BIOPHYSICS DIVISION

6.BIOPHYSICS DIVISION

    Programmed cell death (PCD) represented by apoptosis plays essential roles in the maintenance of homeostasis in multicellular organisms. Therefore, disruption of the control mechanism of PCD causes serious diseases such as cancer and neurodegenerative diseases. PCD plays the most crucial role in the prevention of full malignant transformation of cells concurrently and may occasionally induce tumor regression through elimination of harmful cells. PCD is regulated by many gene products, including onco-proteins and tumor suppressors. An understanding of the regulatory mechanism of PCD and identification of the cell-type-specific factors determining cellular survival or death should lead to fundamental advances in the therapy of human diseases. Thus, in the Biophysics Division, studies on the molecular mechanism of cell death signaling for induction of PCD have been strongly promoted.
Molecular signaling for c-Myc-mediated apoptosis

    c-Myc-mediated cell death signaling is sensitized by various extracellular stimuli including anticancer agents and UV-irradiation. The Biophysics Division has reported that c-Myc is directly and selectively phosphorylated by c-Jun N-terminal kinases (JNKs) at Ser-62 and Ser-71 and this phosphorylation is required for cell sensitization to UV- and Taxol-triggered apoptosis. It has been shown that a new MAP kinase kinase kinase, ASK1 activates both the MKK4/7-JNK and MKK3/6-p38 MAP kinase pathways and plays pivotal roles in apoptosis induction by tumor necrosis factor and stress stimuli. The stress-responsive phosphorylation/de-phosphorylation cascade was next examined in UV-irradiated cells to understand the activation mechanism of JNK required for sensitization of cells to c-Myc-dependent apoptosis triggered by UV-irradiation. Analysis of the stress-responsive protein kinase pathway demonstrated that the ASK1-JNK signaling pathway was activated in UV-irradiated cells and promoted the proapoptotic activity of c-Myc by modulating c-Myc protein stability through phosphorylation at both Ser-62 and Ser-71.⁽¹⁰³⁾
Caspase-independent non-apoptotic programmed cell death and its pathophysiological significance

    The Biophysics Division has reported that human cells have a unique cell death program which causes caspaseindependent non-apoptotic cell death regulated by a molecular mechanism distinct from that of apoptosis. The ras proto-oncogene is a central component of mitogenic signal-transduction pathways involved in cell growth and development. However, in some types of human cancer cells such as malignant glioma cells, neuroblastoma cells and gastric cancer cells, oncogenic H-Ras expression induces non-apoptotic PCD with necrotic-like morphology. This Ras-mediated PCD is executed even in the absence of p53 tumor suppressor gene expression and is not suppressed by Bcl-xL and pancaspase inhibitors. Similar caspase-independent cell death with necrotic-like morphology is also induced in human glioma cells by ceramide. This ceramide-induced non-apoptotic cell death is effectively inhibited by the activation of the Akt/PKB signaling pathway. These observations suggest that the multiple death signaling pathways are involved in the regulation of non-apoptotic cell death induction as well as that of apoptosis.
Molecular mechanism of stop codon recognition

    Translation termination is signaled by the presence of an in-frame stop codon at the aminoacyl (A) site of the ribosome and mediated by protein release factors (RFs) in the presence of GTP. The Biophysics Division has reported that ciliates including Tetrahymena and Paramecium use both translation termination codons UAA and UAG to encode glutamine and only UGA as the stop codon. Recent studies have also indicated that the ciliate Euplotes octocarinatus uses the UGA stop codon as a cysteine codon together with the conventional cysteine codons, UGU and UGC. To elucidate the molecular mechanism by which RFs decode stop codons in eukaryotes, the Biophysics Division isolated and cloned the RF genes from Tetrahymena and Euplotes and determined their nucleotide sequences.⁽¹⁰⁴⁾ Comparison of the amino acid sequences of ciliate RFs deduced from their genomic sequences with that of human RF whose crystal structure was recently analysed reveals that the domain structure proposed to be responsible for codon recognition of human RF differs from that of ciliate RFs.⁽¹⁰⁵⁾




6.BIOPHYSICS DIVISION



	Programmed cell death (PCD) represented by apoptosis plays essential roles in the maintenance of homeostasis in multicellular organisms. Therefore, disruption of the control mechanism of PCD causes serious diseases such as cancer and neurodegenerative diseases. PCD plays the most crucial role in the prevention of full malignant transformation of cells concurrently and may occasionally induce tumor regression through elimination of harmful cells. PCD is regulated by many gene products, including onco-proteins and tumor suppressors. An understanding of the regulatory mechanism of PCD and identification of the cell-type-specific factors determining cellular survival or death should lead to fundamental advances in the therapy of human diseases. Thus, in the Biophysics Division, studies on the molecular mechanism of cell death signaling for induction of PCD have been strongly promoted. Molecular signaling for c-Myc-mediated apoptosis c-Myc-mediated cell death signaling is sensitized by various extracellular stimuli including anticancer agents and UV-irradiation. The Biophysics Division has reported that c-Myc is directly and selectively phosphorylated by c-Jun N-terminal kinases (JNKs) at Ser-62 and Ser-71 and this phosphorylation is required for cell sensitization to UV- and Taxol-triggered apoptosis. It has been shown that a new MAP kinase kinase kinase, ASK1 activates both the MKK4/7-JNK and MKK3/6-p38 MAP kinase pathways and plays pivotal roles in apoptosis induction by tumor necrosis factor and stress stimuli. The stress-responsive phosphorylation/de-phosphorylation cascade was next examined in UV-irradiated cells to understand the activation mechanism of JNK required for sensitization of cells to c-Myc-dependent apoptosis triggered by UV-irradiation. Analysis of the stress-responsive protein kinase pathway demonstrated that the ASK1-JNK signaling pathway was activated in UV-irradiated cells and promoted the proapoptotic activity of c-Myc by modulating c-Myc protein stability through phosphorylation at both Ser-62 and Ser-71.⁽¹⁰³⁾ Caspase-independent non-apoptotic programmed cell death and its pathophysiological significance The Biophysics Division has reported that human cells have a unique cell death program which causes caspaseindependent non-apoptotic cell death regulated by a molecular mechanism distinct from that of apoptosis. The ras proto-oncogene is a central component of mitogenic signal-transduction pathways involved in cell growth and development. However, in some types of human cancer cells such as malignant glioma cells, neuroblastoma cells and gastric cancer cells, oncogenic H-Ras expression induces non-apoptotic PCD with necrotic-like morphology. This Ras-mediated PCD is executed even in the absence of p53 tumor suppressor gene expression and is not suppressed by Bcl-xL and pancaspase inhibitors. Similar caspase-independent cell death with necrotic-like morphology is also induced in human glioma cells by ceramide. This ceramide-induced non-apoptotic cell death is effectively inhibited by the activation of the Akt/PKB signaling pathway. These observations suggest that the multiple death signaling pathways are involved in the regulation of non-apoptotic cell death induction as well as that of apoptosis. Molecular mechanism of stop codon recognition Translation termination is signaled by the presence of an in-frame stop codon at the aminoacyl (A) site of the ribosome and mediated by protein release factors (RFs) in the presence of GTP. The Biophysics Division has reported that ciliates including Tetrahymena and Paramecium use both translation termination codons UAA and UAG to encode glutamine and only UGA as the stop codon. Recent studies have also indicated that the ciliate Euplotes octocarinatus uses the UGA stop codon as a cysteine codon together with the conventional cysteine codons, UGU and UGC. To elucidate the molecular mechanism by which RFs decode stop codons in eukaryotes, the Biophysics Division isolated and cloned the RF genes from Tetrahymena and Euplotes and determined their nucleotide sequences.⁽¹⁰⁴⁾ Comparison of the amino acid sequences of ciliate RFs deduced from their genomic sequences with that of human RF whose crystal structure was recently analysed reveals that the domain structure proposed to be responsible for codon recognition of human RF differs from that of ciliate RFs.⁽¹⁰⁵⁾