Lipid Rafts in Exosome Biogenesis.

Exosomes (secreted extracellular vesicles formed in the intracellular vesicular transport system) play a crucial role in distant cell-cell communication. Exosomes transfer active forms of various biomolecules; the molecular composition of the exosomal cargo is a result of targeted selection and depends on the type of producer cells. The mechanisms underlying exosome formation and cargo selection are poorly understood. It is believed that there are several pathways for exosome biogenesis, although the questions about their independence and simultaneous coexistence in the cell still remain open. The least studied topic is the recently discovered mechanism of exosome formation associated with lipid rafts, or membrane lipid microdomains. Here, we present modern concepts and basic hypotheses on the mechanisms of exosome biogenesis and secretion and summarize current data on the involvement of lipid rafts and their constituent molecules in these processes. Special attention is paid to the analysis of possible role in the exosome formation of raft-forming proteins of the SPFH family, components of planar rafts, and caveolin, the main component of caveolae.

Upregulation of c-FLIP-short in response to TRAIL promotes survival of NSCLC cells, which could be suppressed by inhibition of Ca2+/calmodulin signaling.

TNF-related apoptosis-inducing ligand (TRAIL) is a promising cytokine for killing tumor cells. However, a number of studies have demonstrated that different cancer cells resist TRAIL treatment and, moreover, TRAIL can promote invasion and metastasis in resistant cells. Here we report that TRAIL rapidly activates caspase-8 in a panel of non-small-cell lung carcinomas (NSCLCs). Adenocarcinomas derived from the lung in addition to high caspase-8 expression are characterized by increased expression of DR4 compared with adjacent non-neoplastic tissues. Blocking DR4 or lowering caspase-8 expression significantly reduced apoptosis in NSCLC cell lines, indicating the importance of DR4 and signifying that higher levels of caspase-8 in lung adenocarcinomas make them more susceptible to TRAIL treatment. Despite rapid and robust initial responsiveness to TRAIL, surviving cells quickly acquired resistance to the additional TRAIL treatment. The expression of cellular-FLIP-short (c-FLIPS) was significantly increased in surviving cells. Such upregulation of c-FLIPS was rapidly reduced and TRAIL sensitivity was restored by treatment with cycloheximide. Silencing of c-FLIPS, but not c-FLIP-long (c-FLIPL), resulted in a remarkable increase in apoptosis and significant reduction of clonogenic survival. Furthermore, chelation of intracellular Ca(2+) or inhibition of calmodulin caused a rapid proteasomal degradation of c-FLIPS, a significant increase of the two-step processing of procaspase-8, and reduced clonogenicity in response to TRAIL. Thus, our results revealed that the upregulation of DR4 and caspase-8 expression in NSCLC cells make them more susceptible to TRAIL. However, these cells could survive TRAIL treatment via upregulation of c-FLIPS, and it is suggested that blocking c-FLIPS expression by inhibition of Ca(2+)/calmodulin signaling significantly overcomes the acquired resistance of NSCLC cells to TRAIL.

Suppression and restoration of v-SRC expression in rsv transformed-cells after transfection with N-ras and its antagonist.

Previously, it was shown that hamster cells transformed by Rous Sarcoma Virus (RSV) exhibited a decreased expression of the RSV products (including the pp60 src oncogene) when these cells were supertransfected with the N-ras oncogene. To assess the responsibility of the activated N-ras in the modulation of the RSV viral products, a strategy based on two ras antagonists was used; i.e. i) a rap1A/K-rev1 expression vector known for its capacity to revert the K-ras induced transformed phenotype and ii) a plasmid containing antisense N-ras sequence. We present data showing only the plasmid construct containing the N-ras antisense sequense could inhibit expression or N-ras and, at the same time, restore the expression of v-src, up to a level comparable to that of the parental cells. Our results support the idea that some biological switches, triggered and activated through the N-ras oncogene pathway, might modulate the promoter activity of the RSV LTR.