Microtubules are versatile and ubiquitous polymers that play an essential part in a wide range of fundamenta1 cellular events. Both the mitotic spindle and the interphase cytoskeleton are formed from rapidly tuning-over microtubule populations with half lives of less than a few minutes, which grow from and shrink towards the microtubule organizing centres.
Microtubules are cytoskeletal proteins critical for cell growth, division, motility, signaling, and the development and maintenance of cell shape. Microtubules are highly dynamic structures and their dynamic instability is responsible for many microtubule-dependent processes in cells. The characterization and study of drugs that bind to either free tubulin or formed microtubules have provided new tools with which to study essential cellular functions such as mitosis. Microtubule interacting agents, including members of the taxane and vinca alkaloid families, have been useful clinically as chemotherapeutic agents. These drugs bind to tubulin and cause either microtubule depolymerization or excessive microtubule assembly (bundling) resulting in altered microtubule polymer mass and indiscriminant cell death in normal and transformed cells. Microtubule-targeting agents that only modulate microtubule dynamics and do not alter total polymer mass may be clinically useful and be associated with fewer side effects. Recent findings from our laboratory have shown that the opium alkaloid, noscapine, binds to tubulin and arrests HeLa cells in mitosis. This document outlines experiments that further characterize the effect of the tubulin interacting agent, noscapine, in cells of neuroectodermal origin including normal and immortalized/transformed cells derived from the neural crest such as melanocytes and neuroglia. When transformed, these cells are responsible for malignant melanoma and glioblastoma. These studies will provide insight into cellular functions sensitive to small changes in microtubule dynamics and lays the groundwork for evaluating the therapeutic potential of noscapine to treat two neoplastic diseases, melanoma and glioblastoma.
Microtubules are composed of α- and β-tubulin heterodimers assembled into tubular structures that have inherent structural and kinetic polarity. Microtubule assembly is initiated at a critical subunit concentration, and elongation proceeds by the reversible addition of tubulin dimers to microtubule ends. The addition of tubulin subunits to microtubule ends is known as polymerization, whereas the loss of subunits is known ad depolymerization. One end of a microtubule grows and shortens faster than the other end and hence it is called the plus end to distinguish it from the slower minus end. The assembly of microtubules depends on several parameters including: (1) polymerization rate, (2) depolymerization rate, (3) rescue rate (disassembly to assembly transition), and (4) catastrophe rate (assembly to disassembly rate). In addition to the net addition and loss of tubulin dimers from microtubule ends, GTP binds exchangeably to tubulin and is irreversibly hydrolyzed during tubulin dimer addition to microtubule ends. The energy input from GTP hydrolysis allows for nonequilibrium polymerization dynamics creating a behavior in which individual microtubule ends alternate stochastically between prolonged phases of polymerization and depolymerization. The rates at which these events occur (1-4 above) and the hydrolysis of GTP accounts for the dynamic instability seen in microtubules. These behaviors are known as collectively referred to as microtubule dynamics. These parameters can be best represented as microtubule dynamicity defined as the overall rate of microscopically detectable tubulin exchange at the microtubule end. Microtubules are essential for a variety of cellular processes such as cell motility, intracellular transport, maintenance of cellular morphology, and cell division. Thus, any agent that changes the assembly or disassembly of microtubules can potentially prevent cell division by interfering with essential cellular functions.