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metaphase and induced endoreduplication and apoptosis. Normal human fibroblasts, a human lung carcinoma cell line (A549) and its derivative cell line which ...
Biochemical SocietyTransactions(1 996) 24 533s C41 ROTENONE INDUCED MITOSIS,

ENDOREDUPLICATION A N D APOPTOSIS ARE NOT MEDIATED VIA MITOCHONDRUL RESPIRATORY CHAIN INHIBITION. M. Gu,A.H.V. Schapira and J.M. Cooper Department of Clinical Neurosciences, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF. Exposure of cells to the mitochondrial complex I inhibitor rotenone (1.0 pM), caused an increase in the proportion of cells in metaphase and induced endoreduplication and apoptosis. Normal human fibroblasts, a human lung carcinoma cell line (A549) and its derivative cell line which lacked mitochondrial DNA (A549po) were equally sensitive to the actions of rotenone indicating these effects are neither cell specific nor the result of rotenone acting via the mitochondrial respiratory chain. Prolonged incubation with rotenone resulted in a decrease in the number of mitotic cells while the number of endoreduplicated and apoptotic cells increased. The rotenone induced effects on the cell cycle did not involve disruption of microtubule formation. The levels of bcl-2 were markedly increased after rotenone treatment, but the bcl-2 was restricted to cells in metaphase and correlatedwith the increased number of mitotic cells. Other mitochondrial respiratory chain inhibitors (piericidin A and antimycin A) did not cause endoreduplication or increase the number of cells in metaphase but did induce apoptosis which was more pronounced in A549 than in A549p" cells. These results suggested that although the mitochondrial respiratory chain inhibitors piericidin A and antimycin A induce apoptosis via a mechanism that involves inhibition of the mitochondrial respiratory chain the predominant toxic effects of rotenone are mediated via a pathway independent of the mitochondrial respiratory chain possibly involving cytoskeletal changes.

C42 PHOTOSENSITIZATION

INDUCES APOPTOTIC OR NECROTIC-LIKE CELL DEATH DEPENDING ON INTRACELLULAR PHOTOSENSITIZER (PHOTOFRINa) DISTRIBUTION

Marc Dellinger, Laboratoire d e biophysique et Laboratoire de Photobiologie, M u h m National dHistoire Naturelle, INSERM U201, CNRS URA 481,43, rue Cuvier, F-75231 Paris Cedex OS, FRANCE. Photofin@, a complex mixture of porphyrins, has been used to eradicate superficial tumors upon red light activation. Porphyrin photosensitization has been shown to produce singlet oxygen leading to the impairment of cellular functions such as respiration, calcium homeostasis, or plasma membrane transport. Apoptosis and necrosis have both been reported, depending on the cell type. However, as Photofrin contains various porphyrins that incorporate differently into cells, the modulation of intracellular porphyrin localization by using different incubation protocols may influence the cell death process. In this study, CV-1 cells were treated first with a low porphyrin concentration during one day to favor intracellularmembrane loading, then illuminated to induce cell death. The cells underwent typical apoptotic morphological changes: retraction from the substrate, rounding-up and fractionation in vesicles. The nucleus was fragmented and DNA degraded into oligonucleosomic sizes, which are also key features of apoptosis. In a second type of experiment, CV-1 cells were treated with a high dose of Photofrin in saline medium during only one hour to increase plasma membrane loading, then illuminated to induce cell death. The cells underwent the same early morphological changes (retraction and rounding-up), but finally lysed instead of fragmented. Moreover, neither nucleus fragmentation, nor DNA degradation in oligonucleosomicsizes were observed.This type of cell death appeared to be necrotic, although it could also be interpreted as apoptosis arrested by membrane leakage (stopped-apoptosis). As the porphyrin accumulation in vivo is closer to the one obtained by using the first protocol, it is suggested that Photofrin photosensitization mainly induce apoptosis. It is also suggested that plasma membrane damage may prevent the appearance of the characteistics of apoptosis by stopping the process at an early stage.

C43 PREVENTION OF HYDROGEN PEROXIDE- AND CISPLATIN-INDUCED APOPTOSIS BY CATALASE OVEREXPRESSION Haluk B. OralLb,Andrew J.T. George' and Dorian 0. Haskardb 'Department of Immunology and bCardiovascular Medicine, University of London, Royal Postgraduate Medical School, London W12 0 " . U.K.

Programmed Cell Death (PCD) induced by H2@ was investigated in Chinese Hamster Ovarian Cells (CHO), and compared to PCD i n d u d by Cisplatin (25-50 pM), which is a well-known apoptosis- inducing agent.

In p r e l i experiments, CHO cells were treated with low concentrations of Hz02 (250-500 pM) continuously or for 3 hours followed by washing and further incubation in complete growtb medium for 1- 10 days. Fluorescence microscopy of &dine orangestained cells indicated morphological changes of PCD, including membrane blebbing, nuclear condensation and hgmentation. Similar changes were observed in cisplatin-treated cells. DNA laddering also revealed that DNA hgmemtation began to occur in both adherent and non-adherent cells by day 2. To prevent H202-mediated PCD, the catalase gene was isolated and ligated into a mammalian expression vector @CDMS). Transiently lransfected cells contained approximately 17-fold increased catalase levels, as measured by ELISA. Overexpressionof intracelldar catalase provided protection of the CHO cells from H2@ as measured by chromium-51 release and MTS assays. Furthermore, Cisplatin-mediated PCD was prevented by intracelldar catalase overexpression. The protective effect of catalase overexpression on PCD induced by other agents is being investigated further.

c44 ADP-RIBOSYLATIONS IN OXIDATIVE STRESSINDUCED APOPTOSIS SAumha. C. Colussi, C. Albeaini? S. Rovidati? F. Canestrari', .. L . Ghibelli Dipartimenro di Biologia, Universird di Roma Tor Vergara *Isriruro di Biochim'ca G. Fomini, Universird di Vrbino Cell reactions to oxidative stress, which may lead to the induction of apoptosis, involve post-uanslational modifications of proteins by ADP-ribosylation. Indeed, the nuclear enzyme poly(ADPRib0Syl)Polymerase(PARF'), activated by stress-induced DNA breaks, may have a role in apoptosis, since PAW inhibitors modulate the extent of apoptosis induced by oxidative stress [l, 2). In some systems, the inhibition of ADP-ribosylation events with high doses of the inhibitor 3-Aminobenzamide (3ABA) leads to cellular fragmentation upon oxidative stress, due to the promotion of a strong cell blebbing: this is possibly due to the inhibition of some cytoskeletal-related mono-ADP-ribosylation events [3]. This leads to postulate that some mono-ADP-ribosylations may be part of a mechanism of self-protection lhat the stressed cells activate in order to protect themselves from the oxidative damage-induced suicide pathway. ADP-ribosylated proteins are usually inactive: thus, the self-protective mechanism could conceivably be a reversible "stand by". We are now looking for possible targets of mono-ADP-ribosylations which may be involved in protecting from stress-inducedapoptosis; in particular we have evidences that stressed cells may actively block, via ADPribosylation,their metabolism as a part of a self-defensiveaction. Experimentally induced thermotolerance and oxytolerancehave allowed to discover that cells are able to build self-protective reactions to stress. We have evidences that these cell reactions protect cells not only from hyperthermic or oxidative stress, but also from many types of unrelated apoptogenic agents, possibly via ADP-ribosylation events. [I] C. Nosseri, S. Coppola, L. Ghibelli., 1994, Exp. Cell Res. 212, 367-373 [21 S.Coppola, C. Nosseri, V. Maresca,L. Ghibelli, 1995, Exp. Cell Res. 221.46249 [31 L. Ghibelli, C. Nosseri, S. Coppola, V. Maresca. L. Dini. 1995, Exp. Cell Res. 221. 470-477