Geldanamycin, an inhibitor of the chaperone activity of HSP90, induces MAPK-independent cell cycle arrest

Abstract

The effects of GA, an ansamycin antibiotic in development as a lead anticancer drug, were studied in mouse BP-A31 fibroblasts and in human cancer-derived cell lines. GA and related molecules act by inhibiting the chaperone function of the Hsp90 protein through competition for ATP binding. The antiproliferative effects of GA have been attributed to destabilization of the Raf-1 protein, one of the targets of Hsp90, and to the resulting inhibition of MAPK. Addition of GA to BP-A31 cells, synchronously progressing through the G(1) phase, inhibited Rb hyperphosphorylation and G(1)/S transition irrespective of the time of addition. The G(1) arrest was accompanied by a progressive decrease in Raf-1 content, especially of the phosphorylated form; however, GA caused only partial inhibition of MAPK phosphorylation. We show that GA triggers a rapid and marked decrease in the kinase activity of the cyclin E/cdk2 complex coupled with a decline in both total and cdk2-associated cyclin E. In transient transfection experiments, inhibition of cyclin E expression by GA was correlated with inhibition of the transcriptional activity of the cyclin E gene promoter. Inhibition of cdk4 activity by GA was observed 3 hr after addition of the drug to late G(1) cells but not after a short (1 hr) exposure, as revealed by the phosphorylation of Rb on the Ser(780) residue. In human cancer-derived cell lines expressing or not a functional Rb protein, GA blocked proliferation and inhibited the transcriptional activity of the cyclin E gene promoter. In these cell lines, the antiproliferative effect of GA was not limited to the G(1) phase, suggesting the existence of multiple cellular targets of the drug.