Biophysics Division

6. Biophysics Division

　The isolation and characterization of tumor suppressors and cell death inducers, which might be applied to therapy of human cancer, is the main subject of the Biophysics Division. Programmed cell death (PCD) represented by apoptosis plays an important role in the maintenance of homeostasis in multicellular organisms, and is regulated by many genes including oncogenes and tumor suppressor genes. In tumor cells, many of these genes are frequently mutated, deleted or abnormally expressed, leading to resistance to PCD. In the Biophysics Division, studies on regulation of PCD at a cellular and molecular level, including how PCD may contribute to malignancy, and how PCD may be exploited in cancer therapy are in progress.
Regulation of Apoptosis Signaling

　Unique intronless myc genes in mammals including mouse, hamster, rat, monkey and human have been identified in the Biophysics Division. The intronless myc gene, ms-myc, isolated from a mouse genomic library effectively induced apoptosis via activation of caspase-3-like proteases in serum-deprived Rat-1 fibroblasts in the same manner as rat s-Myc. However, in contrast to c-Myc, ms-Myc expression induced apoptotic cell death even in human glioma cells cultured in medium containing 10% FCS in a wild-type p53-independent manner. It has been reported that Myc-induced apoptosis is inhibited by a restricted group of cytokines such as IGFs. In human glioma cells a member of EGF family, heparin-binding EGF-like growth factor, is highly expressed and acts as an autocrine growth factor to stimulate their proliferation.⁽¹⁰⁷⁾ However, anti-HB-EGF blocking antibody failed to induce apoptosis in glioma cells, although the antibody reduced the growth rate of the tumor cells. In apoptosis a family of cysteine proteases called caspases participate in a cascade that is activated by apoptotic signals and cleave a set of substrate proteins. To characterize the substrate specificity of proteases, a novel peptide library method was developed.⁽¹⁰⁸⁾ In this method, the concentration of each substrate peptide hydrolyzed is measured by reverse-phase HPLC and then relative k_cat/K_M values are calculated from the initial velocities of the fraction of substrate peptide hydrolyzed. In studies on apoptosis regulation, PCR technique is also frequently used for cloning of desired genes. However, in the DNA polymerization reaction, Taq DNA polymerase catalyzes two unwanted reactions, the slow process of polymerization of dATP and dTTP causing the formation of oligo d(A-T) in the absence of primer/template and the rapid process of elongation of the oligo d(A-P) by self-priming.⁽¹⁰⁹⁾ These polymerization reactions interfere with DNA synthesis requiring templates.
The Role of Mitochondria in Cell Suicide System

　Mitochondria play a crucial role in execution of the cell suicide program through a variety of key events including the release of caspase activators such as cytochrome C, altered cellular oxidation-reduction and changes in electron transport. Chinese hamster cell lines were established that were resistant to H₂O₂, an inducer of genotoxic stress.^(110,111) The frequency of DNA single strand breaks in mitochondrial DNA induced by H₂O₂ was examined in these cell lines, and the prediction that the sensitivity of mitochondria to DNA strand breaks, but not that of nuclei, in CHO cells may correlate with the cellular sensitivity to H₂O₂ was proven.
Analysis of Three-dimensional (3D) Structure of Biological Molecules by MO Calculation

　Elucidation of the 3D structures of biologically active molecules is imortant to understand their function. This approach, however, is not always straightforward, since those molecules are usually too flexible to assume any unique structures. To perform molecular dynamics (MD) simulations of biological molecules, a set of analytical potentials based on the ab initio MO method was developed. Using this set of potentials, the 3D structures of Pd(110)-c(4x2)-benzene imaged by scanning tunneling microscopy were shown by MO calculations.⁽¹¹²⁾ In particular, the existence of a benzene derived state near Fermi level was proven. The normal modes of vibration and their associated polarizability oscillation of L-tyrosine were also calculated by the ab initio MO method.⁽¹¹³⁾ The energy minimization procedure was used to ensure the 3D-conformation of Leu-enkephalin dihydrate determined on the basis of six sets of interatomic distances accurately determined by REDOR NMR and the conformation-dependent ¹³C chemical shift.⁽¹¹⁴⁾ Thymine dimers generated in DNA molecules by far-UV irradiation are toxic and mutagenic in living cells. The reaction mechanism of fragmentation of the thymine dimer radical cation was shown by the ab initio MO method.⁽¹¹⁵⁾ The dynamics of chemical reactions, such as the hydrolysis of CH₃Cl in water, were also demonstrated by MO calculations.^(116,117)




	6. Biophysics Division



		The isolation and characterization of tumor suppressors and cell death inducers, which might be applied to therapy of human cancer, is the main subject of the Biophysics Division. Programmed cell death (PCD) represented by apoptosis plays an important role in the maintenance of homeostasis in multicellular organisms, and is regulated by many genes including oncogenes and tumor suppressor genes. In tumor cells, many of these genes are frequently mutated, deleted or abnormally expressed, leading to resistance to PCD. In the Biophysics Division, studies on regulation of PCD at a cellular and molecular level, including how PCD may contribute to malignancy, and how PCD may be exploited in cancer therapy are in progress. Regulation of Apoptosis Signaling 　Unique intronless myc genes in mammals including mouse, hamster, rat, monkey and human have been identified in the Biophysics Division. The intronless myc gene, ms-myc, isolated from a mouse genomic library effectively induced apoptosis via activation of caspase-3-like proteases in serum-deprived Rat-1 fibroblasts in the same manner as rat s-Myc. However, in contrast to c-Myc, ms-Myc expression induced apoptotic cell death even in human glioma cells cultured in medium containing 10% FCS in a wild-type p53-independent manner. It has been reported that Myc-induced apoptosis is inhibited by a restricted group of cytokines such as IGFs. In human glioma cells a member of EGF family, heparin-binding EGF-like growth factor, is highly expressed and acts as an autocrine growth factor to stimulate their proliferation.⁽¹⁰⁷⁾ However, anti-HB-EGF blocking antibody failed to induce apoptosis in glioma cells, although the antibody reduced the growth rate of the tumor cells. In apoptosis a family of cysteine proteases called caspases participate in a cascade that is activated by apoptotic signals and cleave a set of substrate proteins. To characterize the substrate specificity of proteases, a novel peptide library method was developed.⁽¹⁰⁸⁾ In this method, the concentration of each substrate peptide hydrolyzed is measured by reverse-phase HPLC and then relative k_cat/K_M values are calculated from the initial velocities of the fraction of substrate peptide hydrolyzed. In studies on apoptosis regulation, PCR technique is also frequently used for cloning of desired genes. However, in the DNA polymerization reaction, Taq DNA polymerase catalyzes two unwanted reactions, the slow process of polymerization of dATP and dTTP causing the formation of oligo d(A-T) in the absence of primer/template and the rapid process of elongation of the oligo d(A-P) by self-priming.⁽¹⁰⁹⁾ These polymerization reactions interfere with DNA synthesis requiring templates. The Role of Mitochondria in Cell Suicide System 　Mitochondria play a crucial role in execution of the cell suicide program through a variety of key events including the release of caspase activators such as cytochrome C, altered cellular oxidation-reduction and changes in electron transport. Chinese hamster cell lines were established that were resistant to H₂O₂, an inducer of genotoxic stress.^(110,111) The frequency of DNA single strand breaks in mitochondrial DNA induced by H₂O₂ was examined in these cell lines, and the prediction that the sensitivity of mitochondria to DNA strand breaks, but not that of nuclei, in CHO cells may correlate with the cellular sensitivity to H₂O₂ was proven. Analysis of Three-dimensional (3D) Structure of Biological Molecules by MO Calculation 　Elucidation of the 3D structures of biologically active molecules is imortant to understand their function. This approach, however, is not always straightforward, since those molecules are usually too flexible to assume any unique structures. To perform molecular dynamics (MD) simulations of biological molecules, a set of analytical potentials based on the ab initio MO method was developed. Using this set of potentials, the 3D structures of Pd(110)-c(4x2)-benzene imaged by scanning tunneling microscopy were shown by MO calculations.⁽¹¹²⁾ In particular, the existence of a benzene derived state near Fermi level was proven. The normal modes of vibration and their associated polarizability oscillation of L-tyrosine were also calculated by the ab initio MO method.⁽¹¹³⁾ The energy minimization procedure was used to ensure the 3D-conformation of Leu-enkephalin dihydrate determined on the basis of six sets of interatomic distances accurately determined by REDOR NMR and the conformation-dependent ¹³C chemical shift.⁽¹¹⁴⁾ Thymine dimers generated in DNA molecules by far-UV irradiation are toxic and mutagenic in living cells. The reaction mechanism of fragmentation of the thymine dimer radical cation was shown by the ab initio MO method.⁽¹¹⁵⁾ The dynamics of chemical reactions, such as the hydrolysis of CH₃Cl in water, were also demonstrated by MO calculations.^(116,117)