DZ is a naturally existing compound that has been recently synthesized to evaluate its potential as a cancer therapeutic. In our study, DZ treatment demonstrated a disruption of microtubules from the plus end, consistent with its proven ability to bind tubulin [12
], indicating that the mechanism of action of DZ is similar to that of other classical microtubule disruptors. However, DZ treatment seemed to show stronger mitotic slippage than VBL in RPE-hTERT cells (Figure ), suggesting that DZ can act differently than some other microtubule destabilizers.
Previous studies of microtubule inhibitors' effects on non-transformed cells have been limited to a DNA content-based analysis of cell cycle distribution. Researchers have observed an accumulation of cells with a 4N DNA content in the second cell cycle following mitosis, referred to as mitotic slippage [22
]. However, the cause of this phenotype is not fully understood. Recently, Brito and Rieder showed that mitotic slippage after nocodazole treatment occurs when the spindle-assembly checkpoint (SAC) fails to prevent a slow but continuous proteolysis of cyclin B [16
]. In the current study, we observed that mitotic slippage after DZ treatment was also correlated with cyclin B degradation.
p53 has been demonstrated to play a major role in cell cycle control [23
]; thus it may also be involved in mitotic slippage. In the p53-knockdown RPE-hTERT cells there was no loss of fragmentation; indicating that p53 was not required for mitotic slippage in the presence of this microtubule inhibitor. In fact, there was a consistent increase
in mitotic slippage in the transient p53 knockdown cells. This could be due to p53 down-regulation of cdc20 [24
], which is demonstrated to be required for the proteolysis of cyclin B by activating anaphase-promoting complex, a mitotic ubiquitin ligase [25
]. By knocking down p53, cdc20 levels may increase, thus activating more ubiquitin ligase complexes and promoting cyclin B degradation. Alternatively, the observed increase in the percentage of cells undergoing mitotic slippage may result from cells proliferating faster after p53 knockdown, which can increase the frequency of cells passing through mitosis. However, we did not see an increase in mitotic slippage with the stable p53 knockdown and this relationship will thus require further study.
It has been demonstrated that after mitotic slippage, non-cancer cells can be arrested in the next G1 stage in a p53-dependent manner [15
]. Similarly, after DZ treatment, we also observed an accumulation of cells with a 4N DNA content (Figure ), as well as an induction of p53 (Figure ), which is similar to the postmitotic G1 arrest of TXL-treated non-cancer cells [15
]. Hence, we suggest that when non-cancer cells are treated with microtubule inhibitors, for example DZ, they may have two "checkpoints" sequentially to ensure the fidelity of cell division. The first one is SAC, which can cause mitotic arrest if microtubules are disrupted [25
]. If SAC is not effective enough, cells may slip out from mitotic arrest and become arrested in the next G1 stage [15
]. Based on our data and previous studies, it is plausible that p53 facilitates both of these "checkpoints" after microtubule disruption as described in the model (Figure ).
Figure 7 Model: response of non-cancer cells to microtubule inhibitors. Disorazole is proposed to activate two sequential cell cycle checkpoints. The first is the APC-dependent arrest at mitosis; when that fails, a second arrest at G1 is observed. p53 stabilization (more ...)
p53 has often been found compromised in human cancer tissues [26
]. In our study, p53 is either truncated (UPCI:SCC103) or undetectable (HeLa) in cancer cell lines (data not shown). This indicates that the apoptosis triggered by DZ in cancer cells may be p53-independent.
Finally, our data suggest that after exposure to a certain concentration of DZ (10 nM in this study), cancer cells tend to execute apoptosis while non-cancer cells undergo mitotic slippage without activating the apoptotic pathways, indicating that non-cancer and cancer cells respond differently after DZ treatment. Those cells that do not die from apoptosis may become senescent, a phenotype discussed previously [[12
] and references within].