RESUMEN
Recent studies have shown that paclitaxel leads to activation of Raf-1 kinase and have suggested that this activation is essential for bcl-2 phosphorylation and apoptosis. In the present study, we demonstrate that, in addition to paclitaxel, other agents that interact with tubulin and microtubules also induce Raf-1/bcl-2 phosphorylation, whereas DNA-damaging drugs, antimetabolites, and alkylating agents do not. Activation of Raf-1 kinase by paclitaxel is linked to tubulin polymerization; the effect is blunted in paclitaxel-resistant cells, the tubulin of which does not polymerize following the addition of paclitaxel. In contrast, vincristine and vinblastine, drugs to which the paclitaxel-resistant cells retain sensitivity were able to bring about Raf-1 phosphorylation. The requirement for disruption of microtubules in this signaling cascade was strengthened further using paclitaxel analogues by demonstrating a correlation between tubulin polymerization, Raf-1/bcl-2 phosphorylation, and cytotoxicity. Inhibition of RNA or protein synthesis prevents Raf-1 activation and bcl-2 phosphorylation, suggesting that an intermediate protein(s) acts upstream of Raf-1 in this microtubule damage-activating pathway. A model is proposed that envisions a pathway of Raf-1 activation and bcl-2 phosphorylation following disruption of microtubular architecture, serving a role similar to p53 induction following DNA damage.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Microtúbulos/efectos de los fármacos , Paclitaxel/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Tubulina (Proteína)/efectos de los fármacos , Antineoplásicos/farmacología , Resistencia a Medicamentos , Activación Enzimática , Femenino , Humanos , Fosforilación , Polímeros/metabolismo , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-raf , Tubulina (Proteína)/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
The anticancer drug paclitaxel (Taxol) has been converted to a large number of 2-debenzoyl-2-aroyl derivatives by three different methods. The bioactivities of the resulting analogues were determined in both tubulin polymerization and cytotoxicity assays, and several analogues with enhanced activity as compared with paclitaxel were discovered. Correlation of cytotoxicity in three cell lines with tubulin polymerization activity showed reasonable agreement. Among the cell lines examined, the closest correlation with antitubulin activity was observed with a human ovarian carcinoma cell line.
Asunto(s)
Antineoplásicos Fitogénicos , Paclitaxel , Antineoplásicos Fitogénicos/síntesis química , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Biopolímeros , Catálisis , División Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Paclitaxel/análogos & derivados , Paclitaxel/síntesis química , Paclitaxel/química , Paclitaxel/farmacología , Polietilenglicoles/química , Relación Estructura-Actividad , Tubulina (Proteína)/metabolismo , Células Tumorales CultivadasRESUMEN
The cytoplasmic membrane proteins ExbB and ExbD support TonB-dependent active transport of iron siderophores and vitamin B12 across the essentially unenergized outer membrane of Escherichia coli. In this study, in vivo formaldehyde cross-linking analysis was used to investigate the interactions of T7 epitope-tagged ExbB or ExbD proteins. ExbB and ExbD each formed two unique cross-linked complexes which were not dependent on the presence of TonB, the outer membrane receptor protein FepA, or the other Exb protein. Cross-linking analysis of ExbB- and ExbD-derived size variants demonstrated instead that these ExbB and ExbD complexes were homodimers and homotrimers and suggested that ExbB also interacted with an unidentified protein(s). Cross-linking analysis of epitope-tagged ExbB and ExbD proteins with TonB antisera afforded detection of a previously unrecognized TonB-ExbD cross-linked complex and confirmed the composition of the TonB-ExbB cross-linked complex. The implications of these findings for the mechanism of TonB-dependent energy transduction are discussed.