Inhibition of Hsp90 function by ansamycins causes downregulation of cdc2 and cdc25c and G(2)/M arrest in glioblastoma cell lines.
Ansamycins exert their effects by binding heat shock protein 90 (Hsp90) and targeting important signalling molecules for degradation via the proteasome pathway. We wanted to study the effect of geldanamycin (GA) and its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) on glioblastoma cell lines. We show that these cells are growth inhibited by ansamycins by being arrested in G(2)/M and, subsequently, cells undergo apoptosis. The protein levels of cell division cycle 2 (cdc2) kinase and cell division cycle 25c (cdc25c) were downregulated upon GA and 17-AAG treatment and cdc2 kinase activity was inhibited. However, other proteins involved in the G(2)/M checkpoint were not affected. The cdc2 and cdc25c mRNA levels did not show significant differences upon ansamycin treatment, but the stability of cdc2 protein was reduced. The association of cdc2 and cdc25c with p50(cdc37), an Hsp90 co-chaperone, decreased, but the interaction of cdc2 and cdc25c with the Hsp70 co-chaperone increased after ansamycin treatment. Proteasome inhibitors were able to rescue the cdc2 downregulation, but not the cdc25c reduction. However, calpain inhibitors were able to rescue the cdc25c downregulation, suggesting that cdc25c is proteolysed by calpains in the presence of ansamycins, and not by the proteasome. We conclude that ansamycins downregulate cdc2 and cdc25c by two different mechanisms.
García-Morales
P
P
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Alicante, Spain.
Carrasco-García
E
E
Ruiz-Rico
P
P
Martínez-Mira
R
R
Menéndez-Gutiérrez
M P
MP
Ferragut
J A
JA
Saceda
M
M
Martínez-Lacaci
I
I
D001932:Brain Neoplasms / Q000378:metabolism* / Q000473:pathology*; D042846:CDC2-CDC28 Kinases / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors; D002455:Cell Division; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000187:drug effects*; D016195:G2 Phase; D005909:Glioblastoma / Q000378:metabolism* / Q000473:pathology*; D018841:HSP90 Heat-Shock Proteins / Q000502:physiology*; D006801:Humans; D017828:Rifabutin / Q000494:pharmacology*; D020687:cdc25 Phosphatases / Q000378:metabolism*
D018841:HSP90 Heat-Shock Proteins; D017828:Rifabutin; D042846:CDC2-CDC28 Kinases; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases; D002154:Calpain