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O6-Alkylguanine-DNA alkyltransferase: Influence on susceptibility to the genetic effects of alkylating agents
4
Symposium 2. Disruption of Cell Signalling
be discussed as well as aspects of the molecular biochemistry of the function of the gene product, CSB. CSB appears to playa role in both basal transcription and DNA repair. Another premature aging disorder is Wemers syndrome. These human disorders provide excellent model systems for studies on aging. Wemers patients also appear to have defects in DNA repair and transcription, and the protein is a helicase, The clinical and molecular phenotypes will be discussed.
The focus of this presentation will be on the effects cannabinoids elicit on events within the cAMP cascade and related signaling pathways critical to the regulation of cytokine genes. Special emphasis will be given to the effects cannabinoids exert on the regulation of interleukin-2 and the transcription factors that regulate this gene in T-cells. (This work was supported by funds from NIDA Grants DA07908 and DA09171).
TRANSIENT BRAIN ISCHEMIA- AND BACTERIAL ENDOTOXIN (LPS}-INDUCED GLIAL iNOS EXPRESSION AND NO-INDUCED NEURONAL APOPTOSIS
IS1/L41 06·ALKYLGUANINE·DNA ALKYLTRANSFERASE: INFLUENCE ON SUSCEPTIBILITY TO THE GENETIC EFFECTS OF ALKYLATING AGENTS
S.A. Kyrtopoulos. National Hellenic Research Foundation, Athens 11635, Greece
Hokkaido University, Sapporo 060, Japan
Repair of 06-alkylguanine, a critical lesion in mutagenesis and toxicity caused by alkylating agents, takes place via a pathway involving self-inactivating demethylation by 06-alkylguanine-DNA alkyltransferase (AGT). Hence AGT is important in determining cellular susceptibility to genotoxins acting via attack on position 0 6 of guanine, as exemplified especially by the case of the cross-linking nitrosoureas. As far as susceptibility to methylating agents is concerned, its relationship to AGT content has generally been studied using relatively high doses which result in significant disturbance of the total cellular pool of this suicide enzyme. Recent observations on the kinetics of repair of 06-methylguanine under a wider range of exposure conditions, including exposure to low doses causing minimal disturbance of the AGT pool, suggest a relatively complex relationship between AGT levels and 06-meG repair, possibly related to the existence of kinetically distinct "active" and "backup" enzyme pools in mammalian cells, a suggestion compatible with other recent findings on the DNA binding and intracellular distribution and transport properties of AGT. The implications of these new lines of biochemical and kinetic evidence for the determination of cellular susceptibility to methylating agents will be discussed.
Glial cells play important roles in neuronal survival/death by secreting neurotrophins, cytokines and so on. We found that an inducible type of NO synthase (iNOS) was expressed in glial cells at 3 and 7 days after a transient 4 vessel occiusionJreperfusion (4VO) in male, adult rats. Approximately the same day after 4VO, we observed the appearance of TUNEL staining positive neurons and nuclear fragmentation in neurons by the Hoechst 33258 staining, suggesting the 4VO-induced neuronal apoptosis by iNOSINO derived from glial cells. We also obtained results that bacterial endotoxin (LPS) enhanced INFy-induced iNOS expression in cultured rat glial cells and C6 glioma cells. This enhancement seems to be due to activation and intranuclear translocation of NF-KB. NO generated by LPS-induced glial iNOS caused DNA fragmentation by (apoptosis) in NG108-15 cells. NO donors (sodium nitroprusside) resulted in apoptosis in NG I08-15 cells, inhibited glycenaldehyde-3-phosphate dehydrogenese (GAPDH) activity and increased [32p]_NAD binding/[ 32p] ADP-ribosylation of GAPDH. Interestingly, koningic acid (KA), a selective and potent inhibitor of GAPDH, induced the apoptosis too. Taken together, we presume that NO-elicited disruption of GAPDH, a key enzyme in glycolytic convension and a protein involved in RNA transport, could be involved in neuronal apoptosis.
S2. Disruption of Cell Signalling
IS2/L31
IS2/L1 I
INHIBITION OF THE cAMP SIGNALLING CASCADE VIA THE CANNABINOID RECEPTORS: A PUTATIVE MECHANISM OF IMMUNE MODULATION BY CANNABINOID COMPOUNDS
N.E. Kaminski. Michigan State University. East Lansing, MI, USA Immune modulation by cannabinoids has been widely established over the past three decades. In spite of this, the mechanism of action responsible for immune modulation and other well described biological effects attributed to cannabinoid compounds has been elusive. The identification and cloning of two novel G-protein coupled receptors, CB I and CB2, both of which bind cannabimimetic agents has served as the basis for a putative mechanism of action. CB I, which is also referred to as the central cannabinoid receptor is the primary form expressed within the central nervous system (CNS). Conversely, the peripheral cannabinoid receptor, CB2, does not appear to be expressed with in the CNS but is the predominant form of the receptor expressed within the immune system. Both CB I and CB2 negatively regulate adenylate cyclase activity through a pertussis toxin sensitive GTP-binding protein. Recent investigations addressing the mechanism by which cannabinoids disrupt leukocyte function have demonstrated that in the presence of cannabinoids the cAMP signalling cascade is markedly inhibited as evidenced by decreased adenylate cyclase and protein kinase A activity and decreased DNA binding by cAMP response element binding proteins.
Yasuyuki Nomura. Graduate School of Pharmaceutical Sciences,
EPIGENETIC TOXICOLOGY AS TOXICANT-INDUCED CHANGES IN INTRACELLULAR SIGNALLING LEADING TO ALTERED GAP JUNCTIONAL COMMUNICATION
J.E. Trosko. Michigan State University, Dept. Pediatrics and Human Devel., Natl. Food Safety and Toxicology Center. East Lansing, MI, USA Communication mechanisms [extra-, intra- and gap junctional inter-cellular communication (GJIC)] control, from the fertilized egg, through embryo-genesis to maturity and aging, whether a cell (a) proliferates, (b) differentiates, (c) dies by apoptosis. or (d) if differentiated, adaptively responds to exogenous and endogenous signals. From the egg to the I()() trillion cells in the human body, health is maintained when these communication processes between stem, progenitor and terminally differentiated cells are integrated. Each cell choice involves "epigenetic" mechanisms to alter the expression of genes at the transcriptional, translational or posttranslational levels. Disruption of any of the communication mechanisms can be either adaptive or maladaptive. Modulation of extra-cellular communication, either by genetic imbalances of growth factors, hormones or neuro-transmitters or by environmental, exogenous chemicals can trigger signal transducting intracellular communication mechanisms. These intracellular-cellular signals can modulate gene expression at the transcriptional, translational, or posttranslational levels while also modulating GJIc. Untimely or chronic disruption of GJIC during embryonic or fetal development could lead to embryonic lethality or terato-genesis. By modulation of GJIC, homeostatic control of cell growth, differentiation or apoptosis could lead to specific diseases,
Symposium 2. Disruption of Cell Signalling
be discussed as well as aspects of the molecular biochemistry of the function of the gene product, CSB. CSB appears to playa role in both basal transcription and DNA repair. Another premature aging disorder is Wemers syndrome. These human disorders provide excellent model systems for studies on aging. Wemers patients also appear to have defects in DNA repair and transcription, and the protein is a helicase, The clinical and molecular phenotypes will be discussed.
The focus of this presentation will be on the effects cannabinoids elicit on events within the cAMP cascade and related signaling pathways critical to the regulation of cytokine genes. Special emphasis will be given to the effects cannabinoids exert on the regulation of interleukin-2 and the transcription factors that regulate this gene in T-cells. (This work was supported by funds from NIDA Grants DA07908 and DA09171).
TRANSIENT BRAIN ISCHEMIA- AND BACTERIAL ENDOTOXIN (LPS}-INDUCED GLIAL iNOS EXPRESSION AND NO-INDUCED NEURONAL APOPTOSIS
IS1/L41 06·ALKYLGUANINE·DNA ALKYLTRANSFERASE: INFLUENCE ON SUSCEPTIBILITY TO THE GENETIC EFFECTS OF ALKYLATING AGENTS
S.A. Kyrtopoulos. National Hellenic Research Foundation, Athens 11635, Greece
Hokkaido University, Sapporo 060, Japan
Repair of 06-alkylguanine, a critical lesion in mutagenesis and toxicity caused by alkylating agents, takes place via a pathway involving self-inactivating demethylation by 06-alkylguanine-DNA alkyltransferase (AGT). Hence AGT is important in determining cellular susceptibility to genotoxins acting via attack on position 0 6 of guanine, as exemplified especially by the case of the cross-linking nitrosoureas. As far as susceptibility to methylating agents is concerned, its relationship to AGT content has generally been studied using relatively high doses which result in significant disturbance of the total cellular pool of this suicide enzyme. Recent observations on the kinetics of repair of 06-methylguanine under a wider range of exposure conditions, including exposure to low doses causing minimal disturbance of the AGT pool, suggest a relatively complex relationship between AGT levels and 06-meG repair, possibly related to the existence of kinetically distinct "active" and "backup" enzyme pools in mammalian cells, a suggestion compatible with other recent findings on the DNA binding and intracellular distribution and transport properties of AGT. The implications of these new lines of biochemical and kinetic evidence for the determination of cellular susceptibility to methylating agents will be discussed.
Glial cells play important roles in neuronal survival/death by secreting neurotrophins, cytokines and so on. We found that an inducible type of NO synthase (iNOS) was expressed in glial cells at 3 and 7 days after a transient 4 vessel occiusionJreperfusion (4VO) in male, adult rats. Approximately the same day after 4VO, we observed the appearance of TUNEL staining positive neurons and nuclear fragmentation in neurons by the Hoechst 33258 staining, suggesting the 4VO-induced neuronal apoptosis by iNOSINO derived from glial cells. We also obtained results that bacterial endotoxin (LPS) enhanced INFy-induced iNOS expression in cultured rat glial cells and C6 glioma cells. This enhancement seems to be due to activation and intranuclear translocation of NF-KB. NO generated by LPS-induced glial iNOS caused DNA fragmentation by (apoptosis) in NG108-15 cells. NO donors (sodium nitroprusside) resulted in apoptosis in NG I08-15 cells, inhibited glycenaldehyde-3-phosphate dehydrogenese (GAPDH) activity and increased [32p]_NAD binding/[ 32p] ADP-ribosylation of GAPDH. Interestingly, koningic acid (KA), a selective and potent inhibitor of GAPDH, induced the apoptosis too. Taken together, we presume that NO-elicited disruption of GAPDH, a key enzyme in glycolytic convension and a protein involved in RNA transport, could be involved in neuronal apoptosis.
S2. Disruption of Cell Signalling
IS2/L31
IS2/L1 I
INHIBITION OF THE cAMP SIGNALLING CASCADE VIA THE CANNABINOID RECEPTORS: A PUTATIVE MECHANISM OF IMMUNE MODULATION BY CANNABINOID COMPOUNDS
N.E. Kaminski. Michigan State University. East Lansing, MI, USA Immune modulation by cannabinoids has been widely established over the past three decades. In spite of this, the mechanism of action responsible for immune modulation and other well described biological effects attributed to cannabinoid compounds has been elusive. The identification and cloning of two novel G-protein coupled receptors, CB I and CB2, both of which bind cannabimimetic agents has served as the basis for a putative mechanism of action. CB I, which is also referred to as the central cannabinoid receptor is the primary form expressed within the central nervous system (CNS). Conversely, the peripheral cannabinoid receptor, CB2, does not appear to be expressed with in the CNS but is the predominant form of the receptor expressed within the immune system. Both CB I and CB2 negatively regulate adenylate cyclase activity through a pertussis toxin sensitive GTP-binding protein. Recent investigations addressing the mechanism by which cannabinoids disrupt leukocyte function have demonstrated that in the presence of cannabinoids the cAMP signalling cascade is markedly inhibited as evidenced by decreased adenylate cyclase and protein kinase A activity and decreased DNA binding by cAMP response element binding proteins.
Yasuyuki Nomura. Graduate School of Pharmaceutical Sciences,
EPIGENETIC TOXICOLOGY AS TOXICANT-INDUCED CHANGES IN INTRACELLULAR SIGNALLING LEADING TO ALTERED GAP JUNCTIONAL COMMUNICATION
J.E. Trosko. Michigan State University, Dept. Pediatrics and Human Devel., Natl. Food Safety and Toxicology Center. East Lansing, MI, USA Communication mechanisms [extra-, intra- and gap junctional inter-cellular communication (GJIC)] control, from the fertilized egg, through embryo-genesis to maturity and aging, whether a cell (a) proliferates, (b) differentiates, (c) dies by apoptosis. or (d) if differentiated, adaptively responds to exogenous and endogenous signals. From the egg to the I()() trillion cells in the human body, health is maintained when these communication processes between stem, progenitor and terminally differentiated cells are integrated. Each cell choice involves "epigenetic" mechanisms to alter the expression of genes at the transcriptional, translational or posttranslational levels. Disruption of any of the communication mechanisms can be either adaptive or maladaptive. Modulation of extra-cellular communication, either by genetic imbalances of growth factors, hormones or neuro-transmitters or by environmental, exogenous chemicals can trigger signal transducting intracellular communication mechanisms. These intracellular-cellular signals can modulate gene expression at the transcriptional, translational, or posttranslational levels while also modulating GJIc. Untimely or chronic disruption of GJIC during embryonic or fetal development could lead to embryonic lethality or terato-genesis. By modulation of GJIC, homeostatic control of cell growth, differentiation or apoptosis could lead to specific diseases,