Inhibition of Hsp90 function by ansamycins causes retinoblastoma gene product-dependent G1 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 degradation of a subset of signaling molecules. These include proteins known to associate with Hsp90, e.g., the steroid receptors and Raf, as well as certain transmembrane tyrosine kinases, such as the ErbB receptor family. In a variety of tumor cell lines, treatment with HA potently inhibited cellular proliferation by inducing G1 arrest. This arrest was accompanied by hypophosphorylation of the retinoblastoma gene product (RB) and rapid down-regulation of cyclin D- and E-associated kinase activities. Inhibition of kinase activity was found to result from loss in expression of cyclins D1, D3, and E, as well as the associated cyclin-dependent kinases, cyclin-dependent kinase 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 other effects of HA, suggesting that it might be the primary cause of G1 arrest. To determine whether the effects of HA are mediated by selective inhibition 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 elicit a G1 arrest. In addition, after release from synchronization with nocodazole, Rb-negative but not Rb-positive cell lines were able to progress through G1 into S phase in the presence of HA. Together, these findings suggest that induction of G1 arrest by HA results from down-regulation of cyclin D expression and its associated kinase activity. Furthermore, these findings imply that Hsp90 selectively regulates signaling pathways upstream of RB.
Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
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M
Srethapakdi
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F
Liu
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R
Tavorath
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N
Rosen
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D016227:Benzoquinones; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects; D003110:Colonic Neoplasms / Q000378:metabolism; D019925:Cyclin A / Q000378:metabolism; D019938:Cyclin D1 / Q000378:metabolism; D056743:Cyclin D3; D019927:Cyclin E / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D005434:Flow Cytometry; D016193:G1 Phase / Q000187:drug effects*; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors*; D006801:Humans; D047029:Lactams, Macrocyclic; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D011809:Quinones / Q000494:pharmacology*; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
D000903:Antibiotics, Antineoplastic; D016227:Benzoquinones; C534696:CCND3 protein, human; D018797:Cell Cycle Proteins; D019925:Cyclin A; D056743:Cyclin D3; D019927:Cyclin E; D016213:Cyclins; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D008869:Microtubule-Associated Proteins; D011518:Proto-Oncogene Proteins; D011809:Quinones; D016160:Retinoblastoma Protein; D025521:Tumor Suppressor Proteins; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D017828:Rifabutin; C020754:herbimycin; D017346:Protein Serine-Threonine Kinases; C495900:CDK4 protein, human; C495911:CDK6 protein, human; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases