Saccharomyces cerevisiae contains two structural genes, ACS1 and ACS2, each encoding an active acetylcoenzyme A synthetase. Characterization of enzyme activities in cell-free extracts from strains expressing either of the two genes revealed differences in the catalytic properties of the two enzymes. The K m for acetate of Acs1p was about 30-fold lower than that of Acs2p and Acs1p, but not Acs2p, could use propionate as a substrate. Enzyme activity measurements and mRNA analyses showed that ACS1 and ACS2 were both expressed during carbon-limited growth on glucose, ethanol, and acetate in aerobic chemostat cultures. In anaerobic glucose-limited cultures, only the ACS2 gene was expressed. Based on these facts, the products of the ACS1 and ACS2 genes were identified as the previously described "aerobic" and "non-aerobic" forms of acetylcoenzyme A synthetase, respectively. Batch and glucosepulse experiments revealed that transcription of ACS1 is subject to glucose repression. A mutant strain lacking Acs2p was unable to grow on glucose in batch cultures, but grew readily in aerobic glucose-limited chemostat cultures, in which the low residual glucose concentration alleviated glucose repression. Experiments in which ethanol was pulsed to aerobic ethanol-limited chemostat cultures indicated that, in addition to glucose, ethanol also repressed ACS1 transcription, although to a lesser extent. In contrast, transcription of ACS2 was slightly induced by ethanol and glucose. Absence of ACS2 prevented complete glucose repression of ACS1, indicating that ACS2 (in)directly is involved in the transcriptional regulation of ACS1.When Saccharomyces cerevisiae grows on acetate or ethanol, ATP-dependent activation of acetate to acetyl-coenzyme A is catalyzed by acetyl-coenzyme A synthetase (EC 6.2.1.1). In addition to serving as the fuel for the citric acid cycle, acetylcoenzyme A is an essential building block for the synthesis of lipids and some amino acids. During growth on glucose, direct formation of acetyl-coenzyme A from pyruvate is catalyzed by the mitochondrial pyruvate dehydrogenase complex. Alternatively, conversion of pyruvate into acetyl-coenzyme A can be accomplished by the concerted action of the enzymes of the pyruvate dehydrogenase bypass: pyruvate decarboxylase, acetaldehyde dehydrogenase, and acetyl-coenzyme A synthetase (1, 2). Recent work has indicated that the latter pathway is essential for growth, probably for the provision of cytosolic acetylcoenzyme A required for lipid synthesis (3, 4).In S. cerevisiae, a completely respiratory sugar metabolism is only observed at relatively low growth rates in aerobic, sugarlimited cultures (e.g. chemostat cultures). Upon exposure of such cultures to high sugar concentrations, metabolism becomes respirofermentative and pyruvate metabolism occurs predominantly via pyruvate decarboxylase (5, 6). Under such conditions acetate is formed as a by-product, indicating that the in vivo activity of acetaldehyde dehydrogenase exceeds that of acetyl-coenzyme A synthetase. Acetate produ...

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Summary We examined the sensitivity for cisplatin-induced apoptosis in a panel of four testicular germ cell tumour (TGCT) cell lines and monitored the cellular expression of the apoptosis-related proteins p53, Bcl-2 and Bax. Three of four TGCT cell lines (NT2, NCCIT and S2) were hypersensitive for cisplatin-induced apoptosis, while the TGCT cell line 2102 EP appeared to be resistant for cisplatin-induced apoptosis, even at relatively high drug concentrations (12.5 IIM). For all four cell lines, the induction of apoptosis by cisplatin correlated with drug sensitivity in the MTT assay. The differences in chemosensitivity and induction of apoptosis could not be attributed to differences in cellular platinum accumulation, DNA platination or platinum-DNA adduct removal. We next analysed the relationship between p53 status and cisplatin-induced up-regulation of p53, and the susceptibility to cisplatin-induced apoptosis. Wild-type p53 containing NT2 and 2102 EP cells showed p53 up-regulation upon drug treatment, and NCCIT (mutant p53) and S2 (no p53 protein) cells did not. Consistently, the increase in wild-type p53 protein in NT2 and 2102 EP cells led to an increase in mRNA level of the p53 downstream gene p21/WA/F/C/P, whereas mutant p53-containing NCCIT cells and p53-non-expressing S2 cells could not transactivate this p53-responsive gene. As NT2, NCCIT and S2 were readily triggered into apoptosis, while 2102 EP cells failed to undergo cisplatin-induced apoptosis, our data suggest that the presence of wildtype and/or transactivation-competent p53 might not be an absolute prerequisite for efficient induction of apoptosis in TGCT cell lines. Also endogenous levels of Bcl-2 and Bax expression did not correlate with cisplatin-induced apoptosis. In addition, the endogenous Bcl-2 and Bax expression was not affected by cisplatin treatment. The present study suggests that, at least in our panel of TGCT cell lines, hypersensitivity for cisplatin-induced apoptosis might not be necessarily correlated with the presence of wild-type p53 and is probably not associated with Bcl-2 and Bax expression.

Expression of the apoptosis‐associated proteins Bcl‐2 and Bax was quantitated by flow cytometry (FCM) in chemosensitive testicular germ‐cell tumor NT2 cells, and the results were compared with those obtained by Western blotting. NT2 cells were incubated with cisplatin (3.1 μM for 2 h at 37°C), and 24, 48, and 72 h later were analyzed for induction of apoptosis, and for modulation of the expression of cell death suppressing protein Bcl‐2, as well as cell death promoting protein Bax. Apoptosis was quantitated by binding of annexin V conjugated with fluorescein isothiocyanate (FITC) to the cell membrane. Cisplatin‐treatment induced apoptosis in NT2 cells. The apoptotic cell population increased in time, and at t = 72 h after drug incubation, about 90% of cells that were present in the cell culture were apoptotic. Subsequently, we determined the expression of the Bcl‐2 and Bax proteins by FCM and Western blotting before and after drug treatment. NT2 cells had low constitutive expression levels of Bcl‐2 and elevated constitutive expression levels of Bax protein, as determined by both methods. At t = 24 h and 48 h after drug treatment, no changes were observed in the expression of the Bcl‐2 protein, as quantitated by FCM and Western blotting. Also, the expression of the Bax protein had not changed, based on Western blotting. However, FCM revealed that in a specific subpopulation of drug‐treated NT2 cells, Bax expression was increased. On the basis of forward and perpendicular light‐scatter this subpopulation, which consisted of large, early apoptotic, swollen cells with increased internal complexity, was sorted, and showed abundant Bax protein by FCM and Western blotting. Our results demonstrate that the chemosensitivity of NT2 cells is probably due to a low intrinsic threshold for drug‐induced apoptosis that is accompanied by overexpression of the death‐promoting Bax protein during the early stages of the apoptotic process. We conclude that FCM is superior to Western blotting for the detection of heterogeneous expression of Bax in a given cell population. Cytometry 27:275–282, 1997. © 1997 Wiley‐Liss, Inc.