Stathmin regulation of interphase cells: Implications for cancer research

Cells are complex systems with numerous functions regulated in time and space. Cell function and organization depend on the net activity of various biomolecules; understanding how these biomolecules function in the context of the cellular environment is necessary to understand the system failures in disease states. The cytoskeleton, polymers of protein subunits, are one example of a biological system with major functions in cell organization. My thesis research explored regulation of the microtubule cytoskeleton by stathmin, a protein expressed in all mammalian cells and highly expressed in many human cancers. In my first experiments, I examined how stathmin controls the amount of microtubule polymer in the cell.;Several potential mechanisms could regulate partitioning of tubulin between dimer and polymer pools. I experimentally tested mechanisms of tubulin dimer/polymer partitioning through study of the effects of changing stathmin protein level on parameters of microtubule dynamics and microtubule polymer density via live cell imaging and confocal microscopy. I found that changes to stathmin protein levels through genetic deletion of one or both alleles and over-expression only marginally influenced microtubule dynamics. Surprisingly, stathmin protein level had a profound effect on tubulin partitioning through the regulation of microtubule nucleation rate at the centrosome.;Changes in stathmin expression level could impact expression of other components and thus influence cell organization. To expand our understanding of stathmin function in normal cells, we compare gene expression profiles of mouse embryo fibroblasts isolated from STMN1+/+ and STMN1 -/- mice to determine the transcriptome level changes present in the genetic knock-out of stathmin Approximately 50% of genes up or down regulated by ≥2.0 fold in STMN1-/- mouse embryo fibroblasts function broadly in cell adhesion and motility. These results support models indicating a role for stathmin in regulating cell locomotion, but also suggest that this functional activity may involve changes to the cohort of proteins expressed in the cell, rather than as a direct consequence of stathmin-dependent regulation of the microtubule cytoskeleton.;Successful cancer therapeutics, including paclitaxel, target the microtubule network of the mitotic spindle. An alternative to targeting microtubules directly is to target proteins associating with, or regulating, the microtubule cytoskeleton. Two phenotypically similar cancer treatments, paclitaxel and stathmin-siRNA, were studied in human colon cancer cells. Messenger RNA isolated 6-72 h after treatment with either paclitaxel or siRNA was analyzed by microarray to compare genes expression fold changes ≥2.0. The analysis was extended by comparing 24 h samples in triplicate with fold changes ≥1.2. The regulatory changes observed were different, and in opposite directions, between the two treatments for most genes probed. Quantitative RT-PCR measurement of apoptosis pathway-specific changes between treatments in triplicate samples confirmed opposite regulation of most significantly changed genes. Therefore, paclitaxel treatment and stathmin-siRNA generate different transcriptome level changes, indicating that therapies based on stathmin depletion may initiate unique changes to the target cell compared to those generated by paclitaxel.