the head. Less basic N- and C-terminal portions of bull sperm histone molecules are digested quickly. The central, very arginine-rich portions of the molecules degrade gradually, yielding an heterogeneous series of arginine-rich peptides (molecular weight, 400-1500). Evidence suggests that the protease which is responsible for the degradation of sperm chromatin is a small fraction of acrosin. This fraction of acrosin appears to be arranged along the nuclear surface and to become associated with sperm chromatin during structural changes of the nuclear surface. A similar proteolysis of rabbit, hamster and guinea pig sperm chromatin has also been observed. The resulting pattern of dissolution of the sperm nucleus is proposed as a model of some of the steps involved in male pronucleus formation from the sperm head after fertilization. Histones H2a, H2b, H3, and H4 associated with DNA are relatively resistant to acrosin.