Document Type



Doctor of Philosophy


Molecular Biology

First Adviser

Cassimeris, Lynne U.

Other advisers/committee members

Lowe-Krentz, Linda; Skibbens, Robert; Luca, Frank


For any cell within a multicellular organism, a multitude of environmental signals influence cell fate. The context and timing of these often redundant and overlapping signaling cascades govern whether a cell will differentiate, reproduce or die. Some of these signaling networks form checkpoints that sense and respond to the needs of the individual cell. In cancer, often one or more of these signals is missing or mutated which results in uncontrolled cell growth, usually to the detriment of the organism. Understanding the signaling events that normally control cell proliferation and death is important for controlling and eliminating cancerous cells. Microtubule targeting drugs have been used to disrupt mitotic progression and cause cell death in cancer cells because of their typically high proliferation rates. However, these drugs kill normal dividing cells and damage healthy tissues. Therefore, there have been great efforts to find selective ways to target cancerous cells without harming normal cells. One of the major regulators of genomic stability in cells is p53. Mutations or gene deletions of p53 are present in over half of all cancers. Many studies have demonstrated that p53-deficient cells are uniquely vulnerable to depletion of a microtubule regulator protein called stathmin. Stathmin depletion slows proliferation via a mitotic entry delay (including results presented in Chapter 2) and increases cell death in p53-deficient cells. Research presented in this dissertation addresses the mechanisms by which stathmin loss delays mitotic entry and triggers cell death. In order to know why cells are slow to enter mitosis we investigated the activity of the enzymes that govern mitotic entry. We previously found stathmin-depleted cells have decreased active CDK1. The level of active CDC25, which removes CDK1 inhibition, was also reduced in these cells. However, none of the upstream regulators that might inhibit CDC25 activation were changed upon stathmin depletion. Therefore, we hypothesized that loss of stathmin directly affects the core enzymes in the mitotic entry complex. We found that stathmin depletion decreases activation of Aurora A kinase (AURKA) and Polo-like Kinase 1 (PLK1), two key enzymes of the mitotic entry feedback loop that control CDK1 activation kinetics. Since stathmin depletion delays mitotic entry and causes apoptotic cell death, we hypothesized that the cell cycle delay and cell death were linked. We found that inducing a mitotic entry delay with the use of enzyme inhibitors to AURKA and PLK1 was sufficient to trigger cell death but only in cells lacking functional p53. To understand the mechanism of the cell death in these cells we looked for activation of initiator caspases 8 and 9, the primary drivers of apoptosis. We found caspase 8 activation in both stathmin-depleted and mitotic entry-delayed cells. Additionally, we were able to rescue viability of these cells by inhibiting caspase 8 activity indicating that cell death occurs via a caspase 8 dependent pathway. CDK1 is known to inhibit a number of caspases including caspase 8 via a protective phosphorylation. We found that phosphorylation of caspase 8 at Serine 387 was decreased in stathmin-depleted cells, suggesting an increased sensitivity of caspase 8 to activation. In order to understand the role of p53 for survival in stathmin depletion we hypothesized that p53 may regulate a caspase 8 inhibitor. We found the level of cFLIP, a major inhibitor of caspase 8, to be decreased in the absence of p53, consistent with the observations of others. We were able to rescue viability of stathmin-depleted cells by restoring cFLIP level. Therefore we concluded that in the absence of p53, decreased cFLIP levels lower the threshold for caspase 8 activation. The absence of stathmin and p53 results in loss of two inhibitors of caspase 8. The sum of inhibition may be sufficient to either stochastically trigger cell death or increase susceptibility to other death signals. Regardless of the mechanism, modulating stathmin or disrupting mitotic entry directly represents a potential selective and potent means of targeting p53 deficient cancers.