Abstract 664: Polymerization of human βIII-tubulin is distinct from βI-tubulin in a cell-free system

Abstract

Abstract Tubulins are a family of proteins consisting of 7 α- and 6 β-tubulin isotypes. Many studies have demonstrated that tumors overexpressing βIII-tubulin have a poor response to chemotherapy. Suppression of βIII-tubulin sensitizes non-small cell lung cancer (NSCLC) cells to microtubule-interacting agents, such as Taxol. βIII-tubulin has also been shown to mediate the incidence and progression of lung cancer. Tubulin isotype content of βI-, βIII-, βIV- and βV-tubulin in the human lung carcinoma cell line A549 is 50%, 8%, 36.5%, and 5.5%, respectively. In our study, the ability of six microtubule stabilizing agents, Taxol, epothilone B (EpoB), discodermolide (Disco), Ixabepilone, laulimalide and peloruside A, to polymerize tubulin in a 100,000 × g supernatant prepared from A549 cells was examined. The level of different tubulin isotypes in the drug-polymerized tubulin was determined by Western blot analysis with tubulin isotype specific antibodies. In the absence of drug, the level of polymerized βI-, βIII-, βIV- and βV-tubulin was 55%, 9%, 25%, and 48%, respectively. Ten µM Taxol caused a smalll increase (<15%) in βI- and βV- tubulin polymerization, but it induced a 367% and 228% increase in βIII- and βIV-tubulin polymerization. Among the six drugs tested, Disco and EpoB exhibited the greatest ability to polymerize all isotypes. The differential βI- and βIII-tubulin polymerization effects also were observed when intact A549 cells were lysed in the presence of drugs, followed by centrifugation. In the absence of drug, the level of polymerized βI- and βIII-tubulin was 9% and 0.2%, respectively. In the presence of 10 µM Taxol, the level of polymerized βI- and βIII-tubulin was increased to 28% and 7% which corresponded to a 3- and 35-fold increase in βI- and βIII-tubulin polymerization, respectively. However, when purified bovine brain tubulin in the absence of all endogenous proteins was used, there was no differential effect of drugs on the polymerization of βI+II- and βIII-tubulin. The βI-, βII-, βIII-, and βIV-tubulin content in bovine brain is 3%, 58%, 25% and 14%, respectively. Disco and EpoB also have the strongest effects on the polymerization of all bovine brain tubulin isotypes. Our results indicate that polymerization of βIII-tubulin is distinct from that of βI-tubulin in a cell-free system and that endogenous cellular components may influence the polymerization of tubulin isotypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 664. doi:10.1158/1538-7445.AM2011-664