We show here that JNK1 activity is rapidly up-regulated and prolonged by specific mechanisms during apoptosis induced by paclitaxel-or ginsenoside-Rh2 in SK-HEP-1 cells. The early phase of JNK1 activation is prevented in cells expressing the dominant negative SEK1 mutant, although this JNK1 perturbation does not prevent apoptotic cell death. The later phase of JNK1 activation, which is temporally coincided with caspasedependent cleavage of JNK1-associated p21 WAF1/CIP1 , is efficiently prevented by expressing p21D112N, an uncleavable mutant of p21 WAF1/CIP1 and this perturbation of JNK1 activation results in prevention of apoptosis. The later JNK1 activation and apoptotic progression are also prevented by co-treatments of cells with rottlerin, a PKC-␦ inhibitor or z-VAD-fmk, a pan caspase inhibitor. We also provide evidence that apoptotic cell death is significantly promoted in cells expressing JNK1, while this apoptotic cell death is effectively suppressed in cells expressing the dominant negative JNK1 mutant (DN-JNK1) or JBD, a JNK inhibitor protein. Thus, the later phase of JNK1 activation, which is linked to a caspase-dependent mechanism that requires PKC-␦ activity, is associated with the induction of apoptosis, while the early JNK1 activation that is associated with a SEK1-mediated mechanism is not directly involved in apoptotic progression.
We show that caspase-3 cleaves Cdc6 at D290/S and D442/G sites, producing p32-tCdc6 (truncated Cdc6) and p49-tCdc6, respectively, during etoposide- or tumor necrosis factor (TNF)-α–induced apoptosis. The expression of these tCdc6 proteins, p32- and p49-tCdc6, promotes etoposide-induced apoptosis. The expression of tCdc6 perturbs the loading of Mcm2 but not Orc2 onto chromatin and activates ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) kinase activities with kinetics similar to that of the phosphorylation of Chk1/2. The activation kinetics are consistent with elevated cellular levels of p53 and mitochondrial levels of Bax. The tCdc6-induced effects are all suppressed to control levels by expressing a Cdc6 mutant that cannot be cleaved by caspase-3 (Cdc6-UM). Cdc6-UM expression attenuates the TNF-α–induced activation of ATM and caspase-3 activities. When ATM or ATR is down-expressed by using the small interfering RNA technique, the TNF-α– or tCdc6-induced activation of caspase-3 activities is suppressed in the cells. These results suggest that tCdc6 proteins act as dominant-negative inhibitors of replication initiation and that they disrupt chromatin structure and/or induce DNA damage, leading to the activation of ATM/ATR kinase activation and p53–Bax-mediated apoptosis.
We show here that Ca 2ϩ and reactive oxygen species (ROS) are involved in the up-regulation of c-Jun NH 2 -terminal kinase 1 (JNK1) activity during apoptosis induced by ginsenoside Rh2 (G-Rh2) in HeLa, MCF10A-ras, and MCF7 cells. Addition of antioxidants such as N-acetyl-L-cysteine or catalase attenuates G-Rh2-induced ROS generation, JNK1 activation, and apoptosis. The overexpression of catalase down-regulates caspase-3 and JNK1 activities. G-Rh2 treatment of cells results in mitochondrial depolarization, second mitochondrial activator of caspase release, and translocation of Bax into the mitochondria, and these events are inhibited by antioxidants. Ca 2ϩ is also involved in mitochondrial depolarization during G-Rh2-induced apoptosis. These results suggest that ROS and Ca 2ϩ are important signaling intermediates leading to stress-activated protein kinase/extracellular signal-regulated kinase kinase 1/JNK1 activation and depolarization of the mitochondrial membrane potential in G-Rh2-induced apoptosis.
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