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1.
Cell Death Dis ; 1: e60, 2010 Jul 29.
Article in English | MEDLINE | ID: mdl-21364665

ABSTRACT

Recently developed heavy ion irradiation therapy using a carbon beam (CB) against systemic malignancy has numerous advantages. However, the clinical results of CB therapy against glioblastoma still have room for improvement. Therefore, we tried to clarify the molecular mechanism of CB-induced glioma cell death. T98G and U251 human glioblastoma cell lines were irradiated by CB, and caspase-dependent apoptosis was induced in both cell lines in a dose-dependent manner. Knockdown of Bax (BCL-2-associated X protein) and Bak (BCL-2-associated killer) and overexpression of Bcl-2 or Bcl-xl (B-cell lymphoma-extra large) showed the involvement of Bcl-2 family proteins upstream of caspase activation, including caspase-8, in CB-induced glioma cell death. We also detected the activation of extracellular signal-regulated kinase (ERK) and the knockdown of ERK regulator mitogen-activated protein kinase kinase (MEK)1/2 or overexpression of a dominant-negative (DN) ERK inhibited CB-induced glioma cell death upstream of the mitochondria. In addition, application of MEK-specific inhibitors for defined periods showed that the recovery of activation of ERK between 2 and 36 h after irradiation is essential for CB-induced glioma cell death. Furthermore, MEK inhibitors or overexpression of a DN ERK failed to significantly inhibit X-ray-induced T98G and U251 cell death. These results suggested that the MEK-ERK cascade has a crucial role in CB-induced glioma cell death, which is known to have a limited contribution to X-ray-induced glioma cell death.


Subject(s)
Apoptosis , Caspases/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Glioma/enzymology , Mitogen-Activated Protein Kinase Kinases/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Caspase 8/metabolism , Cell Line, Tumor , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/genetics , Glioma/radiotherapy , Humans , Mitochondria/metabolism , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/genetics , RNA Interference , RNA, Small Interfering/metabolism , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism , bcl-X Protein/genetics , bcl-X Protein/metabolism
2.
Neurosurg Rev ; 31(2): 173-6; discussion 176-7, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18092184

ABSTRACT

Central nervous system (CNS) germ cell tumors constitute a unique class of rare tumors that mainly affect children and adolescents. These tumors are believed to originate from displaced primordial germ cells. Recently, results of treatment of germ cell tumors have improved with use of radiotherapy and combination chemotherapy. However, some tumors have proven refractory to intensive treatment with surgery, radiation, and combination chemotherapy. Nestin is an intermediate filament protein expressed in undifferentiated cells during CNS development and in CNS tumors and is used as a marker of immature elements of tumors, including brain tumor stem cells. In this study, we examined for the first time nestin expression in 19 CNS germ cell tumors (nine pure germinomas, five germinomas with syncytiotrophoblastic giant cells, one yolk sac tumor, one choriocarcinoma, one embryonal carcinoma, and two mature teratomas). Nestin was expressed in 14 cases but was not expressed in three pure germinomas and two mature teratomas. Clinically, nestin-negative tumors did not exhibit dissemination, while all tumors that exhibited dissemination also strongly expressed nestin protein. These findings suggest that the detection of nestin expression could be useful in the management of CNS germ cell tumors, as an auxiliary predictor of dissemination and/or progression.


Subject(s)
Biomarkers, Tumor/biosynthesis , Biomarkers, Tumor/genetics , Central Nervous System Neoplasms/genetics , Intermediate Filament Proteins/biosynthesis , Intermediate Filament Proteins/genetics , Neoplasms, Germ Cell and Embryonal/genetics , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Adolescent , Adult , Central Nervous System Neoplasms/metabolism , Central Nervous System Neoplasms/pathology , Child , Choriocarcinoma/genetics , Choriocarcinoma/metabolism , Choriocarcinoma/pathology , Endodermal Sinus Tumor/genetics , Endodermal Sinus Tumor/metabolism , Endodermal Sinus Tumor/pathology , Female , Germinoma/genetics , Germinoma/metabolism , Germinoma/pathology , Giant Cell Tumors/genetics , Giant Cell Tumors/metabolism , Giant Cell Tumors/pathology , Humans , Hypopituitarism/etiology , Immunoenzyme Techniques , Magnetic Resonance Imaging , Male , Neoplasms, Germ Cell and Embryonal/metabolism , Neoplasms, Germ Cell and Embryonal/pathology , Nestin , Teratoma/genetics , Teratoma/metabolism , Teratoma/pathology , Vision Disorders/etiology
3.
Cell Death Differ ; 11(7): 771-81, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15031724

ABSTRACT

Bcl-2 homology domain (BH) 3-only proteins of the proapoptotic Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis. To date, at least 10 mammalian BH3-only proteins have been identified, and it is now being realized that they have different roles and mechanisms of regulation in the transduction of apoptotic signals to mitochondria. Hrk/DP5 is one of the mammalian BH3-only proteins implicated in a variety of physiological and pathological apoptosis, yet the molecular mechanism involved in Hrk-mediated apoptosis remains poorly understood. In an attempt to identify cellular proteins participating in Hrk-mediated apoptosis, we have conducted yeast two-hybrid screening for Hrk-interacting proteins and isolated p32, a mitochondrial protein that has been shown to form a channel consisting of its homotrimer. In vitro binding, co-immunoprecipitation, as well as immunocytochemical analyses verified specific interaction and colocalization of Hrk and p32, both of which depended on the presence of the highly conserved C-terminal region of p32. Importantly, Hrk-induced apoptosis was suppressed by the expression of p32 mutants lacking the N-terminal mitochondrial signal sequence (p32(74-282)) and the conserved C-terminal region (p32 (1-221)), which are expected to inhibit binding of Hrk competitively to the endogenous p32 protein and to disrupt the channel function of p32, respectively. Furthermore, small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis. Altogether, these results suggest that p32 may be a key molecule that links Hrk to mitochondria and is critically involved in the regulation of Hrk-mediated apoptosis.


Subject(s)
Mitochondrial Proteins/metabolism , Neuropeptides/metabolism , Nuclear Proteins/metabolism , Peptide Fragments/metabolism , Proto-Oncogene Proteins/metabolism , Amino Acid Sequence , Animals , Apoptosis , Apoptosis Regulatory Proteins , Astrocytoma/pathology , Binding Sites , COS Cells , Carrier Proteins , Cell Line, Tumor , Chlorocebus aethiops , Conserved Sequence , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Glutathione Transferase/metabolism , Humans , Immunohistochemistry , Microscopy, Fluorescence , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/isolation & purification , Precipitin Tests , Protein Binding , RNA, Small Interfering/metabolism , Recombinant Fusion Proteins/metabolism , Rhodamines , Sequence Deletion , Two-Hybrid System Techniques
4.
Biochem Biophys Res Commun ; 287(1): 257-63, 2001 Sep 14.
Article in English | MEDLINE | ID: mdl-11549284

ABSTRACT

Rat neuronal leucine-rich repeat protein-3 (rNLRR-3) gene was isolated and cloned from fibrosarcoma cells overexpressing c-Ha-ras. Stable expression of constitutively active forms of Ras (H-Ras(V12) or v-H-Ras) led to a two- to fourfold increase in rNLRR-3 mRNA in rat normal fibroblasts (3Y1). When cells expressing H-Ras(V12) were treated with mitogen activated protein kinase (MAPK) kinase inhibitors (U0126, PD98059), suppression of rNLRR-3 mRNA correlated well with a reduction in MAPK activity. Epidermal growth factor (EGF) led to elevation of rNLRR-3 gene expression about 4 h after stimulation of normal fibroblasts. U0126 completely suppressed the induction by EGF of rNLRR-3 mRNA with abrogation of MAPK phosphorylation. U0126 inhibited the basal transcription of rNLRR-3. LY294002, a PI3 kinase inhibitor, showed a lesser effect on expression of the gene. These results indicate that rNLRR-3 gene expression is regulated mainly through the Ras-MAPK signaling pathway in fibroblasts.


Subject(s)
Gene Expression Regulation , Nerve Tissue Proteins/genetics , Amino Acid Sequence , Animals , Cell Line, Transformed , Cells, Cultured , Cloning, Molecular , DNA, Complementary/analysis , Humans , Mitogen-Activated Protein Kinases/metabolism , Molecular Sequence Data , Rats , Sequence Homology, Amino Acid , Up-Regulation , ras Proteins/metabolism
5.
Biochem Biophys Res Commun ; 281(5): 1313-20, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11243879

ABSTRACT

We previously reported that JNK is involved in the regulation of c-Myc-mediated apoptosis triggered by UV irradiation and anticancer drug treatment. Here we show that ASK1 is an upstream regulator for c-Myc-mediated apoptosis triggered by UV, and we found a direct role for Ser-62 and Ser-71 in the regulation of protein stability and function of c-Myc. The ASK1-JNK pathway enhanced the protein stability of c-Myc through phosphorylation at Ser-62 and Ser-71, which was required for c-Myc-dependent apoptosis by ASK1-signaling. Interestingly, ASK1-signaling attenuated the degradation of ubiquitinated c-Myc without affecting the ubiquitination process. Together, these findings indicate that the ASK1-JNK pathway promotes the proapoptotic activity of c-Myc by modulating c-Myc protein stability through phosphorylation at Ser-62 and Ser-71.


Subject(s)
Apoptosis , Arabidopsis Proteins , Plant Proteins/physiology , Proto-Oncogene Proteins c-myc/metabolism , 3T3 Cells , Animals , Humans , Kinetics , Mice , Mitogen-Activated Protein Kinase 8 , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Phosphoserine/metabolism , Plant Proteins/genetics , Signal Transduction , Transfection , Tumor Cells, Cultured , Ubiquitins/metabolism
6.
FEBS Lett ; 488(3): 105-9, 2001 Jan 19.
Article in English | MEDLINE | ID: mdl-11163755

ABSTRACT

We propose that the amino acid residues 57/58 and 60/61 of eukaryotic release factors (eRF1s) (counted from the N-terminal Met of human eRF1) are responsible for stop codon recognition in protein synthesis. The proposal is based on amino acid exchanges in these positions in the eRF1s of two ciliates that reassigned one or two stop codons to sense codons in evolution and on the crystal structure of human eRF1. The proposed mechanism of stop codon recognition assumes that the amino acid residues 57/58 interact with the second and the residues 60/61 with the third position of a stop codon. The fact that conventional eRF1s recognize all three stop codons but not the codon for tryptophan is attributed to the flexibility of the helix containing these residues. We suggest that the helix is able to assume a partly relaxed or tight conformation depending on the stop codon recognized. The restricted codon recognition observed in organisms with unconventional eRF1s is attributed mainly to the loss of flexibility of the helix due to exchanged amino acids.


Subject(s)
Anticodon/metabolism , Codon, Terminator/metabolism , Models, Genetic , Peptide Termination Factors/metabolism , Protein Biosynthesis , Amino Acid Sequence , Animals , Anticodon/chemistry , Anticodon/genetics , Codon, Terminator/chemistry , Codon, Terminator/genetics , Eukaryotic Cells/metabolism , Evolution, Molecular , Genetic Code/genetics , Genetic Code/physiology , Humans , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Peptide Termination Factors/chemistry , Peptide Termination Factors/genetics , Protein Structure, Tertiary , Sequence Alignment , Substrate Specificity , Tetrahymena thermophila/genetics , Tetrahymena thermophila/metabolism
7.
Biochem Biophys Res Commun ; 267(1): 221-7, 2000 Jan 07.
Article in English | MEDLINE | ID: mdl-10623602

ABSTRACT

The s-Myc is similar to c-Myc in its ability to induce apoptosis requiring caspase activation. However, s-Myc is distinct from c-Myc in that it has activity to suppress tumor growth and does not require wild-type p53 to induce apoptosis. These facts suggest differential regulation between s-Myc and c-Myc. Here we showed that s-Myc-mediated apoptosis triggered by UV was not inhibited by the inactive form mutant JNK (APF), though c-Myc-mediated apoptosis was. Moreover, we found that JNK did not affect the transactivation activity of s-Myc, but stimulated that of c-Myc. In contrast, both Myc-mediated apoptosis and caspase-3-like protease activation were suppressed by kinase-negative MKK6 and an inactive form mutant p38(AGF). Our results indicate that s-Myc does not require the JNK signaling unlike c-Myc during UV-triggered apoptosis, but the MKK6/p38MAPK pathway might regulate common apoptotic machinery for both s-Myc and c-Myc upstream of caspase.


Subject(s)
Apoptosis/physiology , Calcium-Calmodulin-Dependent Protein Kinases , Mitogen-Activated Protein Kinases/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Animals , Apoptosis/radiation effects , COS Cells , Caspases/metabolism , Cell Line , Chloramphenicol O-Acetyltransferase/genetics , Genes, Reporter , HeLa Cells , Humans , JNK Mitogen-Activated Protein Kinases , Kinetics , MAP Kinase Kinase 6 , Mitogen-Activated Protein Kinase Kinases/metabolism , Rats , Recombinant Proteins/metabolism , Transfection , Ultraviolet Rays , p38 Mitogen-Activated Protein Kinases
9.
J Biol Chem ; 274(46): 32580-7, 1999 Nov 12.
Article in English | MEDLINE | ID: mdl-10551811

ABSTRACT

The expression of c-myc promotes cell proliferation and also sensitizes cells to various extracellular apoptotic stimuli. However, signal pathways regulating the function of Myc proteins during apoptosis are unknown. c-Jun N-terminal kinase (JNK) is activated by various apoptotic stimuli, but neither the target molecule(s) or the action of JNK has been identified in Myc-mediated apoptosis. Here, we found that JNK selectively interacted with, and phosphorylated, c-Myc at Ser-62 and Ser-71 as confirmed with phospho-c-Myc-specific antibodies. Interestingly, dominant negative mutant JNK(APF) impaired the c-Myc-dependent apoptosis, but not mutated c-Myc (S62A/S71A)-dependent apoptosis triggered by UV irradiation. Furthermore, c-Myc (S62A/S71A)-expressing NIH3T3 cells were not sensitized like wild type c-Myc-expressing NIH3T3 cells to JNK-activating apoptotic stimuli, such as UV and Taxol. These results indicate that the JNK pathway is selectively involved in the c-Myc-mediated apoptosis and that the apoptotic function of c-Myc is directly regulated by JNK pathway through phosphorylation at Ser-62 and Ser-71.


Subject(s)
Mitogen-Activated Protein Kinases/metabolism , Proto-Oncogene Proteins c-myc/metabolism , 3T3 Cells , Animals , Apoptosis , Gene Expression Regulation , HeLa Cells , Humans , JNK Mitogen-Activated Protein Kinases , Mice , Mutagenesis , Paclitaxel/pharmacology , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-myc/genetics , Recombinant Fusion Proteins , Signal Transduction , Ultraviolet Rays
10.
Cell Death Differ ; 6(6): 508-15, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10381653

ABSTRACT

Cell death is generally classified into two large categories: apoptosis represents active, programmed cell death, while necrosis represents passive cell death without underlying regulatory mechanisms. Recent progress revealed that caspases, a family of cysteine proteases, play a central role in the regulation of apoptosis. Unexpectedly, however, caspase inhibition occasionally turns the morphology of programmed cell death from apoptotic into necrotic without inhibiting death itself. In this article, we review different models of caspase-independent programmed cell death showing necrotic-like morphology, including our Ras-mediated caspase-independent cell death. Based on these findings, we suggest the existence of a necrotic-like cell death regulated by cellular intrinsic death programs distinct from that of apoptosis. Even though type 2 physiological cell death, or autophagic degeneration, has been recognized as a necrotic-like programmed cell death for a long time, the underlying molecular mechanisms have not been identified despite its physiological significance. This has been in part due to the previous absence of adequate caspase-independent cellular models to study, recent efforts may now help to elucidate these mechanisms.


Subject(s)
Apoptosis , Caspases , Animals , Cell Death , Humans , Intracellular Fluid , Necrosis , Signal Transduction , ras Proteins/metabolism
11.
J Biol Chem ; 274(26): 18659-66, 1999 Jun 25.
Article in English | MEDLINE | ID: mdl-10373478

ABSTRACT

The pim-1 oncogene encodes a serine/threonine kinase (Pim-1) involved in the transduction of cytokine-triggered mitogenic signals. Pim-1 is unique in that it closely cooperates with c-Myc not only in oncogenesis, but also in apoptosis induction. However, the molecular basis of Pim-1 function remains poorly understood, largely because the downstream effector molecule(s) for Pim-1 kinase has not been identified. Here we provide several lines of evidence that Cdc25A cell cycle phosphatase, a direct transcriptional target for c-Myc, is a substrate for Pim-1 kinase and functions as an effector for Pim-1. We found that Pim-1 physically interacts with Cdc25A both in vitro and in vivo and phosphorylates Cdc25A. We also observed that Pim-1-mediated phosphorylation of Cdc25A increases its phosphatase activity. In addition, wild-type Pim-1, but not kinase-inactive Pim-1, enhanced Cdc25A-mediated cellular transformation and apoptosis. Our results indicate that Cdc25A might be a key molecule that links Pim-1 and c-Myc and that also ties Pim-1-mediated mitogenic signals to cell cycle machinery.


Subject(s)
Protein Serine-Threonine Kinases/metabolism , Protein Tyrosine Phosphatases/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins/metabolism , Signal Transduction , cdc25 Phosphatases , Animals , Apoptosis , COS Cells , Drug Synergism , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-pim-1 , Rats
12.
Oncogene ; 18(13): 2281-90, 1999 Apr 01.
Article in English | MEDLINE | ID: mdl-10327074

ABSTRACT

To prevent neoplasia, cells of multicellular organisms activate cellular disposal programs such as apoptosis in response to deregulated oncogene expression, making the suppression of such programs an essential step for potentially neoplastic cells to become established as clinically relevant tumors. Since the mutation of ras proto-oncogenes, the most frequently mutated proto-oncogenes in human tumors, is very rare in some tumor types such as glioblastomas and gastric cancers, we hypothesized that mutated ras genes might activate a cell death program that cannot be overcome by these tumor types. Here we show that the expression of oncogenically mutated ras gene induces cellular degeneration accompanied by cytoplasmic vacuoles in human glioma and gastric cancer cell lines. Cells dying as a result of oncogenic Ras expression had relatively well-preserved nuclei that were negative for TUNEL staining. An immunocytochemical analysis demonstrated that the cytoplasmic vacuoles are derived mainly from lysosomes. This oncogenic Ras-induced cell death occurred in the absence of caspase activation, and was not inhibited by the overexpression of anti-apoptotic Bcl-2 protein. These observations suggested that oncogenic Ras-induced cell death is most consistent with a type of programmed cell death designated 'type 2 physiological cell death' or 'autophagic degeneration', and that this cell death is regulated by a molecular mechanism distinct from that of apoptosis. Our findings suggest a possible role for this non-apoptotic cell death in the prevention of neoplasia, and the activation of the non-apoptotic cell death program may become a potential cancer therapy complementing apoptosis-based therapies. In addition, the approach used in this study may be a valuable way to find genetically-regulated cell suicide programs that cannot be overcome by particular tumor types.


Subject(s)
Caspases/physiology , Cell Death/genetics , Cell Transformation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic , Genes, ras , Neoplasm Proteins/physiology , Neoplastic Stem Cells/cytology , Proto-Oncogene Proteins p21(ras)/physiology , Brain Neoplasms/pathology , Cell Death/physiology , Cell Nucleus/ultrastructure , Cysteine Proteinase Inhibitors/pharmacology , DNA Fragmentation , Glioblastoma/pathology , Glioma/pathology , Humans , Lysosomes/ultrastructure , Neoplastic Stem Cells/metabolism , Phagocytosis , Proto-Oncogene Proteins c-bcl-2/physiology , Recombinant Fusion Proteins/physiology , Signal Transduction/genetics , Stomach Neoplasms/pathology , Transfection , Urinary Bladder Neoplasms/pathology , Vacuoles/ultrastructure
13.
Gene ; 226(2): 273-83, 1999 Jan 21.
Article in English | MEDLINE | ID: mdl-9931502

ABSTRACT

Our previous findings suggest that the activation of the rat intronless myc gene provides a selective advantage in tumor suppression through apoptosis induction. In the present study, to examine whether intronless myc gene acting as an apoptosis inducer is evolutionarily conserved in mammalian cells, we isolated the mouse intronless myc gene and characterized it. A sequence analysis demonstrated that mouse intronless myc gene, ms-myc, has a linearly opened translatable frame consisting of 1293bp with 90% homology with that of rat s-myc. The chromosomal locus of ms-myc was identified on chromosome 19B by a fluorescent in situ hybridization (FISH) analysis. Gene transfection experiments showed that the transient overexpression of ms-Myc with transactivation activity effectively induces cell death in a wild-type p53-independent manner. In addition, cells stably expressing transfected ms-myc became more susceptible to apoptosis induced by genotoxic stress such as UV-irradiation and hydrogen peroxide compared with untransfected control cells. These observations suggest that the rodents commonly contain an s-myc-type of intronless myc gene with apoptosis-inducing activity.


Subject(s)
Apoptosis/genetics , Chromosome Mapping , Genes, myc , Introns , Transcription Factors , Amino Acid Sequence , Animals , Base Sequence , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Basic-Leucine Zipper Transcription Factors , Cell Line , Cloning, Molecular , DNA , DNA-Binding Proteins/metabolism , Dimerization , HeLa Cells , Humans , In Situ Hybridization, Fluorescence , Mice , Molecular Sequence Data , Open Reading Frames , Rats , Transcriptional Activation
14.
Acta Neuropathol ; 96(4): 322-8, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9796995

ABSTRACT

We previously reported that schwannoma-derived growth factor (SDGF), a member of heparin-binding epidermal growth factor (EGF) family, participates in autocrine pathways and promotes rat glioma cell growth. To investigate the potential role of similar molecules in human gliomas, we examined 7 human glioma cell lines and 11 glioblastoma specimens for expression of the human homologue of SDGF, amphiregulin (AR), as well as heparin-binding EGF-like growth factor (HB-EGF). Northern blot analysis revealed that only one cell line and no tumor specimens expressed AR mRNA. In contrast, HB-EGF mRNA was expressed in all human glioma cell lines and its level of expression was two- to five-fold higher than that of control brain tissues in 8 of 11 glioblastoma cases. Immunohistochemistry demonstrated that membrane-anchored HB-EGF (proHB-EGF) and EGFR were co-expressed in 44% of 34 human malignant gliomas. Introduction of exogenous HB-EGF (10 ng/ml) increased human glioma cell proliferation, and anti-HB-EGF blocking antibodies reduced the growth of glioma cells by 30-40%, confirming the presence of an autocrine loop. When added to the medium, transforming growth factor-alpha, basic fibroblast growth factor, or HB-EGF rapidly induced HB-EGF mRNA expression. These results indicate that HB-EGF and proHB-EGF contribute to the growth of human malignant glioma cells, most likely through autocrine and juxtacrine mechanisms.


Subject(s)
Epidermal Growth Factor/physiology , Glioma/metabolism , Intercellular Signaling Peptides and Proteins , Mitosis/physiology , Signal Transduction/physiology , Amphiregulin , Cell Division/drug effects , EGF Family of Proteins , Epidermal Growth Factor/genetics , Epidermal Growth Factor/pharmacology , ErbB Receptors/metabolism , Glioblastoma/metabolism , Glioblastoma/pathology , Glioma/pathology , Glycoproteins/metabolism , Growth Substances/metabolism , Heparin-binding EGF-like Growth Factor , Humans , RNA, Messenger/metabolism , Recombinant Proteins , Transforming Growth Factor alpha/pharmacology , Transforming Growth Factor beta/pharmacology , Tumor Cells, Cultured/drug effects
15.
Oncogene ; 15(15): 1763-72, 1997 Oct 09.
Article in English | MEDLINE | ID: mdl-9362442

ABSTRACT

Bcl-2 family proteins and ICE/CED-3 family proteases (caspases) are regarded as the basic regulators of apoptotic cell death. They are evolutionarily conserved and implicated in a variety of apoptosis. However, the precise mechanism by which these two families interact to regulate cell death is not yet known. In this study, we found that the overexpression of the Bcl-2 family member Bax induced apoptotic cell death in COS-7 cells through the activation of CPP32 (caspase-3)-like proteases that cleaved the DEVD tetrapeptide. This apoptotic cell death was suppressed by the viral proteins CrmA and p35, as well as by the chemically synthesized caspase inhibitors Z-Asp-CH2-DCB and zVAD-fmk. We also found that the Bax-induced apoptosis of COS-7 cells was suppressed by Bcl-xL and Bcl-2, though both Bcl-xL and Bcl-2 similarly prevented etoposide-induced apoptosis in COS-7 cells. In addition, Bcl-xL inhibited the activation of caspase-3-like proteases accompanying Bax-induced COS-7 cell death but Bcl-2 did not. These results indicate that the caspase activation is essential for Bax-induced apoptosis, and that the ability of Bcl-2 and Bcl-xL to prevent the Bax-induced caspase activation and apoptosis in COS-7 cells could be differentially regulated. Our results also suggest that Bcl-2 family proteins function upstream of caspase activation and control apoptosis through the regulation of caspase activity.


Subject(s)
Apoptosis/genetics , Cysteine Endopeptidases/metabolism , Proto-Oncogene Proteins/genetics , Animals , COS Cells , Enzyme Activation , Gene Expression , Protease Inhibitors/pharmacology
16.
Mol Cell Biol ; 17(11): 6736-45, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9343438

ABSTRACT

Upon activation, cell surface death receptors, Fas/APO-1/CD95 and tumor necrosis factor receptor-1 (TNFR-1), are attached to cytosolic adaptor proteins, which in turn recruit caspase-8 (MACH/FLICE/Mch5) to activate the interleukin-1 beta-converting enzyme (ICE)/CED-3 family protease (caspase) cascade. However, it remains unknown whether these apoptotic proteases are generally involved in apoptosis triggered by other stimuli such as Myc and p53. In this study, we provide lines of evidence that a death protease cascade consisting of caspases and serine proteases plays an essential role in Myc-mediated apoptosis. When Rat-1 fibroblasts stably expressing either s-Myc or c-Myc were induced to undergo apoptosis by serum deprivation, a caspase-3 (CPP32)-like protease activity that cleaves a specific peptide substrate, Ac-DEVD-MCA, appeared in the cell lysates. Induction of s-Myc- and c-Myc-mediated apoptotic cell death was effectively prevented by caspase inhibitors such as Z-Asp-CH2-DCB and Ac-DEVD-CHO. Furthermore, exposing the cells to a serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), also significantly inhibited s-Myc- and c-Myc-mediated apoptosis and the appearance of the caspase-3-like protease activity in vivo. However, AEBSF did not directly inhibit caspase-3-like protease activity in the apoptotic cell lysates in vitro. Together, these results indicate that caspase-3-like proteases play a critical role in both s-Myc- and c-Myc-mediated apoptosis and that caspase-3-like proteases function downstream of the AEBSF-sensitive step in the signaling pathway of Myc-mediated apoptosis.


Subject(s)
Apoptosis/physiology , Caspases , Cysteine Endopeptidases/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Animals , Caspase 3 , Cells, Cultured , Culture Media, Serum-Free , Enzyme Activation , Enzyme Induction , Fibroblasts/cytology , Genes, myc , Proto-Oncogene Proteins c-myc/genetics , Rats , Recombinant Proteins/metabolism , Serine Proteinase Inhibitors/pharmacology , Signal Transduction , Sulfones/pharmacology
17.
Oncogene ; 15(12): 1471-80, 1997 Sep 18.
Article in English | MEDLINE | ID: mdl-9333023

ABSTRACT

Pim-1 oncoprotein is a serine/threonine kinase that can closely cooperate with c-Myc in lymphomagenesis, as does Bcl-2. Although the molecular mechanism of this cooperative transformation remains unknown, it is speculated that, similar to Bcl-2, Pim-1 contributes to transformation by inhibiting apoptosis. In this study, therefore, we examined the effect of Pim-1 expression on c-Myc-mediated apoptosis of Rat-1 fibroblasts triggered by serum deprivation. Our results showed that, rather than inhibiting apoptosis, Pim-1 expression stimulated c-Myc-mediated apoptosis in Rat-1 fibroblasts. Pim-1 stimulated c-Myc-mediated apoptosis through an enhancement of the c-Myc-mediated activation of caspase-3 (CPP32)-like proteases, since the suppression of this activity by a specific caspase inhibitor abolished the apoptosis stimulation by Pim-1. A kinase-defective Pim-1 mutant failed to stimulate c-Myc-mediated apoptosis, and Pim-1 expression alone in the absence of c-Myc overexpression did not induce apoptosis of serum-deprived Rat-1 cells, indicating that the kinase activity of Pim-1 and the activated c-Myc signaling pathway were required for apoptosis stimulation by Pim-1. Together, these results suggest that Pim-1 oncoprotein stimulates as a serine/threonine kinase the death signaling elicited by c-Myc at a step upstream of caspase-3-like protease activation in Rat-1 fibroblasts. Our results also suggest that Pim-1 kinase might function cooperatively with c-Myc through the phosphorylation of a factor(s) which regulates the common signaling pathway involved in c-Myc-mediated apoptosis and transformation.


Subject(s)
Caspases , Cysteine Endopeptidases/metabolism , Oncogenes , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Apoptosis/physiology , Caspase 3 , Cell Line , Cell Transformation, Neoplastic , Endopeptidases/metabolism , Enzyme Precursors/metabolism , Polymerase Chain Reaction , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins c-myc/biosynthesis , Proto-Oncogene Proteins c-pim-1 , Rats , Recombinant Fusion Proteins/metabolism , Signal Transduction , Transfection
18.
J Neurooncol ; 32(2): 103-9, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9120539

ABSTRACT

Central neurocytoma is considered to be a benign intracranial neoplasm, but little is known about the biological behavior of this type of tumor. Proliferative activity of central neurocytoma was measured in 10 cases using MIB-1 staining for Ki-67 antigen. The MIB-1 staining value varied from < 0.1% to 5.6%, to indicating that some of these tumors have proliferative potential similar to that of anaplastic astrocytoma or malignant meningioma. The bromodeoxyuridine labeling index (BUdR LI, BrdU LI) was measured in 2 cases and the results correlated well with those of the MIB-1 analysis. Tumor volume doubling time (Td) measured in one case was 358 days which is similar to that of malignant meningioma. In one case, the MIB-1 value taken before and after 58 Gy of radiation treatment decreased markedly from 5.6% to 0.2%. The other 9 cases were also treated by radiation therapy (50-60 Gy) and no tumor recurrence was observed during follow-up periods ranging from 23 to 160 months. Another two patients with partially removed and 3 with subtotally removed tumors showing relatively high MIB-1 values might also have benefited from radiation therapy.


Subject(s)
Brain Neoplasms/pathology , Neurocytoma/pathology , Adult , Antibodies, Monoclonal , Brain Neoplasms/diagnostic imaging , Brain Neoplasms/radiotherapy , Bromodeoxyuridine , Cell Division , Female , Humans , Ki-67 Antigen/immunology , Magnetic Resonance Imaging , Male , Neurocytoma/diagnostic imaging , Neurocytoma/radiotherapy , Staining and Labeling , Tomography, X-Ray Computed
19.
Hum Cell ; 9(3): 223-8, 1996 Sep.
Article in English | MEDLINE | ID: mdl-9183653

ABSTRACT

Apoptosis is a genetically encoded cell death program and plays an important role in the regulation of both normal and malignant processes. Recently, many factors involved in the control of apoptosis have been isolated and characterized, and thereby studies on the molecular mechanism of the signaling pathway of apoptosis have made rapid progress. In this article, we discuss the role of apoptosis in carcinogenesis and the possible ways to apply apoptosis to cancer therapy.


Subject(s)
Apoptosis/physiology , Neoplasms/genetics , Genes, p53/physiology , Genetic Therapy , Humans , Neoplasms/therapy
20.
Nihon Rinsho ; 54(7): 1869-75, 1996 Jul.
Article in Japanese | MEDLINE | ID: mdl-8741680

ABSTRACT

Recent studies have revealed that c-Myc is involved not only in cellular transformation but also in apoptosis induction. However, the intronless myc family genes such as s-myc, N-myc2, mycL2 lack transforming ability and retain only apoptosis inducing activity. Although much remains to be clear regarding the molecular mechanism of apoptosis induction by myc gene expression, our recent results suggest that the intronless myc genes significantly differ from that of the c-myc gene in their requirement for wild-type p53 activity in apoptosis induction. We introduce here recent reports on Myc-mediated apoptosis and discuss its molecular mechanism.


Subject(s)
Apoptosis , Genes, myc , Animals , Apoptosis/genetics , Cyclins , Genes, myc/physiology , Humans , Ornithine Decarboxylase , Transcription Factors
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