MAP kinase-dependent degradation of p27Kip1 by calpains in choroidal melanoma cells. Requirement of p27Kip1 nuclear export

We investigated the status and the regulation of the cyclin-dependent kinases (CDK) inhibitor p27(Kip1) in a choroidal melanoma tumor-derived cell line (OCM-1). By contrast to normal choroidal melanocytes, the expression level of p27(Kip1) was low in these cells and the mitogen-activated protein (MAP) kinase pathway was constitutively activated. Genetic or chemical inhibition of this pathway induced p27(Kip1) accumulation, whereas MAP kinase reactivation triggered a down-regulation of p27(Kip1) that could be partially reversed by calpain inhibitors. In good accordance, ectopic expression of the cellular calpain inhibitor calpastatin led to an increase of endogenous p27(Kip1) expression. In vitro, p27(Kip1) was degraded by calpains, and OCM-1 cell extracts contained a calcium-dependent p27(Kip1) degradation activity. MAP kinase inhibition partially inhibited both calpain activity and calcium-dependent p27(Kip1) degradation by cellular extracts. Immunofluorescence labeling and subcellular fractionation revealed that p27(Kip1) was in part localized in the cytoplasmic compartment of OCM-1 cells but not of melanocytes, and accumulated into the nucleus upon MAP kinase inhibition. MAP kinase activation triggered a cytoplasmic translocation of the protein, as well as a change in its phosphorylation status. This CRM-1-dependent cytoplasmic translocation was necessary for MAP kinase- and calpain-dependent degradation. Taken together, these data suggest that in tumor-derived cells, p27(Kip1) could be degraded by calpains through a MAP kinase-dependent process, and that abnormal cytoplasmic localization of the protein, probably linked to modifications of its phosphorylation state, could be involved in this alternative mechanism of degradation.     

Organotypic heart slices for cell transplantation and physiological studies

Recent studies have significantly improved our ability to investigate cell transplantation and study the physiology of transplanted cells in cardiac tissue. Several previous studies have shown that fully-immersed heart slices can be used for electrophysiological investigations. Additionally, ischemic heart slices induced by glucose and oxygen deprivation offer a useful tool to investigate mechanical integration and to measure forces of contraction of engrafted cells, at least for short term analysis. A recent and novel model of heart slices, prepared from rat and human tissues, can be maintained in culture for up to two months. This new heart slice model can be used for long term in vitro cell transplantation studies and for pharmacological evaluation. This review will focus on describing these models and demonstrating the use of organotypic heart slices as a novel tool for drugs for studying electrophysiology and developing cellular therapeutic approaches to alleviate cardiac tissue damage.Key words: heart, organotypic, culture, stem cells, transplantation, electrophysiology, pharmacology     

Synthesis and Cytotoxic Evaluation of Novel Thiazolocarbazoles

Novel thiazolocarbazole derivatives 4 and 5 have been synthesized via the corresponding imino-1,2,3-dithiazoles 3. In vitro antitumor activity of these polyheterocyclic compounds was studied and the results show that 2-cyano derivatives exhibit a medium in vitro antiproliferative effect.