thapsigargin and monorden

Genome-wide transcript profiling to elucidate responses to HSP90 inhibition revealed strong induction of HSPA6 in MCF-7 cells treated with 17-AAG. Time- and dose dependent induction of HSPA6 (confirmed by qPCR and Western Blots) occurred also upon treatment with Radicicol, another HSP90 inhibitor. HSPA6 was not detectable in untreated cells or cells treated with toxins that do not inhibit HSP90, or upon applying oxidative stress. Thus, HSPA6 induction is not a general response to cytotoxic insults. Modulation of HSPA6 levels by siRNA-mediated inhibition or recombinant expression did not influence 17-AAG mediated cell death. HSPA6 induction as a consequence of HSP90 inhibition occurs in various (but not all) cell lines and may be a more specific marker for HSP90 inhibition than induction of other HSP70 proteins.

Topics: Amino Acid Sequence; Benzoquinones; Blotting, Western; Brefeldin A; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Hot Temperature; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Leupeptins; Macrolides; MCF-7 Cells; Molecular Sequence Data; RNA Interference; Sequence Homology, Amino Acid; Thapsigargin; Time Factors; Transcriptional Activation

We studied the phosphorylation (activation status) of c-Src and CaMKII in MEFs either wild type for calreticulin, calreticulin-null, or rescued with full-length calreticulin. We found that calreticulin-null cells were poorly spread on the substratum and formed few, if any, focal contacts. Fibronectin expression and deposition were lower in calreticulin-null MEFs compared to calreticulin-expressing cells, which also exhibited increased c-Src and CaMKII phosphorylation (activity). Plating MEFs on preformed fibronectin rescued the poor adhesive phenotype of calreticulin-null cells, and caused a decrease in c-Src Y418 phosphorylation (activity). c-Src inhibition caused the calreticulin-null MEFs to become well spread on the substratum and to make many prominent focal contacts. Calmodulin and CaMKII inhibition caused similar results, along with a notable increase in paxillin phosphorylation (activation). To test if the calcium storage function of calreticulin was responsible for these effects, we manipulated intracellular [Ca(2+)]. Lowering [Ca(2+)](ER) caused an increase in c-Src phosphorylation and a decrease in fibronectin abundance. Conversely, increasing [Ca(2+)] caused opposite effects. These results suggest that calreticulin regulates both the c-Src and calmodulin/CaMKII pathways, enabling cells to be better spread on the substratum by allowing greater fibronectin deposition and increased focal contact formation.

Topics: Actins; Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calmodulin; Calreticulin; Cell Adhesion; Cell Movement; Egtazic Acid; Embryo, Mammalian; Fibroblasts; Fibronectins; Focal Adhesions; Macrolides; Mice; Models, Biological; Paxillin; Phenotype; Protein Kinase Inhibitors; Proto-Oncogene Proteins pp60(c-src); Thapsigargin; Vinculin