The process of apoptosis is essential for maintaining the physiologic balance between cell death and cell growth. This complex process is executed by two major pathways that participate in activating an executioner mechanism leading to chromatin disintegration and nuclear fragmentation. Dysregulation of these pathways often contributes to cancer development and resistance to cancer therapy. Here, we review the most recent discoveries in apoptosis regulation and possible mechanisms for resensitizing tumor cells to therapy.

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Paraquat, N-methyl-4-phenyl-1,2,3,6 tetrahydropyridine, and rotenone have been shown to reproduce several features of Parkinson's disease in animal and cell culture models. Although these chemicals are known to perturb dopamine homeostasis and induce dopaminergic cell death, their molecular mechanisms of action are not well defined. We have previously shown that paraquat does not require functional dopamine transporter and does not inhibit mitochondrial complex I in order to mediate its toxic action (Richardson et al., 2005). In this study, we show that paraquat specifically oxidized the cytosolic form of thioredoxin and activated Jun N-terminal kinase (JNK), followed by caspase-3 activation. Conversely, 1-methyl-4-phenylpyridinium (MPP(+)) and rotenone oxidized the mitochondrial form of thioredoxin but did not activate JNK-mitogen-activated protein kinase and caspase-3. Loading cells with exogenous dopamine did not exacerbate the toxicity of any of these compounds. These data suggest that oxidative modification of cytosolic proteins is critical to paraquat toxicity, while oxidation of mitochondrial proteins is important for MPP(+) and rotenone toxicity. In addition, intracellular dopamine does not seem to exacerbate the toxicity of these dopaminergic neurotoxicants in this model.

Monks. EGFR-independent activation of p38 MAPK and EGFR-dependent activation of ERK1/2 are required for ROS-induced renal cell death. Am J Physiol Renal Physiol 287: F1049 -F1058, 2004. First published June 29, 2004 doi:10.1152/ajprenal.00132.2004,5-Tris-(glutathion-S-yl)hydroquinone (TGHQ), a reactive metabolite of the nephrotoxicant hydroquinone, induces the ROS-dependent activation of MAPKs, followed by histone H3 phosphorylation and oncotic cell death in renal proximal tubule epithelial cells (LLC-PK 1). Cell death and histone H3 phosphorylation are attenuated by pharmacological inhibition of p38 MAPK or ERK1/2 pathways. Because TGHQ, but not epidermal growth factor (EGF), induces histone H3 phosphorylation and cell death in LLC-PK 1 cells, we hypothesized that there are differences in the mechanisms by which TGHQ and EGF induce activation of the EGF receptor (EGFR). We therefore compared the relative ability of TGHQ, H 2O2, and EGF to activate EGFR and MAPKs and found that p38 MAPK activation is EGFR independent, whereas ERK1/2 activation occurs mainly through EGFR activation. TGHQ, H 2O2, and EGF induce different EGFR tyrosine phosphorylation profiles that likely influence the subsequent differential kinetics of MAPK activation. We next transfected LLC-PK 1 cells with a dominant negative p38 MAPK-expressing plasmid (pcDNA3-DNp38). TGHQ failed to induce phosphorylation of p38 MAPK and its substrate, MK-2, in pcDNA3-DNp38-transfected cells, indicating loss of function of p38 MAPK. In untransfected, pcDNA3 or pcDNA3-p38 (native)-transfected LLC-PK 1 cells, Hsp27 was intensively phosphorylated after TGHQ treatment, whereas in pcDNA3-DNp38-transfected cells, TGHQ failed to induce Hsp27 phosphorylation. Thus EGFR-independent p38 MAPK and EGFR-dependent ERK1/2 activation by TGHQ lead to the activation of two downstream signaling factors, i.e., histone H3 and Hsp27 phosphorylation, which have in common the potential ability to remodel chromatin. reactive oxygen species; epidermal growth factor receptor; mitogenactivated protein kinase; histone H3; heat shock protein 27 REACTIVE OXYGEN SPECIES (ROS) are associated with a variety of human diseases and toxicities associated with exposure to redox-active chemicals and/or their metabolites (4). 2,3,5-Tris-(glutathion-S-yl)hydroquinone (TGHQ) is a metabolite of hydroquinone (HQ) and contributes to HQ-mediated nephrotoxicity and nephrocarcinogenesis (21). Renal proximal tubule epithelial cells are especially susceptible to TGHQ-induced toxicity, probably due to the high activity of ␥-glutamyl transpeptidase (␥-GT), which catalyzes the metabolism of TGHQ activity and facilitates the subsequent cellular uptake of

Extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNK/SAPK), and p38 mitogen-activated protein kinase (MAPK) were all rapidly activated in a ROS-dependent manner during 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ)-mediated oxidative stress and oncotic cell death in renal proximal tubule epithelial cells (LLC-PK1). TGHQ-induced phosphorylation of ERK1/2 and JNK MAPKs required epidermal growth factor receptor (EGFR) activation, whereas p38 MAPK activation was EGFR independent. In contrast to their established roles in cell survival, TGHQ-activated ERK1/2 and p38 MAPK (but not JNK) appear to contribute to cell death, since inhibition of ERK1/2 or p38 MAPKs with PD098059 or SB202190 respectively, attenuated TGHQ-mediated cell death. TGHQ increased AP-1 and NFkappaB DNA-binding activity, but whereas pharmacological inhibition of ERK1/2 or p38 MAPKs attenuated AP-1 DNA binding activity, it potentiated TGHQ-mediated NFkappaB activation. Consistent with a role for NFkappaB activation in the cytoprotective response to ROS in renal epithelial cells, an anti-NFkappaB peptide SN50 suppressed the protective effects of ERK inhibition (PD098059 treatment). The data provide evidence that the activation of MAPKs by ROS in renal epithelial cells plays an important role in oncotic cell death, and NF-kB is involved in the cytoprotective effects of PD098059.

A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.

Prior work using allogeneic bone marrow transplantation (allo-BMT) models showed that peritransplant administration of flagellin, a toll-like receptor 5 (TLR5) agonist protected murine allo-BMT recipients from CMV infection while limiting graft-vs-host disease (GvHD). However, the mechanism by which flagellin-TLR5 interaction promotes anti-CMV immunity was not defined. Here, we investigated the anti-CMV immunity of NK cells in C57BL/6 (B6) mice treated with a highly purified cGMP grade recombinant flagellin variant CBLB502 (rflagellin) followed by murine CMV (mCMV) infection. A single dose of rflagellin administered to mice between 48 to 72 hours prior to MCMV infection resulted in optimal protection from mCMV lethality. Anti-mCMV immunity in rflagellin-treated mice correlated with a significantly reduced liver viral load and increased numbers of Ly49H+ and Ly49D+ activated cytotoxic NK cells. Additionally, the increased anti-mCMV immunity of NK cells was directly correlated with increased numbers of IFN-γ, granzyme B- and CD107a producing NK cells following mCMV infection. rFlagellin-induced anti-mCMV immunity was TLR5-dependent as rflagellin-treated TLR5 KO mice had ∼10-fold increased liver viral load compared with rflagellin-treated WT B6 mice. However, the increased anti-mCMV immunity of NK cells in rflagellin-treated mice is regulated indirectly as mouse NK cells do not express TLR5. Collectively, these data suggest that rflagellin treatment indirectly leads to activation of NK cells, which may be an important adjunct benefit of administering rflagellin in allo-BMT recipients.

. Tuberous sclerosis-2 tumor suppressor modulates ERK and B-Raf activity in transformed renal epithelial cells. Am J Physiol Renal Physiol 286: F417-F424, 2004. First published November 11, 2003 10.1152/ ajprenal.00234.2003.-The tuberous sclerosis-2 (Tsc-2) gene is a suppressor of renal tumorigenesis and an early target of reactive oxygen species-induced renal cancer. Tuberin, the protein product of the Tsc-2 gene, participates in the regulation of cell proliferation, although the mechanism by which it suppresses proliferation is unknown. Quinol-thioether-transformed rat renal epithelial (QT-RRE) cell lines, derived from quinol-thioether-transformed primary renal epithelial cells from Eker rats, lack tuberin expression due to loss of heterozygosity of the Tsc-2 gene. These cell lines were used to examine the mechanism by which tuberin exerts its antiproliferative action. Loss of tuberin function correlates with high ERK activity (39), which could contribute to the formation of renal tumors. In this study, we sought to identify possible downstream effectors regulated by tuberin, using QT-RRE cells transfected with Tsc-2 cDNA to restore tuberin expression. Constitutively high ERK, B-Raf, and Raf-1 activities were observed in QT-RRE cells. However, restoration of tuberin expression in QT-RRE cells by transient transfection with Tsc-2 cDNA substantially decreased both ERK and B-Raf activity, with only modest changes in Raf-1 activity, suggesting tuberin functions as an upstream negative regulator of the ERK pathway. High ERK activity was not mediated through EGF receptor activation, but treatment with genistein demonstrated that protein kinases are involved in ERK cascade activation. The data indicate that loss of tuberin results in the upregulation of the ERK signaling pathway with subsequent increases in new DNA synthesis, and ultimately, tumor formation.cell cycle extracellular signal-regulated kinase; quinol-thioether reactive oxygen species THE TUBEROUS SCLEROSIS-2 (Tsc-2) gene functions as a renal tumor suppressor (33), although the mechanism of this effect remains elusive (25,35,38). Tuberin, the Tsc-2-encoded protein, regulates cell growth and proliferation (1,9,23,24,27) and cell cycle progression (25,26). Loss of tuberin expression induces quiescent G 0 -arrested cells to enter the cell cycle and prevents cells from reentering a quiescent state (25). Similarly, a reduction in the level of tuberin increases cyclin D 1 protein expression and promotes the transition from G 0 /G 1 to S phase. Inactivation of gigas, the Drosophila homolog of Tsc-2, results in the formation of tissues composed of enlarged cells with a repeating S phase without entering the M phase (8).The mechanism underlying the antiproliferative function of tuberin is still relatively uncharacterized. Recent studies showed that the Akt pathway regulates the formation of the tuberous sclerosis complex (7). Components of this complex, in turn, inhibit signaling mediated by the mammalian target of rapamycin (28). Tuberin is a structurally compl...