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

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Induction of C-Anaphase and Diplochromosome Through Dysregulation of Spindle Assembly Checkpoint by Sodium Arsenite in Human Fibroblasts1 [CANCER RESEARCH 63, 6680–6688, October 15, 2003] 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 18). Accumulated evidence supports the view that cytogenetic alter- ations play a crucial role in cancer development (19–22). Cytogenetic alterations induced by arsenite are associated with its Cancer cells are genetically unstable. A variety of cytogenetic or carcinogenic activity. Cytogenetic analysis revealed first that arsenite chromosomal alterations including losses and/or gains of whole or induced c-anaphases in a time- and dose-dependent manner in human large portions of chromosomes are frequently observed in cancer cells fibroblasts (HFW). With additional incubation of arsenite-arrested mi- totic cells in drug-free medium for 0–48 h, approximately 35% exited (23). To maintain the integrity during genome transmission, the proc- from mitosis without cell division. This was confirmed by the appearance ess of mitosis is stringently monitored by means of mitotic check- of tetraploid metaphase, mainly diplochromosomes, in the subsequent cell points (24, 25). Mitotic checkpoint defects can result in chromosomal division. Treatment of HFW cells with both nocodazole, a known agent of instability and lead to carcinogenesis (26, 27). Faithful chromosome microtubular depolymerization, and Taxol, which induces tubulin poly- segregation is monitored by the spindle assembly checkpoint that merization and inhibits disassembly of microtubules, resulted in remark- functions to delay the onset of anaphase until the mitotic spindles are able mitotic arrest but induced only negligible c-anaphase, tetraploidy, correctly attached to chromosome kinetochores. There are three major and diplochromosomes. Staurosporine, a kinase inhibitor that could ef- proteins involved in the spindle checkpoint: Mad2,3 p55cdc20, and fectively reduce arsenite-induced c-anaphase, could also decrease the APC/C (24). Mad2 is activated through unattached kinetochore (25) development of diplochromosomes in the subsequent cell division cycle. and is tightly associated with the APC/C and p55cdc20 complex when These results imply that arsenite-induced c-anaphases mainly exited from the spindle assembly checkpoint is activated (28). The association of mitosis without cell division and became tetraploid in the subsequent cell Mad2 with APC/C and p55cdc20 is shown to inhibit the activation of cycle. Antitubulin immunofluorescent staining confirmed no formation of bipolar spindles in nocodazole-arrested mitotic HFW cells, whereas in APC/C, to prevent degradation of Pds1, the anaphase inhibitor, and, arsenite-arrested mitotic cells bipolar spindles were present but distorted hence, to inhibit the initiation of anaphase. Malfunction of spindle in appearance and apparently dysfunctional. Mitotic arrest deficient 2 assembly checkpoint results in the initiation of abnormal anaphase (Mad2) signal was, as expected, clearly visible at centromeres of nocoda- and leads to chromosome mis-segregation. zole-arrested mitotic cells. However, the Mad2 signal at centrosomes Arsenite has been reported to alter microtubule dynamics in 3T3 became insignificant in either arsenite-arrested or nocodazole/arsenite- and HeLa cells (29–31). Our previous studies have also shown that arrested mitotic cells. In addition, the association of Mad2 with the arsenite treatment can induce abnormal chromosome segregation and cdc20 APC/C complex and the accumulation of Pds1, an anaphase inhibitor, chromosome loss in human fibroblasts and HeLa cells (14, 30). were remarkably reduced in arsenite-arrested mitotic cells as compared Arsenite can also induce c-anaphase, which has been frequently with nocodazole-arrested mitotic cells. These results support the observa- observed in solid tumor cells (32) and lymphocytes of patients with tion that nocodazole can inhibit spindle formation and, hence, activate leukemia (33, 34). C-anaphase can lead to formation of aneuploidy spindle assembly checkpoint to arrest cells at metaphase. In contrast, the dysfunctional bipolar spindles in arsenite-arrested mitotic cells could not (35), which is a manifestation of abnormal chromatid separation. The effectively activate spindle assembly checkpoint and, hence, resulted in generation of c-anaphase is possibly caused by the deranged check- formation of c-anaphase and diplochromosomes in the subsequent cell point control of metaphase to anaphase transition. In this study, division. experiments were conducted to further assess the consequences of arsenite-induced c-anaphases and the potential alterations of check- point control in arsenite-arrested mitotic cells. INTRODUCTION Arsenic compounds are known to increase the risks for many forms MATERIALS AND METHODS of cancer. Field studies have shown that ingestion of arsenic-contam- inated well water in the Southeastern area of Taiwan is highly asso- Cell Culture. HFW cells, derived from newborn human foreskin and ciated with increased incidences of cancers of skin, lung, liver, pros- kindly provided by Dr. W. N. Wen (National Taiwan University, Taiwan, Republic of China), were routinely maintained in DMEM (Life Technologies, tate, and bladder (1–6). Laboratory studies have also shown that Inc., Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone arsenite is able to cause DNA strand breaks (7–10) and induces Laboratories, Logan, UT), 0.37% sodium bicarbonate (Life Technologies, various types of cytogenetic alterations, including chromosomal ab- Inc.), 100 units/ml penicillin (Life Technologies, Inc.), and 100 ␮g/ml strep- errations, sister chromatid exchanges (11, 12), aneuploidy (13, 14), tomycin (Life Technologies, Inc.) and cultured at 37°C in an incubator with and micronuclei (15–17) in a variety of cell systems. Arsenite-induced humidity-saturated air and 10% CO2 (36). cytogenetic alterations are closely associated with its induction of Cytogenetic Examination and Mitotic Index. Logarithmically growing morphological transformation in Syrian hamster embryo cells (11, HFW cells were treated with various concentrations of sodium arsenite (0, 1.25, 2.5, and 5 ␮M) for 24 h or selected time intervals. To harvest metaphase cells, 0.05 ␮g/ml colcemid was added during the final 3-h incubation, and the Received 3/10/03; revised 7/1/03; accepted 7/30/03. The costs of publication of this article were defrayed in part by the payment of page cells were subsequently trypsinized, treated with hypotonic solution, fixed, and charges. This article must therefore be hereby marked advertisement in accordance with prepared for chromosome analysis (14). For each treatment, 100 metaphases 18 U.S.C. Section 1734 solely to indicate this fact. were examined for determination of cytogenetic alterations. Chromosomes 1 Supported by the Academia Sinica and by grants from the National Science Council, Republic of China. 2 To whom requests for reprints should be addressed, at at Institute of Biomedical 3 The abbreviations used are: Mad2, mitotic arrest deficient 2; APC/C, anaphase- Sciences, Academia Sinica, Taipei 115, Taiwan, Republic of China. Phone: 886-2- promoting complex/cyclosome; DAPI, 4,6-diamino-2-phenyl-indole; HFW, human fibro- 7899014; Fax: 886-2-7825573; E-mail: [email protected]. blast; SP, staurosporine. 6680 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2003 American Association for Cancer Research. ARSENITE INDUCES C-ANAPHASE AND DIPLOCHROMOSOME Fig. 1. Chromosome spreadings of HFW cells. A, normal chromosomes from untreated HFW cells. B, c-anaphase chromosomes from HFW cells treated with 5 ␮M arsenite for 24 h. C, diplochromosomes from HFW cells treated with 5 ␮M arsenite for 24 h and recovered in drug-free medium for 24 h. Bar,10␮m. exhibiting separated centromeres and splayed chromatids were recognized as RESULTS c-anaphases (Fig. 1B), and those that exhibited duplicated and parallel align- ment as diplochromosomes (Fig. 1C; Ref. 34). The mitotic index, defined as Dose- and Time-dependent Induction of C-Anaphases by Ar- the percentage of mitotic cells in the total cell population, was determined by senite in HFW Cells. Consistent with our previous study (14), ar- the number of mitotic cells per 1000 cells. To examine the effects of various senite treatment resulted in a slightly elevated mitotic index but inhibitors on c-anaphase formation, these inhibitors were, in general, inocu- profoundly induced c-anaphase in HFW cells (Fig. 2, A and B). lated into the cultural medium 6 h before the end of treatment. Induction of c-anaphase by arsenite follows a time- and dose-depen- Analysis of Cell Cycle Progression. Cell cycle progression was monitored dent manner (Fig. 2, A and B). At the end of a 24-h treatment, 18% using the technique of DNA flow cytometry. In brief, the trypsinized cells were and 61% of mitotic cells showed the manifestation of c-anaphases in washed once with PBS, fixed with ice-cold 70% ethanol for 16 h, and stained HFW cells treated with 2.5 ␮M and 5 ␮M arsenite, respectively (Fig. with 4 ␮g/ml propidium iodide in PBS containing 1% Triton X-100 and 0.1 2A). Treatment of HFW cells with 5 ␮M arsenite showed a remarkable mg/ml RNase A. The DNA content of individual cells was analyzed using a amount
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