BIOPHYSICS DIVISION

6.BIOPHYSICS DIVISION

    Programmed cell death (PCD) represented by apoptosis plays a central role in the regulation of both normal and malignant cell proliferation. Disrupted regulation of the control mechanism of PCD may cause serious diseases such as cancer and neurodegenerative diseases. PCD is regulated by many gene products, including onco-proteins and tumor suppressors. Therefore, an understanding of the molecular 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, the studies on the molecular mechanism of cell death signaling for induction of PCD and application of the genetically regulated cell suicide programs to therapies against human cancer have been actively pursued.
Molecular signaling for Myc-mediated apoptosis

    Myc-mediated cell death signaling is sensitized by various stimuli including anticancer agents and UV-irradiation. The Biophysics Division has reported that the activation of caspase-3-like proteases is essential for induction of Myc-mediated apoptosis as well as for Fas- and TNF-a-mediated apoptosis. The signaling pathway upstream of the caspase cascade required for the induction of Myc-mediated apoptosis triggered by extracellular stimuli was examined. GST-pulldown assay and immunochemical analysis demonstrated that c-Jun N-terminal kinases (JNKs) activated by UV irradiation and anticancer drugs selectively interact with c-Myc and phosphorylate the protein at Ser-62 and Ser-71 causing sensitization of cells to c-Myc-mediated apoptotic signals. However, the JNK signaling has no effect on s-Myc- mediated apoptosis. In contrast, the p38 MAPK pathway regulates common apoptotic machinery for both c-Myc and s-Myc upstream of the caspase cascade.⁽¹⁰¹⁾ It has been reported that most anticancer agents have the ability to kill cancer cells through induction of apoptosis. Among them, actinomycin D and staurosporine are potent apoptosis inducers and potentially effective chemotherapeutic agents against glioblastoma cells in vitro.⁽¹⁰²⁾ However, the effectiveness of anticancer agents is frequently hampered by cancer cell chemoresistance. To overcome this advantage and to obtain effective chemotherapeutic protocols for malignant brain tumors, knowledge of the expression levels of chemoresistance-related genes represented by MGMT and mdr 1 might be required.⁽¹⁰³⁾
The role of mitochondria in the cell suicide system

    Mitochondria are cellular organelles which play a crucial role in the execution of the cell suicide program through a variety of key events including the release of cytochrome C as a caspase activator, alteration of oxidation-reduction conditions and changes in membrane potential. In the Biophysics Division, Chinese hamster cell lines resistant to H₂O₂, an inducer of genotoxic stress, were established. Using these cell lines, it was suggested that elevated intracellular Ca²⁺ concentration may protect against H₂O₂-induced mitochondrial damage and cytotoxicity in these cells.⁽¹⁰⁴⁾
    Mitochondria have a unique ATP-dependent protein hydrolyzing system mediated by Lon protease. To understand the biological function of Lon protease, the Biophysics Division isolated and cloned the lon genes and/or its cDNAs from E.coli, Thermus thermophilus, mouse and human, and determined their nucleotide sequences. Using a large amount of recombinant thermophilic Lon protease prepared in an E.coli expression system, structural analyses of Lon protease by X-ray crystallography and by NMR spectroscopy are in progress in the Biophysics Division.⁽¹⁰⁵⁾
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. 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. Comparison of the amino acid sequences of ciliate RFs deduced from their genomic sequences with that of human RF, whose crystal structure was recently determined, revealed that the domain structure proposed to be responsible for codon recognition of human RF differs from that of ciliate RFs.




6.BIOPHYSICS DIVISION



	Programmed cell death (PCD) represented by apoptosis plays a central role in the regulation of both normal and malignant cell proliferation. Disrupted regulation of the control mechanism of PCD may cause serious diseases such as cancer and neurodegenerative diseases. PCD is regulated by many gene products, including onco-proteins and tumor suppressors. Therefore, an understanding of the molecular 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, the studies on the molecular mechanism of cell death signaling for induction of PCD and application of the genetically regulated cell suicide programs to therapies against human cancer have been actively pursued. Molecular signaling for Myc-mediated apoptosis Myc-mediated cell death signaling is sensitized by various stimuli including anticancer agents and UV-irradiation. The Biophysics Division has reported that the activation of caspase-3-like proteases is essential for induction of Myc-mediated apoptosis as well as for Fas- and TNF-a-mediated apoptosis. The signaling pathway upstream of the caspase cascade required for the induction of Myc-mediated apoptosis triggered by extracellular stimuli was examined. GST-pulldown assay and immunochemical analysis demonstrated that c-Jun N-terminal kinases (JNKs) activated by UV irradiation and anticancer drugs selectively interact with c-Myc and phosphorylate the protein at Ser-62 and Ser-71 causing sensitization of cells to c-Myc-mediated apoptotic signals. However, the JNK signaling has no effect on s-Myc- mediated apoptosis. In contrast, the p38 MAPK pathway regulates common apoptotic machinery for both c-Myc and s-Myc upstream of the caspase cascade.⁽¹⁰¹⁾ It has been reported that most anticancer agents have the ability to kill cancer cells through induction of apoptosis. Among them, actinomycin D and staurosporine are potent apoptosis inducers and potentially effective chemotherapeutic agents against glioblastoma cells in vitro.⁽¹⁰²⁾ However, the effectiveness of anticancer agents is frequently hampered by cancer cell chemoresistance. To overcome this advantage and to obtain effective chemotherapeutic protocols for malignant brain tumors, knowledge of the expression levels of chemoresistance-related genes represented by MGMT and mdr 1 might be required.⁽¹⁰³⁾ The role of mitochondria in the cell suicide system Mitochondria are cellular organelles which play a crucial role in the execution of the cell suicide program through a variety of key events including the release of cytochrome C as a caspase activator, alteration of oxidation-reduction conditions and changes in membrane potential. In the Biophysics Division, Chinese hamster cell lines resistant to H₂O₂, an inducer of genotoxic stress, were established. Using these cell lines, it was suggested that elevated intracellular Ca²⁺ concentration may protect against H₂O₂-induced mitochondrial damage and cytotoxicity in these cells.⁽¹⁰⁴⁾ Mitochondria have a unique ATP-dependent protein hydrolyzing system mediated by Lon protease. To understand the biological function of Lon protease, the Biophysics Division isolated and cloned the lon genes and/or its cDNAs from E.coli, Thermus thermophilus, mouse and human, and determined their nucleotide sequences. Using a large amount of recombinant thermophilic Lon protease prepared in an E.coli expression system, structural analyses of Lon protease by X-ray crystallography and by NMR spectroscopy are in progress in the Biophysics Division.⁽¹⁰⁵⁾ 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. 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. Comparison of the amino acid sequences of ciliate RFs deduced from their genomic sequences with that of human RF, whose crystal structure was recently determined, revealed that the domain structure proposed to be responsible for codon recognition of human RF differs from that of ciliate RFs.