Biophysics Division

6. Biophysics Division

The isolation and characterization of tumor suppressors and inducers of apoptosis which might be applied to the therapy of human cancer is the main subject of the Biophysics Division. Apoptosis is a genetically programmed mode of cell death that is regulated by many genes, including oncogenes and tumor suppressor genes. In tumor cells, however, many of these genes are frequently mutated, deleted or abnormally expressed. Thus, the susceptibility to cell death of tumor cells may be altered by modulation of the apoptosis process, causing apoptosis resistance. In the Biophysics Division, experiments addressing the genetic basis of apoptosis, together with the signaling pathways that regulate apoptosis suppression in tumor cells, are in progress.
Signaling Pathway of Apoptosis

The identification of unique intronless myc genes in mammals including mouse, hamster, rat, monkey and human has been performed in the Biophysics Division. The intronless myc genes such as s-myc and mycL2 can induce apoptosis in mammalian cells, regardless of whether the cells retain wild type p53. Recent publications suggest that caspases, a family of cysteine proteases, might be the integral component of the basic regulatory machinery of apoptosis. As well as c-myc, s-myc gene expression can effectively induce apoptosis in serum-deprived Rat-1 fibroblasts via the activation of caspase-3-like proteases (casp-3s). Furthermore, s-Myc- and c-Myc-mediated apoptosis and activation of casp-3s were significantly inhibited by a serine protease inhibitor, AEBSF, implicating that serine proteases which function upstream of casp-3s play a critical role in Myc-mediated apoptosis.⁽¹⁰³⁾ A similar requirement for casp-3s activation was also observed in Bax-induced apoptosis of COS-7 cells.⁽¹⁰⁴⁾ Interestingly, Bcl-xL inhibited Bax-induced COS-7 cell death and the accompanying casp-3s activation more efficiently than Bcl-2. The role of the serine/threonine kinase Pim-1, which has been thought to act as an apoptosis inhibitor like Bcl-2, was examined in c-Myc-mediated apoptosis. Unexpectedly, gene transfection experiments suggested that Pim-1 kinase might function cooperatively with c-Myc not only in transformation but also in apoptosis.⁽¹⁰⁵⁾ When Ras is activated by proteolytic trimming at the C-terminal CAAX motif it can induce programmed cell death and suppress apoptosis induced by Myc and E1A. The protease responsible was partially purified from bovine brain and characterized.⁽¹⁰⁶⁾
A variety of anti-cancer drugs can also induce apoptosis in tumor cells. However, many tumor cells acquire drug-resistance through the expression of drug-resistance genes. For instance, ACNU is a useful drug for the chemotherapy of human glioma cells. However, the cytocidal effect of ACNU is effectively repressed by MGMT drug-resistance gene expression. To overcome ACNU resistance in glioma therapy, an MGMT antisense RNA inhibitory approach might be useful.⁽¹⁰⁷⁾ EGF growth factor protects hippocampal neurons from apoptotic cell death induced by anoxia and nitric oxide exposure. Immunohisochemical analysis demonstrated that HB-EGF, a member of the EGF family, is ubiquitously expressed in neurons and glial cells in white matter together with its cognitive receptor, EGFR.⁽¹⁰⁸⁾ This finding may provide a clue towards understanding the mechanism of cell death of hippocampal neurons induced by ischemia.
Transcriptional Regulation of UAG Suppressor tRNA Genes

Transcriptional expression of the UAG suppressor tRNA gene in rat and mouse cells was negatively regulated by binding of an E-box binding nuclear protein named STGBP, which recognizes a CATGTG sequence located 40nts upstream of the transcription initiation site of the tRNA gene.⁽¹⁰⁹⁾ Northern blot analysis and RT-PCR analysis suggested that the suppressor tRNA gene is located in an intron region of a gene encoding a 7 kb mRNA. Taq DNA polymerase used in PCR can polymerize dATP and dTTP to poly d(A-T) in a primer/template-independent manner depending on the experimental conditions.⁽¹¹⁰⁾
Three-dimensional (3D) Structures and Vibrational Spectra of Biological Molecules

Elucidation of the 3D structures of biologically active peptides can provide insights into their functions. This approach, however, is not always straightforward, since those molecules are usually too flexible to assume any specific unique structures. To perform molecular dynamics (MD) simulations of biological molecules, a set of analytical potentials based on the abinitio MO method was developed.⁽¹¹¹⁾ Using this set of potentials, 3D structures of N-Ac-PGF in the crystalline states were shown by MD simulations.⁽¹¹²⁾ Vibrational modes and Raman scattering tensors in thymine molecule were also calculated by the ab initio MO method.^{(113, 114)} The formation of free radicals and active oxygen species from carbonyl sulfide and nicotine in cigarette smoke, and from steroid hormones was detected using a spin trapping agent and ESR.^{(115, 116)}




	6. Biophysics Division



		The isolation and characterization of tumor suppressors and inducers of apoptosis which might be applied to the therapy of human cancer is the main subject of the Biophysics Division. Apoptosis is a genetically programmed mode of cell death that is regulated by many genes, including oncogenes and tumor suppressor genes. In tumor cells, however, many of these genes are frequently mutated, deleted or abnormally expressed. Thus, the susceptibility to cell death of tumor cells may be altered by modulation of the apoptosis process, causing apoptosis resistance. In the Biophysics Division, experiments addressing the genetic basis of apoptosis, together with the signaling pathways that regulate apoptosis suppression in tumor cells, are in progress. Signaling Pathway of Apoptosis The identification of unique intronless myc genes in mammals including mouse, hamster, rat, monkey and human has been performed in the Biophysics Division. The intronless myc genes such as s-myc and mycL2 can induce apoptosis in mammalian cells, regardless of whether the cells retain wild type p53. Recent publications suggest that caspases, a family of cysteine proteases, might be the integral component of the basic regulatory machinery of apoptosis. As well as c-myc, s-myc gene expression can effectively induce apoptosis in serum-deprived Rat-1 fibroblasts via the activation of caspase-3-like proteases (casp-3s). Furthermore, s-Myc- and c-Myc-mediated apoptosis and activation of casp-3s were significantly inhibited by a serine protease inhibitor, AEBSF, implicating that serine proteases which function upstream of casp-3s play a critical role in Myc-mediated apoptosis.⁽¹⁰³⁾ A similar requirement for casp-3s activation was also observed in Bax-induced apoptosis of COS-7 cells.⁽¹⁰⁴⁾ Interestingly, Bcl-xL inhibited Bax-induced COS-7 cell death and the accompanying casp-3s activation more efficiently than Bcl-2. The role of the serine/threonine kinase Pim-1, which has been thought to act as an apoptosis inhibitor like Bcl-2, was examined in c-Myc-mediated apoptosis. Unexpectedly, gene transfection experiments suggested that Pim-1 kinase might function cooperatively with c-Myc not only in transformation but also in apoptosis.⁽¹⁰⁵⁾ When Ras is activated by proteolytic trimming at the C-terminal CAAX motif it can induce programmed cell death and suppress apoptosis induced by Myc and E1A. The protease responsible was partially purified from bovine brain and characterized.⁽¹⁰⁶⁾ A variety of anti-cancer drugs can also induce apoptosis in tumor cells. However, many tumor cells acquire drug-resistance through the expression of drug-resistance genes. For instance, ACNU is a useful drug for the chemotherapy of human glioma cells. However, the cytocidal effect of ACNU is effectively repressed by MGMT drug-resistance gene expression. To overcome ACNU resistance in glioma therapy, an MGMT antisense RNA inhibitory approach might be useful.⁽¹⁰⁷⁾ EGF growth factor protects hippocampal neurons from apoptotic cell death induced by anoxia and nitric oxide exposure. Immunohisochemical analysis demonstrated that HB-EGF, a member of the EGF family, is ubiquitously expressed in neurons and glial cells in white matter together with its cognitive receptor, EGFR.⁽¹⁰⁸⁾ This finding may provide a clue towards understanding the mechanism of cell death of hippocampal neurons induced by ischemia. Transcriptional Regulation of UAG Suppressor tRNA Genes Transcriptional expression of the UAG suppressor tRNA gene in rat and mouse cells was negatively regulated by binding of an E-box binding nuclear protein named STGBP, which recognizes a CATGTG sequence located 40nts upstream of the transcription initiation site of the tRNA gene.⁽¹⁰⁹⁾ Northern blot analysis and RT-PCR analysis suggested that the suppressor tRNA gene is located in an intron region of a gene encoding a 7 kb mRNA. Taq DNA polymerase used in PCR can polymerize dATP and dTTP to poly d(A-T) in a primer/template-independent manner depending on the experimental conditions.⁽¹¹⁰⁾ Three-dimensional (3D) Structures and Vibrational Spectra of Biological Molecules Elucidation of the 3D structures of biologically active peptides can provide insights into their functions. This approach, however, is not always straightforward, since those molecules are usually too flexible to assume any specific unique structures. To perform molecular dynamics (MD) simulations of biological molecules, a set of analytical potentials based on the abinitio MO method was developed.⁽¹¹¹⁾ Using this set of potentials, 3D structures of N-Ac-PGF in the crystalline states were shown by MD simulations.⁽¹¹²⁾ Vibrational modes and Raman scattering tensors in thymine molecule were also calculated by the ab initio MO method.^{(113, 114)} The formation of free radicals and active oxygen species from carbonyl sulfide and nicotine in cigarette smoke, and from steroid hormones was detected using a spin trapping agent and ESR.^{(115, 116)}