Natural products have historically been the source of most of the microtubule (MT)-targeting small molecules whose properties have allowed them to become useful drugs. That remains true of most but not all of the compounds that we have used in this study. These include the clinically established MT-active drugs colchicine, combretastatin, vincristine, taxol, and others. Almost all such agents were developed first in pre-clinical research that included in vitro studies of the effect of the compounds on polymerization of tubulin to microtubules as well as the effect of such compounds on cell behavior, especially examining the ability of the compounds to disrupt mitosis through effects on the MT arrays that comprise the mitotic spindle. The in vitro studies with purified tubulin are a critical way in which compounds are evaluated, and indeed this is often used as a screening method to discover new active compounds as well as to quantitatively evaluate established ones. To further this, we have reviewed approaches to quantitating MT assembly by optical methods, with the goal of maximizing the information that may be obtained and minimizing the errors that the system can produce. We used both optical density-based and fluorescence-based approaches, and evaluated sources of error as well as underappreciated sources of extra information on the polymerization process. While the roles of MT extend throughout the life of the cell, it is nonetheless true that mitosis is a central role for MTs. Dysfunction of the MT-based mitotic spindle leads cells to abnormal separation of chromosomes, resulting in abnormal chromosome numbers in daughter cells. This aneuploidy is commonly found in human cancers. Cells have multiple mechanisms to avoid aneuploidy, focusing on maintaining proper functioning of the mitotic spindle. Some of these mechanisms involve proteins that are only expressed and active during mitosis. One of these proteins is known as CKAP2, whose expression is altered in some cancers. We studied this protein to understand how it helps to stabilize mitotic spindle function, and how alterations in its expression in cells can lead to aneuploidy in the daughter cells. We discovered that CKAP2 regulates the functioning of the two poles of the MT arrays in the spindle. Abnormal amounts of CKAP2 reduce the ability of cells to form two and only two spindle poles. When there are multiple poles, it is impossible to divide the chromosomes evenly. Thus failure, or deficit, of CKAP2 function leads to attachment failure in some fraction of the chromosomes and to accumulation of aneuploidy in the cell population.

Project Start
Project End
Budget Start
Budget End
Support Year
11
Fiscal Year
2013
Total Cost
$354,673
Indirect Cost
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State
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Lee, Hee-Sheung; Lee, Nicholas C O; Kouprina, Natalay et al. (2016) Effects of Anticancer Drugs on Chromosome Instability and New Clinical Implications for Tumor-Suppressing Therapies. Cancer Res 76:902-11
Montecinos-Franjola, Felipe; Schuck, Peter; Sackett, Dan L (2016) Tubulin Dimer Reversible Dissociation: AFFINITY, KINETICS, AND DEMONSTRATION OF A STABLE MONOMER. J Biol Chem 291:9281-94
Sheldon, Kely L; Gurnev, Philip A; Bezrukov, Sergey M et al. (2015) Tubulin Tail Sequences and Posttranslational Modifications Regulate Closure of Mitochondrial Voltage-Dependent Anion Channel (VDAC). J Biol Chem :
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Poruchynsky, Marianne S; Komlodi-Pasztor, Edina; Trostel, Shana et al. (2015) Microtubule-targeting agents augment the toxicity of DNA-damaging agents by disrupting intracellular trafficking of DNA repair proteins. Proc Natl Acad Sci U S A 112:1571-6
Mukherjee, Kamalika; Chio, Tak Ian; Sackett, Dan L et al. (2015) Detection of oxidative stress-induced carbonylation in live mammalian cells. Free Radic Biol Med 84:11-21
Case, Chanelle M; Sackett, Dan L; Wangsa, Danny et al. (2013) CKAP2 ensures chromosomal stability by maintaining the integrity of microtubule nucleation sites. PLoS One 8:e64575
Komlodi-Pasztor, Edina; Sackett, Dan L; Fojo, Tito (2013) Tales of how great drugs were brought down by a flawed rationale--response. Clin Cancer Res 19:1304
Mirigian, Matthew; Mukherjee, Kamalika; Bane, Susan L et al. (2013) Measurement of in vitro microtubule polymerization by turbidity and fluorescence. Methods Cell Biol 115:215-29

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