Mitotic slippage in non-cancer cells induced by a microtubule disruptor, disorazole C1
© Xu et al. 2010
Received: 23 February 2009
Accepted: 11 February 2010
Published: 11 February 2010
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© Xu et al. 2010
Received: 23 February 2009
Accepted: 11 February 2010
Published: 11 February 2010
Disorazoles are polyene macrodiolides isolated from a myxobacterium fermentation broth. Disorazole C1 was newly synthesized and found to depolymerize microtubules and cause mitotic arrest. Here we examined the cellular responses to disorazole C1 in both non-cancer and cancer cells and compared our results to vinblastine and taxol.
In non-cancer cells, disorazole C1 induced a prolonged mitotic arrest, followed by mitotic slippage, as confirmed by live cell imaging and cell cycle analysis. This mitotic slippage was associated with cyclin B degradation, but did not require p53. Four assays for apoptosis, including western blotting for poly(ADP-ribose) polymerase cleavage, microscopic analyses for cytochrome C release and annexin V staining, and gel electrophoresis examination for DNA laddering, were conducted and demonstrated little induction of apoptosis in non-cancer cells treated with disorazole C1. On the contrary, we observed an activated apoptotic pathway in cancer cells, suggesting that normal and malignant cells respond differently to disorazole C1.
Our studies demonstrate that non-cancer cells undergo mitotic slippage in a cyclin B-dependent and p53-independent manner after prolonged mitotic arrest caused by disorazole C1. In contrast, cancer cells induce the apoptotic pathway after disorazole C1 treatment, indicating a possibly significant therapeutic window for this compound.
Microtubules are dynamic polymers that facilitate transport and movement within the cell . Microtubule dynamics are a critical aspect of mitosis, ensuring accurate chromosome capture and segregation [1, 2]. Factors that interfere with microtubule attachment keep the mitotic spindle checkpoint unsatisfied, thus causing mitotic arrest and inhibition of cell proliferation .
Microtubule dynamics can be modified by two groups of chemical inhibitors. The first group, represented by taxanes, stabilizes microtubules and is clinically used to treat breast, lung, bladder and head and neck cancers . The second group of modifiers include vinblastine, vincristine, and vinorelbine, disrupt microtubules and are used in the treatment of leukemia, lymphoma, small cell lung and breast cancer, and other malignancies . Although these chemotherapeutic drugs are efficacious, new drug development is still needed due to intrinsic and acquired drug resistance and untoward actions of existing therapeutics.
Intensive research has focused on the responses of cancer cells to microtubule inhibitors, and it is known that apoptosis is a common outcome triggered by either p53-dependent or p53-independent pathways [[6, 7] and references within]. However, the response of non-transformed cells to microtubule inhibitors is less well understood. Contrasting the response of non-cancer cells and tumor cells should enhance our understanding of the effects of microtubule inhibitors on cancer patients.
The disorazoles are a family of natural compounds isolated from the fermentation broth of the myxobacteria Sorangium cellulosum . Disorazole A1, one of the major components, was demonstrated to disrupt microtubules, leading to mitotic arrest and eventually apoptosis . Disorazole C1 (DZ), a relatively minor constituent of the fermentation mixture, was obtained synthetically since it was considered to have greater therapeutic potential due to the absence of the chemically reactive divinyl oxirane and (E, Z)-dienyl oxazole moieties [8, 10, 11] (Additional file 1). DZ was found to cause microtubule depolymerization and mitotic arrest during a small molecule screen . In this paper, we further examined the cellular responses to DZ and compared our results to two known microtubule inhibitors, vinblastine (VBL) and taxol (TXL).
One property that microtubule inhibitors share is that they cause mitotic arrest by activating or preventing the satisfaction of the spindle assembly checkpoint . DAPI-staining of chromosomes was used to demonstrate an increase in the mitotic index in both non-cancer and cancer cells 16 hours after DZ treatment (Figure 1b). Similar results were observed using phosphorylated histone H3 staining (data not shown).
Finally, cell proliferation is typically blocked by microtubule disruption. After treatment with 10 nM of DZ, the proliferation of immortalized human foreskin fibroblast cells (HFF-hTERT) was inhibited, as determined by the MTS assay (Figure 1c). Similar results were also obtained in RPE-hTERT cells (Additional file 2). In summary, DZ disrupts microtubules, arrests cells in mitosis and inhibits cell proliferation, similar to classical tubulin inhibitors.
To confirm that mitotic slippage was the source of the fragmented nuclei in the majority of the population, serum starvation was used to arrest cells in G1. After a 38-hour treatment with 10 nM of DZ, less than 5% of the serum-starved RPE-hTERT cells showed fragmented nuclei, indicating that the observed nuclear fragmentation required progression through the cell cycle (Figure 2d). Similar results were obtained in hydroxyurea-treated cells, which were arrested in S phase, confirming that the nuclear fragmentation required transit through mitosis (Figure 2d). Although the cycling cells were able to escape the DZ-induced arrest, they did not resume a normal cell cycle but arrested again in G1, as indicated by FACS analysis at 38 hours of DZ exposure (Figure 2e).
To study whether protein degradation was required for the observed mitotic slippage, we used the protease inhibitor MG132 to block cyclin B degradation. In MG132-treated cells, the percentage of cells undergoing mitotic slippage was dramatically reduced while the mitotic index remained high, indicating that cells blocked for proteasomal protein degradation were less likely to undergo mitotic slippage from DZ (Figure 4c). Our data support the conclusion that cyclin B degradation was correlated with mitotic slippage in non-cancer cells after DZ treatment.
Even when cells were treated with DZ for 68 or 72 hours, very little apoptosis was observed (data not shown). Furthermore, other non-cancer cell lines, such as HFF-hTERT (immortalized human foreskin fibroblasts) and UP3 cells (primary human oral cells grown in culture), also showed very little or no PARP cleavage after 38 hours of 10 nM DZ treatment (Additional file 5). Additionally, DNA ladder formation was not observed in RPE-hTERT cells exposed to DZ (Additional file 6). Taken together, our results indicate that the apoptotic pathway was not activated in the RPE-hTERT cells exposed to DZ. However, PARP cleavage was strongly observed in all the tested cancer cell lines (Figure 6d), suggesting apoptosis pathway was activated in malignant cells by DZ.
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 , 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 2d), 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 . 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 . 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 ; 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 , which is demonstrated to be required for the proteolysis of cyclin B by activating anaphase-promoting complex, a mitotic ubiquitin ligase . 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.
p53 has often been found compromised in human cancer tissues . 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 [ and references within].
In this study, we analyzed the cellular response to a synthesized natural product, DZ. Although mitotic arrest induced by DZ was observed in both non-cancer and cancer cells, these cells had different fates. Non-cancer cells escaped from mitotic arrest (mitotic slippage) in a cyclin B-dependent and p53-independent manner without apoptosis. In contrast, cancer cells activated the apoptosis pathway. These differential responses observed after DZ treatment were similar to the effects of vinblastine or taxol, suggesting a promising therapeutic potential of DZ.
HFF-hTERT vector only (human foreskin fibroblast immortalized with human telomerase reverse transcriptase) and HFF-hTERT shp53 (stable-knockdown of p53) cells (gifts from Dr. Edward Prochownik, University of Pittsburgh) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine. RPE-HTERT cells (retinal pigmented epithelial cell line stably transfected with human telomerase reverse transcriptase) were maintained in DMEM/F12 supplemented with 10% FBS. All UPCI:SCC cells (oral squamous carcinomas cells, gifts from Dr. Susanne Gollin, University of Pittsburgh) were maintained in MEM supplemented with 10% FBS and 1% non-essential amino acid. UP3 (uvulopalatopharyngoplasty) cells (gift from Dr. Susanne Gollin) were maintained in KGM2 medium. U2OS cells (ATCC) were maintained in McCoy's 5A supplemented with 10% FBS. Generally, cells were treated with 10 nM DZ or 100 nM vinblastine or 1 μM taxol for 38 hours at 37°C before analysis, unless where indicated. Vinblastine concentration was chosen to demonstrate a similar level of microtubule disruption compared with 10 nM of DZ. Cell permeable caspase inhibitors (caspase-3/7, -8, -9 inhibitors) (Calbiochem) were used as in reference Jennings JJ et al., 2006 .
Cells were fixed in 3.7% paraformaldehyde at room temperature for 15 minutes followed by 7 minute-incubation of 0.1% triton X-100 at 4°C. Primary α-tubulin antibodies (Abcam, Cambridge, MA) or cytochrome C antibodies (Santa Cruz) were detected with the application of Alexa Fluor488 or 568 (Molecular Probes, Invitrogen) as the secondary antibodies. All antibodies were diluted in 1% bovine serum albumin (BSA)/PBS. DNA-specific fluorescent dye 4,6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis) was used to visualize DNA. The samples were examined using a 40× or 100× oil immersion objective (UPlanApo) at an Olympus BX60 epifluorescence microscope. Images were taken using a Hamamatsu Argus-20 CCD camera (Hamamatsu Co., Bridgewater, NJ)
Cells were seeded on 35-mm glass-bottom culture dish (MatTek Corporation, Ashland, MA) and treated with DZ at a final concentration of 10 nM for 16 hours before analysis. Epifluorescence microscopy was performed on a Nikon TE2000-U inverted microscope with a CoolSNAP HQ digital camera (Roper Scientific Photometrics) while cells were maintained at 37°C in a moisturized and air-heated microscope chamber (Life Imaging Services, Reinach, Switzerland). Images were taken and analyzed using MetaMorph software (Molecular Devices).
Antibodies against cyclin B (BD, San Jose, CA), actin (Cytoskeleton, Denver, CO), p53 (Santa Cruz Biotechnology, Santa Cruz, CA), PARP (Cell Signaling Technology, Danvers, MA) were used as primary antibodies and all diluted in 5% milk/Tris-buffered Saline with 0.5% Tween-20 (TBST). Anti-mouse or anti-rabbit IgG-HRP-linked secondary antibodies (Amersham, GE Healthcare, UK) were also diluted in 5% milk/TBST. Results were visualized using enhanced chemiluminescent kit (Pierce).
Cell proliferation was measured using the MTS assay kit (Promega; tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS]) following the manufacture's instruction. In annexin-V assays, Tetramethyl Rhodamine Methyl Ester (TMRM) diluted in regular buffer was used to stain mitochondria at 37°C for 10 minutes before the addition of annexin-V (Molecular Probes, Invitrogen, CA) following the manufacture's instruction. MitoTracker (Invitrogen, CA) staining was performed following the manufacture's protocol.
Cells were fixed with cold 70% EtOH and stained with propidium iodide (Fluka: BioChemika, Switzerland) at a final concentration of 50 μg/ml. Analysis of flow cytometry was performed in Flow Cytometry Facility in University of Pittsburgh Cancer Institute (UPCI).
Cells were seeded onto 60-mm culture dishes five hour before siRNA transfection. sip53 (Qiagen) was transfected into cells using HiPerFect Transfection Reagent (Qiagen) following the manufacture's instruction.
Cells grown on plastic dishes were fixed in 2.5% glutaraldehyde or paraformaldehyde in 0.1 M Na-cacodylate (30 min), washed with 0.1 M Na-cacodylate, post-fixed with 1% OsO4, washed with PBS and stained for 30 min with 2% uranyl acetate. Following dehydration in 30% to 100% ethanol, the samples were embedded in resin by immersion in 30% to 100% resin:propylene oxide mixtures. Fixed samples were mounted on grids and analyzed with a JEOL 100CX transmission electron microscope.
We thank Dr. Edward Prochownik (University of Pittsburgh) for HFF-hTERT vector only cells and HFF-hTERT shp53 cells, Dr. Susanne Gollin (University of Pittsburgh) for all the UPCI:SCC cell lines and UP3 cells. We thank E. Michael Meyer at Flow Cytometry Facility in University of Pittsburgh Cancer Institute for the help in flow cytometry analysis. We also thank Dr. Thomas Graham for synthesizing DZ and Kristen M. Bartoli for help with this manuscript. This study was supported by the National Institute of Health (NIH) grant ROI DE 016086-01 to Dr. William S. Saunders and NIH grant (National Cancer Institute) CA78039 to Dr. John S. Lazo.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.