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[Cancer Research 63, 6680-6688, October 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Induction of C-Anaphase and Diplochromosome through Dysregulation of Spindle Assembly Checkpoint by Sodium Arsenite in Human Fibroblasts1

Ling-Huei Yih and Te-Chang Lee2

Institutes of Zoology [L-H. Y.] and Biomedical Sciences [T-C. L.], Academia Sinica, and Institute of BioPharmaceutical Sciences, National Yang Ming University [T-C. L.], Taipei 115, Taiwan, Republic of China


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytogenetic alterations induced by arsenite are associated with its carcinogenic activity. Cytogenetic analysis revealed first that arsenite induced c-anaphases in a time- and dose-dependent manner in human fibroblasts (HFW). With additional incubation of arsenite-arrested mitotic cells in drug-free medium for 0–48 h, approximately 35% exited from mitosis without cell division. This was confirmed by the appearance of tetraploid metaphase, mainly diplochromosomes, in the subsequent cell division. Treatment of HFW cells with both nocodazole, a known agent of microtubular depolymerization, and Taxol, which induces tubulin polymerization and inhibits disassembly of microtubules, resulted in remarkable mitotic arrest but induced only negligible c-anaphase, tetraploidy, and diplochromosomes. Staurosporine, a kinase inhibitor that could effectively reduce arsenite-induced c-anaphase, could also decrease the development of diplochromosomes in the subsequent cell division cycle. These results imply that arsenite-induced c-anaphases mainly exited from mitosis without cell division and became tetraploid in the subsequent cell cycle. Antitubulin immunofluorescent staining confirmed no formation of bipolar spindles in nocodazole-arrested mitotic HFW cells, whereas in arsenite-arrested mitotic cells bipolar spindles were present but distorted in appearance and apparently dysfunctional. Mitotic arrest deficient 2 (Mad2) signal was, as expected, clearly visible at centromeres of nocodazole-arrested mitotic cells. However, the Mad2 signal at centrosomes became insignificant in either arsenite-arrested or nocodazole/arsenite-arrested mitotic cells. In addition, the association of Mad2 with the APC/Ccdc20 complex and the accumulation of Pds1, an anaphase inhibitor, were remarkably reduced in arsenite-arrested mitotic cells as compared with nocodazole-arrested mitotic cells. These results support the observation that nocodazole can inhibit spindle formation and, hence, activate spindle assembly checkpoint to arrest cells at metaphase. In contrast, the dysfunctional bipolar spindles in arsenite-arrested mitotic cells could not effectively activate spindle assembly checkpoint and, hence, resulted in formation of c-anaphase and diplochromosomes in the subsequent cell division.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Arsenic compounds are known to increase the risks for many forms of cancer. Field studies have shown that ingestion of arsenic-contaminated well water in the Southeastern area of Taiwan is highly associated with increased incidences of cancers of skin, lung, liver, prostate, and bladder (1, 2, 3, 4, 5, 6) . Laboratory studies have also shown that arsenite is able to cause DNA strand breaks (7, 8, 9, 10) and induces various types of cytogenetic alterations, including chromosomal aberrations, sister chromatid exchanges (11 , 12) , aneuploidy (13 , 14) , and micronuclei (15, 16, 17) in a variety of cell systems. Arsenite-induced cytogenetic alterations are closely associated with its induction of morphological transformation in Syrian hamster embryo cells (11 , 18) . Accumulated evidence supports the view that cytogenetic alterations play a crucial role in cancer development (19, 20, 21, 22) .

Cancer cells are genetically unstable. A variety of cytogenetic or chromosomal alterations including losses and/or gains of whole or large portions of chromosomes are frequently observed in cancer cells (23) . To maintain the integrity during genome transmission, the process of mitosis is stringently monitored by means of mitotic checkpoints (24 , 25) . Mitotic checkpoint defects can result in chromosomal instability and lead to carcinogenesis (26 , 27) . Faithful chromosome segregation is monitored by the spindle assembly checkpoint that functions to delay the onset of anaphase until the mitotic spindles are correctly attached to chromosome kinetochores. There are three major proteins involved in the spindle checkpoint: Mad2,3 p55cdc20, and APC/C (24) . Mad2 is activated through unattached kinetochore (25) and is tightly associated with the APC/C and p55cdc20 complex when the spindle assembly checkpoint is activated (28) . The association of Mad2 with APC/C and p55cdc20 is shown to inhibit the activation of APC/C, to prevent degradation of Pds1, the anaphase inhibitor, and, hence, to inhibit the initiation of anaphase. Malfunction of spindle assembly checkpoint results in the initiation of abnormal anaphase and leads to chromosome mis-segregation.

Arsenite has been reported to alter microtubule dynamics in 3T3 and HeLa cells (29, 30, 31) . Our previous studies have also shown that arsenite treatment can induce abnormal chromosome segregation and chromosome loss in human fibroblasts and HeLa cells (14 , 30) . Arsenite can also induce c-anaphase, which has been frequently observed in solid tumor cells (32) and lymphocytes of patients with leukemia (33 , 34) . C-anaphase can lead to formation of aneuploidy (35) , which is a manifestation of abnormal chromatid separation. The generation of c-anaphase is possibly caused by the deranged checkpoint control of metaphase to anaphase transition. In this study, experiments were conducted to further assess the consequences of arsenite-induced c-anaphases and the potential alterations of checkpoint control in arsenite-arrested mitotic cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
HFW cells, derived from newborn human foreskin and kindly provided by Dr. W. N. Wen (National Taiwan University, Taiwan, Republic of China), were routinely maintained in DMEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 0.37% sodium bicarbonate (Life Technologies, Inc.), 100 units/ml penicillin (Life Technologies, Inc.), and 100 µg/ml streptomycin (Life Technologies, Inc.) and cultured at 37°C in an incubator with humidity-saturated air and 10% CO2 (36) .

Cytogenetic Examination and Mitotic Index.
Logarithmically growing HFW cells were treated with various concentrations of sodium arsenite (0, 1.25, 2.5, and 5 µM) for 24 h or selected time intervals. To harvest metaphase cells, 0.05 µg/ml colcemid was added during the final 3-h incubation, and the cells were subsequently trypsinized, treated with hypotonic solution, fixed, and prepared for chromosome analysis (14) . For each treatment, 100 metaphases were examined for determination of cytogenetic alterations. Chromosomes exhibiting separated centromeres and splayed chromatids were recognized as c-anaphases (Fig. 1B)Citation , and those that exhibited duplicated and parallel alignment as diplochromosomes (Fig. 1CCitation ; Ref. 34 ). The mitotic index, defined as the percentage of mitotic cells in the total cell population, was determined by the number of mitotic cells per 1000 cells. To examine the effects of various inhibitors on c-anaphase formation, these inhibitors were, in general, inoculated into the cultural medium 6 h before the end of treatment.



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  		Fig. 1. Chromosome spreadings of HFW cells. A, normal chromosomes from untreated HFW cells. B, c-anaphase chromosomes from HFW cells treated with 5 µM arsenite for 24 h. C, diplochromosomes from HFW cells treated with 5 µM arsenite for 24 h and recovered in drug-free medium for 24 h. Bar, 10 µm.

 
Analysis of Cell Cycle Progression.
Cell cycle progression was monitored using the technique of DNA flow cytometry. In brief, the trypsinized cells were washed once with PBS, fixed with ice-cold 70% ethanol for 16 h, and stained with 4 µg/ml propidium iodide in PBS containing 1% Triton X-100 and 0.1 mg/ml RNase A. The DNA content of individual cells was analyzed using a fluorescence-activated cell sorter (FACStar, Becton Dickinson Immunocytometry Systems), as described previously (14) .

Immunofluorescence Staining of Mitotic Spindles.
HFW cells were seeded onto glass coverslips. After treatment, cells on the coverslips were washed twice with PBS and then fixed in situ with 90% methanol at -20°C for 10 min. The coverslips were washed twice with PBS and incubated with anti-ß-tubulin monoclonal antibody (Sigma Chemical Co., St. Louis, MO), anti-Mad2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-centromere antibody (Antibodies Incorporated, Davis, CA) at 4°C for 1 h. The unbound antibody was removed by extensively washing with PBS containing 0.2% Tween 20. The coverslips were incubated further with FITC- or rhodamine-conjugated secondary antibody (Organon Teknika-Cappel, Belgium) in the dark for 30 min. Chromosomes were counterstained with 0.1 µg/ml DAPI (Sigma Chemical Co.). After thoroughly rinsing with PBS containing 0.2% Tween 20, the coverslips were mounted with a 90% glycerol solution containing 1 mg/ml phenylenediamine (pH 8.0; Merck, Gibbstown, NJ). For each treatment, 1000 cells were selected randomly and examined under a fluorescence microscope (Olympus, Tokyo, Japan).

Immunoprecipitation and Immunoblot Analysis of Spindle Checkpoint Proteins.
The levels of cyclin B, Pds1, cdc27, Mad2, and p55cdc20 in total cell extracts were examined by Western blotting technique as described previously (14) . To pull down spindle checkpoint-associated proteins, an aliquot of 500-µg cellular proteins was incubated with 0.5 µg of anti-cdc27 antibody for 1 h at 4°C with agitation. Protein A-Sepharose beads (Sigma Chemical Co.) were added to the mixtures that were incubated for another 1 h at 4°C. The immuno-complex was spun down by centrifugation, washed with lysis buffer five times, and boiled in SDS-PAGE sample buffer (37) . The clear supernatants were subjected to immunoblot analysis for cdc27, Mad2, and p55cdc20 as described previously (14) . ß-Actin was used as loading control in total protein extracts. Anti-cdc27 monoclonal antibody and anti-p55cdc20 antibody were purchased from Transduction Laboratories (BD Biosciences, San Diego, CA); anti-Mad2, anti-cyclin B1, and ß-actin antibodies from Santa Cruz Biotechnology; and anti-Pds1 monoclonal antibodies form NeoMarkers Inc. (Fremont, CA), respectively. Protein concentrations were determined by Bradford analysis (38) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dose- and Time-dependent Induction of C-Anaphases by Arsenite in HFW Cells.
Consistent with our previous study (14) , arsenite treatment resulted in a slightly elevated mitotic index but profoundly induced c-anaphase in HFW cells (Fig. 2, A and B)Citation . Induction of c-anaphase by arsenite follows a time- and dose-dependent manner (Fig. 2, A and B)Citation . At the end of a 24-h treatment, 18% and 61% of mitotic cells showed the manifestation of c-anaphases in HFW cells treated with 2.5 µM and 5 µM arsenite, respectively (Fig. 2A)Citation . Treatment of HFW cells with 5 µM arsenite showed a remarkable amount of c-anaphases when the treatment exceeded 18 h (Fig. 2B)Citation . Without colcemid to accumulate mitotic cells, mitotic cells arrested by arsenite treatment also manifested mainly as c-anaphases. Alternatively, mitotic poisons that target microtubular stability such as nocodazole and Taxol arrested a large portion of cells at the mitotic stage but exhibited much less c-anaphase than arsenite in HFW cells. As shown in Fig. 3Citation , treatment of HFW with 0.4 µM nocodazole or 2 µM Taxol for 24 h resulted in 31% and 53% of cells arrested in mitotic stage, respectively, whereas among these mitotic cells only 7% and 12% showed the manifestation of c-anaphases, respectively. However, with the addition of 5 µM arsenite in nocodazole- or Taxol-treated cultures during the last 6 h of treatment (designated as nocodazole/arsenite or Taxol/arsenite treatment, respectively), the frequency of c-anaphase increased to 84% and 70%, respectively (Fig. 3)Citation . These results indicated that arsenite plays a distinct role in conversion of the metaphase cells arrested by nocodazole or Taxol into c-anaphase.



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  		Fig. 2. Time- and dose-dependent induction of c-anaphase by arsenite in HFW cells. Logarithmically growing HFW cells were treated with various concentrations of arsenite (0–5 µM), 0.4 µM nocodazole, or 2 µM Taxol for 24 h, or 5 µM arsenite (for 24 h) and 40 nM SP added during the last 6 h of treatment (A), or with 5 µM arsenite for various time length (0–24 h; B). Three hours before the end of treatment, 0.05 µg/ml colcemid was added into the culture medium. The mitotic cells were harvested for assessment of mitotic index and karyotype as described in "Materials and Methods." Data presented are the means ± SD of four independent experiments.

 


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  		Fig. 3. Effects of arsenite and SP on c-anaphase induction in nocodazole- or Taxol-arrested mitotic cells. HFW cells were treated with 0.4 µM nocodazole or 2 µM Taxol for 24 h. During the last 6 h, 5 µM arsenite alone (As), 40 nM SP alone, or 5 µM arsenite plus 40 nM SP (As + SP) were added to cultures. The mitotic cells were harvested and analyzed as described in Fig. 2Citation . Data presented are the means ± SD of four independent experiments.

 
Induction of Tetraploidy and Diplochromosomes in Arsenite-arrested Mitotic HFW Cells.
To follow the outcome of HFW cells with c-anaphase, the mitotic cells from cultures treated with arsenite or nocodazole for 24 h were shaken off and replated in drug-free medium. Similar to our previous observation in HeLa S3 cells (30) , nocodazole- and arsenite-arrested mitotic cells showed differential kinetics of mitotic exit. As shown in Fig. 4Citation , the number of nocodazole-arrested mitotic cells (0.2 µM for 24 h) was quickly decreased to 60% within 1 h and to 17% within 3 h after release from nocodazole blockage. In contrast, arsenite-arrested mitotic cells (5 µM for 24 h) were only slightly decreased to 85% within 3 h and 24% within 6 h after discontinuation of arsenite treatment. The time required for arsenite-arrested mitotic cells to exit from mitotic stage was delayed approximately 4 h compared with that for nocodazole-arrested mitotic cells. These results confirmed that nocodazole-induced blockage of spindle assembly could be recovered quickly after the removal of nocodazole and, hence, exit from mitosis. The interference and delay of mitosis caused by arsenite are consistent with our previous observations (14) .



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  		Fig. 4. Kinetics of arsenite- and nocodazole-arrested mitotic HFW cells to exit from mitosis. HFW cells were treated with 5 µM arsenite ({blacktriangledown}) or 0.2 µM nocodazole ({bullet}) for 24 h. Mitotic cells were shaken off and reincubated in drug-free medium for 0–8 h. Cells were then harvested for mitotic index analysis. Data presented are the average of two independent experiments.

 
With additional culturing of the arsenite- and nocodazole-arrested mitotic cells in drug-free media, obvious differences emerged. As summarized in Table 1Citation , with incubation in drug-free medium for 8 h, the mitotic index of the shaken-off nocodazole-arrested mitotic cells dropped from 78% to 1.7%, whereas the distribution of the G2-M phases was 9% in the total population. The reappearance of mitotic index peak at 24 h after culturing in drug-free medium suggested that most nocodazole-arrested cells not only completed the cell division but also progressed forward to the second division cycle by 24 h. As for arsenite-arrested mitotic cells, after culturing in drug-free medium for 8 h, the mitotic index and the frequency of c-anaphase cells decreased from 83% to 6.1% and from 61% to 21%, respectively (Table 1)Citation . However, flow cytometry analysis revealed that the distribution of cells with 4N DNA population remained as high as 35%. These results indicate that arsenite-arrested mitotic cells, mainly c-anaphase cells, could exit from mitosis, but approximately 35% of the cells exited from mitosis without cell division. We, therefore, suspected the appearance of tetraploid cells within subsequent mitosis.


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  		Table 1 Consequence of mitotic arrest cells induced by arsenite

HFW cells were treated with 5 µM arsenite or 0.4 µM nocodazole for 24 h. The mitotic cells were shaken off and replated in drug-free medium for the times indicated. Cells were then harvested for analysis of mitotic index, c-anaphases, cell cycle stages, and karyotypes. Data presented are the average of two independent experiments.

 
To demonstrate the induction of tetraploid cells in arsenite-arrested mitotic cells, the karyotype was examined at 24 and 48 h subsequent to incubation of the shaking-off cells in drug-free medium. As shown in Table 1Citation , 39% and 21% of metaphases were tetraploidy (mainly 92 chromosomes). Among these tetraploid cells, 70% manifested diplochromosomes or endoreduplicated chromosomes (i.e., the duplicated chromosomes were aligned in parallel; Fig. 1CCitation ). The appearance of 39% of tetraploid cells after a 24-h incubation in drug-free medium was consistent in that the DNA content of 35% of cells remained as 4N after an 8-h incubation in drug-free medium. The decrease of the frequency of tetraploidy after a 48-h incubation in drug-free medium may be caused by the comparatively slower growth rate of tetraploid cells to diploid cells. By reincubating the arsenite-arrested mitotic cells for 20 and 40 h in drug-free medium, 81% and 89% of cells were mononucleated cells (Fig. 5B)Citation and only 11% and 3% were binucleated cells (Fig. 5C)Citation , respectively, as examined microscopically. These results imply that most arsenite-arrested mitotic cells exited from mitosis after an adaptation period. In contrast, few tetraploid cells were observed with incubation of nocodazole-arrested mitotic cells in drug-free medium for 24 and 48 h, indicating that nocodazole-arrested mitotic cells could complete their cell division after nocodazole was removed.



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  		Fig. 5. Morphology of HFW cells. A, normal HFW cells. B, mononucleated HFW cells. The arsenite-arrested mitotic HFW cells were reincubated in drug-free medium for 20 h. C, binucleated HFW cells. The arsenite-arrested mitotic cells were reincubated in drug-free medium for 20 h. Bar, 50 µm.

 
To further confirm whether the arsenite-induced c-anaphase could exit mitosis without cell division and lead to the formation of tetraploidy, we harvested the mitotic cells (84% of them were c-anaphase) from nocodazole/arsenite-treated HFW cells (as described in Fig. 3Citation ) and incubated them for an additional 30 h in drug-free media. As expected, 41% of metaphase cells manifested as tetraploidy and 21% as diplochromosomes (Table 2)Citation . As a control, mitotic cells from treatment with nocodazole alone showed a low frequency of c-anaphase (Fig. 3)Citation as well as very little tetraploidy (Table 2)Citation . As similar to that shown in Table 1Citation , mitotic cells from treatment with arsenite alone showed 46% tetraploidy and 33% diplochromosomes. Furthermore, several kinase and phosphatase inhibitors were tested for their effects on arsenite induction of c-anaphases, including vanadate, okadaic acid, genistein, dimethyl aminopurine, wortmannin, and SP. Only SP could effectively reduce arsenite-induced c-anaphases to background level (Fig. 2A)Citation . Additionally, SP could abrogate c-anaphase induction in nocodazole- or Taxol-arrested mitotic cells that were treated with arsenite during the last 6 h of their incubation (Fig. 3)Citation . In addition to reducing the frequency of arsenite-induced c-anaphases, SP treatment significantly reduced the frequency of diplochromosomes induced by arsenite in the subsequent cell division (Table 2)Citation . These results indicate that the SP-sensitive kinase(s) might be involved in the induction of c-anaphases by arsenite and suggest that arsenite-induced c-anaphases might exit from mitosis without cell division and, thus, lead to formation of tetraploidy with diplochromosomes in their subsequent cell division.


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  		Table 2 Induction of tetraploidy by arsenite in nocodazole-arrested mitotic cells

HFW cells were treated with 5 µM arsenite or 0.2 µM nocodazole for 24 h. During the last 6 h of treatment, 5 µM arsenite or 40 nM SP or 5 µM arsenite plus 40 nM SP were added to the cultures. The mitotic cells were shaken off and incubated in drug-free medium for 30 h. Cells were then harvested for analysis of mitotic index and karyotype. Data presented are the average of two independent experiments.

 
Induction of Abnormal Mitotic Spindles and Disturbance of Chromosome Segregation by Arsenite in HFW Cells.
To explore how c-anaphase is formed by arsenite treatment, we conducted experiments to examine the effects of arsenite on mitosis progression in HFW cells. In nocodazole-arrested mitotic cells (0.2 µM for 24 h), in which tubulin polymerization was inhibited, only short aster fibers nucleated from spindle pole were observed by immunostaining with anti-ß-tubulin antibodies (Fig. 6A)Citation . Chromosomes of nocodazole-arrested mitotic cells were morphologically categorized as pro-metaphase chromosomes, as revealed by counterstaining with DAPI (Fig. 6B)Citation . In arsenite-arrested mitotic cells (5 µM for 24 h), the bipolar mitotic spindles were formed, but with distortion and aggregation (Fig. 6C)Citation , similar to those observed in our previous study (14 , 30) . Chromosomes in arsenite-arrested mitotic cells were either scattered within the cytoplasm (70%) or aggregated at the metaphase plate (21%, Fig. 6DCitation ). A few anaphases with laggards and dividing cells with micronuclei were also observed (Fig. 6, E and F)Citation . The present results confirm our previous findings that arsenite can disturb the progress of mitosis. Although distorted bipolar spindles were formed in arsenite-arrested mitotic HFW cells, these cells could eventually adapt to the perturbation caused by arsenite and exit from mitosis with or without cell division.



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  		Fig. 6. Effects of arsenite on the assembly of bipolar spindles and congregation of metaphase chromosomes in HFW cells. HFW cells were treated with 0.2 µM nocodazole (A and B) or 5 µM arsenite (C–F) for 24 h, fixed in situ with 90% methanol for 10 min at -20°C, immunostained with anti-ß-tubulin antibodies, and visualized with rhodamine-conjugated secondary antibodies (A, C, and E). Nuclei and chromosomes were counterstained with DAPI (B, D, and F). Bar, 10 µm.

 
Attenuation of Mad2 Interaction with Kinetochores by Arsenite.
The mitotic spindles in arsenite-arrested mitotic cells were bipolar, but with distorted and aggregated morphology, suggesting that these spindle fibers could not attach normally to chromosomes and perform their functions of chromosomal congression and segregation. In general, defective bipolar spindles will activate spindle assembly checkpoint to prevent the mitotic cells from incorrect chromosome segregation (24) . Mad2, the inhibitory regulator of anaphase initiation, is recruited by unattached kinetochores (39 , 40) and released into cytoplasm to activate spindle assembly checkpoint (28 , 41 , 42) . Using an immunohistochemical staining technique, Mad2 signal (Fig. 7B)Citation was found to be invisible at centromeres (Fig. 7A)Citation of normal metaphase HFW cells but became significantly visible at centromeres of all nocodazole-arrested mitotic cells (Fig. 7, D, E, and G)Citation . In contrast, most arsenite-arrested mitotic cells (83%), although with laggard chromosomes (Fig. 7K)Citation , showed negligible Mad2 signal at centromeres (Fig. 7J)Citation . The remaining 17% of the arsenite-arrested mitotic cells showed a positive Mad2 immunoreactivity at the centromere, but with attenuated signal (data not shown). Moreover, by nocodazole/arsenite treatment, the intensity of Mad2 signal at centromeres of these nocodazole-arrested mitotic cells was attenuated (Fig. 7M)Citation . Mad2, activated through interaction with the unattached kinetochores and released into cytoplasm, was associated with the APC/C and p55cdc20 (APC/Ccdc20) complex to inhibit the proteolytic events required for the onset of chromatid separation. Our results suggest that abnormal and dysfunctional mitotic spindles induced by arsenite might interfere with the interaction between Mad2 and kinetochores and, hence, were unable to fully activate Mad2 and spindle checkpoint. Therefore, chromatid separation was initiated.



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  		Fig. 7. Effects of sodium arsenite on cellular Mad2 localization in HFW cells. HFW cells were untreated (A–C) and treated with 0.2 µM nocodazole for 24 h (D–H), 5 µM arsenite for 24 h (I–K), or 0.2 µM nocodazole (for 24 h) and 5 µM arsenite added during the last 6 h of treatment (L and N). Cells were then fixed in 90% methanol for 10 min at -20°C. Localization of Mad2 at centromeres in nocodazole-arrested mitotic cells was demonstrated by double staining cells with anti-centromere antibody (A and D) and Mad2 antibodies (B and E). Mitotic spindles were visualized by immunostaining with anti-ß-tubulin antibodies and the FITC-conjugated secondary antibodies (F, I, and L), whereas Mad2 was localized by immunostaining with anti-Mad2 antibodies and rhodamine-conjugated secondary antibodies (G, J, and M). Chromosomes were counterstained with DAPI (C, F, H, K, and N).

 
Analysis of APC/C and p55cdc20 Complexes in Nocodazole-, Arsenite-, and Nocodazole/Arsenite-treated Cells.
Because the activated APC/Ccdc20 complex is a crucial factor for triggering chromatid separation, we, therefore, examined the components of the APC/Ccdc20 complex by immunoblot analysis. HFW cells were treated with arsenite (5 µM for 24 h), nocodazole (0.2 µM for 24 h), or nocodazole/arsenite. By shaking off the round-up cells, 81, 93, and 89% of harvested cells from nocodazole-, arsenite-, and nocodazole/arsenite-treated cultures, respectively, were mitotic cells examined microscopically. As shown in Fig. 8ACitation , cyclin B was highly abundant in the shaken-off cells from arsenite-, nocodazole-, and nocodazole/arsenite-treated cultures, whereas little cyclin B was detected in the attached cells. These results confirmed that the shaken-off cells were mainly mitotic cells, whereas the attached cells were the interphase cells. In the shaken-off cells from nocodazole-treated cultures, cdc27, a component of APC/C, was super-shifted to a higher molecular weight as compared with that in the attached cells in which cdc27 was maintained as a Mr 90,000 protein (Fig. 8A)Citation . These results are consistent with previous reports that have shown that cdc27 is hyperphosphorylated during mitosis and its mobility retarded on SDS-PAGE (43 , 44) . The supershift of cdc27 was also observed in arsenite-arrested mitotic cells, but the phosphorylation patterns of cdc27 apparently differed from that in nocodazole-arrested mitotic cells. As estimated by densitometry, 37% of cdc27 in arsenite-arrested mitotic cells moved faster than the hyperphosphorylated forms on the SDS-PAGE, indicative of incomplete phosphorylation. Similar results were also observed in the mitotic cells from nocodazole/arsenite-treated cultures, indicating that arsenite might also alter cdc27 phosphorylation in nocodazole-arrested mitotic cells. Alternatively, p55cdc20 and Mad2 remained at a constant level in cycling, attached, and shaken-off cells from untreated or treated cultures. During the transition from metaphase to anaphase, activated APC/Ccdc20 should cleave the anaphase inhibitor Pds1. As shown in Fig. 8ACitation , Pds1 showed a striking appearance in the mitotic cells from nocodazole-treated cultures, indicating that APC/Ccdc20 was inactivated by the spindle checkpoint and, hence, unable to cleave Pds1 in nocodazole-arrested mitotic cells. In contrast, the levels of Pds1 were remarkably reduced in arsenite-arrested mitotic cells, indicating that APC/Ccdc20 remains active and is able to cleave Pds1. Therefore, no significant amount of Pds1 is accumulated in arsenite-arrested mitotic cells. Moreover, in nocodazole/arsenite-treated cultures, the Pds1 level was significantly reduced to 41% as compared with the band intensity to that of nocodazole-treated mitotic cells. These results indicated that arsenite, in addition to altering mitotic spindles, might also attenuate the function of spindle checkpoint activated by nocodazole.



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  		Fig. 8. Immunoprecipitation and immunoblot analysis of proteins associated with spindle assembly checkpoint. A, immunoblots of cdc27, p55cdc20, Mad2, cyclin B1, Pds-1, and ß-actin of total cell extract from logarithmically growing HFW cells (cycling), interphase cells (A) remaining attached in culture dishes, and mitotic cells (F) shaken off from HFW cells treated with 5 µM arsenite for 24 h (As), 0.2 µM nocodazole for 24 h (Noco), or 0.2 µM nocodazole (for 24 h) and arsenite added during the last 6 h of treatment (Noco/As). The boxes represent the regions subjected for densitometry analysis (Densitometer 300S; Molecular Dynamics, Sunnyvale, CA). The numbers on the left indicated the percentage of band intensity. B, immunoblots of cdc27, p55cdc20, and Mad2 of cdc27 immunoprecipitates from HFW cells treated as in A. The ratio of Mad2 to total cdc27 was determined by quantifying the band intensities with a densitometer. Data presented are one of two independent experiments.

 
Decreased Association of Mad2 and APC/Ccdc20 in Arsenite-arrested Mitotic Cells.
The interaction of activated Mad2 with APC/Ccdc20 complex is a crucial factor for the activation of spindle assembly checkpoint that prevents the chromatid separation before mitotic spindles are properly attached on the kinetochores (45) . To confirm whether arsenite could alter the function of spindle checkpoint, we analyzed the complex formation of Mad2 with APC/Ccdc20 by immunoprecipation and immunoblot analysis. As shown in Fig. 8BCitation , immunoblotting of the cdc27-immunoprecipitates by anti-cdc27 antibodies revealed similar supershift patterns of cdc27 in the shaken-off mitotic cells from nocodazole-, arsenite-, and nocodazole/arsenite-treated cultures as shown in Fig. 8ACitation . Immunoblotting analysis of the cdc27-immunoprecipitates with anti-p55cdc20 antibodies indicated that p55cdc20 forms stable complexes with hyperphosphorylated cdc27 from all of the drug-treated mitotic cells, whereas only few were associated with hypophosphorylated cdc27 from the untreated cycling cells. However, immunoblotting of the cdc27 immunoprecipitates with anti-Mad2 antibodies showed that significantly less Mad2 protein could be observed in the mitotic cells from arsenite-treated cultures than that from nocodazole-treated cultures. Moreover, the levels of Mad2 associated with cdc27 were significantly decreased in nocodazole/arsenite-treated cultures. As analyzed by a densitometer, the ratio of band intensities of Mad2 to total cdc27 in mitotic cells from arsenite-, nocodazole-, and nocodazole/arsenite-treated cultures was 0.07, 0.54, and 0.21, respectively. The association of Mad2 and the APC/Ccdc20 complex confirmed the activation of spindle assembly checkpoint in nocodazole-arrested mitotic cells, whereas reduced association of Mad and APC/Ccdc20 complex supported no full activation of spindle assembly checkpoint in arsenite-arrested mitotic cells. Furthermore, arsenite treatment decreased the association of Mad2 to the APC/Ccdc20 complex, indicating that arsenite might attenuate the activation of spindle checkpoint induced by nocodazole.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mitotic cells manifested as c-anaphases indicate that they overcome the inhibitory constraint of colcemid and initiate the anaphase onset (46) . Cytogenetic analysis in the present study revealed that 61% of arsenite-arrested mitotic HFW cells manifested as c-anaphases. Induction of c-anaphase by arsenite suggests that arsenite treatment is unable to fully terminate the mitotic process during the transition of metaphase to anaphase (i.e., separation of two chromatids). In addition to c-anaphase induction, arsenite treatment also results in overcondensed chromosomes with separated centromeres, as reported previously (14) . The correlation of appearance of c-anaphase and diplochromosome supports the view that a portion of arsenite-induced c-anaphase could exit mitosis without chromosome segregation and, hence, form diplochromosomes in their subsequent cell division. Parallel abrogation of arsenite-induced c-anaphases and diplochromosomes in nocodazole-arrested mitotic cells by kinase inhibitor SP (Fig. 1CCitation and Table 2Citation ) further confirms that diplochromosomes are derived from c-anaphase cells. Because clinical studies have shown that diplochromosomes evolve to form hyperdiploidy during the subsequent development of tumor (47 , 48) , the induction of c-anaphase may underlie the ability of arsenite to cause chromosome instability.

The appearance of c-anaphase indicated the premature onset of anaphase in arsenite-arrested mitotic HFW cells. Our previous results demonstrated that arsenite could interfere with the functions of mitotic spindles by attenuating the dynamics of tubulin and, hence, induce mitotic arrest in HeLa cells (30) . In this study, we demonstrated that arsenite-induced abnormal mitotic spindles were unable to effectively block the onset of metaphase to anaphase transition, indicating that spindle checkpoint was not properly activated in arsenite-arrested mitotic cells. Thereafter, the defective bipolar spindles would not allow completion of normal chromosome segregation and cytokinesis. The induction of c-anaphase in nocodazole-arrested mitotic cells by arsenite further implies that arsenite could disrupt the constraint of metaphase arrest hold by spindle checkpoint. To trigger the transition of metaphase into anaphase, the exit from mitosis, and cytokinesis is generally known to be via the activation of APC/C, a multi-subunit complex with ubiquitin-ligase activity (49) , and by its association with activator proteins p55cdc20 or cdh1 to form the APC/Ccdc20 or APC/Ccdh1 complex (50) . The APC/Ccdc20 complex is required for the onset of anaphase by triggering the proteolytic degradation of Pds1 (51) , whereas the APC/Ccdh1 complex is needed for cytokinesis and mitotic exit by complete degradation of mitotic cyclins (52) . The sequential activation of APC/Ccdc20 and APC/Ccdh1 plays a key role in establishing the temporal order of cell cycle control (53) . Disruption of the temporal order of cell cycle progression might severely alter chromosome segregation and lead to loss of genomic integrity (26 , 53, 54, 55) . Improper interaction between mitotic spindles and chromosomes is known to activate the spindle assembly checkpoint and, hence, to inhibit APC/C activity and prevent mis-segregation of chromosomes (56) . Nocodazole treatment in the present study resulted in inhibition of spindle assembly, activation of spindle assembly checkpoint, and mitotic arrest at the prometaphase stage. As compared with that of nocodazole-arrested mitotic cells, a lesser amount of Mad2 was associated with the APC/Ccdc20 complex, and a negligibly low level of the anaphase inhibitor Pds1 in arsenite-arrested mitotic cells (Fig. 8)Citation indicated that spindle assembly checkpoint was probably not activated in arsenite-arrested mitotic cells. Through a binding/release model (28) , Mad2 is activated at unattached kinetochores, released into cytoplasm, and interacts with the APC/Ccdc20 complex to prevent from the onset of anaphase. After spindle attachment to kinetochores, Mad2 is no longer able to be recruited to kinetochores and loses its binding activity to the APC/Ccdc20 complex. Our results showing the decreased interaction of Mad2 and the APC/Ccdc20 complex indicated that spindle assembly checkpoint was not fully activated and, hence, the chromatid separation was not inhibited in arsenite-arrested mitotic cells. Furthermore, the reduced interaction between Mad2 and the APC/Ccdc20 complex by arsenite in the mitotic cells from nocodazole plus arsenite-treated culture further implied that arsenite could attenuate the activity of spindle checkpoint induced by nocodazole, resulting in a high frequency of c-anaphase formation, the onset of premature anaphase. The mechanism underlying which arsenite decreases the interaction of Mad2 with the APC/Ccdc20 complex is currently unknown.

It has been reported that the aberrant spindles do not themselves induce an arrest, whereas unattached kinetochores do lead to a delay of anaphase onset (57) . Hut et al. (58) reported that cells entering mitosis in the presence of impaired DNA integrity would lead to inactivation of centrosomes and formation of aberrant spindles, which delayed mitosis progression, however, did not block cytokinesis and mitotic exit. We have previously demonstrated that arsenite could induce DNA strand breaks in HFW cells (9) and elongated bipolar spindles by attenuating the dynamics of tubulin in HeLa cells (30) . Arsenite, thus, might induce abnormal mitotic spindles through induction of DNA damages and inactivation of centrosomes while not affecting the bipolar attachment of kinetochores to microtubules. Therefore, in arsenite-arrested mitotic cells, the spindle checkpoint is probably not activated, and, hence, premature onset of anaphase is initiated. However, the result that arsenite could reduce the activity of spindle checkpoint induced by nocodazole indicated that arsenite treatment could also inactivate the function of the preactivated spindle checkpoint. As expected, cdc27, a component of APC/C, was hyperphosphorylated in nocodazole-arrested mitotic cells (Fig. 8)Citation . However, 37% cdc27 was not fully phosphorylated in arsenite-arrested mitotic cells. Although it is not clear whether the altered phosphorylation pattern of cdc27 in arsenite-arrested mitotic cells can explain why spindle assembly checkpoint was not activated, arsenite treatment apparently disturbed some cascades of protein phosphorylation and dephosphorylation. A number of mitotic kinases (such as cdc2 kinase, polo kinase, and aurora kinase) have been shown to phosphorylate many spindle-associated proteins and regulate the microtubule dynamics (53) . Overexpression of aurora-A, commonly found in epithelial cancers, was shown to override the mitotic spindle checkpoint and, hence, enable the onset of inappropriate anaphase despite defective spindle formation and the persistence of Mad2 at the kinetochores (59) . Similarly, aurora-B overexpression caused increased lagging chromosomes during mitosis (60) . On the contrary, inhibition of cyclin-dependent kinases by flavipiridol can prevent endoreduplication and polyploidy formation induced by nocodazole in cells with defective G1 checkpoint (61) . Among several kinase and phosphatase inhibitors used in this study, only SP could remarkably reduce the induction of c-anaphases and diplochromosomes in arsenite-arrested mitotic cells. This result provides a possible explanation in that arsenite may inactivate spindle checkpoint by modulating a SP-sensitive kinase.

The effect of arsenite on mitosis of HFW cells apparently differs from that in a variety of tumor cell lines. It has been shown that arsenite only slightly increases the mitotic index in HFW cells but mimics spindle poisons to remarkably arrest tumor cell lines at metaphase within a similar dose range (<5 µM; Refs. 30 , 31 , 62 ). Furthermore, although mitotic cells arrested by arsenite in those tumor cell lines underwent apoptosis after arsenite exposure and were removed, apoptosis was seldom observed in HFW cells. HFW cells are normal fibroblasts with the wild-type p53 gene. Treatment of HFW cells with 5 µM arsenite can cause DNA damage and p53 protein accumulation (9) . In p53 wild-type cells, rapid activation of G1-S checkpoints in response to DNA injury halts the cell cycle progression for repair or triggers apoptosis to prevent any chance of potentiating or fixing the damage later in the cell cycle (63) . Therefore, by arsenite treatment, the chance of p53 wild-type HFW cells reaching the late stage of mitosis was less than HeLa cells that might be unable to sense the presence of damaged DNA because lack of functional p53. Alternatively, the failure of G1-S and S phase checkpoints in tumor cells does not prevent the damaged cells from progressing to the final stages of mitosis. Mitotic apoptosis is then remarkably triggered (30 , 31 , 62) . In HeLa cells, the induction of apoptosis by arsenite has been associated with delayed cyclin B degradation and altered mitotic exit (64) . The mitotic apoptosis induced by arsenite or arsenic trioxide could possibly allow trivalent arsenic compounds to be used as anticancer drugs in checkpoint-deficient tumor cells (65) . The ability of arsenite to abrogate spindle checkpoint indicated that arsenite could be further applied to cancer therapy in combination with other chemotherapeutic agents.

Chromosome instability is now recognized as a major cause of cancers (23) . Tetraploidy is frequently observed even at an early stage of tumor development and is considered to induce increased instability of the genome with the likelihood of further chromosome changes (66) . We have previously showed that arsenite prolongs the duration of mitosis and disturbs the progression of mitosis in HFW cells. Among the survival clones, 21% developed aneuploidy (14) . In this study, we have shown that re-initiation of mitotic progression occurs in arsenite-arrested HFW cells after a period of adaptation. However, the distorted bipolar spindles perturbs the faithful segregation of chromosomes and results in c-anaphases that then became tetraploidy, especially in the form of diplochromosomes, in their subsequent cell division. The cells with diplochromosmes potentially increase the chance to producing offspring cells with manifestation of unstable karyotype.


   ACKNOWLEDGMENTS
 
We thank Dr. Cyprian Weaver for carefully reading this manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the Academia Sinica and by grants from the National Science Council, Republic of China. Back

2 To whom requests for reprints should be addressed, at at Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, Republic of China. Phone: 886-2-7899014; Fax: 886-2-7825573; E-mail: bmtcl{at}ibms.sinica.edu.tw Back

3 The abbreviations used are: Mad2, mitotic arrest deficient 2; APC/C, anaphase-promoting complex/cyclosome; DAPI, 4,6-diamino-2-phenyl-indole; HFW, human fibroblast; SP, staurosporine. Back

Received 3/10/03. Revised 7/ 1/03. Accepted 7/30/03.


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 DISCUSSION
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