BIOPHYSICS DIVISION

6.BIOPHYSICS DIVISION

　Isolation and characterization of tumor suppressors and cell death inducers which might be useful for therapy of human cancer is the main subject of the Biophysics Division. Programmed cell death represented by apoptosis plays an important role in the maintenance of homeostasis in muliticellular organisms, and is regulated by many genes, including oncogenes and tumor suppressor genes. In the course of studies on how apoptosis is regulated at the cellular and molecular levels, and how apoptosis may contribute to malignancy, the existence of a unique cell death program which causes caspase- independent non- apoptotic cell death was demonstrated in mammalian cells. In the Biophysics Division, the studies on the molecular mechanism of cell death signaling for induction of non- apoptotic as well as apoptotic cell death and application of these genetically regulated cell suicide programs to therapies against human cancer have been strongly promoted.
Molecular Signaling for Apoptotic Cell Death

　The Biophysics Division has identified unique intronless myc genes in mammals including mouse, hamster, rat, monkey and human.⁽¹²¹⁾ In addition, it has been 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. To understand the physiological meaning of Myc- mediated apoptosis, the signaling pathway upstream of the caspase cascade required for the induction of Myc- mediated apoptosis triggered by extracellular stimuli was examined. The GST- pulldown assay and immunochemical analysis demonstrated that c- Jun N- terminal kinases (JNKs)activated by various extracellular apoptotic stimuli such as 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 signal.⁽¹²²⁾ Similarly, it has been shown that Pim- 1 serine/threonine kinase stimulates not only c- Myc- mediated apoptosis signaling upstream of the caspase- 3- like protease cascade, but also c- Myc- mediated cell transformation through phosphorylation of Cdc25A cell cycle phosphatase as a direct transcriptional target for c- Myc.⁽¹²³⁾ The signaling pathway for neuronal cell death triggered by high level expression of calcineurin was also considered essential for cytochrome c/caspase- 3- mediated apoptosis.⁽¹²⁴⁾
Characterization of Ras-activated Cell Suicide Program

　The ras proto- oncogene is a central component of mitogenic signal- transduction pathways involved in cell growth and development. In apoptosis, Ras has been thought to act as a suppressor through the activation of either the PI3K- Akt or Raf- ERK signaling pathways. However, surprisingly, gene transfection experiments demonstrated that the expression of oncogenic Ras induces cellular degeneration with necrotic- like morphology in a PI3K- dependent manner in human cancer cells such as malignant glioma cells and gastric cancer cells. This Ras- induced cell death was not inhibited by caspase inhibitors and cells were negative for the TUNEL assay.^(125,¹²⁶⁾ Taken together these findings indicated that Ras- induced caspase- independent cell death may be regulated by a molecular mechanism distinct from that of apoptosis.
Analysis of Functions and Reactivity of Biological Molecules

　Pyrimidine dimers generated in DNA molecules by far- UV irradiation are toxic and mutagenic in living cells. The conformations of various pyrimidine dimers and their radical cations, and the mechanism of their fragmentation were shown by the ab initio MO method.⁽¹²⁷⁾ The dynamics of chemical reactions, such as hydrolysis of CH₃Cl in water,⁽¹²⁸⁾ and the ET/SN2 boundary reaction,⁽¹²⁹⁾ were demonstrated by ab initio MO calculations and ab inito MD simulations. Vibrational modes and Raman scattering tensors in the thymidine molecule were also calculated by the ab initio MO method.⁽¹³⁰⁾ DNA- protein recognition plays a central role in regulation of expression of genomic information. To understand the specificity of the base- amino acid interactions, we proposed a method to examine the interaction energetics by taking account of the structural flexibility.⁽¹³¹⁾ Modification of the anticodon of tRNA has an essential role in recognition of the genetic codes. On the basis of NMR analysis together with mass spectrometry, the chemical structure of modified nucleoside located at the first position of the anticodon of E. coli tRNA^Leu₅ was determined and considered to be essential for selective recognition of the leucine codons UUA and UUG. ⁽¹³²⁾




6.BIOPHYSICS DIVISION



	Isolation and characterization of tumor suppressors and cell death inducers which might be useful for therapy of human cancer is the main subject of the Biophysics Division. Programmed cell death represented by apoptosis plays an important role in the maintenance of homeostasis in muliticellular organisms, and is regulated by many genes, including oncogenes and tumor suppressor genes. In the course of studies on how apoptosis is regulated at the cellular and molecular levels, and how apoptosis may contribute to malignancy, the existence of a unique cell death program which causes caspase- independent non- apoptotic cell death was demonstrated in mammalian cells. In the Biophysics Division, the studies on the molecular mechanism of cell death signaling for induction of non- apoptotic as well as apoptotic cell death and application of these genetically regulated cell suicide programs to therapies against human cancer have been strongly promoted. Molecular Signaling for Apoptotic Cell Death 　The Biophysics Division has identified unique intronless myc genes in mammals including mouse, hamster, rat, monkey and human.⁽¹²¹⁾ In addition, it has been 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. To understand the physiological meaning of Myc- mediated apoptosis, the signaling pathway upstream of the caspase cascade required for the induction of Myc- mediated apoptosis triggered by extracellular stimuli was examined. The GST- pulldown assay and immunochemical analysis demonstrated that c- Jun N- terminal kinases (JNKs)activated by various extracellular apoptotic stimuli such as 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 signal.⁽¹²²⁾ Similarly, it has been shown that Pim- 1 serine/threonine kinase stimulates not only c- Myc- mediated apoptosis signaling upstream of the caspase- 3- like protease cascade, but also c- Myc- mediated cell transformation through phosphorylation of Cdc25A cell cycle phosphatase as a direct transcriptional target for c- Myc.⁽¹²³⁾ The signaling pathway for neuronal cell death triggered by high level expression of calcineurin was also considered essential for cytochrome c/caspase- 3- mediated apoptosis.⁽¹²⁴⁾ Characterization of Ras-activated Cell Suicide Program 　The ras proto- oncogene is a central component of mitogenic signal- transduction pathways involved in cell growth and development. In apoptosis, Ras has been thought to act as a suppressor through the activation of either the PI3K- Akt or Raf- ERK signaling pathways. However, surprisingly, gene transfection experiments demonstrated that the expression of oncogenic Ras induces cellular degeneration with necrotic- like morphology in a PI3K- dependent manner in human cancer cells such as malignant glioma cells and gastric cancer cells. This Ras- induced cell death was not inhibited by caspase inhibitors and cells were negative for the TUNEL assay.^(125,¹²⁶⁾ Taken together these findings indicated that Ras- induced caspase- independent cell death may be regulated by a molecular mechanism distinct from that of apoptosis. Analysis of Functions and Reactivity of Biological Molecules 　Pyrimidine dimers generated in DNA molecules by far- UV irradiation are toxic and mutagenic in living cells. The conformations of various pyrimidine dimers and their radical cations, and the mechanism of their fragmentation were shown by the ab initio MO method.⁽¹²⁷⁾ The dynamics of chemical reactions, such as hydrolysis of CH₃Cl in water,⁽¹²⁸⁾ and the ET/SN2 boundary reaction,⁽¹²⁹⁾ were demonstrated by ab initio MO calculations and ab inito MD simulations. Vibrational modes and Raman scattering tensors in the thymidine molecule were also calculated by the ab initio MO method.⁽¹³⁰⁾ DNA- protein recognition plays a central role in regulation of expression of genomic information. To understand the specificity of the base- amino acid interactions, we proposed a method to examine the interaction energetics by taking account of the structural flexibility.⁽¹³¹⁾ Modification of the anticodon of tRNA has an essential role in recognition of the genetic codes. On the basis of NMR analysis together with mass spectrometry, the chemical structure of modified nucleoside located at the first position of the anticodon of E. coli tRNA^Leu₅ was determined and considered to be essential for selective recognition of the leucine codons UUA and UUG. ⁽¹³²⁾