Depletion of Securin Increases Arsenite-Induced Chromosome Instability
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《毒物学科学杂志》
Molecular Anticancer Laboratory, Institute of Pharmacology and Toxicology, College of Life Sciences, Tzu Chi University, Hualien, Taiwan
Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Kaohsiung, Taiwan
ABSTRACT
Arsenic is a pathologic factor of cardiovascular diseases and cancers; nevertheless, it also acts as an anticancer agent effective on acute promyelocytic leukemia and multiple myeloma. Securin, a proposed proto-oncogene, regulates cell proliferation and tumorigenesis. However, roles of securin on the arsenic-induced cell cycle arrest and apoptosis remain unknown. In this study, the effects of sodium arsenite on the expression of securin in two tissue types of cell lines, the vascular endothelial and colorectal epithelial cells, were investigated. Arsenite (8–16 μM, 24 h) increased the cytotoxicity, apoptosis, and growth inhibition in both endothelial and epithelial cells. The levels of phospho-CDC2 (threonine-161), CDC2, and cyclin B1 proteins were decreased, and the G2/M fractions were increased by arsenite. Concomitantly, arsenite markedly diminished the securin protein expression and induced the abnormal sister chromatid separation. The depletion of securin proteins increased the induction of mitotic arrest, aberrant chromosome segregation, and apoptosis after arsenite treatment. p53, a tumor suppressor protein, balances the cell survival and apoptosis. Arsenite raised the levels of phospho-p53 (serine-15) and p53 (DO-1) proteins in both the securin-wild-type and -null cells. The p53-functional cells were more susceptible than the p53-mutational cells to arsenite on the cytotoxicity and apoptosis. Besides, arsenite decreased the levels of securin proteins to a similar degree in both the p53-functional and -mutational cells. Together, it is the first time to demonstrate that the inhibition of securin expression induced by arsenite increases the chromosomal instability and apoptosis via a p53-independent pathway.
Key Words: arsenite; apoptosis; securin; separase; mitosis; p53.
INTRODUCTION
Securin has been proposed as an oncogene that is overexpressed in various cancer cells (Bernal et al., 2002; Dominguez et al., 1998; Hamid et al., 2005). It consists of a homologous family of proteins expressed in different species that includes Cut2 in fission yeast, Pds1 in budding yeast, Pimples in Drosophila, and hSecurin in human (Dominguez et al., 1998; Nagao et al., 2004; Zou et al., 1999). In vertebrates, securin is also termed as the vSecurin or pituitary-tumor transforming gene (Dominguez et al., 1998; Pei and Melmed, 1997; Saez et al., 1999; Zou et al., 1999). Securin can promote the cell proliferation and tumorigenesis (Bernal et al., 2002; Hamid et al., 2005; Zhang et al., 1999; Zou et al., 1999). In general conditions, securin acts as a secure protein to prevent abnormal sister chromatid separation in the mitosis progression by binding with separase, an enzyme that cleaves the chromosomal cohesin (Jallepalli et al., 2001; Nasmyth, 2001; Stemmann et al., 2001). Securin can prevent the aberrant chromosome segregation when cellular DNA and spindle are damaged (Funabiki et al., 1996; Yamamoto et al., 1996). Recently, it has been shown that securin regulates DNA repair following UV and X-ray damages (Nagao et al., 2004). However, overexpression of securin induces aneuploidy (Christopoulou et al., 2003), genetic instability (Kim et al., 2005), and apoptosis (Hamid and Kakar, 2004). Thus, the precise roles of securin on the regulation of cell cycle progression and apoptosis are still ambiguous.
p53 is a tumor suppressor protein that regulates cell cycle arrest, apoptosis, and DNA repair (Amundson et al., 2005; Bates and Vousden, 1996; Hofseth et al., 2004; Levine, 1997). The expression of downstream proteins regulated by p53 includes p21 to mediate cell cycle arrest and Bax to induce apoptosis (Agarwal et al., 1995; Bates and Vousden, 1996; Hofseth et al., 2004). The various phosphorylation sites of p53 have been shown to play different roles on the regulation of cellular stresses (Canman and Lim, 1998; Dumaz and Meek, 1999; Meek, 1999). For example, the phosphorylation site of serine-15 on p53 mediates the activation and stabilization of p53 proteins (Canman and Lim, 1998; Dumaz and Meek, 1999; Meek, 1999). Moreover, it has been shown that p53 can inhibit the securin expression following treatment with anticancer drugs such as doxorubicin and bleomycein (Zhou et al., 2003). The overexpressed securin proteins activate p53 to induce the Bax expression and to mediate apoptosis (Hamid and Kakar, 2004). However, securin interacts with p53 to block the specific binding of p53 to DNA and inhibits the latter's transcriptional activity resulting in the prevention of apoptosis (Bernal et al., 2002). Accordingly, the interaction of securin and p53 on the balance of cell survival and apoptosis remains unclear.
Arsenic is a ubiquitous environmental toxicant that has been classified as a human carcinogen (IARC, 1987). It has been shown that arsenic is able to cause DNA and chromosome damages (Lynn et al., 2000; Oya-Ohta et al., 1996; Yih et al., 1997). Nevertheless, arsenic has been used for the treatment of certain types of cancer including acute promyelocytic leukemia and multiple myeloma (Hayashi et al., 2003; Perkins et al., 2000; Shen et al., 1997; Zhao et al., 2001). Chronic exposure to the low levels of arsenic (0.5–5 μM) may increase the incidences of several different human cancers (Chen et al., 1992; Chen and Wang, 1990; Chiou et al., 1995). Furthermore, higher concentrations of arsenic may induce apoptosis in a variety of cancer cells (Ivanov and Hei, 2004; Nimmanapalli et al., 2003; Scholz et al., 2005). Epidemiological studies demonstrated a high association of peripheral vascular diseases with arsenic exposure (Chen et al., 1995; Engel et al., 1994; Tseng et al., 1997). The vascular endothelial cells are an important target for the vasculopathy following arsenic exposure (Engel et al., 1994; Lee et al., 2003; Liu and Jan, 2000; Tseng et al., 1997). However, the regulation of securin and p53 in the arsenic-induced chromosome damage and apoptosis is inconclusive.
Two tissue types of cell lines including the mouse vascular endothelial and human colorectal epithelial cells were examined on the possible roles of securin and p53 proteins in the arsenite-induced cell injury. Arsenite markedly decreased the securin protein expression but increased the p53 protein expression in both endothelial and epithelial cells. Furthermore, the blockage of securin enhanced the induction of cytotoxicity, apoptosis, mitotic arrest, and aberrant sister chromatid segregation after treatment with arsenite. Moreover, we found that the depletion of securin proteins increased the abnormal chromosomal segregation and apoptosis from arsenite exposure via a p53-independent pathway, and the p53-mediated cell cycle arrest and apoptosis induced by arsenite was not related to securin.
MATERIALS AND METHODS
Chemicals and antibodies.
Hoechst 33258, propidium iodide, 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT), sodium arsenite (NaAsO2), and the Cy3-labeled mouse anti--tubulin (c-4585) were purchased from Sigma Chemical (St. Louis, MO). Anti-phospho-CDC2 (threonine-161) (9114S), anti-phospho-p53 (serine-15) (9284S), and goat anti-rabbit IgG-horseradish peroxidase (#7074) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Anti-ERK-2 (C-14), anti-p53 (DO-1), goat anti-mouse IgG-horseradish peroxidase (sc-2005), and the FITC (fluorescein isothiocyanate)-labeled goat anti-mouse IgG antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The monoclonal anti-securin (ab-3305) and polyclonal anti-separase (ab-3762) were purchased from Abcam (Cambridgeshire, UK). Anti-CDC2 (Ab-1) and anti-cyclin B1 (Ab-2) were purchased from Oncogene Sciences Products (Boston, MA). The Cy5-labeled goat anti-rabbit IgG was purchased from Amersham Biosciences.
Cell lines and cell culture.
The SVEC4–10 cell line (ATCC, #CRL-2181) was derived from mouse vascular endothelium that was purchased from Food Industry Research and Development Institute (Hsinchu, Taiwan). The securin-wild-type and -null HCT116 colorectal cancer cell lines (Jallepalli et al., 2001) were kindly provided by Dr. B. Vogelstein of the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins. RKO (ATCC, #CRL-2577) was a colorectal carcinoma cell line that expresses the wild-type p53 proteins (Bhat et al., 1997). SW480 (ATCC, #CRL-228) was established from the colorectal adenocarcinoma of a white male whose p53 gene contains a G to A mutation in codon 273 and a C to T mutation in codon 309 (Rodrigues et al., 1990; Weiss et al., 1993). The SVEC4–10, RKO, and SW480 cells were routinely maintained in DMEM medium (Invitrogen Co., Carlsbad, CA). The DMEM-complete medium was supplemented with 10% fetal bovine serum, L-glutamine (0.03%, w/v), 100 unit/ml penicillin, and 100 μg/ml streptomycin. The HCT116 cells were cultured in the complete Macoy's 5A medium (Sigma Chemical). These cell lines were maintained at 37°C and 5% CO2 in a humidified incubator (310/Thermo, Forma Scientific, Inc., Marietta, OH).
Cytotoxicity assay.
To analyze the induction of cytotoxicity, the cell viability was determined by the MTT colorimetric assay as described (Kuo et al., 2004; Plumb et al., 1989). The cells were plated in 96-well plates at a density of 1 x 104 cells/well for 16–20 h. Then the cells were treated with arsenite in complete medium. After treatment, the cells were carefully and slightly washed with isotonic phosphate-buffered saline (PBS) to avoid the loss of mitotically round-up cells, and were recultured in complete medium for 2 days. Subsequently, the medium was replaced, and the cells were incubated with 0.5 mg/ml of MTT in complete medium for 4 h. The surviving cells converted MTT to formazan that produced a blue-purple color when dissolved in dimethyl sulfoxide. The intensity was measured at 565 nm using a 96-well plate reader (OPTImax; GE Healthcare). The relative percentage of viability was calculated by dividing the absorbance of treated cells by that of the control in each experiment.
Apoptosis analysis.
To evaluate the level of apoptosis, the adherent cells were cultured on coverslip in a 60-mm Petri dish for 16–20 h before treatment. After treatment, the cells were carefully and slightly washed with isotonic PBS (pH 7.4), and incubated with 4% paraformaldehyde solution in PBS for 1 h at 37°C. Then the nuclei were stained with 2.5 μg/ml Hoechst 33258 for 30 min. The number of apoptotic nuclei was counted by a hemocytometer under a fluorescence microscope. In addition, the cell morphology of apoptosis was confirmed by observation of the cell membrane blebbing and the formation of apoptotic bodies under phase contrast microscope. At least 200 cells were examined from the random fields for the calculation of apoptotic percentage in each treatment. Furthermore, the person counting cells was blinded as to which cell line and treatment were being counted.
Cell growth assay.
The cells were plated at a density of 5 x 105 cells per 100-mm Petri dish in complete medium for 16–18 h. After drug treatment, the cells were carefully and slightly washed with PBS and recultured in complete medium. Thereafter, the cells were incubated for various times before they were counted by a hemocytometer.
Cell cycle analysis.
For analysis of cell cycle, the cells were plated at a density of 1 x 106 cells per 60-mm Petri dish in complete medium for 16–20 h. At the end of treatment, the cultured medium was collected, and the cells were trypsinized before being centrifuged. After centrifugation, the cells were collected and fixed with ice-cold 70% ethanol overnight at –20°C. Thereafter, the cell pellets were treated with 4 μg/ml propidium iodide solution containing 1% Triton X-100 and 100 μg/ml RNase for 30 min. To avoid cell aggregation, the cell solutions were filtered through nylon membrane (BD Biosciences, San Jose, CA). Subsequently, the samples were analyzed in a BD LSR system (BD Biosciences) using CellQuest software. A total of 1 x104 cells were analyzed for DNA content, and the percentage of cell cycle phases was quantified by a ModFit LT software (Ver. 3.0, BD Biosciences).
Mitotic index and the ratio of anaphase/mitosis.
The adherent cells were cultured on coverslip in a 60-mm Petri dish for 16–20 h before treatment. After treatment, the cells were carefully and slightly washed with PBS (pH 7.4) to avoid the loss of mitotically round-up cells, and then fixed with 4% paraformaldehyde solution in PBS for one hour at 37°C. Thereafter, the -tubulin was stained with the Cy3-labeled mouse anti--tubulin (1:50) for 30 min at 37°C. Finally, the nuclei were stained with 2.5 μg/ml Hoechst 33258 for 30 min. The cells were counted by a hemocytometer under a fluorescence microscope. At least 500 cells were examined for the calculation of mitotic index. For analysis of the ratio of anaphase/mitosis, at least 100 mitotic cells were counted in each treatment. The mitotic cells showed round-up morphology, compact chromosomes, spindle formation, and contained intact cell membrane but did not produce the cell membrane blebbing and the formation of apoptotic bodies. The cells of anaphase were going through the stage of chromosome separation.
Immunofluorescence staining and confocal microscopy.
To view the location and expression of proteins, the cells were subjected to immunofluorescence staining and confocal microscopy as described (Kuo et al., 2004). After fixation, the cells were washed three times with PBS, and nonspecific binding sites were blocked in PBS containing 10% normal bovine serum and 0.3% Triton X-100 for 1 h. Briefly, the cells were incubated with rabbit anti-separase (1:250), rabbit anti-phospho-p53 (serine-15) (1:250), mouse anti-p53 (DO-1) (1:50), or mouse anti-securin (1:50) antibodies in PBS containing 10% FBS for overnight at 4°C, and washed three times with 0.3% Triton X-100 in PBS. Then the cells were incubated with goat anti-rabbit Cy5 (1:250) or goat anti-mouse FITC (1:50) in PBS containing 10% normal bovine serum for 3 h at 37°C. Thereafter, the -tubulin was stained with the Cy3-labeled mouse anti--tubulin (1:50) for 30 min at 37°C. Finally, the nuclei were stained with 2.5 μg/ml Hoechst 33258 for 30 min. The samples were immediately examined under a Leica confocal laser scanning microscope (Leica, Wetzlar, Germany) that was equipped with a UV laser (351/364 nm), an Ar laser (457/488/514 nm), and a HeNe laser (543 nm/633 nm).
Western blot analysis.
The total cellular protein extracts were prepared as described (Chao et al., 2004; Kuo et al., 2004). The protein concentration was determined by using the BCA protein assay kit (Pierce, Rockford, IL). Western analyses of cyclin B1, CDC2, phospho-CDC2 (threonine-161), securin, separase, and ERK-2 were performed using specific antibodies. Briefly, equivalent amounts (50–100 μg/well) were separated on 10–12% sodium dodecyl sulfate-polyacrylamide gels and electrophoretic transfer of proteins onto polyvinylidene difluoride membranes. The membranes were sequentially hybridized with primary antibody and followed with a horseradish peroxidase-conjugated secondary antibody. Finally, the protein bands were visualized on the X-ray film using the enhanced chemiluminescence detection system (PerkinElmer Life and Analytical Sciences, Boston, MA). A gel-digitizing software, Un-Scan-It gel (ver. 5.1; Silk Scientific, Inc., Orem, UT), was used to quantify the intensity of each band on the X-ray film.
Statistical analysis.
Data were analyzed by one-way or two-way analysis of variance (ANOVA), and further post-hoc tests using the statistic software of GraphPad Prism 4 (GraphPad software, Inc. San Diego, CA). Differences among control and arsenite-treated samples were compared by one-way ANOVA with post-hoc Tukey's tests. Differences among the securin-wild-type and securin-null HCT116 cells (or RKO and SW480 cells) from the control and arsenite treatments were compared by two-way ANOVA with Bonferroni post-tests. A p value of <0.05 was considered as statistically significant in each experiment.
RESULTS
Arsenite Decreases the Cell Viability, Inhibits the Cell Growth, and Increases the G2/M Fractions in the SVEC4–10 Endothelial Cells
The vascular endothelial cells are the important targets of cardiovascular diseases resulting from arsenic exposure (Engel et al., 1994; Tseng et al., 1997). A vascular endothelial cell line, SVEC4–10, was used to study the effects of arsenite on the endothelial cell injury. Treatment with 4–16 μM arsenite for 24 h significantly decreased the cell viability in a concentration-dependent manner in the SVEC4–10 cells (Fig. 1A). Moreover, the growth ability of SVEC4–10 cells was inhibited by arsenite (Fig. 1B). To further investigate the effect of arsenite on the cell cycle arrest, the arsenite-treated cells were analyzed by flow cytometer. As shown in Figure 2A, arsenite (8–16 μM, 24 h) decreased the G1 fractions, while increasing the G2/M fractions in the SVEC4–10 cells. The level of G1 fractions was diminished 25%, and the G2/M fractions were increased 10% after treatment with 16 μM arsenite for 24 h; at the same time, the sub-G1 fractions (apoptotic fractions) were increased 20% by arsenite in the SVEC4–10 cells (Fig. 2A). Immunoblot analysis showed that arsenite reduced the levels of cell cycle-regulated proteins including phospho-CDC2 (threonine-161), total CDC2, and cyclin B1 proteins (Figs. 2B and 2C). In contrast, the level of p53 (DO-1) proteins was markedly elevated in the arsenite-treated cells in a concentration- and time-dependent manner (Figs. 2B and 2C). ERK-2 was used as an internal control that was not altered by arsenite. Previously, ERK-2 has been used as an internal control after treatment with a variety of chemicals (Chao et al., 2004; Kuo et al., 2004). Furthermore, the level of actin, a common internal protein, was not changed in the arsenite-treated cells (data not shown).
Arsenite Decreases the Securin Protein Expression in Both the Vascular Endothelial and Securin-Wild-Type Epithelial Cells
The SVEC4–10 endothelial and securin-wild-type HCT116 colorectal epithelial cells were subjected to immunofluorescence staining and confocal microscopy to examine the expression and location of securin proteins. As shown in Figure 3A, the securin and separase proteins were expressed and colocalized on the nuclei of SVEC4–10 cells (Fig. 3A, arrows). Moreover, the securin proteins were highly expressed at the prophase and metaphase in the securin-wild-type HCT116 cells (Fig. 4A, arrows). After metaphase, the securin proteins were reduced during the anaphase and telophase (Fig. 4A). To further investigate the effects of arsenite on the expression of securin and separase proteins, the cells were subjected to immunoblot analysis. Treatment with 12–16 μM arsenite for 24 h significantly decreased the level of securin proteins in the SVEC4–10 cells (Fig. 3B). Furthermore, arsenite time-dependently inhibited the securin protein expression, but the level of separase proteins was slightly increased by arsenite (Fig. 3C). Two major bands (120 kD and 76 kD) of separase proteins were detected in the immunoblot (Fig. 3C). It has been shown that separase proteins can be auto-cleaved, and the enzyme activity is preserved for anaphase entry (Chestukhin et al., 2003; Peters, 2002). The level of securin proteins was diminished with 4–16 μM arsenite in the securin-wild-type HCT116 cells (Fig. 4B). Consistently, the green fluorescence (FITC) exhibited by securin was significantly decreased by arsenite in the securin-wild-type HCT116 cells (Fig. 4C). However, the securin-null HCT116 cells did not express any securin proteins following arsenite exposure (Fig. 4B).
Loss of Securin Enhances the Cytotoxicity and Apoptosis in the Arsenite-Exposed Cells
The cell survival and apoptosis in the securin-wild-type and -null HCT116 cells were compared after arsenite treatment. As shown in Figure 5A, arsenite (4–16 μM, 24 h) concentration-dependently induced cytotoxicity in both the securin-wild-type and -null HCT116 cells. The securin-null cells were more susceptible to cell death than the securin-wild-type cells treated with 8–16 μM arsenite for 24 h (Fig. 5A). The percentage of apoptotic nuclei was counted under a fluorescence microscope by nuclear staining. Arsenite significantly increased the level of apoptosis in both the securin-wild-type and -null cells (Fig. 5B). However, the loss of securin proteins enhanced the induction of apoptosis by arsenite (Fig. 5B).
Blockade of Securin Increases the G2/M Phases, Mitotic Index, and Abnormal Anaphase in the Arsenite-Treated Cells
We also examined the role of securin on the cell cycle progression treated with arsenite. Arsenite significantly decreased the G1 fractions (Figs. 6A and 6D) and increased the G2/M fractions (Figs. 6C and 6F) in both the securin-wild-type and -null HCT116 cells. However, arsenite produced a large increase in the G2/M fractions of the securin-null HCT116 cells (Fig. 6F). To further determine whether G2 or M phases were increased by arsenite, the cells were analyzed by morphological observation and mitotic index as described in "Materials and Methods." The mitotic cells were increased following treatment with 16 μM arsenite for 24 h in both the securin-wild-type and -null cells (Fig. 7A, arrows). Moreover, the increase of mitotic phases was greater in the securin-null cells than the securin-wild-type cells after treatment with 16 μM arsenite for 24 h (Fig. 7B). As shown in Figure 8A, the abnormal anaphase (aberrant chromosome separation) was increased by arsenite in both the securin-wild-type and -null HCT116 cells. Comparing the ratio of anaphase/mitosis, the securin-null cells were more susceptible than the securin-wild-type cells to the increase of the percentage of aberrant anaphase after arsenite treatment (Fig. 8B). Eventually, cells with arsenite-induced abnormal chromosome separation went into apoptosis (Fig. 8C). The arrows of Figure 8C indicated the formation of apoptotic bodies.
Activation of p53 via Securin-Independent Pathway Following Arsenite Exposure
To investigate the expression and activation of p53 proteins in the securin-wild-type and -null cells after arsenite exposure, the cells were subjected to immunoblot analysis and immunofluorescence staining. The levels of phospho-p53 (serine-15) and p53 (DO-1) proteins were increased after exposure to 4–16 μM arsenite for 24 h in both securin-wild-type and -null HCT116 cells (Fig. 9A). As shown in Figure 9B, the intensities of green fluorescence (FITC) exhibited by p53 (DO-1) and the red fluorescence (Cy5) exhibited by phospho-p53 (serine-15) proteins were elevated by arsenite (Fig. 9B, arrows). Besides, the phospho-p53 (serine-15) and p53 (DO-1) proteins were concentrated on the nuclei in both securin-wild-type and -null cells following treatment with 16 μM arsenite for 24 h (Fig. 9B, arrows).
Inhibition of Securin Protein Expression Is Independent of p53 after Arsenite Treatment
As shown in Figure 10A, the p53-functional RKO cells were more susceptible than the p53-mutational SW480 cells to the cytotoxicity after treatment with 16–32 μM arsenite for 24 h. Additionally, the increase in the levels of sub-G1 and G2/M fractions was significantly greater for the p53-functional cells than the p53-mutational cells after arsenite treatment (data not shown). However, treatment with 8–16 μM arsenite for 24 h decreased the level of securin proteins to a similar extent in both the p53-functional and -mutational cells (Fig. 10B).
DISCUSSION
The cytotoxic anti-tumor agents such as arsenic and X-ray offer an important therapeutic strategy to treat cancer, but also can become a pathogenic factor on carcinogenesis from the induction of aberrant chromosomes. Chromosome instability is associated with the progression of carcinogenesis (Lengauer et al., 1997, 1998). However, if the chromosome damage is beyond repair, the cells will enter into apoptotic pathway leading to cell death. Accordingly, the balance of chromosome instability and apoptosis may be involved in the pathogenesis of cancer or may be used for cancer therapy.
To maintain the integrity during chromosome transmission, the process of mitosis is strictly monitored by spindle checkpoint (Musacchio and Hardwick, 2002). It controls sister chromatid separation, progression from metaphase to anaphase; its defects can result in chromosomal instability (Jallepalli and Lengauer, 2001; Millband et al., 2002; Musacchio and Hardwick, 2002). Securin has been proposed to be required for genome stability in mitosis (Jallepalli et al., 2001; Yanagida, 2000). In a recent study, securin was found to regulate DNA repair following UV and X-ray damage (Nagao et al., 2004). DNA and chromosome damage will activate defense proteins to mediate cell cycle arrest, allowing for repair of the damage. In the present study, we found that arsenite markedly diminished the securin protein expression in both the vascular endothelial and colorectal epithelial cells. Moreover, the securin-null cells were more susceptible than the securin-wild-type cells to the induction of aberrant anaphase following arsenite treatment. Thus, we suggest that the depletion of securin protein promotes the aberrant chromosomal segregation and induces chromosome instability in the arsenite-treated cells.
Activation of CDC2/cyclin B1 complex is required for the mitotic entry, and its inactivation occurs at late anaphase. The activation of CDC2 is through phosphorylation of threonine-161 by CDC2 activating kinase (Pines, 1999). Arsenite decreased the levels of phospho-CDC2 (threonine-161), total CDC2, and cyclin B1 proteins. However, the level of separase proteins was slightly increased by arsenite. The CDC2/cyclin B1 complex can mediate the phosphorylation and inactivation of separase (Nasmyth, 2002). Moreover, the activity of separase is inhibited by binding with securin in metaphase (Nasmyth, 2001; Stemmann et al., 2001). At the metaphase-anaphase transition, the securin is degraded, and separase is released to mediate the separation of sister chromatids through cleavage of the chromosomal cohesin (Nasmyth, 2001; Stemmann et al., 2001). Accordingly, we suggest that arsenite-induced the reduction of phospho-CDC2 (threonine-161), CDC2, cyclin B1, and securin proteins results in the release of the separase, allowing it to digest the sister chromatid cohesion; however, it causes the formation of abnormal chromosome segregation. The anaphase-promoting complex (APC) is one of important proteins participated in the regulation of the spindle checkpoint, which can mediate the cyclin B degradation during the metaphase to anaphase transition (Nasmyth, 2002). Securin can be degraded via ubiquitylation by APC (Cohen-Fix et al., 1996; Funabiki et al., 1996). The imbalance of APC-dependent proteolysis in the cell cycle regulation may lead to genomic instability of cancer cells (Wasch and Engelbert, 2005). Thus, it is possible that the decrease of securin and cyclin B1 proteins elicited by arsenite may be via the APC-mediated protein degradation. Nevertheless, the precise mechanisms of separase and APC on the regulation of arsenite-induced chromosome instability need further investigation.
Securin has been proposed as an oncogene that is expressed abundantly in most cancer cells (Dominguez et al., 1998; Pei and Melmed, 1997; Saez et al., 1999; Zou et al., 1999). It promotes cell proliferation and tumorigenesis (Hamid et al., 2005; Zhang et al., 1999; Zou et al., 1999). Therefore, the inhibition of securin would block the cell survival and proliferation in tumor cells, providing important strategy in cancer therapy. Interestingly, arsenite markedly decreased the levels of securin proteins and increased the cytotoxicity, apoptosis, and cell growth inhibition in both the vascular endothelial and colorectal carcinoma cells. The inhibition of endothelial cell growth may block tumor growth through anti-angiogenesis and is useful in cancer therapy (Marx, 2003). Furthermore, the securin-null colorectal carcinoma cells were more susceptible to the induction of cell growth inhibition and cell death than the securin-wild-type colorectal carcinoma cells after arsenite treatment. Recently, treatment with anticancer agents, such as UV, doxorubicin, and bleomycin, was observed to decrease the securin expression in cancer cells (Romero et al., 2004; Zhou et al., 2003). Consequently, we suggest that the inhibition of securin protein expression by arsenite may increase the cell growth inhibition and apoptosis in vascular endothelial and cancer cells.
p53 is a cellular gatekeeper for the cell growth and division (Bates and Vousden, 1996; Hofseth et al., 2004; Kuo et al., 2004; Levine, 1997). It has been shown that p53 protein mediates the G1 and G2/M checkpoints (Agarwal et al., 1995). The phosphorylation of p53 at serine-15 participates in the p53 activation and stabilization (Canman and Lim, 1998; Dumaz and Meek, 1999; Meek, 1999). We found that arsenite increased the level of phospho-p53 (serine-15) and total p53 proteins in both endothelial and epithelial cells. Moreover, the loss of functional p53 reduced the cytotoxicity, apoptosis, and G2/M fractions in the arsenite-treated cells. These data suggest that the activation of p53 mediates the cell cycle arrest and apoptosis following arsenite treatment. Interaction of p53 and securin has been investigated in recent years; however, it is not unambiguous. p53 can mediate the inhibition of securin expression after DNA damage agents such as doxorubicin and bleomycein (Zhou et al., 2003). Overexpression of securin activates the p53 expression to mediate the Bax activation, leading to the induction of apoptosis (Hamid and Kakar, 2004). In contrast, securin can also interact with p53 to block the transcriptional activity of p53, which can prevent apoptosis (Bernal et al., 2002). In this study, the loss of securin expression increased the cell death and G2/M arrest in the arsenite-treated cells. However, arsenite similarly raised the levels of phospho-p53 (serine-15) and p53 (DO-1) proteins in the securin-wild-type and -null cells. Besides, the securin proteins were reduced by arsenite in both the p53-functional and -mutational cells. Accordingly, the activation of p53 may be via a securin-independent pathway, and the inhibition of securin protein expression is not associated with p53 in the arsenite-treated cells.
In conclusion, we propose that the securin proteins play a critical role in the maintenance of chromosome stability and cell survival following arsenite exposure. Reduction of securin proteins by arsenite may increase the chromosome instability and apoptosis. Conversely, the activation of p53 by arsenite mediates cell cycle arrest and apoptosis. However, there is no cross-talk between p53 and securin in the arsenite-treated cells. The investigation of securin and p53 proteins on the arsenite-induced chromosome instability and apoptosis may elucidate the mechanism of carcinogenesis and offer further application on cancer therapy.
SUPPLEMENTARY DATA
Supplementary data are available online at http://toxsci.oxfordjournals.org/.
ACKNOWLEDGMENTS
We are indebted to Dr. T. H. Chiu (Institute of Pharmacology and Toxicology, Tzu Chi University) for careful reading of the manuscript. We also thank Dr. B. Vogelstein of the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins for permission to use the securin-wild-type and securin-null HCT116 colorectal cancer cell lines. This work was supported by National Science Council, Taiwan, Grant NSC 93–2320-B-320–014.
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Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Kaohsiung, Taiwan
ABSTRACT
Arsenic is a pathologic factor of cardiovascular diseases and cancers; nevertheless, it also acts as an anticancer agent effective on acute promyelocytic leukemia and multiple myeloma. Securin, a proposed proto-oncogene, regulates cell proliferation and tumorigenesis. However, roles of securin on the arsenic-induced cell cycle arrest and apoptosis remain unknown. In this study, the effects of sodium arsenite on the expression of securin in two tissue types of cell lines, the vascular endothelial and colorectal epithelial cells, were investigated. Arsenite (8–16 μM, 24 h) increased the cytotoxicity, apoptosis, and growth inhibition in both endothelial and epithelial cells. The levels of phospho-CDC2 (threonine-161), CDC2, and cyclin B1 proteins were decreased, and the G2/M fractions were increased by arsenite. Concomitantly, arsenite markedly diminished the securin protein expression and induced the abnormal sister chromatid separation. The depletion of securin proteins increased the induction of mitotic arrest, aberrant chromosome segregation, and apoptosis after arsenite treatment. p53, a tumor suppressor protein, balances the cell survival and apoptosis. Arsenite raised the levels of phospho-p53 (serine-15) and p53 (DO-1) proteins in both the securin-wild-type and -null cells. The p53-functional cells were more susceptible than the p53-mutational cells to arsenite on the cytotoxicity and apoptosis. Besides, arsenite decreased the levels of securin proteins to a similar degree in both the p53-functional and -mutational cells. Together, it is the first time to demonstrate that the inhibition of securin expression induced by arsenite increases the chromosomal instability and apoptosis via a p53-independent pathway.
Key Words: arsenite; apoptosis; securin; separase; mitosis; p53.
INTRODUCTION
Securin has been proposed as an oncogene that is overexpressed in various cancer cells (Bernal et al., 2002; Dominguez et al., 1998; Hamid et al., 2005). It consists of a homologous family of proteins expressed in different species that includes Cut2 in fission yeast, Pds1 in budding yeast, Pimples in Drosophila, and hSecurin in human (Dominguez et al., 1998; Nagao et al., 2004; Zou et al., 1999). In vertebrates, securin is also termed as the vSecurin or pituitary-tumor transforming gene (Dominguez et al., 1998; Pei and Melmed, 1997; Saez et al., 1999; Zou et al., 1999). Securin can promote the cell proliferation and tumorigenesis (Bernal et al., 2002; Hamid et al., 2005; Zhang et al., 1999; Zou et al., 1999). In general conditions, securin acts as a secure protein to prevent abnormal sister chromatid separation in the mitosis progression by binding with separase, an enzyme that cleaves the chromosomal cohesin (Jallepalli et al., 2001; Nasmyth, 2001; Stemmann et al., 2001). Securin can prevent the aberrant chromosome segregation when cellular DNA and spindle are damaged (Funabiki et al., 1996; Yamamoto et al., 1996). Recently, it has been shown that securin regulates DNA repair following UV and X-ray damages (Nagao et al., 2004). However, overexpression of securin induces aneuploidy (Christopoulou et al., 2003), genetic instability (Kim et al., 2005), and apoptosis (Hamid and Kakar, 2004). Thus, the precise roles of securin on the regulation of cell cycle progression and apoptosis are still ambiguous.
p53 is a tumor suppressor protein that regulates cell cycle arrest, apoptosis, and DNA repair (Amundson et al., 2005; Bates and Vousden, 1996; Hofseth et al., 2004; Levine, 1997). The expression of downstream proteins regulated by p53 includes p21 to mediate cell cycle arrest and Bax to induce apoptosis (Agarwal et al., 1995; Bates and Vousden, 1996; Hofseth et al., 2004). The various phosphorylation sites of p53 have been shown to play different roles on the regulation of cellular stresses (Canman and Lim, 1998; Dumaz and Meek, 1999; Meek, 1999). For example, the phosphorylation site of serine-15 on p53 mediates the activation and stabilization of p53 proteins (Canman and Lim, 1998; Dumaz and Meek, 1999; Meek, 1999). Moreover, it has been shown that p53 can inhibit the securin expression following treatment with anticancer drugs such as doxorubicin and bleomycein (Zhou et al., 2003). The overexpressed securin proteins activate p53 to induce the Bax expression and to mediate apoptosis (Hamid and Kakar, 2004). However, securin interacts with p53 to block the specific binding of p53 to DNA and inhibits the latter's transcriptional activity resulting in the prevention of apoptosis (Bernal et al., 2002). Accordingly, the interaction of securin and p53 on the balance of cell survival and apoptosis remains unclear.
Arsenic is a ubiquitous environmental toxicant that has been classified as a human carcinogen (IARC, 1987). It has been shown that arsenic is able to cause DNA and chromosome damages (Lynn et al., 2000; Oya-Ohta et al., 1996; Yih et al., 1997). Nevertheless, arsenic has been used for the treatment of certain types of cancer including acute promyelocytic leukemia and multiple myeloma (Hayashi et al., 2003; Perkins et al., 2000; Shen et al., 1997; Zhao et al., 2001). Chronic exposure to the low levels of arsenic (0.5–5 μM) may increase the incidences of several different human cancers (Chen et al., 1992; Chen and Wang, 1990; Chiou et al., 1995). Furthermore, higher concentrations of arsenic may induce apoptosis in a variety of cancer cells (Ivanov and Hei, 2004; Nimmanapalli et al., 2003; Scholz et al., 2005). Epidemiological studies demonstrated a high association of peripheral vascular diseases with arsenic exposure (Chen et al., 1995; Engel et al., 1994; Tseng et al., 1997). The vascular endothelial cells are an important target for the vasculopathy following arsenic exposure (Engel et al., 1994; Lee et al., 2003; Liu and Jan, 2000; Tseng et al., 1997). However, the regulation of securin and p53 in the arsenic-induced chromosome damage and apoptosis is inconclusive.
Two tissue types of cell lines including the mouse vascular endothelial and human colorectal epithelial cells were examined on the possible roles of securin and p53 proteins in the arsenite-induced cell injury. Arsenite markedly decreased the securin protein expression but increased the p53 protein expression in both endothelial and epithelial cells. Furthermore, the blockage of securin enhanced the induction of cytotoxicity, apoptosis, mitotic arrest, and aberrant sister chromatid segregation after treatment with arsenite. Moreover, we found that the depletion of securin proteins increased the abnormal chromosomal segregation and apoptosis from arsenite exposure via a p53-independent pathway, and the p53-mediated cell cycle arrest and apoptosis induced by arsenite was not related to securin.
MATERIALS AND METHODS
Chemicals and antibodies.
Hoechst 33258, propidium iodide, 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT), sodium arsenite (NaAsO2), and the Cy3-labeled mouse anti--tubulin (c-4585) were purchased from Sigma Chemical (St. Louis, MO). Anti-phospho-CDC2 (threonine-161) (9114S), anti-phospho-p53 (serine-15) (9284S), and goat anti-rabbit IgG-horseradish peroxidase (#7074) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Anti-ERK-2 (C-14), anti-p53 (DO-1), goat anti-mouse IgG-horseradish peroxidase (sc-2005), and the FITC (fluorescein isothiocyanate)-labeled goat anti-mouse IgG antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The monoclonal anti-securin (ab-3305) and polyclonal anti-separase (ab-3762) were purchased from Abcam (Cambridgeshire, UK). Anti-CDC2 (Ab-1) and anti-cyclin B1 (Ab-2) were purchased from Oncogene Sciences Products (Boston, MA). The Cy5-labeled goat anti-rabbit IgG was purchased from Amersham Biosciences.
Cell lines and cell culture.
The SVEC4–10 cell line (ATCC, #CRL-2181) was derived from mouse vascular endothelium that was purchased from Food Industry Research and Development Institute (Hsinchu, Taiwan). The securin-wild-type and -null HCT116 colorectal cancer cell lines (Jallepalli et al., 2001) were kindly provided by Dr. B. Vogelstein of the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins. RKO (ATCC, #CRL-2577) was a colorectal carcinoma cell line that expresses the wild-type p53 proteins (Bhat et al., 1997). SW480 (ATCC, #CRL-228) was established from the colorectal adenocarcinoma of a white male whose p53 gene contains a G to A mutation in codon 273 and a C to T mutation in codon 309 (Rodrigues et al., 1990; Weiss et al., 1993). The SVEC4–10, RKO, and SW480 cells were routinely maintained in DMEM medium (Invitrogen Co., Carlsbad, CA). The DMEM-complete medium was supplemented with 10% fetal bovine serum, L-glutamine (0.03%, w/v), 100 unit/ml penicillin, and 100 μg/ml streptomycin. The HCT116 cells were cultured in the complete Macoy's 5A medium (Sigma Chemical). These cell lines were maintained at 37°C and 5% CO2 in a humidified incubator (310/Thermo, Forma Scientific, Inc., Marietta, OH).
Cytotoxicity assay.
To analyze the induction of cytotoxicity, the cell viability was determined by the MTT colorimetric assay as described (Kuo et al., 2004; Plumb et al., 1989). The cells were plated in 96-well plates at a density of 1 x 104 cells/well for 16–20 h. Then the cells were treated with arsenite in complete medium. After treatment, the cells were carefully and slightly washed with isotonic phosphate-buffered saline (PBS) to avoid the loss of mitotically round-up cells, and were recultured in complete medium for 2 days. Subsequently, the medium was replaced, and the cells were incubated with 0.5 mg/ml of MTT in complete medium for 4 h. The surviving cells converted MTT to formazan that produced a blue-purple color when dissolved in dimethyl sulfoxide. The intensity was measured at 565 nm using a 96-well plate reader (OPTImax; GE Healthcare). The relative percentage of viability was calculated by dividing the absorbance of treated cells by that of the control in each experiment.
Apoptosis analysis.
To evaluate the level of apoptosis, the adherent cells were cultured on coverslip in a 60-mm Petri dish for 16–20 h before treatment. After treatment, the cells were carefully and slightly washed with isotonic PBS (pH 7.4), and incubated with 4% paraformaldehyde solution in PBS for 1 h at 37°C. Then the nuclei were stained with 2.5 μg/ml Hoechst 33258 for 30 min. The number of apoptotic nuclei was counted by a hemocytometer under a fluorescence microscope. In addition, the cell morphology of apoptosis was confirmed by observation of the cell membrane blebbing and the formation of apoptotic bodies under phase contrast microscope. At least 200 cells were examined from the random fields for the calculation of apoptotic percentage in each treatment. Furthermore, the person counting cells was blinded as to which cell line and treatment were being counted.
Cell growth assay.
The cells were plated at a density of 5 x 105 cells per 100-mm Petri dish in complete medium for 16–18 h. After drug treatment, the cells were carefully and slightly washed with PBS and recultured in complete medium. Thereafter, the cells were incubated for various times before they were counted by a hemocytometer.
Cell cycle analysis.
For analysis of cell cycle, the cells were plated at a density of 1 x 106 cells per 60-mm Petri dish in complete medium for 16–20 h. At the end of treatment, the cultured medium was collected, and the cells were trypsinized before being centrifuged. After centrifugation, the cells were collected and fixed with ice-cold 70% ethanol overnight at –20°C. Thereafter, the cell pellets were treated with 4 μg/ml propidium iodide solution containing 1% Triton X-100 and 100 μg/ml RNase for 30 min. To avoid cell aggregation, the cell solutions were filtered through nylon membrane (BD Biosciences, San Jose, CA). Subsequently, the samples were analyzed in a BD LSR system (BD Biosciences) using CellQuest software. A total of 1 x104 cells were analyzed for DNA content, and the percentage of cell cycle phases was quantified by a ModFit LT software (Ver. 3.0, BD Biosciences).
Mitotic index and the ratio of anaphase/mitosis.
The adherent cells were cultured on coverslip in a 60-mm Petri dish for 16–20 h before treatment. After treatment, the cells were carefully and slightly washed with PBS (pH 7.4) to avoid the loss of mitotically round-up cells, and then fixed with 4% paraformaldehyde solution in PBS for one hour at 37°C. Thereafter, the -tubulin was stained with the Cy3-labeled mouse anti--tubulin (1:50) for 30 min at 37°C. Finally, the nuclei were stained with 2.5 μg/ml Hoechst 33258 for 30 min. The cells were counted by a hemocytometer under a fluorescence microscope. At least 500 cells were examined for the calculation of mitotic index. For analysis of the ratio of anaphase/mitosis, at least 100 mitotic cells were counted in each treatment. The mitotic cells showed round-up morphology, compact chromosomes, spindle formation, and contained intact cell membrane but did not produce the cell membrane blebbing and the formation of apoptotic bodies. The cells of anaphase were going through the stage of chromosome separation.
Immunofluorescence staining and confocal microscopy.
To view the location and expression of proteins, the cells were subjected to immunofluorescence staining and confocal microscopy as described (Kuo et al., 2004). After fixation, the cells were washed three times with PBS, and nonspecific binding sites were blocked in PBS containing 10% normal bovine serum and 0.3% Triton X-100 for 1 h. Briefly, the cells were incubated with rabbit anti-separase (1:250), rabbit anti-phospho-p53 (serine-15) (1:250), mouse anti-p53 (DO-1) (1:50), or mouse anti-securin (1:50) antibodies in PBS containing 10% FBS for overnight at 4°C, and washed three times with 0.3% Triton X-100 in PBS. Then the cells were incubated with goat anti-rabbit Cy5 (1:250) or goat anti-mouse FITC (1:50) in PBS containing 10% normal bovine serum for 3 h at 37°C. Thereafter, the -tubulin was stained with the Cy3-labeled mouse anti--tubulin (1:50) for 30 min at 37°C. Finally, the nuclei were stained with 2.5 μg/ml Hoechst 33258 for 30 min. The samples were immediately examined under a Leica confocal laser scanning microscope (Leica, Wetzlar, Germany) that was equipped with a UV laser (351/364 nm), an Ar laser (457/488/514 nm), and a HeNe laser (543 nm/633 nm).
Western blot analysis.
The total cellular protein extracts were prepared as described (Chao et al., 2004; Kuo et al., 2004). The protein concentration was determined by using the BCA protein assay kit (Pierce, Rockford, IL). Western analyses of cyclin B1, CDC2, phospho-CDC2 (threonine-161), securin, separase, and ERK-2 were performed using specific antibodies. Briefly, equivalent amounts (50–100 μg/well) were separated on 10–12% sodium dodecyl sulfate-polyacrylamide gels and electrophoretic transfer of proteins onto polyvinylidene difluoride membranes. The membranes were sequentially hybridized with primary antibody and followed with a horseradish peroxidase-conjugated secondary antibody. Finally, the protein bands were visualized on the X-ray film using the enhanced chemiluminescence detection system (PerkinElmer Life and Analytical Sciences, Boston, MA). A gel-digitizing software, Un-Scan-It gel (ver. 5.1; Silk Scientific, Inc., Orem, UT), was used to quantify the intensity of each band on the X-ray film.
Statistical analysis.
Data were analyzed by one-way or two-way analysis of variance (ANOVA), and further post-hoc tests using the statistic software of GraphPad Prism 4 (GraphPad software, Inc. San Diego, CA). Differences among control and arsenite-treated samples were compared by one-way ANOVA with post-hoc Tukey's tests. Differences among the securin-wild-type and securin-null HCT116 cells (or RKO and SW480 cells) from the control and arsenite treatments were compared by two-way ANOVA with Bonferroni post-tests. A p value of <0.05 was considered as statistically significant in each experiment.
RESULTS
Arsenite Decreases the Cell Viability, Inhibits the Cell Growth, and Increases the G2/M Fractions in the SVEC4–10 Endothelial Cells
The vascular endothelial cells are the important targets of cardiovascular diseases resulting from arsenic exposure (Engel et al., 1994; Tseng et al., 1997). A vascular endothelial cell line, SVEC4–10, was used to study the effects of arsenite on the endothelial cell injury. Treatment with 4–16 μM arsenite for 24 h significantly decreased the cell viability in a concentration-dependent manner in the SVEC4–10 cells (Fig. 1A). Moreover, the growth ability of SVEC4–10 cells was inhibited by arsenite (Fig. 1B). To further investigate the effect of arsenite on the cell cycle arrest, the arsenite-treated cells were analyzed by flow cytometer. As shown in Figure 2A, arsenite (8–16 μM, 24 h) decreased the G1 fractions, while increasing the G2/M fractions in the SVEC4–10 cells. The level of G1 fractions was diminished 25%, and the G2/M fractions were increased 10% after treatment with 16 μM arsenite for 24 h; at the same time, the sub-G1 fractions (apoptotic fractions) were increased 20% by arsenite in the SVEC4–10 cells (Fig. 2A). Immunoblot analysis showed that arsenite reduced the levels of cell cycle-regulated proteins including phospho-CDC2 (threonine-161), total CDC2, and cyclin B1 proteins (Figs. 2B and 2C). In contrast, the level of p53 (DO-1) proteins was markedly elevated in the arsenite-treated cells in a concentration- and time-dependent manner (Figs. 2B and 2C). ERK-2 was used as an internal control that was not altered by arsenite. Previously, ERK-2 has been used as an internal control after treatment with a variety of chemicals (Chao et al., 2004; Kuo et al., 2004). Furthermore, the level of actin, a common internal protein, was not changed in the arsenite-treated cells (data not shown).
Arsenite Decreases the Securin Protein Expression in Both the Vascular Endothelial and Securin-Wild-Type Epithelial Cells
The SVEC4–10 endothelial and securin-wild-type HCT116 colorectal epithelial cells were subjected to immunofluorescence staining and confocal microscopy to examine the expression and location of securin proteins. As shown in Figure 3A, the securin and separase proteins were expressed and colocalized on the nuclei of SVEC4–10 cells (Fig. 3A, arrows). Moreover, the securin proteins were highly expressed at the prophase and metaphase in the securin-wild-type HCT116 cells (Fig. 4A, arrows). After metaphase, the securin proteins were reduced during the anaphase and telophase (Fig. 4A). To further investigate the effects of arsenite on the expression of securin and separase proteins, the cells were subjected to immunoblot analysis. Treatment with 12–16 μM arsenite for 24 h significantly decreased the level of securin proteins in the SVEC4–10 cells (Fig. 3B). Furthermore, arsenite time-dependently inhibited the securin protein expression, but the level of separase proteins was slightly increased by arsenite (Fig. 3C). Two major bands (120 kD and 76 kD) of separase proteins were detected in the immunoblot (Fig. 3C). It has been shown that separase proteins can be auto-cleaved, and the enzyme activity is preserved for anaphase entry (Chestukhin et al., 2003; Peters, 2002). The level of securin proteins was diminished with 4–16 μM arsenite in the securin-wild-type HCT116 cells (Fig. 4B). Consistently, the green fluorescence (FITC) exhibited by securin was significantly decreased by arsenite in the securin-wild-type HCT116 cells (Fig. 4C). However, the securin-null HCT116 cells did not express any securin proteins following arsenite exposure (Fig. 4B).
Loss of Securin Enhances the Cytotoxicity and Apoptosis in the Arsenite-Exposed Cells
The cell survival and apoptosis in the securin-wild-type and -null HCT116 cells were compared after arsenite treatment. As shown in Figure 5A, arsenite (4–16 μM, 24 h) concentration-dependently induced cytotoxicity in both the securin-wild-type and -null HCT116 cells. The securin-null cells were more susceptible to cell death than the securin-wild-type cells treated with 8–16 μM arsenite for 24 h (Fig. 5A). The percentage of apoptotic nuclei was counted under a fluorescence microscope by nuclear staining. Arsenite significantly increased the level of apoptosis in both the securin-wild-type and -null cells (Fig. 5B). However, the loss of securin proteins enhanced the induction of apoptosis by arsenite (Fig. 5B).
Blockade of Securin Increases the G2/M Phases, Mitotic Index, and Abnormal Anaphase in the Arsenite-Treated Cells
We also examined the role of securin on the cell cycle progression treated with arsenite. Arsenite significantly decreased the G1 fractions (Figs. 6A and 6D) and increased the G2/M fractions (Figs. 6C and 6F) in both the securin-wild-type and -null HCT116 cells. However, arsenite produced a large increase in the G2/M fractions of the securin-null HCT116 cells (Fig. 6F). To further determine whether G2 or M phases were increased by arsenite, the cells were analyzed by morphological observation and mitotic index as described in "Materials and Methods." The mitotic cells were increased following treatment with 16 μM arsenite for 24 h in both the securin-wild-type and -null cells (Fig. 7A, arrows). Moreover, the increase of mitotic phases was greater in the securin-null cells than the securin-wild-type cells after treatment with 16 μM arsenite for 24 h (Fig. 7B). As shown in Figure 8A, the abnormal anaphase (aberrant chromosome separation) was increased by arsenite in both the securin-wild-type and -null HCT116 cells. Comparing the ratio of anaphase/mitosis, the securin-null cells were more susceptible than the securin-wild-type cells to the increase of the percentage of aberrant anaphase after arsenite treatment (Fig. 8B). Eventually, cells with arsenite-induced abnormal chromosome separation went into apoptosis (Fig. 8C). The arrows of Figure 8C indicated the formation of apoptotic bodies.
Activation of p53 via Securin-Independent Pathway Following Arsenite Exposure
To investigate the expression and activation of p53 proteins in the securin-wild-type and -null cells after arsenite exposure, the cells were subjected to immunoblot analysis and immunofluorescence staining. The levels of phospho-p53 (serine-15) and p53 (DO-1) proteins were increased after exposure to 4–16 μM arsenite for 24 h in both securin-wild-type and -null HCT116 cells (Fig. 9A). As shown in Figure 9B, the intensities of green fluorescence (FITC) exhibited by p53 (DO-1) and the red fluorescence (Cy5) exhibited by phospho-p53 (serine-15) proteins were elevated by arsenite (Fig. 9B, arrows). Besides, the phospho-p53 (serine-15) and p53 (DO-1) proteins were concentrated on the nuclei in both securin-wild-type and -null cells following treatment with 16 μM arsenite for 24 h (Fig. 9B, arrows).
Inhibition of Securin Protein Expression Is Independent of p53 after Arsenite Treatment
As shown in Figure 10A, the p53-functional RKO cells were more susceptible than the p53-mutational SW480 cells to the cytotoxicity after treatment with 16–32 μM arsenite for 24 h. Additionally, the increase in the levels of sub-G1 and G2/M fractions was significantly greater for the p53-functional cells than the p53-mutational cells after arsenite treatment (data not shown). However, treatment with 8–16 μM arsenite for 24 h decreased the level of securin proteins to a similar extent in both the p53-functional and -mutational cells (Fig. 10B).
DISCUSSION
The cytotoxic anti-tumor agents such as arsenic and X-ray offer an important therapeutic strategy to treat cancer, but also can become a pathogenic factor on carcinogenesis from the induction of aberrant chromosomes. Chromosome instability is associated with the progression of carcinogenesis (Lengauer et al., 1997, 1998). However, if the chromosome damage is beyond repair, the cells will enter into apoptotic pathway leading to cell death. Accordingly, the balance of chromosome instability and apoptosis may be involved in the pathogenesis of cancer or may be used for cancer therapy.
To maintain the integrity during chromosome transmission, the process of mitosis is strictly monitored by spindle checkpoint (Musacchio and Hardwick, 2002). It controls sister chromatid separation, progression from metaphase to anaphase; its defects can result in chromosomal instability (Jallepalli and Lengauer, 2001; Millband et al., 2002; Musacchio and Hardwick, 2002). Securin has been proposed to be required for genome stability in mitosis (Jallepalli et al., 2001; Yanagida, 2000). In a recent study, securin was found to regulate DNA repair following UV and X-ray damage (Nagao et al., 2004). DNA and chromosome damage will activate defense proteins to mediate cell cycle arrest, allowing for repair of the damage. In the present study, we found that arsenite markedly diminished the securin protein expression in both the vascular endothelial and colorectal epithelial cells. Moreover, the securin-null cells were more susceptible than the securin-wild-type cells to the induction of aberrant anaphase following arsenite treatment. Thus, we suggest that the depletion of securin protein promotes the aberrant chromosomal segregation and induces chromosome instability in the arsenite-treated cells.
Activation of CDC2/cyclin B1 complex is required for the mitotic entry, and its inactivation occurs at late anaphase. The activation of CDC2 is through phosphorylation of threonine-161 by CDC2 activating kinase (Pines, 1999). Arsenite decreased the levels of phospho-CDC2 (threonine-161), total CDC2, and cyclin B1 proteins. However, the level of separase proteins was slightly increased by arsenite. The CDC2/cyclin B1 complex can mediate the phosphorylation and inactivation of separase (Nasmyth, 2002). Moreover, the activity of separase is inhibited by binding with securin in metaphase (Nasmyth, 2001; Stemmann et al., 2001). At the metaphase-anaphase transition, the securin is degraded, and separase is released to mediate the separation of sister chromatids through cleavage of the chromosomal cohesin (Nasmyth, 2001; Stemmann et al., 2001). Accordingly, we suggest that arsenite-induced the reduction of phospho-CDC2 (threonine-161), CDC2, cyclin B1, and securin proteins results in the release of the separase, allowing it to digest the sister chromatid cohesion; however, it causes the formation of abnormal chromosome segregation. The anaphase-promoting complex (APC) is one of important proteins participated in the regulation of the spindle checkpoint, which can mediate the cyclin B degradation during the metaphase to anaphase transition (Nasmyth, 2002). Securin can be degraded via ubiquitylation by APC (Cohen-Fix et al., 1996; Funabiki et al., 1996). The imbalance of APC-dependent proteolysis in the cell cycle regulation may lead to genomic instability of cancer cells (Wasch and Engelbert, 2005). Thus, it is possible that the decrease of securin and cyclin B1 proteins elicited by arsenite may be via the APC-mediated protein degradation. Nevertheless, the precise mechanisms of separase and APC on the regulation of arsenite-induced chromosome instability need further investigation.
Securin has been proposed as an oncogene that is expressed abundantly in most cancer cells (Dominguez et al., 1998; Pei and Melmed, 1997; Saez et al., 1999; Zou et al., 1999). It promotes cell proliferation and tumorigenesis (Hamid et al., 2005; Zhang et al., 1999; Zou et al., 1999). Therefore, the inhibition of securin would block the cell survival and proliferation in tumor cells, providing important strategy in cancer therapy. Interestingly, arsenite markedly decreased the levels of securin proteins and increased the cytotoxicity, apoptosis, and cell growth inhibition in both the vascular endothelial and colorectal carcinoma cells. The inhibition of endothelial cell growth may block tumor growth through anti-angiogenesis and is useful in cancer therapy (Marx, 2003). Furthermore, the securin-null colorectal carcinoma cells were more susceptible to the induction of cell growth inhibition and cell death than the securin-wild-type colorectal carcinoma cells after arsenite treatment. Recently, treatment with anticancer agents, such as UV, doxorubicin, and bleomycin, was observed to decrease the securin expression in cancer cells (Romero et al., 2004; Zhou et al., 2003). Consequently, we suggest that the inhibition of securin protein expression by arsenite may increase the cell growth inhibition and apoptosis in vascular endothelial and cancer cells.
p53 is a cellular gatekeeper for the cell growth and division (Bates and Vousden, 1996; Hofseth et al., 2004; Kuo et al., 2004; Levine, 1997). It has been shown that p53 protein mediates the G1 and G2/M checkpoints (Agarwal et al., 1995). The phosphorylation of p53 at serine-15 participates in the p53 activation and stabilization (Canman and Lim, 1998; Dumaz and Meek, 1999; Meek, 1999). We found that arsenite increased the level of phospho-p53 (serine-15) and total p53 proteins in both endothelial and epithelial cells. Moreover, the loss of functional p53 reduced the cytotoxicity, apoptosis, and G2/M fractions in the arsenite-treated cells. These data suggest that the activation of p53 mediates the cell cycle arrest and apoptosis following arsenite treatment. Interaction of p53 and securin has been investigated in recent years; however, it is not unambiguous. p53 can mediate the inhibition of securin expression after DNA damage agents such as doxorubicin and bleomycein (Zhou et al., 2003). Overexpression of securin activates the p53 expression to mediate the Bax activation, leading to the induction of apoptosis (Hamid and Kakar, 2004). In contrast, securin can also interact with p53 to block the transcriptional activity of p53, which can prevent apoptosis (Bernal et al., 2002). In this study, the loss of securin expression increased the cell death and G2/M arrest in the arsenite-treated cells. However, arsenite similarly raised the levels of phospho-p53 (serine-15) and p53 (DO-1) proteins in the securin-wild-type and -null cells. Besides, the securin proteins were reduced by arsenite in both the p53-functional and -mutational cells. Accordingly, the activation of p53 may be via a securin-independent pathway, and the inhibition of securin protein expression is not associated with p53 in the arsenite-treated cells.
In conclusion, we propose that the securin proteins play a critical role in the maintenance of chromosome stability and cell survival following arsenite exposure. Reduction of securin proteins by arsenite may increase the chromosome instability and apoptosis. Conversely, the activation of p53 by arsenite mediates cell cycle arrest and apoptosis. However, there is no cross-talk between p53 and securin in the arsenite-treated cells. The investigation of securin and p53 proteins on the arsenite-induced chromosome instability and apoptosis may elucidate the mechanism of carcinogenesis and offer further application on cancer therapy.
SUPPLEMENTARY DATA
Supplementary data are available online at http://toxsci.oxfordjournals.org/.
ACKNOWLEDGMENTS
We are indebted to Dr. T. H. Chiu (Institute of Pharmacology and Toxicology, Tzu Chi University) for careful reading of the manuscript. We also thank Dr. B. Vogelstein of the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins for permission to use the securin-wild-type and securin-null HCT116 colorectal cancer cell lines. This work was supported by National Science Council, Taiwan, Grant NSC 93–2320-B-320–014.
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