Defining the Tubulin Cytoskeleton of Drug Resistant Cells By High Resolution MS

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DESCRIPTION (provided by applicant): Project Summary: Microtubules are dynamic protein polymers that play an essential role in cell division, maintenance of cell shape, transport of vesicles, and motility. Microtubules are proven to be an effective target for anticancer drugs, such as the microtubule-stabilizing agents Taxol, the epothiolones and discodermolide. However, there is a recurring problem of resistance to these microtubule-stabilizing drugs and the process by which tumors develop resistance is not well understood. One possible cause of resistance is a change in the tubulin isotype composition of the microtubules. Microtubules are made up of alpha- and beta-tubulin heterodimers, of which there are 6 alpha- and 7 beta-tubulin isotypes in humans. Alterations in tubulin isotype expression can affect the stability of the microtubules leading to reduced efficacy of microtubule-stabilizing drugs. Further contributing to the diversity of microtubules are post- translational modifications (PTMs) to tubulin. Microtubule alterations may also affect the ability of proteins that bind to the microtubule scaffold, another possible mechanism of drug resistance. The objective of this research is to ascertain the changes in tubulin isotype expression and post-translational modification and in proteins associated with microtubules in the development of resistance to microtubule-stabilizing drugs. Specific Aims: (1) To establish a high resolution mass spectrometric methodology for complete characterization of tubulin isotypes, providing a basis for understanding the relationship between tubulin isotype expression and resistance; (2) to examine the expressed tubulins and PTMs that occur in sensitive and drug resistant cell lines; (3) to quantitate changes in the level of expression and extent of modification of proteins that are associated with microtubules in resistant cell lines using top down and bottom up proteomic methodologies. Using high resolution mass spectrometry, we will measure the intact mass of the tubulins present in cell lines sensitive and resistant to microtubule-stabilizing drugs and accurately determine the tubulin isotypes present along with any PTMs or mutations. Also, we will identify proteins associated with the microtubules and determine changes that occur with these proteins in resistant cell lines. Relevance: These studies will contribute to our understanding of how cells become resistant to Taxol and other microtubule-stabilizing drugs and may provide insight as to why certain tumor types respond better to these drugs than others and some do not respond at all. Ultimately, this information will help us to develop new cancer therapeutics and also to understand how to treat specific tumors.