Kwang-Hoon Chun

The c-Jun N-terminal Kinase 1 Activity Is Differentially Regulated by Specific Mechanisms during Apoptosis

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 caspase-dependent cleavage of JNK1-associated p21WAF1/CIP1, is efficiently prevented by expressing p21D112N, an uncleavable mutant of p21WAF1/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-δ 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-δ 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.

Cleavage of Cdc6 by caspase-3 promotes ATM/ATR kinase-mediated apoptosis of HeLa cells.

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.

Protein kinase C-δ regulates the stability of hypoxia-inducible factor-1α under hypoxia

Hypoxia is a state of deficiency of available oxygen in the blood and tissues, and it occurs during several pathophysiological processes, including tumorigenesis. Under hypoxia, hypoxia‐inducible factor‐1 (HIF‐1) plays an essential role in cellular oxygen homeostasis. In the present article protein kinase C‐δ (PKC‐δ) is activated by hypoxia, increases the protein stability and transcriptional activity of HIF‐1α in human cervical adenocarcinoma cells. Moreover, the knockdown of PKC‐δ inhibited vascular endothelial growth factor expression and angiogenic activity under hypoxia. These effects were completely reversed by PKC‐δ overexpression following the knockdown of PKC‐δ. Collectively, these findings demonstrate the role of PKC‐δ as a new regulator of hypoxia‐induced angiogenesis. (Cancer Sci 2007; 98: 1476–1481)

SEK1-dependent JNK1 activation prolongs cell survival during G-Rh2-induced apoptosis

We provide here evidence that c-Jun N-terminal protein kinase 1 (JNK1) activity is differentially up-regulated during apoptosis of SK-HEP-1 cells after treatment with ginsenoside Rh2 (G-Rh2). The G-Rh2-mediated JNK1 activation that occurred for the first 10-30min was associated with SEK1 activity, but thereafter, the sustained activation was associated not with SEK1 activity, but with proteolytic cleavage of JNK1-associated p21(WAF1/CIP1). Supporting this is that the expression of the dominant negative SEK1 mutant effectively blocked the early JNK1 activation phase but did not alter the sustained activation phase or apoptosis. Furthermore, expression of p21D112N, an uncleavable mutant of p21(WAF1/CIP1), suppressed the later JNK1 activation. Moreover, the stable overexpression of ectopic JNK1 suppressed apoptosis while expression of the dominant negative JNK1 mutant promoted it. We propose that the early SEK1-associated JNK1 activation phase acts to prolong cell survival in response to apoptosis-inducing agents, thereby serving as an intervening checkpoint prior to the commitment to apoptosis.

Cyclin-dependent protein kinase 2 activity is required for mitochondrial translocation of Bax and disruption of mitochondrial transmembrane potential during etoposide-induced apoptosis

Previous studies have suggested that upregulation of Cyclin A-dependent protein kinase 2 (Cdk2) activity is an essential event in apoptotic progression and the mitochondrial permeability transition in human cancer cells. Here, we show that upregulated Cyclin A/Cdk2 activity precedes the proteolytic cleavage of PARP and is correlated with the mitochondrial translocation of Bax and the loss of mitochondrial transmembrane potential (Δψm) during etoposide-induced apoptosis in human cervical adenocarcinoma (HeLa) cells. Etoposide-induced apoptotic cell death is efficiently prevented in cells that overexpress a dominant negative mutant of Cdk2 (Cdk2-dn) or p21WAF1/CIP1, a specific Cdk inhibitor. Conversely, apoptotic cell death is promoted in Cyclin A-expressing cells. Disruption of the mitochondrial transmembrane potential in etoposide-induced cells is prevented in cells that overexpress Cdk2-dn or p21WAF1/CIP1, while this transition is prominently promoted in Cyclin A-expressing cells. We screened for mitochondrial Cdk2 targets in the etoposide-induced cells and found that the mitochondrial level of Bax is elevated by more than three fold in etoposide-treated cells and this elevation is effectively prevented in cells expressing Cdk2-dn under the same conditions. Thus, we suggest that Cdk2 activity is involved in the mitochondrial translocation of Bax, which plays an important role in the mitochondrial membrane permeability transition during apoptotic progression.

Electron-beam lithography resist profile simulation for highly sensitive resist

Abstract A very highly sensitive resist is difficult to simulate its resist profile because of its extreme difference of development rate which can be determined from absorbed energy when electron beam is exposed. We developed resist profile simulator named ELIS (Electron-beam LIthography Simulator) that can predict the development profile of a very highly sensitive resist: Hybrid scattering model, Gaussian beam convolution and its superposition were used for Monte Carlo simulation and the ray tracing model for resist development.

Fabrication of integrated double-gated field emission microsource and its electrical characteristics

This paper presents the fabrication and electrical characteristics of the Integrated Field Emission Microsource (IFEMS) with multiple gates. The molybdenum field emission tip was made by filling cusp-like mold formed when a conformal film was deposited on the hole-trench. Each field emitter has a 1.8 μm-diameter extraction gate and a 1.4 μm-diameter focusing gate. To make a path for the emitted electrons, silicon bulk was etched anisotropically in KOH and EDP(Ethylene-Diamine Pyrocatechol) solution successively. The emission characteristics were measured and the influence of the focusing gate has been analyzed.