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Depletion of Securin Induces Senescence After Irradiation and Enhances Radiosensitivity in Human Cancer Cells Regardless of Functional p53 Expression
Int. J. Radiation Oncology Biol. Phys., Vol. 77, No. 2, pp. 566–574, 2010 Copyright Ó 2010 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/$–see front matter
doi:10.1016/j.ijrobp.2009.12.013
BIOLOGY CONTRIBUTION
DEPLETION OF SECURIN INDUCES SENESCENCE AFTER IRRADIATION AND ENHANCES RADIOSENSITIVITY IN HUMAN CANCER CELLS REGARDLESS OF FUNCTIONAL p53 EXPRESSION WEN-SHU CHEN,* YI-CHU YU,* YI-JANG LEE, PH.D.,y JI-HSHIUNG CHEN, PH.D.,z HSUE-YIN HSU, PH.D.,* x AND SHU-JUN CHIU, PH.D.* *Department of Life Science, Tzu Chi University, Hualien, Taiwan; yDepartment of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan; zDepartment of Molecular Biology and Human Genetics, Tzu Chi University, Hualien, Taiwan; and xInstitute of Radiation Sciences, Tzu Chi Technology College, Hualien, Taiwan Purpose: Radiotherapy is one of the best choices for cancer treatment. However, various tumor cells exhibit resistance to irradiation-induced apoptosis. The development of new strategies to trigger cancer cell death besides apoptosis is necessary. This study investigated the role of securin in radiation-induced apoptosis and senescence in human cancer cells. Methods and Materials: Cell survival was determined using clonogenic assays. Western blot analysis was used to analyze levels of securin, caspase-3, PARP, p53, p21, Rb, g-H2AX, and phospho-Chk2. Senescent cells were analyzed using a b-galactosidase staining assay. A securin-expressed vector (pcDNA-securin) was stably transfected into securin-null HCT116 cells. Securin gene knockdown was performed by small interfering RNA and small hairpin RNA in HCT116 and MDA-MB-231 cells, respectively. Results: Radiation was found to induce apoptosis in securin wild type HCT116 cells but induced senescence in securin-null cells. Restoration of securin reduced senescence and increased cell survival in securin-null HCT116 cells after irradiation. Radiation-induced g-H2AX and Chk2 phosphorylation were induced transiently in securin-wildtype cells but exhibited sustained activation in securin-null cells. Securin gene knockdown switches irradiationinduced apoptosis to senescence in both HCT116 p53-null and MDA-MB-231 cells. Conclusions: Our results demonstrated that the level of securin expression plays a determining role in the radiosensitivity and fate of cells. Depletion of securin impairs DNA repair after irradiation, increasing DNA damage and promoting senescence in the residual surviving cells regardless of functional p53 expression. The knockdown of securin may contribute to a novel radiotherapy protocol for the treatment of human cancer cells that are resistant to irradiation. Ó 2010 Elsevier Inc. Securin, Radiosensitivity, Apoptosis, Senescence, p53.
INTRODUCTION
apoptotic cell death are equivalent to long-term cell-cycle arrest (which is frequently associated with cellular senescence) in terms of the suppression of cancer cell proliferation and tumor growth (5, 6). Senescence has been considered a tumor suppressive mechanism that stringently inhibits replication of cancer cells. Senescent cells lack 5-bromo-2-deoxyuridine (BrdU) incorporation and exhibit permanent arrest of cell proliferation, resistance to apoptosis, and changes in gene expression, as well as increased activity of acidic senescence-associated b-galactosidase (SA-b-gal) (6, 7). Senescence regulationrelated proteins vary according to the different sources of DNA damage, stress, and cell type. Cyclin-dependent kinase
Radiotherapy is one of the best therapeutic choices for cancer treatment (1). The cellular response to ionizing radiation produces a spectrum of effects ranging from growth arrest to apoptotic cell death, senescence, necrosis or mitotic catastrophe, and autophagy (2, 3). Apoptosis is the primary cause of cell death induced by radiation; however, solid tumors derived from epithelial cells exhibit resistance to radiationinduced apoptosis (1, 4). Hence, the development of new strategies to trigger cancer cell death besides apoptosis may contribute to improved prognosis in cancer patients. Recent studies have found that the effects of DNA damage-induced Reprint requests to: Shu-Jun Chiu, Ph.D., Department of Life Science, Tzu Chi University, 701, Section 3, Chung-Yang Road, Hualien 970, Taiwan. Tel: 886-3-8565301, ext. 2631; Fax: 8863-8572526; E-mail: [email protected] Wen-Shu Chen and Yi-Chu Yu contributed equally to this work. This work was supported by a grant from the National Science Council, Taiwan (NSC 96-2321-B-320-001-MY3).
Conflict of interest: none. Acknowledgment—We thank Dr. B. Vogelstein, The Johns Hopkins University, for permission to use HCT116 colorectal cancer cell lines. Received Sept 22, 2009, and in revised form Oct 16, 2009. Accepted for publication Dec 10, 2009. 566
Securin determines radiosensitivity and cell fate d W.-S. CHEN et al.
(cdk) inhibitors p21waf1/cip1 (referred to as p21 hereafter) and p16INK4a interfere with the activity of cdk, leading to dephosphorylation of Rb and thereby blocking cell cycle progression (7). Subsequently, Chk2 acts downstream of ATM to induce p21 in a p53-dependent and -independent manner and promotes senescence (8, 9). The p53–p21 pathway has been reported to be important for the onset of senescence and p16INK4a as essential for the maintenance of senescence (10). Nevertheless, there are examples of senescence demonstrating p53-independent and p16-pRB-independent pathways (7, 11–13). The molecular pathways corresponding to cellular senescence remain controversial and are worthy of further study. Securin is known to prevent abnormal sister chromatid segregation during mitosis and maintain genomic stability (14). In addition, securin has been shown to participate in DNA repair through interaction with separase or the Ku70/80 heterodimer of the double-strand breaks (DSBs) nonhomologous DNA end-joining repair machinery after exposure to ultraviolet, X-ray, and gamma-ray irradiation (15, 16). Securin has also been found to be downregulated by DNA-damaging drugs and UV radiation prior to DNA repair (17–19). Furthermore, loss of securin sensitized HCT116 cells to chemotherapeutic agents such as adriamycin and methyl methanesulphonate or to ionizing radiation via proliferative inhibition but not apoptosis (20). In addition, securin has been described as a novel oncogene being expressed abundantly in most cancer cells and overexpressed in colorectal cancer cells (21) and promoting cell proliferation and tumorigenesis (22). Securin overexpression is correlated with poor prognosis in many carcinoma patients (23, 24); however, overexpression of securin has been reported to induce aneuploidy, genetic instability and apoptosis (22). Therefore, the precise role of securin in determining the fate of cells, including apoptosis and senescence, induced by radiation and the correlated radiosensitivity is still unclear. In this study, we investigated the effect of securin expression on radiosensitivity and the fate of cells exerted via different mechanisms induced by irradiation. Radiation was found to induce apoptosis in securin-wild-type HCT116 cells but induced senescence in securin-null cells, and depletion of securin was found to enhance radiosensitivity and induce senescence in the residual surviving cells, regardless of functional p53 expression in human cancer cells. Knockdown of the securin gene may contribute to a novel radiotherapy protocol for human cancer cells that are resistant to irradiation. METHODS AND MATERIALS
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from Calbiochem (San Diego, CA). Anti-caspase-3 was purchased from Imgenex (San Diego, CA).
Cell culture Securin-wild-type, securin-null, and p53-null human HCT116 colorectal cancer cells were routinely maintained in McCoy’s 5A medium (Sigma). MCF-7 and MDA-MB-231 human breast cancer cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) medium (Gibco). The complete medium was supplemented with 10% fetal bovine serum.
Stable transfection for generating a human securinexpressing cell line Securin-null (p53 wild-type) HCT116 cells, a standard cell line provided by Dr. B. Vogelstein at Johns Hopkins University, were transfected with the expression vector human securin-pcDNA3 or pcDNA3 by Lipofectamine, and hygromycin B (100 mg/ml) selection was initiated 48 hr after transfection. Single colonies were picked out, expanded, and then stored in liquid nitrogen, after which human securin expression levels were determined by Western blot analysis.
X-ray irradiation Irradiation was produced by an X-ray machine (RS2000; RAD Source Technologies) operating at 160 kVp (peak kilovoltage) and 25 mA. The dose rate at a source-–subject distance of 38 cm was 1.83 Gy/min. The machine output was routinely calibrated using an air ionization chamber. Briefly, the cells were replenished with fresh medium before irradiation and then subjected to X-ray irradiation immediately at room temperature.
Clonogenic survival assay Cells were seeded at a density of 10,000 cells per well in 2 ml of culture medium in 6-well plates 1 day before treatment. Cells were subsequently exposed to irradiation and incubated for a period of 5 to 7 days to recover and then fixed in 1% crystal violet (containing 30% ethanol) and visualized under an inverted phase-contrast microscope. Twenty random fields were selected for analysis, and isolated clusters of more than 50 cells were counted as a single colony. The relative percentage of surviving cells was calculated by dividing the number of colonies of treated cells by that of the control.
Cell cycle analysis Cells were seeded at a density of 1 x 106 cells per 60-mm culture dish and incubated for 24 hr and then irradiated with X-rays. After treatment, cells were trypsinized and fixed with 70% ethanol at 20 C overnight. Fixed cells were subsequently stained with 20 mg/ml PI staining buffer (containing 1% Triton X-100 and 100 mg/ml RNase A) for 30 min, and cells were analyzed by flow cytometry (FACScalibur; Becton Dickinson, San Jose, CA). The percentage of cells in each of the different cell cycle phases was determined using ModFit LT software (version 2.0; Becton Dickinson).
Chemicals and antibodies Propidium iodide (PI) was purchased from Sigma Chemical Co. (St. Louis, MO). Anti-phospho-histone H2AX (Ser-139) was purchased from Upstate Biological Co. (Lake Placid, NY). Anti-phospho-p53 (Ser-15), anti-phospho-Chk2 (Thr-68), Chk2, Rb, PARP, and an Absolute-S kit were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Monoclonal anti-securin antibody was purchased from Abcam (Cambridgeshire, UK). Anti-actin antibody was purchased from Chemicon International, Millipore (Billerica, MA). Anti-p21waf1/cip1 mouse monoclonal antibody was purchased
SA-b-gal SA-b-gal staining is widely used as a biomarker of cellular senescence in vivo and in vitro (8), the positive blue-colored staining of bgalactosidase at pH 6.0 being remarkably increased in senescent cells. Senescent cells were analyzed using a b-gal staining kit (Cell Signaling) in accordance with the manufacturer’s instructions. The percentage of SA-b-gal-positive cells was then calculated by counting the cells in 10 random fields (at least 100 cells) using bright-field microscopy at a magnification of 200 or 320, in triplicate.
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Fig. 1. Comparison of apoptosis, senescence, and radiosensitivity levels in irradiated securin-wild-type and -null HCT116 cells. (A) Western blot analysis showing levels of securin, caspase-3, and PARP. (B) SA-b-gal staining was performed after irradiation, followed by a postirradiation period of 2 to 7 days. SA-b-gal-positive-stained cells (green) were observed with bright-field microscopy at a magnification of 320. (C) The surviving fractions of securin-wild-type and -null HCT116 cells were measured by clonogenic survival assay.
Western blot analysis Total cellular protein extracts were prepared according to our previous study (25). Briefly, equal amounts of total protein (20–60 mg/ well) were subjected to electrophoresis using 10% to 12% sodium dodecyl sulfate-polyacrylamide gels. Following electrophoretic transfer of proteins onto polyvinylidene fluoride membranes, the proteins were sequentially hybridized with primary antibody, followed by a horseradish peroxidase-conjugated secondary antibody. Finally, the protein bands were visualized on X-ray film, using an enhanced chemiluminescence detection system (Immobilon Western Chemiluminescent horseradish peroxidase substrate, WBKLS0500, Millipore), and gel-digitizing software, Un-Scan-It (version 5.1; Silk Scientific, Inc., Orem, UT) was used to quantify the relative intensity of each band on the X-ray films.
turer’s instructions. Forty-eight hours after transfection, the cells were subjected to irradiation or Western blot analysis as described above.
Establishment of human securin small hairpin RNA knockdown stable MDA-MB-231 cell lines Small fragments of securin small hairpin RNA (shRNA) were cloned into pLKO.1-puro vector. All shRNA clones and two functional plasmids, pMD.G and pCMVdeltaR8.91 (10:1:9), were obtained from the National RNA Interference Core Facility (Academic Sinica, Taiwan). MDA-MB-231 cells were infected with lentiviruses from supernatant media from these clones in DMEM. After cells were infected, they were selected from DMEM culture containing 0.5 mg/ml puromycin dihydro-chloride.
RNA interference
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Securin small interfering RNA (siRNA) (s17655, [5’/3’] sense: GCACCCGUGUGGUUGCUAAtt; antisense: UUAGCAACCACACGGGUGCct) and scrambled siRNA (4390846) were purchased from Ambion (Austin, TX). Transfection of HCT116 cells with the above-described siRNA oligonucleotides was performed with Lipofectamine 2000 (Invitrogen) according to the manufac-
All data are represented by the means standard error of the mean (SEM) of at least three independent experiments. Statistical analysis was performed by one-way analysis of variance, and further post hoc testing was performed using statistical software GraphPad Prism 4 (GraphPad, Inc. San Diego, CA). A p value of <0.05 was considered statistically significant.
Securin determines radiosensitivity and cell fate d W.-S. CHEN et al.
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Fig. 2. Effects of securin expression in securin-null HCT116 cells and knockdown in securin-wild-type cells on radiationinduced senescence and surviving cell fractions. (A) The expression level of securin in stably transfected securin reexpressing HCT116 cells was observed by Western blotting. (B) The percentages of SA-b-gal-positive-stained cells were calculated in securin re-expressing HCT116 cells. (C) The surviving cell fraction in securin re-expressing HCT116 cells was measured by clonogenic survival assay after irradiation. (D) Securin siRNA was transfected into HCT116 securin-wild-type cells, and levels of securin were characterized by Western blot analysis. (E) The percentages of SAb-gal-positive cells transfected with securin siRNA were calculated. (F) The surviving cell fraction of securin siRNAtransfected HCT116 cells was measured by clonogenic survival assay. A p value of <0.01 (**) indicates a significant difference between nonirradiated control and irradiated samples. A p value of <0.01 (##) indicates a significant difference between scrambled and securin siRNA-transfected samples (E).
RESULTS Irradiation induces senescence in securin-null HCT116 cells and apoptosis in securin-wild-type cells Western blot analysis showed that 6 Gy irradiation followed by a postirradiation period of 2 to 7 days reduced the level of securin and induced significant apoptotic hallmarks, specifically caspase-3 activation and PARP cleavage, in securin-wild-type HCT116 cells but not in securin-null cells (Fig. 1A). In addition, as shown in Fig. 1B, securinnull cells exhibited senescence-like morphologic features (enlarged, flattened cells and positive b-gal activities) after irradiation. Notably, irradiation (at 3 and 6 Gy) increased cellular senescence in securin-null cells but not securinwild-type cells. The intrinsic radiosensitivity assessed by
clonogenic survival assay is expressed by the surviving fraction at 2 Gy (SF2). As shown in Fig. 1C, securin-null cells were more susceptible to irradiation than securin-wild-type cells. The SF2 of securin-wild-type and securin-null HCT116 cells exposed to irradiation were about 0.12 and 0.02, respectively. Securin re-expression and gene knockdown oppositely regulate senescence and radiosensitivity in HCT116 cells Western blot analysis with anti-securin antibody showed that the stable cell clone (transfected with a securin-expressed vector) expressed securin protein in securin-null HCT116 cells (Fig. 2A). Radiation-induced senescence was significantly reduced by restoration of securin in securin-null cells exposed to 3 and 6 Gy irradiation followed by a postirradiation
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Fig. 3. Effects of securin expression on the levels of g-H2AX, phospho-Chk2, p53, p21, and Rb in HCT116 cells after irradiation. (A) The levels of g-H2AX, phospho-Chk2, total Chk2, and securin were analyzed by Western blotting in irradiated securin-wild-type, -null, and stably expressing cells. (B) The levels of phospho-Chk2, total Chk2, phosphop53, p53 (DO-1), p21, Rb, and securin were ascertained by Western blot analysis.
duration of 7 days (Fig. 2B). In addition, the securin expressed in the stable cell clone restored the surviving cell fraction (SF2 = 0.32) after irradiation in comparison with securin-null cells (SF2 = 0.03) (Fig. 2C). Additionally, the levels of securin were diminished when HCT116 cells were transfected with 50 nM securin siRNA (Fig. 2D). Transfection of securin siRNA increased senescence in comparison with scrambled siRNA in irradiated HCT116 cells (Fig. 2E). Furthermore, the SF2 values for securin-wild-type HCT116 cells transfected with 50 nM securin siRNA after irradiation were reduced from 0.26 to 0.12 (Fig. 2F). Therefore, transfection with securin siRNA increased the elevation of radiosensitivity in securin-wild-type HCT116 cells. Radiation induces g-H2AX and Chk2 activation in securinnull HCT116 cells but elevates the levels of p53 and p21 proteins, in addition to reducing Rb levels in both securinwild-type and securin -null HCT116 cells The levels of g-H2AX and phospho-Chk2 (Thr-68) were increased by 6-Gy irradiation in securin-null HCT116 cells in a time-dependent manner (Fig. 3A, middle). However, 6-Gy irradiation rapidly and transiently increased the levels of g-H2AX and phospho-Chk2 (Thr-68) in securin-wildtype and securin re-expression cells (Fig. 3A, left and right). Stable transfection with the securin-expressing vector
reduced the radiation-induced g-H2AX and phospho-Chk2 (Thr-68) levels in securin-null HCT116 cells (Fig. 3A, middle and right). Irradiation (6 Gy followed by a postirradiation duration of 2–7 days) also caused a sustained increase in phospho-Chk2 (Thr-68) expression in securin-null HCT116 cells but not in securin-wild-type cells (Fig. 3B, right). As shown in Fig. 3B, the levels of p53 and p21 were elevated in both securin-wild-type and securin-null cells after irradiation followed by a postirradiation period of 2 to 7 days. In contrast, the levels of Rb were reduced, whereas p16 expression was undetectable in both types of cells. Indeed, HCT116 cells are deficient for p16 (26). Securin gene knockdown switches irradiation-induced apoptosis to senescence in HCT116 cells lacking p53 expression Securin levels were found to be decreased by transfection with 25 nM securin siRNA (Fig. 4A). As shown in Fig. 4B, the securin gene knockdown was found to increase radiationinduced senescence-like G2/M arrest in HCT116 p53-null cells after a postirradiation duration of 48 h by flow cytometry. In p53-null HCT116 cells, irradiation reduced the level of securin and increased caspase-3 activation but did not induce p21 protein expression after 6 Gy of irradiation followed by a 5-day recovery period (Fig. 4C). Moreover, caspase-3
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Fig. 4. Effects of the securin gene knockdown on radiation-induced G2/M arrest, apoptosis, senescence, and surviving cell fraction in HCT116 p53-null cells. (A) Securin siRNA was transfected into HCT116 p53-null cells, and levels of securin were characterized by Western blot analysis. (B) G2/M arrest was analyzed by flow cytometry. (C) The levels of securin, p53, p21, and active caspase-3 in cells transfected with securin siRNA were determined by Western blot analysis. (D and E) The percentages of SA-b-gal-positive cells transfected with securin siRNA were calculated by observation with bright-field microscopy at a magnification of 320. (F) The surviving cell fraction was measured by clonogenic survival assay. A p value of <0.01 (**) indicates a significant difference between untreated control and irradiated samples. A p value of <0.01(##) indicates a significant difference between scrambled and securin siRNA-transfected samples.
activation was significantly reduced by knockdown of securin in p53-null HCT116 cells after irradiation (Fig. 4C). As shown in Fig. 4D, irradiation induced significant senescence in the securin gene knockdown p53-null HCT116 cells compared with p53-null cells transfected with scrambled siRNA. Transfection of securin siRNA (25 nM, 48 hr) increased senescence in p53-null HCT116 cells subjected to 3 and 6 Gy of irradiation (Fig. 4E). In addition, transfection with 25 nM securin siRNA enhanced radiosensitivity in p53-null HCT116 cells after 3 Gy of irradiation (Fig. 4F). Securin gene knockdown increases senescence and enhances radiosensitivity in irradiated human breast cancer cells As shown in Fig. 5A, endogenous levels of securin were much higher in MDA-MB-231 cells, a p53 mutant human breast cancer cell line, than in MCF-7 cells, a p53 wildtype human breast cancer cell line. Furthermore, we gener-
ated an MDA-MB-231 clone (MDA-MB-231-2A) in which endogenous securin expression was knocked down by shRNA (Fig. 5A). Irradiation at 4 and 6 Gy induced significant senescence in MCF-7 cells, which expressed lower levels of securin (Fig. 5B and C); on the contrary, senescence was not increased in the MDA-MB-231 cells after 4- and 6Gy irradiation. Securin shRNA knockdown MDA-MB-2312A cells significantly increased senescence in comparison to MDA-MB-231 cells after irradiation (Fig. 5B and C); in addition, MCF-7 cells were found to be more susceptible to irradiation than MDA-MB-231 cells (Fig. 5D). Furthermore, MDA-MB-231 cells transfected with securin shRNA exhibited enhanced radiosensitivity (Fig. 5D). DISCUSSION Securin has been shown to participate in DNA repair after exposure to ultraviolet, X-ray, and gamma-ray irradiation
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Fig. 5. Effects of the securin gene knockdown on radiation-induced senescence and cell survival in human breast cancer cell lines. (A) The levels of securin in MCF-7 and MDA-MB-231 cells and in the shRNA stable transfectant MDA-MB231-2A clone were characterized by Western blot analysis. (B and C) The percentages of SA-b-gal-positive cells (blue) in the three breast cancer cell lines subjected to irradiation were calculated by observation with bright-field microscopy at a magnification of 200. (D) The surviving cell fraction was measured by clonogenic survival assay.
(15, 16). Nevertheless, securin has also been found to be downregulated by DNA-damaging drugs and UV radiation prior to DNA repair (17–19). Loss of securin compromises nonhomologous end joining repair, cell survival, and proliferation after genotoxic stress (20). In this study, radiation was found to downregulate securin expression and induce apoptosis in securin-wild-type HCT116 cells, whereas senescence was induced in the surviving securin-null HCT116 cells (the surviving fractions were about 0.001 to 0.002 after 3-Gy irradiation [Fig. 1C, and 2C and F). It has been reported that securin deletion leads to chromosomal instability, DNA damage, signaling pathway activation, and senescence (27). Moreover, repression of apoptosis triggered by genotoxic stress may shift the cell failsafe to senescence (28). We found that pharmacological blockade of apoptosis by z-VAD-fmk failed to promote senescence in securin-wild-type cells in response
to irradiation (data not shown), which may indicate that senescence is not a compensatory mechanism for apoptosis. Accordingly, we suggest that the gradual downregulation of securin after irradiation leads to repair of radiation-induced DNA damage and maintenance of cell proliferation. However, the damage may be partly repaired; hence, the unrepaired DNA triggers apoptosis while the cell cycle progresses. On the contrary, loss of securin before irradiation impairs DNA repair, thereby causing the accumulation of radiation-induced DNA damage signals and promoting senescence. Therefore, we propose that securin expression plays an important role in the induction of apoptosis or senescence by radiation, i.e., apoptosis is induced by the existence of securin, whereas senescence is promoted when securin is absent. Phosphorylation of H2AX at serine 139, g-H2AX, is one of the responses of mammalian cells to DNA DSBs induced
Securin determines radiosensitivity and cell fate d W.-S. CHEN et al.
by ionizing radiation and a variety of genotoxic drugs (25, 29). g-H2AX is regarded as a checkpoint maintenance factor, dephosphorylation of which enables resumption of the cell cycle after DNA damage is repaired (30, 31). Chk2 is a central component of the signal transduction pathway that is activated by DNA damage, resulting in apoptosis or cell cycle arrest and senescence (32). Threonine 68 phosphorylation by the ataxia telangiectasia mutated (ATM) protein kinase is required for efficient activation of Chk2 (32), which maintains cell cycle arrest in senescent cells (33). We found that radiation could induce transient and prolonged g-H2AX and phospho-Chk2 expression in securin-wild-type and -null HCT116 cells, respectively; however, the prolonged expression of both proteins in securin-null cells was overridden by stable transfection of securin. Chk2 was reported to induce p21 in a p53-dependent and -independent manner and to promote senescence (8, 9). Accordingly, we suggest that the loss of securin impaired DNA repair after irradiation and thereby led to the accumulation of DNA damage signals, which promotes senescence via a Chk2-dependent pathway. It has been reported that p53 and p21 play central roles in the onset of senescence and that p16INK4a expression is essential for the maintenance of senescence (9, 10). The Chk2 protein has been found to regulate p21 expression and senescence through p53-dependent and -independent pathways (8, 9). Interestingly, the levels of p53 and p21 were increased in both securin-wild-type and -null cells following irradiation, and there was no p16 protein expression in HCT116 cell lines with or without irradiation treatment; meanwhile, the Rb protein was downregulated in both cell lines. Accordingly, we suggest that the p53–p21 and p16– pRb pathways may not play a determinant role in deciding the fate of cells after irradiation. A mutation in p53 promotes cell proliferation, cell survival, genomic instability, and resistance to certain types of chemotherapy and radiotherapy (34). We found that cells lacking functional p53 expression were more radioresistant
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than their p53 wild-type counterparts in breast cancer cell lines and HCT116 cell lines (data not shown). Most cells appear to establish and maintain senescence primarily through the p53–p21 tumor suppressor pathway; however, several studies have revealed the possibility of a p53-independent senescent pathway (7, 11, 12). We found that the knockdown of securin resulted in noticeable senescence development and enhanced radiosensitivity in p53-null HCT116 cells and MDA-MB-231 breast cancer cells carrying mutated p53 protein. These findings suggest that irradiation could induce senescence via a p53-independent pathway in securin-deficient human cancer cells. Securin has been considered a novel therapeutic target in vitro and in vivo. Downregulation of securin expression inhibited cell proliferation and tumor formation and increased apoptosis in cancer cells (35–37). On the contrary, depletion of securin diminished pancreatic b cell mass via both apoptosis and senescence (38), reduced bone marrow stem cell proliferation, and enhanced senescent features (27). The actual mechanism (apoptosis or senescence) of the reduction in cell proliferation following securin downregulation remains controversial. In this study, we demonstrated that the loss of securin inhibited proliferation of irradiated colorectal cancer and breast cancer cells and enhanced their radiosensitivity accompanied by an arrest of senescent cell growth. CONCLUSIONS In conclusion, we propose that securin expression plays an important role in determining the proportion of human colorectal and breast cancer cells undergoing radiation-induced apoptosis or senescence. Depletion of securin could enhance radiosensitivity and induce senescence via a p53-independent pathway; thus, knockdown of securin expression to trigger senescence after irradiation may be able to contribute to the radiotherapeutic treatment of cancer cells overexpressing securin, regardless of p53 expression.
REFERENCES 1. Suzuki M, Boothman DA. Stress-induced premature senescence (SIPS): Influence of SIPS on radiotherapy. J Radiat Res (Tokyo) 2008;49:105–112. 2. Roninson IB, Broude EV, Chang BD. If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells. Drug Resist Updat 2001;4:303–313. 3. Verheij M. Clinical biomarkers and imaging for radiotherapyinduced cell death. Cancer Metastasis Rev 2008;27:471–480. 4. Dewey WC, Ling CC, Meyn RE. Radiation-induced apoptosis: Relevance to radiotherapy. Int J Radiat Oncol Biol Phys 1995; 33:781–796. 5. Bhonde MR, Hanski ML, Notter M, et al. Equivalent effect of DNA damage-induced apoptotic cell death or long-term cell cycle arrest on colon carcinoma cell proliferation and tumour growth. Oncogene 2006;25:165–175. 6. Schmitt CA. Cellular senescence and cancer treatment. Biochim Biophys Acta 2007;1775:5–20. 7. Campisi J. d’Adda di Fagagna F. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol 2007; 8:729–740.
8. Aliouat-Denis CM, Dendouga N, Van den Wyngaert I, et al. p53-independent regulation of p21Waf1/Cip1 expression and senescence by Chk2. Mol Cancer Res 2005;3:627–634. 9. Gewirtz DA, Holt SE, Elmore LW. Accelerated senescence: An emerging role in tumor cell response to chemotherapy and radiation. Biochem Pharmacol 2008;76:947–957. 10. Beausejour CM, Krtolica A, Galimi F, et al. Reversal of human cellular senescence: roles of the p53 and p16 pathways. EMBO J 2003;22:4212–4222. 11. Olsen CL, Gardie B, Yaswen P, et al. Raf-1-induced growth arrest in human mammary epithelial cells is p16-independent and is overcome in immortal cells during conversion. Oncogene 2002;21:6328–6339. 12. Michaloglou C, Vredeveld LC, Soengas MS, et al. BRAFE600associated senescence-like cell cycle arrest of human naevi. Nature 2005;436:720–724. 13. Chang BD, Broude EV, Dokmanovic M, et al. A senescencelike phenotype distinguishes tumor cells that undergo terminal proliferation arrest after exposure to anticancer agents. Cancer Res 1999;59:3761–3767.
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14. Jallepalli PV, Waizenegger IC, Bunz F, et al. Securin is required for chromosomal stability in human cells. Cell 2001;105:445– 457. 15. Romero F, Multon MC, Ramos-Morales F, et al. Human securin, hPTTG, is associated with Ku heterodimer, the regulatory subunit of the DNA-dependent protein kinase. Nucleic Acids Res 2001;29:1300–1307. 16. Nagao K, Adachi Y, Yanagida M. Separase-mediated cleavage of cohesin at interphase is required for DNA repair. Nature 2004;430:1044–1048. 17. Zhou Y, Mehta KR, Choi AP, et al. DNA damage-induced inhibition of securin expression is mediated by p53. J Biol Chem 2003;278:462–470. 18. Romero F, Gil-Bernabe AM, Saez C, et al. Securin is a target of the UV response pathway in mammalian cells. Mol Cell Biol 2004;24:2720–2733. 19. Chiu SJ, Hsu TS, Chao JI. Opposing securin and p53 protein expression in the oxaliplatin-induced cytotoxicity of human colorectal cancer cells. Toxicol Lett 2006;167:122–130. 20. Bernal JA, Roche M, Mendez-Vidal C, et al. Proliferative potential after DNA damage and non-homologous end joining are affected by loss of securin. Cell Death Differ 2008;15: 202–212. 21. Heaney AP, Singson R, McCabe CJ, et al. Expression of pituitary-tumour transforming gene in colorectal tumours. Lancet 2000;355:716–719. 22. Tfelt-Hansen J, Kanuparthi D, Chattopadhyay N. The emerging role of pituitary tumor transforming gene in tumorigenesis. Clin Med Res 2006;4:130–137. 23. Solbach C, Roller M, Fellbaum C, et al. PTTG mRNA expression in primary breast cancer: a prognostic marker for lymph node invasion and tumor recurrence. Breast 2004;13:80–81. 24. Solbach C, Roller M, Eckerdt F, et al. Pituitary tumor-transforming gene expression is a prognostic marker for tumor recurrence in squamous cell carcinoma of the head and neck. BMC Cancer 2006;6:242. 25. Chiu SJ, Chao JI, Lee YJ, et al. Regulation of gamma-H2AX and securin contribute to apoptosis by oxaliplatin via a p38 mitogen-activated protein kinase-dependent pathway in human colorectal cancer cells. Toxicol Lett 2008;179:63–70.
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26. Chang BD, Swift ME, Shen M, et al. Molecular determinants of terminal growth arrest induced in tumor cells by a chemotherapeutic agent. Proc Natl Acad Sci U S A 2002;99:389–394. 27. Rubinek T, Chesnokova V, Wolf I, et al. Discordant proliferation and differentiation in pituitary tumor-transforming genenull bone marrow stem cells. Am J Physiol Cell Physiol 2007; 293:C1082–C1092. 28. Di X, Shiu RP, Newsham IF, et al. Apoptosis, autophagy, accelerated senescence and reactive oxygen in the response of human breast tumor cells to adriamycin. Biochem Pharmacol 2009;77: 1139–1150. 29. Rogakou EP, Pilch DR, Orr AH, et al. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 1998;273:5858–5868. 30. Downey M, Durocher D. gammaH2AX as a checkpoint maintenance signal. Cell Cycle 2006;5:1376–1381. 31. Keogh MC, Kim JA, Downey M, et al. A phosphatase complex that dephosphorylates gammaH2AX regulates DNA damage checkpoint recovery. Nature 2006;439:497–501. 32. Antoni L, Sodha N, Collins I, et al. CHK2 kinase: Cancer susceptibility and cancer therapy–two sides of the same coin? Nat Rev Cancer 2007;7:925–936. 33. d’Adda di Fagagna F, Reaper PM, Clay-Farrace L, et al. A DNA damage checkpoint response in telomere-initiated senescence. Nature 2003;426:194–198. 34. Xu Y. Induction of genetic instability by gain-of-function p53 cancer mutants. Oncogene 2008;27:3501–3507. 35. Chen G, Li J, Li F, et al. Inhibitory effects of anti-sense PTTG on malignant phenotype of human ovarian carcinoma cell line SK-OV-3. J Huazhong Univ Sci Technolog Med Sci 2004;24: 369–372. 36. Solbach C, Roller M, Peters S, et al. Pituitary tumor-transforming gene (PTTG): A novel target for anti-tumor therapy. Anticancer Res 2005;25:121–125. 37. El-Naggar SM, Malik MT, Kakar SS. Small interfering RNA against PTTG: A novel therapy for ovarian cancer. Int J Oncol 2007;31:137–143. 38. Chesnokova V, Wong C, Zonis S, et al. Diminished pancreatic beta-cell mass in securin-null mice is caused by beta-cell apoptosis and senescence. Endocrinology 2009;150:2603–2610.
doi:10.1016/j.ijrobp.2009.12.013
BIOLOGY CONTRIBUTION
DEPLETION OF SECURIN INDUCES SENESCENCE AFTER IRRADIATION AND ENHANCES RADIOSENSITIVITY IN HUMAN CANCER CELLS REGARDLESS OF FUNCTIONAL p53 EXPRESSION WEN-SHU CHEN,* YI-CHU YU,* YI-JANG LEE, PH.D.,y JI-HSHIUNG CHEN, PH.D.,z HSUE-YIN HSU, PH.D.,* x AND SHU-JUN CHIU, PH.D.* *Department of Life Science, Tzu Chi University, Hualien, Taiwan; yDepartment of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan; zDepartment of Molecular Biology and Human Genetics, Tzu Chi University, Hualien, Taiwan; and xInstitute of Radiation Sciences, Tzu Chi Technology College, Hualien, Taiwan Purpose: Radiotherapy is one of the best choices for cancer treatment. However, various tumor cells exhibit resistance to irradiation-induced apoptosis. The development of new strategies to trigger cancer cell death besides apoptosis is necessary. This study investigated the role of securin in radiation-induced apoptosis and senescence in human cancer cells. Methods and Materials: Cell survival was determined using clonogenic assays. Western blot analysis was used to analyze levels of securin, caspase-3, PARP, p53, p21, Rb, g-H2AX, and phospho-Chk2. Senescent cells were analyzed using a b-galactosidase staining assay. A securin-expressed vector (pcDNA-securin) was stably transfected into securin-null HCT116 cells. Securin gene knockdown was performed by small interfering RNA and small hairpin RNA in HCT116 and MDA-MB-231 cells, respectively. Results: Radiation was found to induce apoptosis in securin wild type HCT116 cells but induced senescence in securin-null cells. Restoration of securin reduced senescence and increased cell survival in securin-null HCT116 cells after irradiation. Radiation-induced g-H2AX and Chk2 phosphorylation were induced transiently in securin-wildtype cells but exhibited sustained activation in securin-null cells. Securin gene knockdown switches irradiationinduced apoptosis to senescence in both HCT116 p53-null and MDA-MB-231 cells. Conclusions: Our results demonstrated that the level of securin expression plays a determining role in the radiosensitivity and fate of cells. Depletion of securin impairs DNA repair after irradiation, increasing DNA damage and promoting senescence in the residual surviving cells regardless of functional p53 expression. The knockdown of securin may contribute to a novel radiotherapy protocol for the treatment of human cancer cells that are resistant to irradiation. Ó 2010 Elsevier Inc. Securin, Radiosensitivity, Apoptosis, Senescence, p53.
INTRODUCTION
apoptotic cell death are equivalent to long-term cell-cycle arrest (which is frequently associated with cellular senescence) in terms of the suppression of cancer cell proliferation and tumor growth (5, 6). Senescence has been considered a tumor suppressive mechanism that stringently inhibits replication of cancer cells. Senescent cells lack 5-bromo-2-deoxyuridine (BrdU) incorporation and exhibit permanent arrest of cell proliferation, resistance to apoptosis, and changes in gene expression, as well as increased activity of acidic senescence-associated b-galactosidase (SA-b-gal) (6, 7). Senescence regulationrelated proteins vary according to the different sources of DNA damage, stress, and cell type. Cyclin-dependent kinase
Radiotherapy is one of the best therapeutic choices for cancer treatment (1). The cellular response to ionizing radiation produces a spectrum of effects ranging from growth arrest to apoptotic cell death, senescence, necrosis or mitotic catastrophe, and autophagy (2, 3). Apoptosis is the primary cause of cell death induced by radiation; however, solid tumors derived from epithelial cells exhibit resistance to radiationinduced apoptosis (1, 4). Hence, the development of new strategies to trigger cancer cell death besides apoptosis may contribute to improved prognosis in cancer patients. Recent studies have found that the effects of DNA damage-induced Reprint requests to: Shu-Jun Chiu, Ph.D., Department of Life Science, Tzu Chi University, 701, Section 3, Chung-Yang Road, Hualien 970, Taiwan. Tel: 886-3-8565301, ext. 2631; Fax: 8863-8572526; E-mail: [email protected] Wen-Shu Chen and Yi-Chu Yu contributed equally to this work. This work was supported by a grant from the National Science Council, Taiwan (NSC 96-2321-B-320-001-MY3).
Conflict of interest: none. Acknowledgment—We thank Dr. B. Vogelstein, The Johns Hopkins University, for permission to use HCT116 colorectal cancer cell lines. Received Sept 22, 2009, and in revised form Oct 16, 2009. Accepted for publication Dec 10, 2009. 566
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(cdk) inhibitors p21waf1/cip1 (referred to as p21 hereafter) and p16INK4a interfere with the activity of cdk, leading to dephosphorylation of Rb and thereby blocking cell cycle progression (7). Subsequently, Chk2 acts downstream of ATM to induce p21 in a p53-dependent and -independent manner and promotes senescence (8, 9). The p53–p21 pathway has been reported to be important for the onset of senescence and p16INK4a as essential for the maintenance of senescence (10). Nevertheless, there are examples of senescence demonstrating p53-independent and p16-pRB-independent pathways (7, 11–13). The molecular pathways corresponding to cellular senescence remain controversial and are worthy of further study. Securin is known to prevent abnormal sister chromatid segregation during mitosis and maintain genomic stability (14). In addition, securin has been shown to participate in DNA repair through interaction with separase or the Ku70/80 heterodimer of the double-strand breaks (DSBs) nonhomologous DNA end-joining repair machinery after exposure to ultraviolet, X-ray, and gamma-ray irradiation (15, 16). Securin has also been found to be downregulated by DNA-damaging drugs and UV radiation prior to DNA repair (17–19). Furthermore, loss of securin sensitized HCT116 cells to chemotherapeutic agents such as adriamycin and methyl methanesulphonate or to ionizing radiation via proliferative inhibition but not apoptosis (20). In addition, securin has been described as a novel oncogene being expressed abundantly in most cancer cells and overexpressed in colorectal cancer cells (21) and promoting cell proliferation and tumorigenesis (22). Securin overexpression is correlated with poor prognosis in many carcinoma patients (23, 24); however, overexpression of securin has been reported to induce aneuploidy, genetic instability and apoptosis (22). Therefore, the precise role of securin in determining the fate of cells, including apoptosis and senescence, induced by radiation and the correlated radiosensitivity is still unclear. In this study, we investigated the effect of securin expression on radiosensitivity and the fate of cells exerted via different mechanisms induced by irradiation. Radiation was found to induce apoptosis in securin-wild-type HCT116 cells but induced senescence in securin-null cells, and depletion of securin was found to enhance radiosensitivity and induce senescence in the residual surviving cells, regardless of functional p53 expression in human cancer cells. Knockdown of the securin gene may contribute to a novel radiotherapy protocol for human cancer cells that are resistant to irradiation. METHODS AND MATERIALS
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from Calbiochem (San Diego, CA). Anti-caspase-3 was purchased from Imgenex (San Diego, CA).
Cell culture Securin-wild-type, securin-null, and p53-null human HCT116 colorectal cancer cells were routinely maintained in McCoy’s 5A medium (Sigma). MCF-7 and MDA-MB-231 human breast cancer cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) medium (Gibco). The complete medium was supplemented with 10% fetal bovine serum.
Stable transfection for generating a human securinexpressing cell line Securin-null (p53 wild-type) HCT116 cells, a standard cell line provided by Dr. B. Vogelstein at Johns Hopkins University, were transfected with the expression vector human securin-pcDNA3 or pcDNA3 by Lipofectamine, and hygromycin B (100 mg/ml) selection was initiated 48 hr after transfection. Single colonies were picked out, expanded, and then stored in liquid nitrogen, after which human securin expression levels were determined by Western blot analysis.
X-ray irradiation Irradiation was produced by an X-ray machine (RS2000; RAD Source Technologies) operating at 160 kVp (peak kilovoltage) and 25 mA. The dose rate at a source-–subject distance of 38 cm was 1.83 Gy/min. The machine output was routinely calibrated using an air ionization chamber. Briefly, the cells were replenished with fresh medium before irradiation and then subjected to X-ray irradiation immediately at room temperature.
Clonogenic survival assay Cells were seeded at a density of 10,000 cells per well in 2 ml of culture medium in 6-well plates 1 day before treatment. Cells were subsequently exposed to irradiation and incubated for a period of 5 to 7 days to recover and then fixed in 1% crystal violet (containing 30% ethanol) and visualized under an inverted phase-contrast microscope. Twenty random fields were selected for analysis, and isolated clusters of more than 50 cells were counted as a single colony. The relative percentage of surviving cells was calculated by dividing the number of colonies of treated cells by that of the control.
Cell cycle analysis Cells were seeded at a density of 1 x 106 cells per 60-mm culture dish and incubated for 24 hr and then irradiated with X-rays. After treatment, cells were trypsinized and fixed with 70% ethanol at 20 C overnight. Fixed cells were subsequently stained with 20 mg/ml PI staining buffer (containing 1% Triton X-100 and 100 mg/ml RNase A) for 30 min, and cells were analyzed by flow cytometry (FACScalibur; Becton Dickinson, San Jose, CA). The percentage of cells in each of the different cell cycle phases was determined using ModFit LT software (version 2.0; Becton Dickinson).
Chemicals and antibodies Propidium iodide (PI) was purchased from Sigma Chemical Co. (St. Louis, MO). Anti-phospho-histone H2AX (Ser-139) was purchased from Upstate Biological Co. (Lake Placid, NY). Anti-phospho-p53 (Ser-15), anti-phospho-Chk2 (Thr-68), Chk2, Rb, PARP, and an Absolute-S kit were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Monoclonal anti-securin antibody was purchased from Abcam (Cambridgeshire, UK). Anti-actin antibody was purchased from Chemicon International, Millipore (Billerica, MA). Anti-p21waf1/cip1 mouse monoclonal antibody was purchased
SA-b-gal SA-b-gal staining is widely used as a biomarker of cellular senescence in vivo and in vitro (8), the positive blue-colored staining of bgalactosidase at pH 6.0 being remarkably increased in senescent cells. Senescent cells were analyzed using a b-gal staining kit (Cell Signaling) in accordance with the manufacturer’s instructions. The percentage of SA-b-gal-positive cells was then calculated by counting the cells in 10 random fields (at least 100 cells) using bright-field microscopy at a magnification of 200 or 320, in triplicate.
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Fig. 1. Comparison of apoptosis, senescence, and radiosensitivity levels in irradiated securin-wild-type and -null HCT116 cells. (A) Western blot analysis showing levels of securin, caspase-3, and PARP. (B) SA-b-gal staining was performed after irradiation, followed by a postirradiation period of 2 to 7 days. SA-b-gal-positive-stained cells (green) were observed with bright-field microscopy at a magnification of 320. (C) The surviving fractions of securin-wild-type and -null HCT116 cells were measured by clonogenic survival assay.
Western blot analysis Total cellular protein extracts were prepared according to our previous study (25). Briefly, equal amounts of total protein (20–60 mg/ well) were subjected to electrophoresis using 10% to 12% sodium dodecyl sulfate-polyacrylamide gels. Following electrophoretic transfer of proteins onto polyvinylidene fluoride membranes, the proteins were sequentially hybridized with primary antibody, followed by a horseradish peroxidase-conjugated secondary antibody. Finally, the protein bands were visualized on X-ray film, using an enhanced chemiluminescence detection system (Immobilon Western Chemiluminescent horseradish peroxidase substrate, WBKLS0500, Millipore), and gel-digitizing software, Un-Scan-It (version 5.1; Silk Scientific, Inc., Orem, UT) was used to quantify the relative intensity of each band on the X-ray films.
turer’s instructions. Forty-eight hours after transfection, the cells were subjected to irradiation or Western blot analysis as described above.
Establishment of human securin small hairpin RNA knockdown stable MDA-MB-231 cell lines Small fragments of securin small hairpin RNA (shRNA) were cloned into pLKO.1-puro vector. All shRNA clones and two functional plasmids, pMD.G and pCMVdeltaR8.91 (10:1:9), were obtained from the National RNA Interference Core Facility (Academic Sinica, Taiwan). MDA-MB-231 cells were infected with lentiviruses from supernatant media from these clones in DMEM. After cells were infected, they were selected from DMEM culture containing 0.5 mg/ml puromycin dihydro-chloride.
RNA interference
Statistical analysis
Securin small interfering RNA (siRNA) (s17655, [5’/3’] sense: GCACCCGUGUGGUUGCUAAtt; antisense: UUAGCAACCACACGGGUGCct) and scrambled siRNA (4390846) were purchased from Ambion (Austin, TX). Transfection of HCT116 cells with the above-described siRNA oligonucleotides was performed with Lipofectamine 2000 (Invitrogen) according to the manufac-
All data are represented by the means standard error of the mean (SEM) of at least three independent experiments. Statistical analysis was performed by one-way analysis of variance, and further post hoc testing was performed using statistical software GraphPad Prism 4 (GraphPad, Inc. San Diego, CA). A p value of <0.05 was considered statistically significant.
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Fig. 2. Effects of securin expression in securin-null HCT116 cells and knockdown in securin-wild-type cells on radiationinduced senescence and surviving cell fractions. (A) The expression level of securin in stably transfected securin reexpressing HCT116 cells was observed by Western blotting. (B) The percentages of SA-b-gal-positive-stained cells were calculated in securin re-expressing HCT116 cells. (C) The surviving cell fraction in securin re-expressing HCT116 cells was measured by clonogenic survival assay after irradiation. (D) Securin siRNA was transfected into HCT116 securin-wild-type cells, and levels of securin were characterized by Western blot analysis. (E) The percentages of SAb-gal-positive cells transfected with securin siRNA were calculated. (F) The surviving cell fraction of securin siRNAtransfected HCT116 cells was measured by clonogenic survival assay. A p value of <0.01 (**) indicates a significant difference between nonirradiated control and irradiated samples. A p value of <0.01 (##) indicates a significant difference between scrambled and securin siRNA-transfected samples (E).
RESULTS Irradiation induces senescence in securin-null HCT116 cells and apoptosis in securin-wild-type cells Western blot analysis showed that 6 Gy irradiation followed by a postirradiation period of 2 to 7 days reduced the level of securin and induced significant apoptotic hallmarks, specifically caspase-3 activation and PARP cleavage, in securin-wild-type HCT116 cells but not in securin-null cells (Fig. 1A). In addition, as shown in Fig. 1B, securinnull cells exhibited senescence-like morphologic features (enlarged, flattened cells and positive b-gal activities) after irradiation. Notably, irradiation (at 3 and 6 Gy) increased cellular senescence in securin-null cells but not securinwild-type cells. The intrinsic radiosensitivity assessed by
clonogenic survival assay is expressed by the surviving fraction at 2 Gy (SF2). As shown in Fig. 1C, securin-null cells were more susceptible to irradiation than securin-wild-type cells. The SF2 of securin-wild-type and securin-null HCT116 cells exposed to irradiation were about 0.12 and 0.02, respectively. Securin re-expression and gene knockdown oppositely regulate senescence and radiosensitivity in HCT116 cells Western blot analysis with anti-securin antibody showed that the stable cell clone (transfected with a securin-expressed vector) expressed securin protein in securin-null HCT116 cells (Fig. 2A). Radiation-induced senescence was significantly reduced by restoration of securin in securin-null cells exposed to 3 and 6 Gy irradiation followed by a postirradiation
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Fig. 3. Effects of securin expression on the levels of g-H2AX, phospho-Chk2, p53, p21, and Rb in HCT116 cells after irradiation. (A) The levels of g-H2AX, phospho-Chk2, total Chk2, and securin were analyzed by Western blotting in irradiated securin-wild-type, -null, and stably expressing cells. (B) The levels of phospho-Chk2, total Chk2, phosphop53, p53 (DO-1), p21, Rb, and securin were ascertained by Western blot analysis.
duration of 7 days (Fig. 2B). In addition, the securin expressed in the stable cell clone restored the surviving cell fraction (SF2 = 0.32) after irradiation in comparison with securin-null cells (SF2 = 0.03) (Fig. 2C). Additionally, the levels of securin were diminished when HCT116 cells were transfected with 50 nM securin siRNA (Fig. 2D). Transfection of securin siRNA increased senescence in comparison with scrambled siRNA in irradiated HCT116 cells (Fig. 2E). Furthermore, the SF2 values for securin-wild-type HCT116 cells transfected with 50 nM securin siRNA after irradiation were reduced from 0.26 to 0.12 (Fig. 2F). Therefore, transfection with securin siRNA increased the elevation of radiosensitivity in securin-wild-type HCT116 cells. Radiation induces g-H2AX and Chk2 activation in securinnull HCT116 cells but elevates the levels of p53 and p21 proteins, in addition to reducing Rb levels in both securinwild-type and securin -null HCT116 cells The levels of g-H2AX and phospho-Chk2 (Thr-68) were increased by 6-Gy irradiation in securin-null HCT116 cells in a time-dependent manner (Fig. 3A, middle). However, 6-Gy irradiation rapidly and transiently increased the levels of g-H2AX and phospho-Chk2 (Thr-68) in securin-wildtype and securin re-expression cells (Fig. 3A, left and right). Stable transfection with the securin-expressing vector
reduced the radiation-induced g-H2AX and phospho-Chk2 (Thr-68) levels in securin-null HCT116 cells (Fig. 3A, middle and right). Irradiation (6 Gy followed by a postirradiation duration of 2–7 days) also caused a sustained increase in phospho-Chk2 (Thr-68) expression in securin-null HCT116 cells but not in securin-wild-type cells (Fig. 3B, right). As shown in Fig. 3B, the levels of p53 and p21 were elevated in both securin-wild-type and securin-null cells after irradiation followed by a postirradiation period of 2 to 7 days. In contrast, the levels of Rb were reduced, whereas p16 expression was undetectable in both types of cells. Indeed, HCT116 cells are deficient for p16 (26). Securin gene knockdown switches irradiation-induced apoptosis to senescence in HCT116 cells lacking p53 expression Securin levels were found to be decreased by transfection with 25 nM securin siRNA (Fig. 4A). As shown in Fig. 4B, the securin gene knockdown was found to increase radiationinduced senescence-like G2/M arrest in HCT116 p53-null cells after a postirradiation duration of 48 h by flow cytometry. In p53-null HCT116 cells, irradiation reduced the level of securin and increased caspase-3 activation but did not induce p21 protein expression after 6 Gy of irradiation followed by a 5-day recovery period (Fig. 4C). Moreover, caspase-3
Securin determines radiosensitivity and cell fate d W.-S. CHEN et al.
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Fig. 4. Effects of the securin gene knockdown on radiation-induced G2/M arrest, apoptosis, senescence, and surviving cell fraction in HCT116 p53-null cells. (A) Securin siRNA was transfected into HCT116 p53-null cells, and levels of securin were characterized by Western blot analysis. (B) G2/M arrest was analyzed by flow cytometry. (C) The levels of securin, p53, p21, and active caspase-3 in cells transfected with securin siRNA were determined by Western blot analysis. (D and E) The percentages of SA-b-gal-positive cells transfected with securin siRNA were calculated by observation with bright-field microscopy at a magnification of 320. (F) The surviving cell fraction was measured by clonogenic survival assay. A p value of <0.01 (**) indicates a significant difference between untreated control and irradiated samples. A p value of <0.01(##) indicates a significant difference between scrambled and securin siRNA-transfected samples.
activation was significantly reduced by knockdown of securin in p53-null HCT116 cells after irradiation (Fig. 4C). As shown in Fig. 4D, irradiation induced significant senescence in the securin gene knockdown p53-null HCT116 cells compared with p53-null cells transfected with scrambled siRNA. Transfection of securin siRNA (25 nM, 48 hr) increased senescence in p53-null HCT116 cells subjected to 3 and 6 Gy of irradiation (Fig. 4E). In addition, transfection with 25 nM securin siRNA enhanced radiosensitivity in p53-null HCT116 cells after 3 Gy of irradiation (Fig. 4F). Securin gene knockdown increases senescence and enhances radiosensitivity in irradiated human breast cancer cells As shown in Fig. 5A, endogenous levels of securin were much higher in MDA-MB-231 cells, a p53 mutant human breast cancer cell line, than in MCF-7 cells, a p53 wildtype human breast cancer cell line. Furthermore, we gener-
ated an MDA-MB-231 clone (MDA-MB-231-2A) in which endogenous securin expression was knocked down by shRNA (Fig. 5A). Irradiation at 4 and 6 Gy induced significant senescence in MCF-7 cells, which expressed lower levels of securin (Fig. 5B and C); on the contrary, senescence was not increased in the MDA-MB-231 cells after 4- and 6Gy irradiation. Securin shRNA knockdown MDA-MB-2312A cells significantly increased senescence in comparison to MDA-MB-231 cells after irradiation (Fig. 5B and C); in addition, MCF-7 cells were found to be more susceptible to irradiation than MDA-MB-231 cells (Fig. 5D). Furthermore, MDA-MB-231 cells transfected with securin shRNA exhibited enhanced radiosensitivity (Fig. 5D). DISCUSSION Securin has been shown to participate in DNA repair after exposure to ultraviolet, X-ray, and gamma-ray irradiation
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Fig. 5. Effects of the securin gene knockdown on radiation-induced senescence and cell survival in human breast cancer cell lines. (A) The levels of securin in MCF-7 and MDA-MB-231 cells and in the shRNA stable transfectant MDA-MB231-2A clone were characterized by Western blot analysis. (B and C) The percentages of SA-b-gal-positive cells (blue) in the three breast cancer cell lines subjected to irradiation were calculated by observation with bright-field microscopy at a magnification of 200. (D) The surviving cell fraction was measured by clonogenic survival assay.
(15, 16). Nevertheless, securin has also been found to be downregulated by DNA-damaging drugs and UV radiation prior to DNA repair (17–19). Loss of securin compromises nonhomologous end joining repair, cell survival, and proliferation after genotoxic stress (20). In this study, radiation was found to downregulate securin expression and induce apoptosis in securin-wild-type HCT116 cells, whereas senescence was induced in the surviving securin-null HCT116 cells (the surviving fractions were about 0.001 to 0.002 after 3-Gy irradiation [Fig. 1C, and 2C and F). It has been reported that securin deletion leads to chromosomal instability, DNA damage, signaling pathway activation, and senescence (27). Moreover, repression of apoptosis triggered by genotoxic stress may shift the cell failsafe to senescence (28). We found that pharmacological blockade of apoptosis by z-VAD-fmk failed to promote senescence in securin-wild-type cells in response
to irradiation (data not shown), which may indicate that senescence is not a compensatory mechanism for apoptosis. Accordingly, we suggest that the gradual downregulation of securin after irradiation leads to repair of radiation-induced DNA damage and maintenance of cell proliferation. However, the damage may be partly repaired; hence, the unrepaired DNA triggers apoptosis while the cell cycle progresses. On the contrary, loss of securin before irradiation impairs DNA repair, thereby causing the accumulation of radiation-induced DNA damage signals and promoting senescence. Therefore, we propose that securin expression plays an important role in the induction of apoptosis or senescence by radiation, i.e., apoptosis is induced by the existence of securin, whereas senescence is promoted when securin is absent. Phosphorylation of H2AX at serine 139, g-H2AX, is one of the responses of mammalian cells to DNA DSBs induced
Securin determines radiosensitivity and cell fate d W.-S. CHEN et al.
by ionizing radiation and a variety of genotoxic drugs (25, 29). g-H2AX is regarded as a checkpoint maintenance factor, dephosphorylation of which enables resumption of the cell cycle after DNA damage is repaired (30, 31). Chk2 is a central component of the signal transduction pathway that is activated by DNA damage, resulting in apoptosis or cell cycle arrest and senescence (32). Threonine 68 phosphorylation by the ataxia telangiectasia mutated (ATM) protein kinase is required for efficient activation of Chk2 (32), which maintains cell cycle arrest in senescent cells (33). We found that radiation could induce transient and prolonged g-H2AX and phospho-Chk2 expression in securin-wild-type and -null HCT116 cells, respectively; however, the prolonged expression of both proteins in securin-null cells was overridden by stable transfection of securin. Chk2 was reported to induce p21 in a p53-dependent and -independent manner and to promote senescence (8, 9). Accordingly, we suggest that the loss of securin impaired DNA repair after irradiation and thereby led to the accumulation of DNA damage signals, which promotes senescence via a Chk2-dependent pathway. It has been reported that p53 and p21 play central roles in the onset of senescence and that p16INK4a expression is essential for the maintenance of senescence (9, 10). The Chk2 protein has been found to regulate p21 expression and senescence through p53-dependent and -independent pathways (8, 9). Interestingly, the levels of p53 and p21 were increased in both securin-wild-type and -null cells following irradiation, and there was no p16 protein expression in HCT116 cell lines with or without irradiation treatment; meanwhile, the Rb protein was downregulated in both cell lines. Accordingly, we suggest that the p53–p21 and p16– pRb pathways may not play a determinant role in deciding the fate of cells after irradiation. A mutation in p53 promotes cell proliferation, cell survival, genomic instability, and resistance to certain types of chemotherapy and radiotherapy (34). We found that cells lacking functional p53 expression were more radioresistant
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than their p53 wild-type counterparts in breast cancer cell lines and HCT116 cell lines (data not shown). Most cells appear to establish and maintain senescence primarily through the p53–p21 tumor suppressor pathway; however, several studies have revealed the possibility of a p53-independent senescent pathway (7, 11, 12). We found that the knockdown of securin resulted in noticeable senescence development and enhanced radiosensitivity in p53-null HCT116 cells and MDA-MB-231 breast cancer cells carrying mutated p53 protein. These findings suggest that irradiation could induce senescence via a p53-independent pathway in securin-deficient human cancer cells. Securin has been considered a novel therapeutic target in vitro and in vivo. Downregulation of securin expression inhibited cell proliferation and tumor formation and increased apoptosis in cancer cells (35–37). On the contrary, depletion of securin diminished pancreatic b cell mass via both apoptosis and senescence (38), reduced bone marrow stem cell proliferation, and enhanced senescent features (27). The actual mechanism (apoptosis or senescence) of the reduction in cell proliferation following securin downregulation remains controversial. In this study, we demonstrated that the loss of securin inhibited proliferation of irradiated colorectal cancer and breast cancer cells and enhanced their radiosensitivity accompanied by an arrest of senescent cell growth. CONCLUSIONS In conclusion, we propose that securin expression plays an important role in determining the proportion of human colorectal and breast cancer cells undergoing radiation-induced apoptosis or senescence. Depletion of securin could enhance radiosensitivity and induce senescence via a p53-independent pathway; thus, knockdown of securin expression to trigger senescence after irradiation may be able to contribute to the radiotherapeutic treatment of cancer cells overexpressing securin, regardless of p53 expression.
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