Manganese superoxide dismutase (MnSOD) protects against mitochondrial-mediated programmed cell death

256 El METALLOTHIONEIN INHIBITS DOXORUBICIN-INDUCED APOPTOSIS IN MOUSE HEART a-Wu Wang Department of Medicine, University of Louisville, Louisville, Kk 40292 Metallothionein (MT), a low molecular weight and cysteinerich protein, has been shown to protect the heart from oxidative injury by doxorubicin (DOX). To investigate possible mechanisms of this protection, the effect of MT on DOX-induced apoptosis in the heart was determined. Seven-week-old transgenic mice overexpressing MT specifically in the heart about IO-fold higher than normal, along with their non-transgenic littermates, were treated with DOX by iv. injection at 20 mg/kg. Twelve hours after the treatment, the hearts were isolated for analysis of apoptosis by a TUNEL assay, which was further confirmed by an electron microscopic examination. Results showed that DOX induced apoptosis in the mouse heart and MT overexpression markedly suppressed the effect of this drug. Furthermore, a neonatal mouse cardiomyocyte culture was successfully established. The cultured transgenic cardiomyocytes contained higher concentrations of MT than the non-transgenic cells. Other antioxidant activities were comparable between the two cell types. Studies using this cell culture system revealed that preceding the programmed cell death, a delta isoform protein kinase C (PKC,) was activated by DOX. MT inhibited this activation, an event that was likely involved in the MT inhibition of the drug-induced apoptosis. Supported in part by a NIH grant CA68125, and an American Heart Association EI grant (9640091N).

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MANGANESE SUPEROXIDE DISMIJTASE (MnSOD) PROTECTS AGAINST MITOCHONDRIAL-MEDIATED PROGRAMMED CELL DEATH Terry D. 0berley2, and Daret K. St. Cl&* v’, ‘%mduate Center for Toxicology, Univ. of Kentucky and 2Department of Pathology, VA Hospital, Univ. of Wisconsin, Madison, WI. Mitochondria have recently been shown to serve a central role in programmed (apoptotic) cell death. In addition, reactive oxygen species (ROS) have been implicated in cell death pathways upon treatment with a variety of agents. We hypothesize that mitochondria-derived free radicals play a critical role in apoptotic cell death. To directly test this hypothesis, we treated a murine fibrosarcoma (FSa) cell line with a series of respiratory chain inhibitors. Apoptotic cell death was confumed by DNA fragmentation analysis as well as electron microscopy. We found that overexpression of mitochondrial MnSOD specifically protected against cell death by inhibition of complexes I and III. Furthermore. we examined tbe role of bcl-xL, ~53 and poly (ADP-ribose) polymerase (PARP) to identify which pathway might contribute to the ROS-mediated cell death. Cells overexpressing MoSOD were found to have a decrease in the redox sensitive transcription factor AP-1 and its anti-apoptotic target gene, bcl-xL. Therefore, the bcl-XL-linked pathway could be excluded. p53 protein was non-detectable by Western analysis and examination of the pro-apoptotic protein bax, a ~53 target gene, did not increase with treatment. Examination of ICE(interleukin- 1B converting enzyme) family members revealedspecific activation of caspase 3 (CPP-32) in the NE0 control cells independent of cytochrome c release from the mitochondria. PARP, a target protein of CPP-32 activity, was cleaved to a 64 kD fragment in the NE0 cells. Taken together, these findings suggest that mitcchondtid-mediated ROS generation is a key event by which inhibition of mitochondrial respiration causes cell death and identifies CPP32 and the PARP-linked apoptotic pathway as targets of mitochondrialderived ROS-induced apoptotic cell death. (Supported by NIH grants CA 49797, CA 29142, HL 03544. *DKS is the recipient of a NIH Independent Screntist Award).

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INHIBITION OF APOF’IOSIS BY HYPEROXIA S. MpnteE Akn Iin L Mnnr4 Lad S~h&fj+$y$;~~o~ M. Fin, Stwrl Hmwitz. Ventilation with high concentrations of oxygen is a necessary, life-sustaining therapy. However, hyperoxia generates toxic levels of reactive oxygen species, which are themselves cytotoxic, trl ering cell death. Pmviously, we showed that hyperoxia infiguced cell death via necrosis in human lung adenocarcinoma AS49 cells. In contrast, lethal concentrations of hydrogen peroxide caused apoptosis. To teat whether some steps of apoptosis are sensitive to molecular 02 AS49 cells were preexposed to hyperoxia prior to the treatment of hydrogen peroxide. The extent of a ptosis was reduced in the hyperoxia-treated cells re!ztive to the room-air controls. Using mutant epithelial cells that do not suffer from 02 toddty, we further demonstrate that thii inhibition of apoptosis by hyperoxia is not caused by 02 toxicity. Interestingly, inhibition of oxidant induced apoptosis by hyperoxia was also observed when paraquat (the intraceklar superoxide generator)was employed to induce cell death.

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HP Mon$iol, J Pereid, NB Melo’. Vhl Yoshida3,LCB Ch’eira’, A Stem2 Hemocsntro s. Fwo, s. Paub, Brazil; 2. l.Fu-da@oP16&1@~ DeptcfPhannacdogy,NYUM~kalCenter,Ne’.4Yo&USk3. hstiuo do Cua@o HCFMUSP, S.FWo, Brazil.

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