Inhibition of Hsp90 function by ansamycins causes retinoblastoma gene product-dependent G(1) arrest

The ansamycin antibiotics, herbimycin A (HA) and geldanamycin (GM), bind to a conserved pocket in heat shock protein 90 (Hsp90) and alter the function of this chaperone protein. Occupancy of this pocket results in the degrada tion of a subset of signaling molecules. These include proteins known to as sociate with Hsp90, e.g., the steroid receptors and Raf, as well as certain transmembrane tyrosine kinases, such as the ErbB receptor family, In a var iety of tumor cell lines, treatment with HA potently inhibited cellular pro liferation by inducing G(1) arrest. This arrest was accompanied by hypophos phorylation of the retinoblastoma gene product (RB) and rapid down-regulati on of cyclin D- and E-associated kinase activities. Inhibition of kinase ac tivity was found to result from loss in expression of cyclins D1, D3, and E , as well as the associated cyclin-dependent kinases, cyclin-dependent kina se 4 and cyclin-dependent kinase 6. In addition, HA treatment also caused a late induction of p27(Kip1) protein. The loss of cyclin D preceded the oth er effects of HA, suggesting that it might be the primary cause of G(1) arr est. To determine whether the effects of HA are mediated by selective inhib ition of the cyclin D-RB pathway, HA was added to tumor cell lines lacking functional RB. HA treatment of Rb-negative tumor cell lines failed to elici t a G(1) arrest. In addition, after release from synchronization with nocod azole, Rb-negative but not Rb-positive cell lines were able to progress thr ough G(1) into S phase in the presence of HA. Together, these findings sugg est that induction of G(1) arrest by HA results from down-regulation of cyc lin D expression and its associated kinase activity. Furthermore, these fin dings imply that Hsp90 selectively regulates signaling pathways upstream of RB.