Abstract: Paclitaxel has powerful anticancer activity, but some tumors are inherently resistant to the drug, whereas others are initially sensitive but acquire resistance during treatment. To deal with this problem, it will be necessary to understand the mechanisms of drug action and resistance. Recent studies indicate that paclitaxel blocks cell division by inhibiting the detachment of microtubules from centrosomes. Here, we demonstrate that mitotic centromere-associated kinesin (MCAK), a kinesin-related protein that destabilizes microtubules, plays an important role in microtubule detachment. Depletion of MCAK altered mitotic spindle morphology, increased the frequency of lagging chromosomes, and inhibited the proliferation of WT CHO cells, confirming that it is an essential protein for cell division. In contrast, MCAK depletion rescued the proliferation of mutant paclitaxel-dependent cell lines that are unable to divide because of defective spindle function resulting from altered α-tubulin or class III β-tubulin overexpression. In concert with the correction of mitotic defects, loss of MCAK reversed an aberrantly high frequency of microtubule detachment in the mutant cells and increased their sensitivity to paclitaxel. The results indicate that MCAK affects cell sensitivity to mitotic inhibitors by modulating the frequency of microtubule detachment, and they demonstrate that changes in a microtubule-interacting protein can reverse the effects of mutant tubulin expression.
Abstract: Mechanisms to explain tumor cell resistance to drugs that target the microtubule cytoskeleton have relied on the assumption that the drugs act either to suppress microtubule dynamics or to perturb the balance between assembled and nonassembled tubulin. Recently, however, it was found that these drugs also alter the stability of microtubule attachment to centrosomes, and do so at the same concentrations that are needed to inhibit cell division. Based on this new information, a new model is presented that explains resistance resulting from a variety of molecular changes that have been reported in the literature. The improved understanding of drug action and resistance has important implications for chemotherapy with these agents.
Abstract: Abstract
Class VI β-tubulin (β6) is the most divergent tubulin produced in mammals and is found only in platelets and mature megakaryocytes. To determine how this unique tubulin isotype affects microtubule assembly and organization, we expressed the cDNA in tissue culture cells under the control of a tetracycline regulated promoter. The β6 coassembled with other endogenous β-tubulin isotypes into a normal microtubule array; but once the cells entered mitosis it caused extensive fragmentation of the microtubules, disrupted the formation of the spindle apparatus, and allowed entry into G1 phase without cytokinesis to produce large multinucleated cells. The microtubule fragments persisted into subsequent cell cycles and accumulated around the membrane in a marginal band-like appearance. The persistence of the fragments could be traced to a pronounced suppression of microtubule dynamic instability. Impairment of centrosomal nucleation also contributed to the loss of a normal microtubule cytoskeleton. Incorporation of β6 allowed microtubules to resist the effects of colcemid and maytansine, but not vinblastine or paclitaxel; however, cellular resistance to colcemid or maytansine did not occur because expression of β6 prevented cell division. The results indicate that many of the morphological features of megakaryocyte differentiation can be recapitulated in non-hematopoietic cells by β6 expression and they provide a mechanistic basis for understanding these changes.
Abstract: Drugs that target microtubules are thought to inhibit cell division and cell migration by suppressing dynamic instability, a "search and capture" behavior that allows microtubules to probe their environment. Here, we report that subtoxic drug concentrations are sufficient to inhibit plus-end microtubule dynamic instability and cell migration without affecting cell division or microtubule assembly. The higher drug concentrations needed to inhibit cell division act through a novel mechanism that generates microtubule fragments by stimulating microtubule minus-end detachment from their organizing centers. The frequency of microtubule detachment in untreated cells increases at prophase suggesting that it is a regulated cellular process important for spindle assembly and function. We conclude that drugs produce differential dose-dependent effects at microtubule plus and minus-ends to inhibit different microtubule-mediated functions.
Abstract: MCAK, a kinesin related motor protein with microtubule depolymerizing activity, is known to play an important role in spindle assembly and correcting errors in mitotic chromosome alignment. Experiments to determine how cellular levels of the protein are regulated demonstrate that MCAK accumulates during cell cycle progression, reaches a maximum at G(2)/M phase, and is rapidly degraded by the proteasome during mitosis. Immunofluorescence microscopy further indicates that MCAK largely disappears from kinetochores and spindle poles at the metaphase to anaphase transition. A phosphorylated form of MCAK appears during mitosis and seems to be preferentially degraded, but degradation does not appear to depend on Aurora B, a kinase reported to be involved in regulating the error correcting activity of the protein. These studies indicate that MCAK activity is limited during the latter stages of mitosis by protein degradation, and argue against a role for the protein in anaphase chromosome movement.
Abstract: DNA damage (determined by the Comet Assay) and the occurrence of deformed nuclei were measured as endpoints of genotoxicity in male gonad cells of the marine mussel (Perna viridis). Upon exposure of the organism to varying concentrations of extracts of smoked and non-smoked cigar tobacco over a period of 16 days, DNA damage was found to be highest in marine mussels exposed to extracts of smoked cigar tobacco. Conversely, more deformed nuclei were detected in marine mussels exposed to extracts of non-smoked cigar tobacco. The level of DNA damage and the number of deformed nuclei reach a maximum at day 12 of exposure to both extracts but decrease thereafter. This phenomenon is attributed to the organism's capacity to maintain the integrity of its genetic material upon exposure to potential genotoxicants present in the tobacco extracts. A dose response in DNA damage and deformed nuclei was also detected in isolated gonad cells upon in vitro exposure to hydrogen peroxide a known DNA strand breaking agent. The results of this study indicate that the DNA in male gonad cells of the marine mussel is damaged upon exposure to genotoxicants, and suggests the suitability of the organism for future investigations into the effect of such agents on its reproductive capacities.
