Inhibition of telomerase activity by geldanamycin and 17-allylamino, 17-demethoxygeldanamycin in human melanoma cells.
As it has been demonstrated that the heat shock protein 90 (HSP90) is required for the assembly and activation of telomerase in human cells, we investigated the effect exerted by the ansamycin antibiotics geldanamycin (GA) and 17-allylamino,17-demethoxygeldanamycin (17-AAG), two well-known inhibitors of the HSP90 chaperone function, on telomerase activity in JR8 human melanoma cells. Using an antibody to HSP90, we precipitated the telomerase activity associated with the molecular chaperone. The results of TRAP (telomeric repeat amplification protocol) experiments carried out on HSP90 immunoprecipitates showed that exposure to 100 ng/ml GA and 17-AAG induced a significant (P < 0.01) inhibition of telomerase activity, which was observed at earlier time points than drug-induced inhibition of cell proliferation. Superimposable results were obtained from TRAP experiments carried out on total JR8 protein extracts. To investigate whether the basal level of telomerase activity of the tumour cell system plays a role in determining the cellular response to 17-AAG, we compared the cytotoxic activity of the drug in JR8 cells and in two JR8-derived clones that were stably transfected with a hammerhead ribozyme targeting the RNA template of telomerase and were characterized by a markedly lower telomerase activity than the parental cells. The cytotoxicity results indicated that both ribozyme-transfectant clones were almost 2-fold more sensitive to 72 h 17-AAG exposure than JR8 cells as a consequence of a more than double apoptotic response [in terms of the percentage of apoptotic nuclei in cells stained with propidium iodide and the percentage of Tdt-mediated dUTP nick-end labelling (TUNEL)-positive cells]. In summary, our results suggest that (i) telomerase is a target of GA and 17-AAG action and its inhibition may contribute to the cytotoxic activity of the drugs, (ii) the basal level of telomerase activity of the tumour cell system may also have a role in influencing 17-AAG cytotoxicity.
Villa
Raffaella
R
Department of Experimental Oncology, Unit 10, Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian 1, 20133 Milan, Italy.
Folini
Marco
M
Porta
Chiara Della
CD
Valentini
Alessandra
A
Pennati
Marzia
M
Daidone
Maria Grazia
MG
Zaffaroni
Nadia
N
D017209:Apoptosis / Q000187:drug effects; D016227:Benzoquinones; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005434:Flow Cytometry; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D020287:In Situ Nick-End Labeling; D047029:Lactams, Macrocyclic; D008545:Melanoma / Q000201:enzymology* / Q000473:pathology; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011809:Quinones / Q000494:pharmacology*; D017828:Rifabutin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D019098:Telomerase / Q000037:antagonists & inhibitors*; D016615:Telomere / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects
D016227:Benzoquinones; D017931:DNA Primers; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D011809:Quinones; D017828:Rifabutin; C112765:tanespimycin; D011505:Protein-Tyrosine Kinases; D019098:Telomerase; C001277:geldanamycin