Biochemical SocietyTransactions ( 1 996) 24 €41 EFFECT OF ZINC AND CADMIUM ON THE APOPTOTIC CA'+-DEPENDENTENDONUCLEASE Lohmann R.D. & Kortenkamp A. IVth Medical Dep., Charitk University Hospital, Berlin, Germany The Ca"-mediated DNA fragmentation in mammalian cells is due to a Ca2'/Mg2' dependent endonuclease, which is constitutive in nuclei and not lysosomal. The identity of this enzyme remains still unclear. Dnase I is discussed as a possible candidate (I). For the further characterizatioi. of the endonuclease we carried out experiments with whole nuclear protein extracts. Work started with the isolation of nuclei from rat liver tissue as already described for bovine liver (2), followed by the extraction of nuclear protein (3). The ability of the protein fraction to digest DNA was confmed by measuring the induction of single- and double strand breaks in isolated supercoiled DNA. The number of strand breaks was determined using agarose gel electrophoresis and digital imaging video equipment. The nuclear protein showed a Ca2'-dependent DNA digestion, with 100pM free Ca*' exhibiting an optimum activation. In the presence of 100pM Ca" zinc induced a concentration dependent inhibition of the DNA cleavage. Cadmium also displayed an inhibitory effect on the Ca2+-activated DNA digestion. Surprisingly, this effect was smaller when compared to the inhibition observed with zinc. In whole nuclei, cadmium exhibited the stronger effect (2). Further experiments were c q i e d out to address the question whether zinc or cadmium ions could substitute for Ca" in the activation of the nuclear protein. In the absence of Ca*', there was no DNA digestion with zinc. However, cadmium could substitute for Ca", showing an optimum in the activation of the nuclear protein at around lpM free cadmium. Further increases in the concentration of 6ee cadmium led to decreases in the degree of DNA cleavage. These results compare well with the known ability of cadmium to activate and inhibit DNA fragmentation in whole nuclei (2). Under the same conditions DNase I showed its well documented activation by Ca2', but Cd" failed to exhibit stimulatory effects. These results confirm the Ca2'-dependent endonuclease is not identical with DNase 1. w: m: ( I ) Peitsch,M.C. et al. (1993) EMBO J. 12,371-377 ( 2 ) Lohmann,R.D. and Beyersmann,D. (1993) Biochem. Biophys. Res. Commun. 190, 1097-1103 (3) Caron-Les1ie.L.M. et al. (1991) J. Steorid Biochem. Mol. Biol. 40,661-671 E42 CHROMATIN CONDENSATION DURING APOPTOSIS REQUIRES ATP. George E.N. Kassl. John E. Eriksson', Marianne Weis$, Richard Hinton'. Sten Orrenius: and Sek C. Chow". 'School of Biological Sciences, University of Surrey, Guildford, U.K., "Turku Center for Biotechnology, Turku, Finland, $Division of Toxicology, Karolinska Institutet. Stockholm, Sweden, and lkenter for Mechanisms of Human Toxicity. University of Leicester, U.K. The processes leading to the morpho~ogica~changes of the chromatin in cells undergoing apoptosis are presently unclear. We have recently reported that the nuclear morphological changes and chromatin fragmentation typically seen in apoptosis were reproduced in an in id/ro system comprised of isolated rat thymocyte nuclei incubated in the presence of a lysate from FaslAPO-I-stimulated JURKAT cells (S.C. Chow, M. Weis, G.E.N. Kass, T.H. Holmstrom. J.E. Eriksson and S. Orrenius (1995) FEBS Lett. 364, 134-138). Using this h i vifro system, we show here that the presence of ATP was necessary for chromatin condensation and its movement to the nuclear periphery and apoptotic body formation. In clear contrast, chromatin cleavage into high molecular weight and oligonucleosomal-length DNA fragments induced by lysates derived from FadAPO- 1-activated JURKAT cells did not require the presence of ATP. The induction of these morphological changes by ATP could not be substituted by the analogues, AMP-PCP and AMP-CPP, AMP. cyclic AMP and UTP. However. ATP-y-S, and to a lesser degree GTP and ADP could partially replace ATP in inducing nuclear apoptotic morphological changes. It is concluded that ATP is essential for the morphological changes occurring in nuclei during apoptosis but not for DNA fragmentation. 569s E43 ISOLATION OF APOPTOSIS RELATED GENES IN THE RAT OVARY. BodilHarriet As Thelander sa& rHgkan ss Billig. on, Department of Physiology, University of Goteborg, Medicinaregatan 11, S-413 90 Goteborg. Sweden. The ovary is a complex organ, containing follicles of different developing stages. During follicular development most follicles die, a process which is called atresia, and will never ovulate. The intracellular mechanism of follicular atresia is not fully understood. However, ovarian atresia shows a number of morphological, endocrine and biochemical changes that are observed in apoptosis, and it was recently shown that apoptosis is involved in atresia. As with apoptosis in other tissues. ovarian apoptosis can be regulated by hormones and growth factors. In many tissues cell transcription is required for apoptosis to occur. The objective of the present study was to see if apoptosis in the ovarian follicle is dependent on transcription, and, if so, isolate cell specific genes involved in apoptosis. To do this, ovarian cells (granulosa cells) obtained from gonadotropintreated rats were incubated for 24 h in serum-free medium with or without actinomycin-D. In the absence of actinomycin-D, granulosa cells underwent apoptosis, assayed as increased internucleosomal DNA degradation. In the presence of actinomycin-D, apoptosis was inhibited, suggesting that granulosa cell apoptosis in v i m is transcription-dependent. To isolate apoptosis related genes in the ovary, we used differential display of mRNA. The RNA was obtained from hypophysectomized rats treated with estrogen implants (+DES). Removal of the estrogen support (-DES), resulted in increased ovarian apoptosis. 72 candidate mRNA were expressed in (-DES), and 31 in (+DES)-ovaries. These candidates for apoptosis related genes were partially sequenced and hybridized to RNA from apoptotic and non-apoptotic ovarian cells. After sequencing and hybridization, a large number of these genes were shown to be either duplicates or the genes did not show a detectable hybridization signal. Four genes were selected for further studies. These genes showed a clearcut increase in mRNA expression in apoptotic ovarian cells. We are currently isolating the fullength cDNA to study their function. ' E44 APOPTOSIS IN RAT GRANULOSA CELLS ; DEPENDENT ON DIFFERENTIATION AND CELL CULTURE DENSITY. Fva Svensson, Matilda Utter, Harriet Thelander and Hlkan Billig. Department of Physiology, Div. of Endocrinology, Medicinaregatan 11, S-413 90 Goteborg, Sweden In the human ovary, millions of follicles with oocytes are present at birth. Of these, only about 400 will ovulate during female reproductive life. The rest (>99%) will undergo a degenerative process called atresia. The mechanism responsiblefor atresia of ovarian follicular cells (granulosa cells) in rats has been shown to be apoptosis. Isolated rat granulosa cells undergo spontaneousapoptosis in vifro. Many factors regulating ovarian apoptosis in vivo and in vitro have been identified, such as gonadotropins, steroid hormones and growth factors. Using analysis of DNA-fragmentation characteristical for apoptosis (internucleosomal DNA-degradation), we demonstrate that apoptosis in rat granulosa cells in vitro is dependent on cell density as well as the stage of differentiation in vivo prior to cell isolation. High cell density in culture and low cellular differentiation of the granulosa cells in vivo correspondto a higher degree of apoptosis. We propose that free radicals may be responsible for part of the cell culture density-dependent apoptosis development in vitro because the degree of apoptosis is inhibited by addition of scavengers of free radicals such as superoxidedismutase and 1,lo-phenantroline. The mechanisms involved in ovarian differentiation and apoptosis are under further investigation.
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