Immunofluorescence studies have revealed that H2AX is phosphorylated at the sites of DNA double-strand breaks induced by ionizing radiation and is required for recruitment of repair factors into nuclear foci after DNA damage. Therefore, the function of H2AX is believed to be associated primarily with repair of DNA damage. Here, we report a function of H2AX in cellular apoptosis. Our data showed that H2AX is phosphorylated by UVA-activated JNK. We also provided evidence showing that UVA induces caspase-3 and caspase-activated DNase (CAD) activity in both H2AX wild-type and H2AX knockout mouse embryonic fibroblasts (MEFs). However, DNA fragmentation occurred only in H2AX wild-type MEFs. Furthermore, H2AX phosphorylation was critical for DNA degradation triggered by CAD in vitro. Taken together, these data indicated that H2AX phosphorylation is required for DNA ladder formation, but not for the activation of caspase-3; and the JNK/H2AX pathway cooperates with the caspase-3/CAD pathway resulting in cellular apoptosis.

Many beneficial properties have been attributed to (À)Àepi-gallocatechin gallate (EGCG), including chemopreventive, anticarcinogenic, and antioxidant actions. In this study, we investigated the effects of EGCG on the function of glucoseregulated protein 78 (GRP78), which is associated with the multidrug resistance phenotype of many types of cancer cells. Our investigation was directed at elucidating the mechanism of the EGCG and GRP78 interaction and providing evidence about whether EGCG modulates the activity of anticancer drugs through the inhibition of GRP78 function. We found that EGCG directly interacted with GRP78 at the ATP-binding site of protein and regulated its function by competing with ATP binding, resulting in the inhibition of ATPase activity. EGCG binding caused the conversion of GRP78 from its active monomer to the inactive dimer and oligomer forms. Further, we showed that EGCG interfered with the formation of the antiapoptotic GRP78-caspase-7 complex, which resulted in an increased etoposide-induced apoptosis in cancer cells. We also showed that EGCG significantly suppressed the transformed phenotype of breast cancer cells treated with etoposide. Overall, these results strongly suggested that EGCG could prevent the antiapoptotic effect of GRP78, which usually suppresses the caspase-mediated cell death pathways in drugtreated cancer cells, contributing to the development of drug resistance. (Cancer Res 2006; 66(18): 9260-9)

No abstract

Retinoic acid is one of the most promising drugs for chemotherapy and chemoprevention of cancer. Either blocking activator protein-1 (AP-1) activity or activating retinoic acid response element (RARE) have been proposed to be responsible for its antitumor activity. However, evidence for this hypothesis is lacking in vivo studies. To address this issue, we used an AP-1-luciferase transgenic mouse as a carcinogenesis model and new synthetic retinoids that are either selective inhibitors of AP-1 activation or selective activators of the RARE. The results showed that the SR11302, an AP-1 inhibition-specific retinoid, and other AP-1 inhibitors such as trans-retinoic acid and f luocinolone acetonide, markedly inhibit both 12-O-tetradecanoylphorbol-13-acetate-induced papilloma formation and AP-1 activation in 7,12-dimethyl benz(a)anthracene-initiated mouse skin (P < 0.05). In contrast, repeated applications of SR11235, a retinoid with RARE transactivating activity, but devoid of AP-1 inhibiting effect, did not cause significant inhibition of papilloma formation and AP-1 activation (P > 0.05). These results provide the first in vivo evidence that the antitumor effect of retinoids is mediated by blocking AP-1 activity, but not by activation of RARE.

The JB6 mouse epidermal cell system, which includes tumor promotion-sensitive (P ؉ ) and tumor promotion-resistant (P ؊ ) cells, is a well-established and extensively used cell culture model for studying the mechanism of latestage tumor promotion. Tumor promoters, such as 12-Otetradecanoylphorbol 13-acetate (TPA) or epidermal growth factor (EGF), induce high levels of activator protein 1 (AP-1) activity and large, tumorigenic, anchorage-independent colonies in soft agar at a high frequency in JB6 P ؉ cells, but not in JB6 P ؊ cells. We report here a molecular explanation for the defect in the AP-1 activation and promotion-resistant phenotype of P؊ cells. We demonstrate that the lack of AP-1 activation and cell transformation responses to TPA and EGF in P ؊ cells appears attributable to the low level of mitogenactivated protein kinase (MAPK) (extracellular signalregulated protein kinase, Erk) in these cells. TPA and EGF induce transactivation of AP-1 activity in P ؉ cells but not in P ؊ cells. Nonphosphorylated forms and TPA-or EGF-induced phosphorylated forms of Erks (Erk1 and Erk2) in P ؊ cells were much lower than those in P ؉ cells. Stable transfection of wild-type MAPK (Erk2) into P ؊ cells restored its response to TPA and EGF for both AP-1 activation and cell transformation. These results suggest that the shortage of MAPK (Erk1 and Erk2) appears to be an important contributor to the tumor promotion-resistant phenotype in JB6 cells.

No abstract

The endothelial adherens junction is formed by complexes of transmembrane adhesive proteins, of which ␤-catenin is known to connect the junctional protein vascular endothelial (VE)-cadherin to the cytoskeleton and to play a signaling role in the regulation of junctioncytoskeleton interaction. In this study, we investigated the effect of neutrophil activation on endothelial monolayer integrity and on ␤-catenin and VE-cadherin modification. Treatment of cultured bovine coronary endothelial monolayers with C5a-activated neutrophils resulted in an increase in permeability as measured by albumin clearance across the monolayer. Furthermore, large scale intercellular gap formation was observed in coincidence with the hyperpermeability response. Immunofluorescence analysis showed that ␤-catenin and VE-cadherin staining changed from a uniform distribution along the membrane of control cells to a diffuse pattern for both proteins and finger-like projections for ␤-catenin in neutrophil-exposed monolayers. Correlatively, there was an increase in actin stress fiber formation in treated cells. Finally, ␤-catenin and VE-cadherin from neutrophil-treated endothelial cells showed a significant increase in tyrosine phosphorylation. Our results are the first to link neutrophil-mediated changes in adherens junctions with intercellular gap formation and hyperpermeability in microvascular endothelial cells. These data suggest that neutrophils may regulate endothelial barrier function through a process conferring conformational changes to ␤-catenin and VE-cadherin.The wall of exchange vessels consists of a layer of endothelial cells that connect to each other with closely opposed intercellular junctions. A major function of the junctional connection is to maintain the semi-permeable property of the endothelial barrier and to control the transvascular passage of solutes, fluid, and blood cells. Four types of junctions associated with endothelial cells have been identified: adherens junctions (AJ), 1 tight junctions, gap junctions, and complexus adherentes (1, 2). AJ, formed by transmembrane adhesive proteins called cadherins, appear to be the main complex regulating macromolecular permeability in microvascular endothelium. Cadherins, specifically vascular endothelial (VE)-cadherin, are associated with the actin cytoskeleton through a family of proteins called catenins, including ␣-catenin, ␤-catenin, and plakoglobin (3, 4). The endothelial permeability is affected by many agonists including ␣-thrombin, histamine, and phorbol esters (5-9) as well as by a group of inflammatory cells, namely polymorphonuclear leukocytes (PMNs) (10 -14). At the site of injury or inflammation, circulating PMNs often adhere to and subsequently migrate through the endothelium and enter surrounding tissues (15). It has long been documented that the process of PMN adherence and migration is associated with an increase in endothelial permeability (10, 11). Although much work has been dedicated to identify PMN-derived hyperpermeability factors (11-14), little is k...

The activation of glial cells in the spinal dorsal horn and the gracile nucleus by inflammation and nerve injury has been suggested to be involved in neuronal plasticity and central sensitization, hence contributing to tactile allodynia. The aim of this study was to determine the possible intracellular signal transduction pathway associated with glial cells, which have been activated by partial sciatic nerve ligation (PSNL), a well-characterized rat model of neuropathic pain. At 3 weeks post-lesion, PSNL markedly increased glia fibrillary acidic protein (GFAP) immunoreactive (IR) astrocytes in both the L4-5 spinal dorsal horn and the gracile nucleus. Moreover, PSNL increased the phosphorylation of mitogen activated protein (MAP) kinases, including the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but not p38, in glia-like cells in these same areas. Both phosphorylated (p) ERK- and JNK-IR cells were co-localized with GFAP, suggesting their expression in reactive astrocytes. In summary, our data indicate that PSNL activates ERK/MAP and JNK/MAP kinase pathways in astrocytes in the dorsal horn and the gracile nucleus, these events possibly being involved in the pathogenesis of neuropathic pain.