Rodent embryo cells immortalized with temperature-sensitive mutants of simian virus 40 large tumor (T) antigen have a proliferative potential that depends on temperature. At the restrictive temperature, heat-inactivation oflarge T antigen causes p53 release, growth arrest, and cell death.Morphological and molecular analysis indicate that the induced cell death corresponds to apoptosis. Flow cytometric analysis using a combination of forward light scatter and side scatter allows a discrimination of cells committed to apoptosis within the whole population. These cells display a reduction in cell size and a higher cellular density, confirming the apoptotic nature of the cell death. When cells exhibiting the morphological features of apoptosis were stained with a fluorescent probe of the mitochondrial membrane potential, a decreased accumulation of the dye was recorded. Measures of cellular respiration, performed with whole-cell populations, showed that the lower mitochondrial membrane potential (At,) correlates, as expected, with an uncoupling of electron transport from ATP production and is linked to the induction of apoptosis. We also show that this decrease in A'I is associated with a decrease in the rate ofmitochondrial translation. These events are detected at early stages of the apoptotic process, when most of the cells are not irreversibly committed to death, suggesting that mitochondria could be a primary target during apoptosis.Mammalian cells grown in culture exhibit a finite life-span for proliferation. After a variable number of divisions they stop dividing, undergo a variety of changes, and finally die. Some oncogenes have the ability to confer an unlimited proliferative potential (immortality) to primary cells in culture. In the case of polyomavirus-induced immortalization of rodent embryo fibroblasts, it has been shown that the unlimited proliferative capacity is maintained by the large tumor (T) antigen (1)(2)(3)(4) MATERIALS AND METHODSCell Lines and Cell Culture. The REtsAF and RELPB cell lines were isolated at low cell density from a rat embryo fibroblast culture infected with SV40 (2). REtsAF was obtained by using a temperature-sensitive tsA58 mutant and is temperature sensitive for immortalization, whereas RELPB was obtained with wild-type SV40 and is immortal at both 330 and 39.50C. REtsAF-Revl was derived from REtsAF by selection for proliferation at 39.50C (8

Apoptosis, the process whereby cells activate an intrinsic death program, can be induced in HeLa cells by TNF-a treatment. The aims of the present study were (i) to examine the precise role and the origin of Reactive Oxygen Species (ROS) in the TNF-a-induced programmed cell death, (ii) to characterize and order the morphological and mitochondrial changes associated with this process and (iii) to link these events with the activation of caspases. Analyses were performed on TNF-a-treated cells in the presence of an anti-oxidant, or of a general caspase inhibitor. To assess the role of mitochondria in the cell death signal transduction, these studies were also realized on HeLa-variant cell lines lacking functional mitochondrial respiratory chain. We show that at least two separate signaling cascades, both mediated by Z-VAD-sensitive caspase(s), contribute to the TNF-a-induced apoptosis of HeLa cells. One signaling pathway involves an early mitochondriadependent ROS production, the other being ROSindependent.

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In a previous work, we have described the tryptic cleavage of yeast flavocytochrome b, into its two functional domains: a cytochrome h, core and a flavodehydrogenase. The lactate dehydrogenase efficiency of the latter was, however, dramatically low, only about 1 :< that of intact flavocytochrome b,. Our present study concerns a new flavodehydrogenase derivative of Hansenula anomala flavocytochrome 6 , which spontaneously dissociates from the cytochrome domain when the polypeptide bridge connecting thein is cleaved by Staphylococcus aureus V 8 protease I. This flavodehydrogenase was purified and some of its functional and structural properties were studied. It presents an exceptionally high lactate dehydrogenase activity, about 80 "/, that of flavocytochrome b,. This result clearly demonstrates that the cytochrome domain is not necessary for the lactate dehydrogenase function and suggests an autonomous folding for both domains.Our results are discussed in terms of 'gene fusion'.Each chain of yeast flavocytochrome b, has been shown to be folded into three globules: n, E and / I ' joined together in the 'intact' enzyme by two protease-sensitive bridges aa' and cd [I -41 (cf. These two functional domains, flavodehydrogenase and cytochrome, which spontaneously dissociate from each other when their connecting bridge cd is cleaved by trypsin, were isolated as stable, pure, molecular entities, termed FDH, and nT respectively [2, 3, 91. Unfortunately, this polypeptide bridge cd is not the part of the native chain which is most sensitive towards trypsin: the aa' zone is cleaved about 20-times faster [lo], so that FDHT was always found altered by this undesirable inner cleavage, which was achieved with a loss of about 20 amino acid residues [3].From a functional point of view, the first cleavage in aa' results in a 96 %loss of activity [I I]; the isolated 'nicked' FDH, entity itself only retains about 1 % of the ferricyanide reductase activity of native flavocytochrome b2 [lo]. These results did not elucidate whether such a low activity was due to the aa' cleavdge itself or to the release of cytochrome b2 core resulting from the second cleavage at cd.This study presents information concerning this question. Despite the hypersensitivity of the aa' zone towards a number of proteases [I2 -141, we succeeded in obtaining a cleavage in the cd zone faster than in the aa' zone. This was achieved with Hansenula anomala flavocytochrome b2 and the highly specific Staphylococcus aureus V 8 protease 1. We describe a method for separating the so-formed 'intact' flavodehydrogenase part which we currently term 'FDH,,', from the other proteolysis products. Steady-state catalytic parameters of this new flavocytochrome h, derivative have been determined and compared with those of the native flavocytochrome b, and its tryptic derivatives. MATERIALS A N D METHODS Hansenula anomala Flavocytochrome b,This was prepared according to [I51 with the minor modifications described in [I I]. It was stored as an ammonium sulfate precipitate (50 % satur...