- Email: [email protected]
Inhibition by metallothionein of doxorubicin toxicity in cultured mouse cardiomyocytes
98
I 99
I
PROTEASOMAL HISTONE DEGRADATION CELLS AFTER OXIDATIVE STRESS
IN K562
POLY(ADP-RlBOSYL)TRANSFERASE INTERACTS WITH THE 20s PROTEASOME COMPLEX IN VITRO
Oliver Ullrich (I) and Tilman Gnme (2) (1)Instituteof Anatomyand (2) Clinics of Physical Medicine. Medical
Philipp Mayer-Kuckuk (I), Oliver Ulhich (2). Tilman Gnme (3) and Manfred Schweiger (I )
Faculty (CharilC). Hunlboldt-University Berlin. Germany
(I) Institute of Biochemistry, Free University Berlin. (2) Institute of
The nuclear production of oxygen free radicals has been shown to be involved in the cytotoxic action of many antitumor drugs, whereas antioxidative strategies of tumor ceils are supposed to be involved in chemotherapy resistance. Oxidative damaged
histones, particulary HI, are highly susceptible to proteolytic degradation by the 20s proteasome, a 700 kDa cytosolic and nuclear proteinase complex Hydrogen peroxide treatment of K562 cells increases the proteasomal proteolytic activities in isolated nuclei, which is accompanied by and dependent of poly(ADP-ribosyl)ation reactions. Inhibitor experiments, 14Cincorporation assays, immunoblotting and in vitro reconstitution experiments suggest a proteasome-poly(ADP-ribose)-interaction. Poly(ADP-ribosyl)ated native histones are highly resistant towards proteasomal degradation, whereas oxidatively damaged histones are not poly(ADP-ribosyl)ated and therefore are not protected from degradation. Furthermore, the enhanced degradation of metabolically radiolabeled endogenous histones after oxidative stress appears to be proteasome- and poly(ADPribosyl)ation-dependent. We therefore conclude, that the selective degradation of damaged histones after oxidative stress might be regulated by poly(ADP-ribosyl)ation reactions.
Anatomy and (3) Clinics of Physical Medicine, Medical Faculty (Charit@. Humboldt-University Berlin. Germany
poly(ADP-ribosyl)transferase Human nuclear enzyme (pADPRT, E.C. 2.4.2.30) is activated by reactive oxygen species (ROS) via binding on DNA-single strand breaks and catalyses the synthesis of poly(ADP-ribose) from NAD’ and the modification of nuclear proteins and of itself by poly(ADPribose) chains. Whereas no covalent modification of the 20s proteasome could be detected, non-covalent interaction of poly(ADP-ribosyl)ated pADPRT with the isolated 20s proteasome contributes to enhanced sue-LLVY-MCA degradation and an electrophoretic shift in non-denaturating PAGE. There is no interaction between non-modified pADPRT or oligo(ADP-ribosyl)ated pADPRT with the proteasome, but a strong interaction with isolated poly(ADP-ribose) chains, Furthermore the 20s proteasome binds more tightly on poly(ADP-ribosyl)ated pADPRT-Sepharose than on pADPRTSepharose and dissociates with increasing salt concentrations. We therefore conclude that the 20s proteasome is able to interact with poly(ADP-ribosyl)ated pADPRT under oxidative stress conditions.
INHIBITION BY METALLOTHlONElN OF DOXORUBlClb TOXICITY IN CULTURED MOUSE CARDIOMYOCYTES
INHIBITION OF COPPER DEFICIENCY-INDUCED HEART HYPERTROPHY BY METALLOTHIONEIN IN MOUSE
Guana-Wu Wane and Y. James Kang, Department of Medicine, University of Louisville, Louisville, KY 40292
m-Yun WU. Jack T. Saati r& Y. Jm Department of Medicine, University of Louisville, Louisville, KY’40292, and The U.S. Department of Agriculture, Human Nutrition Research Center, Grand Forks, ND 58203
Results obtained in vivo regarding whether metallothionein (MT) protects the heart from oxidative injury by doxorubicin (DOX) were controversial. To define unequivocally the role of MT in this cardioprotection, a primary culture of neonatal mouse heart cells was established. Ventricular cardiomyocytes isolated from lto 3-day neonatal transgenic mice and nontransgenic controls were cultured for 6 days. MT concentrations in these cultured transgenic cardiomyocytes were about 2 fold higher than in the nontransgenic cells. Cell morphology, GSH content and antioxidant enzymatic activities between these two types of cardiomyocytes were not significantly different. DOX was added directly into the cultures at different concentrations for a varying time course. As compared to nontransgenic controls, transgenic cardiomyocytes displayed a significant (pcO.05) resistance to DOX cytotoxicity, as measured by morphological alterations, cell viability (MTT), and lactate dehydrogenase (LDH) leakage from the cells. This cytoprotective effect of MT correlated with its inhibition of DOX-induced lipid peroxidation. The results thus demonstrate unequivocally that elevation of MT concentrations in the cardiomyocytes 2 fold higher than normal provides efficient protection from DOX-induced oxidative injury. Supported in part by a NIH grant CA68 125, and an El Award from the American Heart Association #9640091N.
OXYGEN
Dietary copper restriction causes heart hypertrophy in rodents. Several studies have indicated that this cardiomyopathy is mediated by oxidative stress. Metallothionein (MT), a low molecular weight and cysteine rich protein, has been shown to protect the heart from oxidative injury. It is therefore hypothesized that MT inhibits copper deficiency-induced heart hypertrophy. To test this, a specific cardiac MT overexpressing transgenic mouse model was used. Dams of both transgenic pups and non-transgenic litter-mates were fed copper deficient diet starting on the fourth day post delivery and the weaned mice were continued on the same diet until they were sacrificed. Heart hypertrophy developed in these animals by the fourth week on the copper deficient diet and aggressively progressed until the end of the experiment (6 wks). MT overexpression did not prevent the occurrence of heart hypertrophy, but inhibited the progress of this cardiomyopathy, which correlated with the suppression by MT of the progression of cardiac lipid peroxidation. MT concentrations in the tmnsgenic hearts were about 20-fold higher than that in the non-transgenic littermates. Other antioxidant systems were not different between the two substrains. The results suggest that MT indeed suppresses the development of copper deficiency-induced heart hypertrophy, which is at least partially mediated by oxidative stress. Supported in part by a USDA grant 960453 1.
’ 9
8
s41
I 99
I
PROTEASOMAL HISTONE DEGRADATION CELLS AFTER OXIDATIVE STRESS
IN K562
POLY(ADP-RlBOSYL)TRANSFERASE INTERACTS WITH THE 20s PROTEASOME COMPLEX IN VITRO
Oliver Ullrich (I) and Tilman Gnme (2) (1)Instituteof Anatomyand (2) Clinics of Physical Medicine. Medical
Philipp Mayer-Kuckuk (I), Oliver Ulhich (2). Tilman Gnme (3) and Manfred Schweiger (I )
Faculty (CharilC). Hunlboldt-University Berlin. Germany
(I) Institute of Biochemistry, Free University Berlin. (2) Institute of
The nuclear production of oxygen free radicals has been shown to be involved in the cytotoxic action of many antitumor drugs, whereas antioxidative strategies of tumor ceils are supposed to be involved in chemotherapy resistance. Oxidative damaged
histones, particulary HI, are highly susceptible to proteolytic degradation by the 20s proteasome, a 700 kDa cytosolic and nuclear proteinase complex Hydrogen peroxide treatment of K562 cells increases the proteasomal proteolytic activities in isolated nuclei, which is accompanied by and dependent of poly(ADP-ribosyl)ation reactions. Inhibitor experiments, 14Cincorporation assays, immunoblotting and in vitro reconstitution experiments suggest a proteasome-poly(ADP-ribose)-interaction. Poly(ADP-ribosyl)ated native histones are highly resistant towards proteasomal degradation, whereas oxidatively damaged histones are not poly(ADP-ribosyl)ated and therefore are not protected from degradation. Furthermore, the enhanced degradation of metabolically radiolabeled endogenous histones after oxidative stress appears to be proteasome- and poly(ADPribosyl)ation-dependent. We therefore conclude, that the selective degradation of damaged histones after oxidative stress might be regulated by poly(ADP-ribosyl)ation reactions.
Anatomy and (3) Clinics of Physical Medicine, Medical Faculty (Charit@. Humboldt-University Berlin. Germany
poly(ADP-ribosyl)transferase Human nuclear enzyme (pADPRT, E.C. 2.4.2.30) is activated by reactive oxygen species (ROS) via binding on DNA-single strand breaks and catalyses the synthesis of poly(ADP-ribose) from NAD’ and the modification of nuclear proteins and of itself by poly(ADPribose) chains. Whereas no covalent modification of the 20s proteasome could be detected, non-covalent interaction of poly(ADP-ribosyl)ated pADPRT with the isolated 20s proteasome contributes to enhanced sue-LLVY-MCA degradation and an electrophoretic shift in non-denaturating PAGE. There is no interaction between non-modified pADPRT or oligo(ADP-ribosyl)ated pADPRT with the proteasome, but a strong interaction with isolated poly(ADP-ribose) chains, Furthermore the 20s proteasome binds more tightly on poly(ADP-ribosyl)ated pADPRT-Sepharose than on pADPRTSepharose and dissociates with increasing salt concentrations. We therefore conclude that the 20s proteasome is able to interact with poly(ADP-ribosyl)ated pADPRT under oxidative stress conditions.
INHIBITION BY METALLOTHlONElN OF DOXORUBlClb TOXICITY IN CULTURED MOUSE CARDIOMYOCYTES
INHIBITION OF COPPER DEFICIENCY-INDUCED HEART HYPERTROPHY BY METALLOTHIONEIN IN MOUSE
Guana-Wu Wane and Y. James Kang, Department of Medicine, University of Louisville, Louisville, KY 40292
m-Yun WU. Jack T. Saati r& Y. Jm Department of Medicine, University of Louisville, Louisville, KY’40292, and The U.S. Department of Agriculture, Human Nutrition Research Center, Grand Forks, ND 58203
Results obtained in vivo regarding whether metallothionein (MT) protects the heart from oxidative injury by doxorubicin (DOX) were controversial. To define unequivocally the role of MT in this cardioprotection, a primary culture of neonatal mouse heart cells was established. Ventricular cardiomyocytes isolated from lto 3-day neonatal transgenic mice and nontransgenic controls were cultured for 6 days. MT concentrations in these cultured transgenic cardiomyocytes were about 2 fold higher than in the nontransgenic cells. Cell morphology, GSH content and antioxidant enzymatic activities between these two types of cardiomyocytes were not significantly different. DOX was added directly into the cultures at different concentrations for a varying time course. As compared to nontransgenic controls, transgenic cardiomyocytes displayed a significant (pcO.05) resistance to DOX cytotoxicity, as measured by morphological alterations, cell viability (MTT), and lactate dehydrogenase (LDH) leakage from the cells. This cytoprotective effect of MT correlated with its inhibition of DOX-induced lipid peroxidation. The results thus demonstrate unequivocally that elevation of MT concentrations in the cardiomyocytes 2 fold higher than normal provides efficient protection from DOX-induced oxidative injury. Supported in part by a NIH grant CA68 125, and an El Award from the American Heart Association #9640091N.
OXYGEN
Dietary copper restriction causes heart hypertrophy in rodents. Several studies have indicated that this cardiomyopathy is mediated by oxidative stress. Metallothionein (MT), a low molecular weight and cysteine rich protein, has been shown to protect the heart from oxidative injury. It is therefore hypothesized that MT inhibits copper deficiency-induced heart hypertrophy. To test this, a specific cardiac MT overexpressing transgenic mouse model was used. Dams of both transgenic pups and non-transgenic litter-mates were fed copper deficient diet starting on the fourth day post delivery and the weaned mice were continued on the same diet until they were sacrificed. Heart hypertrophy developed in these animals by the fourth week on the copper deficient diet and aggressively progressed until the end of the experiment (6 wks). MT overexpression did not prevent the occurrence of heart hypertrophy, but inhibited the progress of this cardiomyopathy, which correlated with the suppression by MT of the progression of cardiac lipid peroxidation. MT concentrations in the tmnsgenic hearts were about 20-fold higher than that in the non-transgenic littermates. Other antioxidant systems were not different between the two substrains. The results suggest that MT indeed suppresses the development of copper deficiency-induced heart hypertrophy, which is at least partially mediated by oxidative stress. Supported in part by a USDA grant 960453 1.
’ 9
8
s41