Transactivation of hsp70-1/2 in geldanamycin-treated human non-small cell lung cancer H460 cells: involvement of intracellular calcium and protein kinase C

Geldanamycin is an antitumor drug that binds HSP90 and induces a wide range of heat shock proteins, including HSP70s. In this study we report that the induction of HSP70s is dose-dependent in geldanamycin-treated human non-small cell lung cancer H460 cells. Analysis of the induction of HSP70s specific isoform using LC-ESI-MS/MS analysis and Northern blotting showed that HSP70-1/2 are the major inducible forms under geldanamycin treatment. Transactivation of hsp70-1/2 was determined by electrophoretic mobility-shift assay using heat shock element (HSE) as a probe. The signaling pathway mediators involved in hsp70-1/2 transactivation were screened by the kinase inhibitor scanning technique. Pretreatment with serine/threonine protein kinase inhibitors H7 or H8 blocked geldanamycin-induced HSP70-1/2, whereas protein kinase A inhibitor HA1004, protein kinase G inhibitor KT5823, and myosin light chain kinase inhibitor ML-7 had no effect. Furthermore, the protein kinase C (PKC)-specific inhibitor Ro-31-8425 and the Ca2+-dependent PKC inhibitor G?-6976 diminished geldanamycin-induced HSP70-1/2, suggesting an involvement of the PKC in the process. In addition, geldanamycin treatment causes a transient increase of intracellular Ca2+. Chelating intracellular Ca2+ with BAPTA-AM or depletion of intracellular Ca2+ store with A23187 or thapsigargin significantly decreased geldanamycin-transactivated HSP70-1/2 expression. Taken together, our results demonstrate that geldanamycin-induced specific HSP70-1/2 isoforms expression in H460 cells through signaling pathway mediated by Ca2+ and PKC.     

A comparison of heat shock protein genes from cultured cells of the cabbage armyworm, Mamestra brassicae, in response to heavy metals

Heat shock protein (HSP) genes, hsp90, hsp70, hsc70, hsp20.7, and hsp19.7, were cloned and sequenced from cultured cells of the cabbage armyworm, Mamestra brassicae. Analyses of the cDNA sequences revealed open reading frames of 2,151, 1,914, 1,962, 540, and 465 bp in lengths, which encode proteins with calculated molecular weights of 82.5, 69.9, 71.6, 20.7, and 19.7 kDa, respectively. An increased expression was observed in all five genes after exposure to a high temperature. The induction of gene expression was not observed during a low temperature exposure, but was observed when the cells were recovered at ambient temperature. Expression of hsp90, hsp70, and hsp20.7 was induced after exposure to 2 microM of cadmium, while the minimum cadmium concentration for induction of hsp19.7 was 5 microM. The induction of hsp90 expression was somewhat masked by basal levels of expression. Only hsp20.7 expression was induced by exposure to copper. Lead did not induce expression of any of the HSP genes tested. Cadmium-induced up-regulation of hsp70 expression was lasted longer than heat-induced one. These results suggest that hsp70 could be useful to assess the cellular distress or injury induced by cadmium.