Expression of Cryptosporidium parvum thioredoxin peroxidase in COS-7 cells confers radioprotection.

Cryptosporidium parvum is one of the most radioresistant organisms identified to date. In a previous study, we found that thioredoxin peroxidase (CpTPx) was significantly upregulated in this species following exposure to high dose (10 kGy) of γ-irradiation. To assess the potential of CpTPx to confer radioprotection in mammalian cells, it was expressed in COS-7 African green monkey kidney cells (CpTPx-COS7). For comparison, the thioredoxin peroxidase of Cryptosporidium muris (CmTPx) was also expressed in these cells (CmTPx-COS7 cells), which has been confirmed to have lesser antioxidant activity than CpTPx in the previous study. Notably, the survival rates of CpTPx-COS7 cells were significantly higher (12-22%) at 72 h after 8 Gy irradiation than CmTPx-COS7 or non-transfected COS-7 (ntCOS-7) counterparts. In addition, CpTPx revealed a 50% of ROS reduction in irradiated CpTPx-COS7 cells, while γ-H2AX DNA damage marker expression was not significantly changed. Furthermore, the amount of apoptosis only increased to about 120% after 2-8 Gy irradiation compared to 200-300% increase observed in ntCOS-7 cells. CmTPx was shown to have antioxidant and DNA damage protection activities; however, these activities were always lower than those of CpTPx. These results suggest that the potent antioxidant and protective activities of CpTPx are well conserved in this cell-based system and that CpTPx contributed to the radioprotection of mammalian cells through its exceptional antioxidant activity.

Activation of RSK by UV-light: phosphorylation dynamics and involvement of the MAPK pathway.

Ribosomal S6 kinases (RSKs) are serine/threonine kinases activated by mitogenic signals through the Mitogen-Activated Protein Kinases/Extracellular Signal-Regulated Kinases (MAPK/ERK). RSKs contain two heterologous complete protein kinase domains. Phosphorylation by ERK of the C-terminal kinase domain allows activation of the N-terminal kinase domain, which mediates substrate phosphorylation. In human, there are three isoforms of RSK (RSK1, RSK2, RSK3), whose functional specificity remains undefined. Importantly, we have shown that mutations in the RSK2 gene lead to the Coffin-Lowry syndrome (CLS). In this study, we characterize two monoclonal antibodies raised against phosphorylated forms of the N- and C-terminal domain of RSK2 (P-S227 and P-T577, respectively). Using these two antibodies, we show that stress signals, such as UV light, induce phosphorylation and activation of the three RSKs to an extent which is comparable to Epidermal Growth Factor (EGF)-mediated activation. The use of specific kinase inhibitors indicates that UV-induced phosphorylation and activation of RSK2 is mediated by the MAPK/ERK pathway, but that the Stress-Activated Protein Kinase 2 (SAPK2)/p38 pathway is also involved. These results modify the view of RSKs as kinases restricted to the mitogenic response and reveal a previously unappreciated role of MAPKs in stress induced signaling. Oncogene (2000) 19, 4221 - 4229

Nuclear export of glucocorticoid receptor is enhanced by c-Jun N-terminal kinase-mediated phosphorylation.

The c-Jun N-terminal kinase (JNK) phosphorylates the glucocorticoid receptor (GR) and inhibits GR-mediated transcription. However, the biological effect of the GR phosphorylation remains unknown. Here we demonstrate that activated JNK phosphorylates human GR at Ser226 and enhances its nuclear export after withdrawal of a ligand for GR, dexamethasone. At 1 h after dexamethasone withdrawal, green fluorescent protein-GR molecules were mostly retained at the nucleus, whereas UV exposure enhanced its nuclear export, and approximately 30-40% of cells revealed distinct nuclear export. JNK overexpression alone mimics UV exposure and enhanced GR export accompanied by inhibition of GR-mediated transcription. However, mutation of the Ser226 JNK phosphorylation site in GR abrogated UV-mediated enhancement of GR nuclear export. Furthermore, overexpression of a dominant negative SEK1 mutant also abrogated the effects of UV exposure on GR export. Taken together, these findings suggest that JNK-mediated phosphorylation of the GR-Ser226 enhances GR nuclear export and may contribute to termination of GR-mediated transcription.

Use of an infectious Simian virus 40-based shuttle vector to analyse UV-induced mutagenesis in monkey cells.

SV40 based shuttle vectors able to be packaged as pseudovirions have been used either as naked DNA or as pseudovirus to analyse the mutation frequency and the UV-induced mutation spectra obtained after transfection or infection of COS7 monkey cells. The frequency of supF spontaneous mutants was similar whatever the state of the vector, indicating that the transfection step is not responsible for the high spontaneous mutation frequency when using shuttle vectors. Nevertheless the UV-induced mutation frequency of the supF gene was higher when transfected DNA was replicated into COS7 cells than when pseudovirus infection was performed. The UV induced mutation spectra was basically similar in both situations but a new hot-spot at nucleotide 110 was obtained after pseudovirus infection. UV-pretreated and control COS7 cells were infected with untreated or UV-damaged pi SVPC7 shuttle virus and the survival and the supF mutation frequency were analysed in the progeny. The survival of UV-damaged pseudovirus replicated in 10 J/m2 UV-pretreated cells was 2-fold higher than in untreated cells. This increase in the survival was accompanied by a slight enhancement in the number of supF mutants.

Nuclear localization of cyclin B1 regulates DNA damage-induced apoptosis.

Some cells undergo apoptosis in response to DNA damage, whereas others do not. To understand the biochemical pathways controlling this differential response, we have studied the intracellular localization of cyclin B1 in cell types sensitive or resistant to apoptosis induced by DNA damage. We found that cyclin B1 protein accumulates in the nucleus of cells that are sensitive to gamma radiation-induced apoptosis (thymocytes, lymphoid cell lines), but remains cytoplasmic in apoptosis-resistant cells (primary and transformed fibroblasts). Treatment of both cell types with leptomycin B, an inhibitor of CRM1-dependent cyclin B1 nuclear export, induces apoptosis. Furthermore, ectopic expression of cyclin B1-5xE, a protein that preferentially localizes to the nucleus, is sufficient to trigger apoptosis. Conversely, expression of cyclin B1-5xA, a predominantly cytoplasmic protein, fails to induce apoptosis. This suggests that nuclear accumulation is necessary for cyclin B1-dependent apoptosis. Our observations are consistent with the idea that localization of cyclin B1 is among the factors determining the cellular decision to undergo apoptosis in response to DNA damage.