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Cancer cells acquire mitotic drug resistance properties through beta I-tubulin mutations and alterations in the expression of beta-tubulin isotypes.

ABSTRACT: BACKGROUND: Anti-mitotic compounds (microtubule de-stabilizers) such as vincristine and vinblastine have been shown clinically successful in treating various cancers. However, development of drug-resistance cells limits their efficacies in clinical situations. Therefore, experiments were performed to determine possible drug resistance mechanisms related to the application of anti-mitotic cancer therapy. PRINCIPAL FINDINGS: A KB-derived microtubule de-stabilizer-resistant KB-L30 cancer cell line was generated for this study. KB-L30 cells showed cross-resistance to various microtubule de-stabilizers including BPR0L075, vincristine and colchicine through multiple-drug resistant (MDR)-independent mechanisms. Surprisingly, KB-L30 cells showed hyper-sensitivity to the microtubule-stabilizer, paclitaxel. Results of the RT-PCR analysis revealed that expression of both class II and III ?-tubulin was down-regulated in KB-L30 cells as compared to its parental KB cancer cells. In addition, DNA sequencing analysis revealed six novel mutation sites present in exon four of the ?I-tubulin gene. Computational modeling indicated that a direct relationship exists between ?I-tubulin mutations and alteration in the microtubule assembly and dynamic instability in KB-L30 cells and this predicted model was supported by an increased microtubule assembly and reduced microtubule dynamic instability in KB-L30 cells, as shown by Western blot analysis. CONCLUSIONS AND SIGNIFICANCE: Our study demonstrated that these novel mutations in exon four of the ?I-tubulin induced resistance to microtubule de-stabilizers and hyper-sensitivity to microtubule stabilizer through an alteration in the microtubule assembly and dynamics in cancer cells. Importantly, the current study reveals that cancer cells may acquire drug resistance ability to anti-mitotic compounds through multiple changes in the microtubule networks. This study further provided molecular information in drug selection for patients with specific tubulin mutations.

SUBMITTER: Cheung CH

PROVIDER: S-EPMC2933234 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Cancer cells acquire mitotic drug resistance properties through beta I-tubulin mutations and alterations in the expression of beta-tubulin isotypes.

Cheung Chun Hei Antonio CH Wu Su-Ying SY Lee Tian-Ren TR Chang Chi-Yen CY Wu Jian-Sung JS Hsieh Hsing-Pang HP Chang Jang-Yang JY

PloS one 20100903 9

<h4>Background</h4>Anti-mitotic compounds (microtubule de-stabilizers) such as vincristine and vinblastine have been shown clinically successful in treating various cancers. However, development of drug-resistance cells limits their efficacies in clinical situations. Therefore, experiments were performed to determine possible drug resistance mechanisms related to the application of anti-mitotic cancer therapy.<h4>Principal findings</h4>A KB-derived microtubule de-stabilizer-resistant KB-L30 canc ...[more]

PMID: 20838440

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Project description:BackgroundThe multi-tubulin hypothesis proposes that each tubulin isotype performs a unique role, or subset of roles, in the universe of microtubule function(s). To test this hypothesis, we are investigating the functions of the recently discovered, noncanonical β-like tubulins (BLTs) of the ciliate, Tetrahymena thermophila. Tetrahymena forms 17 distinct microtubular structures whose assembly had been thought to be based on single α- and β-isotypes. However, completion of the macronuclear genome sequence of Tetrahymena demonstrated that this ciliate possessed a β-tubulin multigene family: two synonymous genes (BTU1 and BTU2) encode the canonical β-tubulin, BTU2, and six genes (BLT1-6) yield five divergent β-tubulin isotypes. In this report, we examine the structural features and functions of two of the BLTs (BLT1 and BLT4) and compare them to those of BTU2.Methodology/principal findingsWith respect to BTU2, BLT1 and BLT4 had multiple sequence substitutions in their GTP-binding sites, in their interaction surfaces, and in their microtubule-targeting motifs, which together suggest that they have specialized functions. To assess the roles of these tubulins in vivo, we transformed Tetrahymena with expression vectors that direct the synthesis of GFP-tagged versions of the isotypes. We show that GFP-BLT1 and GFP-BLT4 were not detectable in somatic cilia and basal bodies, whereas GFP-BTU2 strongly labeled these structures. During cell division, GFP-BLT1 and GFP-BLT4, but not GFP-BTU2, were incorporated into the microtubule arrays of the macronucleus and into the mitotic apparatus of the micronucleus. GFP-BLT1 also participated in formation of the microtubules of the meiotic apparatus of the micronucleus during conjugation. Partitioning of the isotypes between nuclear and ciliary microtubules was confirmed biochemically.Conclusion/significanceWe conclude that Tetrahymena uses a family of distinct β-tubulin isotypes to construct subsets of functionally different microtubules, a result that provides strong support for the multi-tubulin hypothesis.

Dataset Information

Cancer cells acquire mitotic drug resistance properties through beta I-tubulin mutations and alterations in the expression of beta-tubulin isotypes.

Publications

Cancer cells acquire mitotic drug resistance properties through beta I-tubulin mutations and alterations in the expression of beta-tubulin isotypes.

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