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.