Abstract 833: Photolabeling of β-tubulin isotypes by radiolabeled 2-(m-azidobenzoyl)taxol

Abstract

Abstract There are seven β-tubulin isotypes present in distinct quantities in cells of different origin. Altered expression of β-tubulin isotypes were found in cells resistant to microtubule stabilizing agents (MSAs), such as Taxol, epothilone B, ixabepilone and discodermolide, all of which bind to β-tubulin. For example, the level of βIII-tubulin was very low in the human ovarian cancer cell line, Hey, but increased 8- to 15-fold in two drug resistant daughter cell lines, EpoB8 and Ixab80, respectively. The βV-tubulin level was increased by 2-fold in EpoB8 cells. In contrast, levels of βI- and βIV-tubulin were unchanged and decreased, respectively, in EpoB8. Therefore, it is important to measure the binding affinity of MSAs to different β-tubulin isotypes and study their influence on the drug resistant phenotype. To study the relative binding affinities of MSAs, tubulins from HeLa cells, bovine brain, porcine brain and chicken erythrocytes were specifically photolabeled with the tritium labeled 2-(m-azidobenzoyl)taxol, in the presence and absence of Taxol, Taxotere, epothilone B, ixabepilone and discodermolide. β-tubulin isotype content from these sources was different, particularly for chicken erythrocytes that contain only one β-tubulin isotype, βVI. The inhibitory effects elicited by these MSAs on photolabeling were distinct for β-tubulins from different sources. Taxol had a minimal inhibitory effect on HeLa, bovine and porcine brain β-tubulin, but a strong inhibitory effect on chicken erythrocyte β-tubulin. Discodermolide exhibited opposite effects on photolabeling, compared to those of Taxol. However, Taxotere appeared to have a stronger inhibitory effect than Taxol on photolabeling of β-tubulins from all sources tested. Ixabepilone had the strongest inhibitory effect on HeLa β-tubulin. The inhibition pattern for β-tubulin from porcine brain correlated very well with that from bovine brain. To determine drug binding affinities for the different β-tubulin isotypes, tubulins from different sources were photolabeled and the isotypes resolved by high resolution isoelectrofocusing (IEF). Approximately 20 bands were seen with bovine and porcine brain tubulin, but only 3-5 bands were resolved with chicken erythrocyte tubulin. Many bands represent posttranslationally modified tubulins. IEF patterns for bovine and porcine brain tubulin were very similar. All bands were analyzed by mass spectrometry following CNBr digestion, and the identity and relative quantity of each β-tubulin isotype determined. After measuring the radioactivity associated with each band, it was found that the binding affinity of 2-(m-azidobenzoyl)taxol to βIII-tubulin was the lowest, compared to other β-tubulin isotypes. Citation Format: Chia-Ping H. Yang, Hui Xiao, Susan Band Horwitz. Photolabeling of β-tubulin isotypes by radiolabeled 2-(m-azidobenzoyl)taxol. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 833. doi:10.1158/1538-7445.AM2014-833