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Silencing the Metallothionein-2A gene inhibits cell cycle progression from G1- to S-phase involving ATM and cdc25A signaling in breast cancer cells
Cancer Letters 276 (2009) 109–117
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Silencing the Metallothionein-2A gene inhibits cell cycle progression from G1- to S-phase involving ATM and cdc25A signaling in breast cancer cells Daina Lim, Koh Mei-Xin Jocelyn, George Wai-Cheong Yip, Boon-Huat Bay * Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, 4 Medical Drive, Blk MD 10, S 117 597 Singapore, Singapore
a r t i c l e
i n f o
Article history: Received 11 July 2008 Received in revised form 15 October 2008 Accepted 28 October 2008
Keywords: Metallothionein Cell cycle Breast cancer ATM cdc25A
a b s t r a c t Metallothioneins (MTs) are a group of metal-binding proteins involved in cell proliferation, differentiation and apoptosis. The MT-2A isoform is generally the most abundant isoform among the 10 known functional MT genes. In the present study, we observed that down-regulation of the MT-2A gene in MCF-7 cells via siRNA-mediated silencing inhibited cell growth by inducing cell cycle arrest in G1-phase (G1-arrest) and a marginal increase in cells in sub-G1-phase. Scanning electron microscopic examination of the cells with silenced expression of MT-2A (siMT-2A cells) revealed essentially normal cell morphology with presence of scattered apoptotic cells. To elucidate the underlying molecular mechanism, we examined the expression of cell cycle related genes in MT-2A-silenced cells and found a higher expression of the ataxia telangiectasia mutated (ATM) gene concomitant with a lower expression of the cdc25A gene. These data suggest that MT-2A could plausibly modulate cell cycle progression from G1- to S-phase via the ATM/Chk2/cdc25A pathway. Ó 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The rapid increase in incidence rate of breast cancer has led to the search and identification of biomarkers which could predict risk, future behavior and aid in the management of this malignancy. A better understanding of the molecular mechanisms of breast carcinogenesis has thus ensued. Several proteins including estrogen receptors, progesterone receptors, bcl-2, E-cadherin, heparan sulfate and metallothionein (MT), have been identified as potential biomarkers in breast cancer [1–5]. MT was first isolated as a zinc and cadmium metal binding, cysteine-rich protein in equine renal cortex [6]. MTs consist of a group of low molecular weight proteins that are involved in heavy metal detoxification, zinc homeostasis [7], scavenging free radicals [8], cell proliferation and apoptosis [9,10]. Moreover, Ostrakhovitch et al. has reported that the p53 tumor suppressor protein interacts with MT and this interaction may be involved in * Corresponding author. Tel.: +65 6516 6139; fax: +65 6778 7643. E-mail address: [email protected] (B.-H. Bay). 0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.10.038
regulating apoptosis in breast cancer cells [11]. Furthermore, MT has been reported to modulate p53 conformation and transcriptional activity by zinc chelation [12]. Hence altered levels of this protein can be expected in abnormal cell growth such as cancer. In humans, there are 10 functional MT mRNA isoforms, viz. MT-1A, 1B, 1E, 1F, 1G, 1H, 1X, 2A, 3 and 4 which encode four MT proteins, MT-1, -2, -3 and -4. MT-3 isoform is specifically located in the brain and has involvement in Alzheimer’s disease [13,14] while MT-4 is found to be involved in the differentiation of certain stratified epithelia [15]. MT-1 and MT-2 isoforms are expressed co-ordinately in most organs but the precise role of these MT isoforms has not been well elucidated. The MT-2A isoform has been reported to be the most abundant MT isoform in breast cancer cell lines and tissues [10,16]. In this work, we evaluated the effect of down-regulation of MT-2A expression on cell cycle progression in breast cancer cells. We observed that silencing of the MT-2A gene in MCF-7 cells induced a block in the G1-phase of the cell cycle. The underlying molecular mechanisms for the observed effects were determined by expression analysis of cell cycle
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related genes and further verified by immunoblotting. Suppression of MT-2A in breast cancer cells lead to an increased expression of ataxia telangiectasia mutated (ATM) with concomitant lowering of cdc25A levels. As cdc25A is necessary for the progression from G1- to S-phase of the cell cycle, the decrease in levels of cdc25A is likely to result in G1phase cell cycle arrest in siMT-2A cells. We therefore postulate a novel pathway by which MT-2A regulates cell cycle progression via ATM and cdc25A. 2. Materials and methods 2.1. Antibodies and reagents MT monoclonal antibody (E9) (Dako Corporation, Carpinteria, CA, USA) was used for the detection of MT proteins. This antibody detects all MT-1 and MT-2 isoforms. Monoclonal antibodies employed are mouse anti-cdc25A (Santa Cruz, CA, USA) and mouse anti-ATM (sc-23922) (Santa Cruz, CA, USA). The two siRNAs against the MT-2A gene were specially designed and manufactured by Qiagen (Hilden, Germany). The MT-2A siRNA sequences used were as follows: siMT2A_1 (S, 50 -CCG GUU CCU GCA AAU GCA A dTdT-30 ; AS, 50 -UUG CAU UUG CAG GAA CCG G dCdG-30 ) and siMT2A_2 (S, 50 -CGC UCC CAG AUG UAA AGA A dTdT-30 ; AS, 50 -UUC UUU ACA UCU GGG AGC G dGdG-30 ). A negative siRNA sequence (S, 50 -UUC UCC GAA CGU GUC ACG U dTdT-30 ; AS, 50 -ACG UGA CAC GUU CGG AGA A dTdT-30 ) that does not target any human genes was used as a negative control.
post-transfection while protein expression was examined at 72 h post-transfection after optimization. 2.4. Real-time RT-PCR The cells were homogenized using the QiaShredder (Qiagen) and the total RNA was extracted from the cells using RNEasy Micro kit (Qiagen) for 24-well plate samples and RNeasy Mini kit (Qiagen, Hilden, Germany) for other larger samples. cDNA was synthesized from 2 lg of RNA using SuperScript III 1st-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) with random hexamer primers. Each cDNA sample equivalent to 10 ng of total RNA was then used in each real-time RT-PCR reaction. The primers specific for the individual MT isoforms were adapted from Mididoddi et al. [17]. Real-time RT-PCR conditions were optimized by adjusting the annealing temperature to 60 °C. PCR was performed using a Light Cycler (Roche Applied Science, Indianapolis, IN, USA) and a program with the following parameters: initial denaturation at 95 °C for 15 min, 45 cycles performed at 94 °C for 15 s, 60 °C for 25 s and 72 °C for 18 s. Melting curve analysis was carried out at 65 °C for 15 s to verify the specificity of the amplification reaction. The amplified products of MT isoforms and G3PDH were removed from the LightCycler reaction capillaries by short spin and analyzed by agarose gel electrophoresis. Relative quantification was calculated using the DDCT and 2DDCT method, where DCT refers to the difference between the CT values of the target gene and the housekeeping gene, G3PDH. 2.5. Immunocytochemistry
2.2. Cell culture MCF-7 breast cancer cells and MCF-12A normal breast epithelial cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). MCF-7 breast cancer cells were maintained in DMEM supplemented with 10% FBS without any antibiotics. MCF-12A cells were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium, supplemented with 20 ng/ml human epidermal growth factor, 100 ng/ml cholera toxin, 0.01 mg/ml bovine insulin, 500 ng/ml hydrocortisone and 5% FBS. Cell cultures were passaged at subconfluency and maintained at 5% carbon dioxide at 37 °C.
Cells grown on coverslips were fixed in 4% paraformaldehyde. Cells were incubated with primary mouse monoclonal anti-MT antibody at 1:200 dilution, 4 °C overnight. Secondary anti-mouse IgG was then added at a dilution of 1:200 and incubated at room temperature for 1 h before incubation with Avidin Biotin Complex (ABC) solution. The cells were then treated with 3,30 -diaminobenzidine (DAB) in Tris buffered saline (TBS) and 1% hydrogen peroxide for 10 min. Subsequently, cells were washed with TBS before rinsing with 0.1 M of acetate buffer pH 4.8, followed by counterstaining with hematoxylin. After dehydrating the cells with increasing concentration of ethanol and Histoclear, the coverslips were mounted on glass slides.
2.3. Silencing of the MT-2A genes in MCF-7 breast cancer cells
2.6. Growth curve analysis
One microgram of siRNA was diluted to a final volume of 100 ll in opti-MEM I Reduced Serum Medium (Gibco, Auckland, New Zealand) in a 24-well plate. Six microliters of RNAiFect Transfection Reagent (Qiagen) was added to the prediluted siRNA and mixed by pipetting. The samples were incubated at room temperature for 15 min before replacing the culture medium with 300 ll of cell culture medium and addition of the transfection complexes drop wise. The plate was incubated at 37 °C in a humidified 5% CO2 atmosphere. The silencing efficiency in MCF-7 cells was optimized using a positive siRNA (siAClamin). Gene expression in the transfected cells was examined at 48 h
Cells were cultured for up to 72 h post-siRNA transfection and analysis was conducted using alamarBlue (Invitrogen, CA, USA) with continuous monitoring of the treated cells. AlamarBlue was added to a concentration of 10% (v/v) of the total volume of the medium bathing the cells 20 h after seeding. The samples were then incubated for 4 h at 37 °C in a 5% CO2 humidified incubator and absorbance was read at 570 nm with 600 nm as the reference wavelength using the Tecan2000 plate reader. Following which, the medium with alamarBlue was removed and fresh medium was added to the cells. The above procedure with alamarBlue was repeated 4 h before
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the 24, 48 and 72 h time points. The experiments were conducted in triplicates. 2.7. Cell proliferation assay Cell proliferation was determined with the CellTiter 96Ò Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI). At 72 h post-siRNA transfection, 150 ll of dye solution was added to the siRNA transfected cells in a 24-well plate, containing 1000 ll of fresh culture medium. The plate was then incubated for 4 h at 37 °C in a humidified, 5% CO2 atmosphere. After incubation, 1000 ll of Solubilization Solution/Stop Mix was added to each well. The plate was then allowed to stand overnight in a humidified incubator at 37 °C. The absorbance was read at 570 nm with 650 nm as the reference wavelength using the Tecan2000 plate reader.
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2.11. Immunoblot analysis Cell lysate was harvested from the monolayer using MPER (Pierce Biotechnology, Rockford, IL, USA) in the presence of EDTA, phosphatase inhibitor cocktail and protease inhibitor cocktail (Pierce Biotechnology) with the use of a cell scrapper. Cell debris was removed by centrifuging the cell lysate at 13,000 rpm at 4 °C for 5 min. Protein concentration was examined using Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). Twenty micrograms of cell extracts were subjected to separation using 10% sodium dodecyl sulfate– polyacrylamide gel by electrophoresis. The separated proteins were then transferred to PVDF membrane
2.8. Flow cytometry analysis of cell cycle Cells were harvested and washed with PBS before fixing in 70% ethanol. The fixed cells were washed once with PBS before suspending in propidium iodide/RNase solution consisting of a final concentration of 20 lg/ml of propidium iodide (Sigma–Aldrich, Saint Louis, MI, USA), 200 lg/ml of RNase A (Roche Applied Science, Indianapolis, IN, USA) solution in 0.1% Triton-X in PBS. The cells were incubated for 30 min at room temperature before analysis. 2.9. Scanning electron microscopy (SEM) MCF-7 cells grown on glass slides were treated with siMT2A_1 or siNeg for 48 h. Cells were then fixed in 3% glutaraldehyde in phosphate buffer (PB) at pH 7.4 for 1 h. After washing with PB, cells were post-fixed with 1% osmium tetraoxide for 30 min. The samples were subsequently dehydrated with increasing concentrations of ethanol followed by drying in a Critical Point Dryer (Baltec CPD 030, Liechtenstein). The cells were mounted on SEM and sputter-coated with gold before viewing in a Philips XL30 SEM (FEI, Holland). 2.10. Superarray analysis cDNA obtained from siMT2A_1 silenced cells at 48 h were used to examine the expression of the various genes related to different cancer pathways using Superarray technology with cell cycle and metastatic genes (Superarray Bioscience, Frederick, MD, USA) according to the manufacturer’s protocol. cDNA of MCF-7 cells treated with siNegative at 48 h was used as a negative control in this experiment. The quality of the RNA was verified by Nanodrop to have a A260:A280 ratio above 2.0 and A260:A230 ratio above 1.7 before further use. The quality of the cDNA of treated and control cells were verified with RT2 RNA QC PCR array (Superarray Bioscience, Frederick, MD, USA) and silencing efficiency by real-time RT-PCR before Superarray analysis. The normalizer used was the average of five housekeeping genes, viz., 18SrRNA, HPRT1, RPL13A, G3PDH and ACTB, for each cDNA sample.
Fig. 1. Differential expression levels of MT isoforms in breast cell lines. (a) MT isoforms in MCF-7 breast cancer cells. A lower DCT value implies a higher gene expression. (b) MT isoforms in MCF-12A normal breast epithelial cells. (c) Gel electrophoresis of the real-time PCR amplification products of MT-2A isoform and housekeeping gene G3PDH from MCF-7 cells. Ten microliters of the PCR product was electrophoresed on 1.2% agarose gel in the presence of ethidium bromide. Lane designation: lane M, molecular weight marker; lane 1, MT-2A; lane 2, G3PDH.
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(Bio-Rad, Hercules, CA, USA) for Western blotting analysis. The membranes were blocked in 5% non-fat milk (in Trisbuffered saline Tween 20 (50 mM Tris, pH 7.6, 150 mM NaCl and 0.1% Tween 20) for 1 h at room temperature before overnight incubation (at 4 °C) with ATM and cdc25A antibodies at 1:200 and 1:100 dilutions, respectively. b-Actin (BioRad, Hercules, CA, USA) was used as the loading control. HRP conjugated goat-anti-mouse secondary antibodies (Amersham Biosciences, Piscataway, NJ) were used. The membranes were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology). Immunoblot bands were analyzed and quantified by densitometer GS-710 (Bio-Rad, Hercules, CA, USA). 2.12. Statistical analysis Values are presented as means ± SEM. An unpaired twotailed t-test or one-way ANOVA followed by a post hoc Tukey test was performed using the GraphPad Prism version 4.00 for Windows. For comparing differences between growth curves, the two-way ANOVA was used. p < 0.05 was considered significant. 3. Results 3.1. Expression of MT isoforms in breast cell lines Of the eight known functional MT-1 and MT-2 mRNA isoforms, MT1A, 1F, 1H, 1X and 2A isoforms were expressed in MCF-7 breast cancer
cells (Fig. 1a). MT-1A and 1H isoforms were minimally expressed in the breast cancer cells with 1F, 1X and 2A being the main isoforms expressed. MCF-12A normal breast epithelial cells additionally expressed the MT-1E isoform which is similar to that previously reported in primary breast myoepithelial cells [15]. The specificity of the MT and G3PDH primers was verified by melting curve analyses (not shown) and running the amplicons on a 1% gel electrophoresis (Fig. 1b). 3.2. Down-regulation of MT-2A at mRNA and protein levels The relative expression of the MT-2A gene following successful downregulation was examined at 48 h and 72 h post-transfection (Fig. 2a). The MT-2A gene was found to be successfully down-regulated at 94% and 96% with siMT2A_1 and siMT2A_2, respectively, at 48 h. The silencing efficiency decreased to 76% and 78% for siMT2A_1 and siMT2A_2, respectively at 72 h. Down-regulation of the MT-2A gene was verified at the protein level with the use of immunohistochemistry (Fig. 2b). The intensity of MT-immunopositive staining in siMT-2A samples was significantly lesser as compared to siNegative or control samples. Although the E9 antibody detects all MT-1 and MT-2 isoforms, MT-2A is known to be the most abundant isoform in MCF-7 cells [9]. Western blotting was not used for the detection of MT protein levels as to-date there is no well-developed protocol for MT due to the small size (6–7 kDa) of this protein. 3.3. Silencing of MT-2A gene inhibited cell proliferation Cell growth was significantly slower in siMT2A_1 cells (p = 0.0056) and siMT2A_2 cells (p = 0.0147) as compared to siNegative treated cells when analyzed with two-way ANOVA (Fig. 3a). There was no significant difference in growth between siMT2A_1 and siMT2A_2 (p = 0.2812). Furthermore, cell proliferation analysis showed reduced cell proliferation at 72 h post-siRNA transfection in siMT2A_1 cells (p < 0.001) and siMT2A_2 cells (p < 0.05) as compared to cells treated with the siNegative control
Fig. 2. Silencing the MT-2A gene with siRNA down-regulates MT-2A expression at mRNA and protein levels. (a) Silencing efficiency of two independent siRNA, namely siMT2A_1 and siMT2A_2 at 48 h and 72 h post-transfection in MCF-7 breast cancer cells. Total RNA was extracted and converted to cDNA before analysis by quantitative real-time RT-PCR. (b) Immunocytochemistry with primary monoclonal anti-MT E9 antibody and DAB staining in control cells and after treatment with siNegative and siMT2A_1. More intense MT-immunopositive staining was observed in the cytoplasm and nuclei of untreated and siNegative-treated cells as compared to cells treated with siMT2A_1. The nuclear staining observed in the siMT2A-1 treated cells was mainly due to the counterstaining by hematoxylin and not DAB staining. Bar = 48 lm.
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4. Discussion
Fig. 3. Silencing of MT-2A gene inhibits cell proliferation in MCF-7 cells. (a) Growth curve analysis up to 72 h post-siRNA transfection using nontoxic alamarBlue. Relative absorbance was determined by the difference between the absorbance of the cells at 570 nm and the absorbance of reference wavelength at 600 nm. (b) Cell proliferation assay at 72 h postsiRNA transfection examined with CellTiter 96Ò Non-Radioactive Cell Proliferation Assay. Relative absorbance was determined by the difference between the absorbance of the cells at 570 nm and the absorbance of reference wavelength at 650 nm. Results are presented as means ± SEM of triplicate experiments.
(Fig. 3b). Both siRNAs against MT-2A demonstrated an increase in the number of cells in the sub-G1- and G1-phase and decrease in the S and G2/M-phases (Figs. 4a and b). There was a slight increase in the percentage of cells in the sub-G1-phase in the siNegative treated cells as compared to the siMT2A_1 (p = 0.0067) and siMT2A_2 (p = 0.0198) treated cells. There was also a significant increase in the percentage of cells in the G1-phase from 71.3% in the siNegative treated cells to 83.7% in siMT2A_1 (p < 0.0001) and 74.4% in the siMT2A_2 (p = 0.0215) treated cells. A decrease in the percentage of cells in the S-phase was observed in the siMT2A_1 (p = 0.0002) and siMT2A_2 (p = 0.012) treated cells as compared to the siNegative treated cells. In the G2/M-phase, siMT2A_1 treated cells showed a significant decrease in the percentage of cells as compared to siNegative treated cells (7.3% compared with 15.0%, p = 0.0008). There was no significant difference between the untreated cells and siNegative treated cells (p > 0.05) for all phases of the cell cycle. SEM micrographs show a marginal increase in the number of apoptotic cells in siMT2A_1 treated cells as compared to siNegative treated cells (Fig. 4c). 3.4. Differential expression of cell cycle genes We also compared the expression of 16 cell cycle related genes in MT-2A down-regulated MCF-7 cells with siNegative treated MCF-7 cells (Table 1). The genes that were differentially regulated include up-regulation of the ATM gene by 1.31-fold (p < 0.05), down-regulation of cdc25A by 2.28-fold (p < 0.05) and Checkpoint homolog 2 (chk2) by 1.35-fold (p < 0.05). The differential regulation of ATM and cdc25A at protein level was verified by immunoblotting and densitometric analysis (Fig. 5).
MTs have been implicated in breast cancer progression as oncogenic proteins, promoting cell proliferation in several types of cancers [18–21]. Clinical studies have documented a relationship between higher aggressiveness and poorer prognosis of breast cancer with increased MT protein expression [10,22,23]. There is still paucity of information with regard to the role of MT isoforms in tumorigenesis. Although down-regulation of MT-2A by anti-sense oligonucleotides in breast cancer cells have revealed the induction of growth arrest and apoptosis [9,10], the exact mechanism by which MT-2A affects cancer progression in breast cancer has not been well elucidated. Our current study shows a significant reduction in the number of cells with an obvious G1-phase cell cycle arrest when the breast cancer cells were treated with MT-2A siRNA. There was a significant increase in the number of cells in the G1-phase of the siMT-2A cells as compared to the siNegative treated cells. In order to better understand the molecular mechanism(s) that may result in a block at the G1-stage of the cell cycle, expression analysis of cell cycle related genes were conducted using the commercially available Superarray kit. From the gene expression study, cdc25A was found to be significantly down-regulated while ATM was found to be significantly up-regulated at both mRNA and protein levels. Both genes encode proteins belonging to the ATM/Chk2/cdc25A pathway which is known to influence the cell cycle. In the early stages of checkpoint activation, DNA damage sensors are known to relay information to a family of phosphoinositide 3-kinase related kinases (PIKKs) [24] which are essentially serine/threonine kinases. These proteins play essential roles in relaying early signals in cell cycle checkpoints. The two PIKK family members in mammalian cells are ATM and ATR (ATM and Rad3-related). One of the relevant substrate in response to DNA damage for both of these kinases is p53 [25–27]. In mammalian cells, ATM controls cell cycle arrest in G1- and G2-phases in response to DNA damage [28]. In p53-dependant G1 checkpoint, phosphorylation of Ser 15 residue on p53 by ATM will eventually lead to its accumulation by the release of p53 from its substrate [29,30]. Activation of p53 will induce transcription of cyclin-dependent kinase (cdk) inhibitor p21 CIP1/WAF1, leading to a G1-growth arrest [31]. Since ATM was found to be up-regulated in siMT-2A cells in our present study, MT-2A could possibly act upstream of ATM and its substrate p53 to induce G1-growth arrest. This is supported by a previous study where MT-2A was reported to bind to p53 and activate its downstream target gene, p21 [11]. However, our results also indicate that MT-2A can mediate G1-growth arrest via ATM/chk2/cdc25A pathway. In human cells, three cdc25 members, viz., cdc25A, cdc25B and cdc25 C have been characterized [32–34]. Cdc25B and cdc25C mainly regulate the transition from G2 to mitosis phase through cdk1 dephosphorylation [35,36] whereas cdc25A is required for progression from G1- to S-phase by phosphorylation of its primary substrate cdk2, which
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Fig. 4. Silencing of MT-2A gene induces G1-phase cell cycle arrest and a marginal rise in apoptosis. (a) Representative cell cycle profile of MCF-7 cells (control) and MCF-7 cells transiently transfected with siNegative, siMT2 A_1 and siMT2A_2. Cells were stained with propidium iodide and analyzed with FACS flow cytometry at 72 h post-transfection. (b) Proportion of cells in various phases of cell cycle for siMT2A_1, siMT2A_2, siNegative and control treated cells. Results are presented as means ± SEM of triplicate experiments. (c) Scanning electron micrographs of MCF-7 cells treated with siMT2A and siNegative at 48 h post-transfection. Bar = 50 lm. Inset shows an apoptotic cell with a shrunken morphology and the presence of cell blebs. Bar = 5 lm.
Table 1 Expression of cell cycle related genes induced and repressed in MCF-7 cells after siMT-2A treatment. Gene name
Description
Cell cycle related genes ATM Ataxia telangiectasia mutated (includes complementation groups A, C and D) BRCA1 Breast cancer 1, early onset CCNE1 Cyclin E1 CDC25A Cell division cycle 25A CDK2 Cyclin-dependent kinase 2 CDK4 Cyclin-dependent kinase 4 CDKN1A Cyclin-dependent kinase inhibitor 1A (p21, Cip1) CDKN2A Cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) CHEK2 CHK2 checkpoint homolog (S. pombe) E2F1 E2F transcription factor 1 MDM2 Mdm2, transformed 3T3 cell double minute 2, p53 binding protein (mouse) RB1 Retinoblastoma 1 (including osteosarcoma) S100A4 S100 calcium binding protein A4 (calcium protein, calvasculin, metastasin, murine placental homolog) TP53 Tumor protein p53 (Li-Fraumeni syndrome)
GenBank Accession No.
Fold change
NM_000051
1.31*
NM_007294 NM_001238 NM_001789 NM_001798 NM_000075 NM_000389 NM_000077 NM_007194 NM_005225 NM_002392 NM_000321 NM_002961
1.46 1.19 2.28* 1.04 1.08 1.38 1.00 1.35* 1.17 1.08 1.08 1.20
NM_000546
1.07
A list of cell-cycle related genes were subjected to real-time RT-PCR analysis by Superarray technology (n = 3). The fold changes of the respective genes in the siMT2A treated sample with respect to siNegative treated sample are shown above. A positive value indicates a positive up-regulation of the gene while a negative value indicates down-regulation of the gene. * p < 0.05.
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Fig. 5. Silencing of MT-2A gene induces an up-regulation in ATM protein expression with a concomitant decrease in cdc25A protein expression. (a) MCF7 cells were treated with siMT-2A and siNegative. Cell lysates were examined by Western blot analysis with antibodies against ATM and the housekeeping protein b-actin. A representative blot is shown along with adjacent densitometric quantitation of ATM and b-actin band intensities. (b) siMT-2A and siNegative-treated MCF7 cell lysates were examined by Western blot analysis with antibodies against cdc25A and b-actin. A representative blot is shown along with adjacent densitometric quantitation of cdc25A and b-actin band intensities. Results are presented as means ± SEM of triplicate experiments.
Fig. 6. Schematic diagram showing that inhibition of MT-2A expression results in G1-cell cycle block mediated via the ATM/Chk2/cdc25A pathway.
is essential for the DNA replication process [37,38]. The abundance of cdc25A is regulated by the ubiquitin–proteasome degradation pathway in response to inhibition of DNA replication or induction of DNA damage [39,40] which is activated by ATM and ataxia telangiectasia-mutated and Rad3-related (ATR) proteins. ATM activates its downstream effector Chk2 by phosphorylation at Thr68 [41–43] while ATR phosphorylates Chk1 at Ser317 and Ser345 [44]. Activated Chk2 phosphorylates downstream substrates, which include p53 tumor suppressor at Ser20 [45], BRCA1 at Ser988 [46] and Cdc25 family of phosphatases which are involved in cell cycle arrest. Chk2 phosphorylates Cdc25A at Ser123 [47] and Cdc25C at Ser216 [48]. Chk2 autophosphorylation has also been reported to
occur both in cis and in trans [49]. We posit that downregulation of MT-2A may lead to increased expression of ATM which in turn regulate phosphorylation of Chk2 and trigger the ubiquitin–proteasome degradation pathway for cdc25A, thereby leading to p53-independent G1-arrest (Fig. 6). In summary, we have shown that MT-2A affects cell proliferation in breast cancer cells. Moreover, cell cycle gene analysis shows that MT-2A regulates the cell cycle through the ATM/p53 and/or ATM/Chk2/cdc25A pathway. One of the recent focuses in cancer therapeutic strategy is to develop molecular cancer therapeutics. To this end, MT-2A appears to be a promising target molecule for the molecular therapy of breast cancer.
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Conflict of Interest None. Acknowledgements This work is supported by Singapore National Medical Research Council Grants NMRC/1019/2005 and NMRC/ 1081/2006. Daina Lim is the recipient of a Research Scholarship from the National University of Singapore. We thank Ms. Y.G. Chan and Ms. S.L. Bay for technical assistance. References [1] A. Nicolini, A. Carpi, G. Tarro, Biomolecular markers of breast cancer, Front Biosci. 11 (2006) 1818–1843. [2] F.J. Esteva, G.N. Hortobagyi, Prognostic molecular markers in early breast cancer, Breast Cancer Res. 6 (2004) 109–118. [3] V. Giancotti, Breast cancer markers, Cancer Lett. 243 (2006) 145– 159. [4] M. Gotte, G.W. Yip, Heparanase, hyaluronan, and CD44 in cancers: a breast carcinoma perspective, Cancer Res. 66 (2006) 10233–10237. [5] M.G. Cherian, A. Jayasurya, B.H. Bay, Metallothioneins in human tumors and potential roles in carcinogenesis, Mutat. Res. 533 (2003) 201–209. [6] J.H. Kagi, B.L. Valee, Metallothionein: a cadmium- and zinccontaining protein from equine renal cortex, J. Biol. Chem. 235 (1960) 3460–3465. [7] M.P. Richards, R.J. Cousins, Mammalian zinc homeostasis: requirement for RNA and metallothionein synthesis, Biochem. Biophys. Res. Commun. 64 (1975) 1215–1223. [8] J. Klimczak, J.M. Wisniewska-Knypl, J. Kolakowski, Stimulation of lipid peroxidation and heme oxygenase activity with inhibition of cytochrome P-450 monooxygenase in the liver of rats repeatedly exposed to cadmium, Toxicology 32 (1984) 267–276. [9] A. Abdel-Mageed, K.C. Agrawal, Antisense down-regulation of metallothionein induces growth arrest and apoptosis in human breast carcinoma cells, Cancer Gene Ther. 4 (1997) 199–207. [10] R. Jin, V.T. Chow, P.H. Tan, et al, Metallothionein 2A expression is associated with cell proliferation in breast cancer, Carcinogenesis 23 (2002) 81–86. [11] E.A. Ostrakhovitch, P.E. Olsson, S. Jiang, et al, Interaction of metallothionein with tumor suppressor p53 protein, FEBS Lett. 580 (2006) 1235–1238. [12] C. Meplan, M.J. Richard, P. Hainaut, Metalloregulation of the tumor suppressor protein p53: zinc mediates the renaturation of p53 after exposure to metal chelators in vitro and in intact cells, Oncogene 19 (2000) 5227–5236. [13] Y. Uchida, K. Takio, K. Titani, et al, The growth inhibitory factor that is deficient in the Alzheimer’s disease brain is a 68 amino acid metallothionein-like protein, Neuron 7 (1991) 337–347. [14] R.D. Palmiter, S.D. Findley, T.E. Whitmore, et al, MT-III a brainspecific member of the metallothionein gene family, Proc. Natl. Acad. Sci. USA 89 (1992) 6333–6337. [15] C.J. Quaife, S.D. Findley, J.C. Erickson, et al, Induction of a new metallothionein isoform (MT-IV) occurs during differentiation of stratified squamous epithelia, Biochemistry 33 (1994) 7250–7259. [16] S.K. Tai, O.J. Tan, V.T. Chow, et al, Differential expression of metallothionein 1 and 2 isoforms in breast cancer lines with different invasive potential: identification of a novel nonsilent metallothionein-1H mutant variant, Am. J. Pathol. 163 (2003) 2009–2019. [17] S. Mididoddi, J.P. McGuirt, M.A. Sens, et al, Isoform-specific expression of metallothionein mRNA in the developing and adult human kidney, Toxicol. Lett. 85 (1996) 17–27. [18] W.G. McCluggage, P. Maxwell, H. Bharucha, Immunohistochemical detection of metallothionein and MIB1 in uterine cervical squamous lesions, Int. J. Gynecol. Pathol. 17 (1998) 29–35. [19] Y. Hishikawa, T. Koji, D.K. Dhar, et al, Metallothionein expression correlates with metastatic and proliferative potential in squamous cell carcinoma of the oesophagus, Br. J. Cancer 81 (1999) 712– 720. [20] Y. Tan, R. Sinniah, B.H. Bay, et al, Metallothionein expression and nuclear size in benign, borderline and malignant serous ovarian tumours, J. Pathol. 189 (1999) 60–65.
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Silencing the Metallothionein-2A gene inhibits cell cycle progression from G1- to S-phase involving ATM and cdc25A signaling in breast cancer cells Daina Lim, Koh Mei-Xin Jocelyn, George Wai-Cheong Yip, Boon-Huat Bay * Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, 4 Medical Drive, Blk MD 10, S 117 597 Singapore, Singapore
a r t i c l e
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Article history: Received 11 July 2008 Received in revised form 15 October 2008 Accepted 28 October 2008
Keywords: Metallothionein Cell cycle Breast cancer ATM cdc25A
a b s t r a c t Metallothioneins (MTs) are a group of metal-binding proteins involved in cell proliferation, differentiation and apoptosis. The MT-2A isoform is generally the most abundant isoform among the 10 known functional MT genes. In the present study, we observed that down-regulation of the MT-2A gene in MCF-7 cells via siRNA-mediated silencing inhibited cell growth by inducing cell cycle arrest in G1-phase (G1-arrest) and a marginal increase in cells in sub-G1-phase. Scanning electron microscopic examination of the cells with silenced expression of MT-2A (siMT-2A cells) revealed essentially normal cell morphology with presence of scattered apoptotic cells. To elucidate the underlying molecular mechanism, we examined the expression of cell cycle related genes in MT-2A-silenced cells and found a higher expression of the ataxia telangiectasia mutated (ATM) gene concomitant with a lower expression of the cdc25A gene. These data suggest that MT-2A could plausibly modulate cell cycle progression from G1- to S-phase via the ATM/Chk2/cdc25A pathway. Ó 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The rapid increase in incidence rate of breast cancer has led to the search and identification of biomarkers which could predict risk, future behavior and aid in the management of this malignancy. A better understanding of the molecular mechanisms of breast carcinogenesis has thus ensued. Several proteins including estrogen receptors, progesterone receptors, bcl-2, E-cadherin, heparan sulfate and metallothionein (MT), have been identified as potential biomarkers in breast cancer [1–5]. MT was first isolated as a zinc and cadmium metal binding, cysteine-rich protein in equine renal cortex [6]. MTs consist of a group of low molecular weight proteins that are involved in heavy metal detoxification, zinc homeostasis [7], scavenging free radicals [8], cell proliferation and apoptosis [9,10]. Moreover, Ostrakhovitch et al. has reported that the p53 tumor suppressor protein interacts with MT and this interaction may be involved in * Corresponding author. Tel.: +65 6516 6139; fax: +65 6778 7643. E-mail address: [email protected] (B.-H. Bay). 0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.10.038
regulating apoptosis in breast cancer cells [11]. Furthermore, MT has been reported to modulate p53 conformation and transcriptional activity by zinc chelation [12]. Hence altered levels of this protein can be expected in abnormal cell growth such as cancer. In humans, there are 10 functional MT mRNA isoforms, viz. MT-1A, 1B, 1E, 1F, 1G, 1H, 1X, 2A, 3 and 4 which encode four MT proteins, MT-1, -2, -3 and -4. MT-3 isoform is specifically located in the brain and has involvement in Alzheimer’s disease [13,14] while MT-4 is found to be involved in the differentiation of certain stratified epithelia [15]. MT-1 and MT-2 isoforms are expressed co-ordinately in most organs but the precise role of these MT isoforms has not been well elucidated. The MT-2A isoform has been reported to be the most abundant MT isoform in breast cancer cell lines and tissues [10,16]. In this work, we evaluated the effect of down-regulation of MT-2A expression on cell cycle progression in breast cancer cells. We observed that silencing of the MT-2A gene in MCF-7 cells induced a block in the G1-phase of the cell cycle. The underlying molecular mechanisms for the observed effects were determined by expression analysis of cell cycle
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related genes and further verified by immunoblotting. Suppression of MT-2A in breast cancer cells lead to an increased expression of ataxia telangiectasia mutated (ATM) with concomitant lowering of cdc25A levels. As cdc25A is necessary for the progression from G1- to S-phase of the cell cycle, the decrease in levels of cdc25A is likely to result in G1phase cell cycle arrest in siMT-2A cells. We therefore postulate a novel pathway by which MT-2A regulates cell cycle progression via ATM and cdc25A. 2. Materials and methods 2.1. Antibodies and reagents MT monoclonal antibody (E9) (Dako Corporation, Carpinteria, CA, USA) was used for the detection of MT proteins. This antibody detects all MT-1 and MT-2 isoforms. Monoclonal antibodies employed are mouse anti-cdc25A (Santa Cruz, CA, USA) and mouse anti-ATM (sc-23922) (Santa Cruz, CA, USA). The two siRNAs against the MT-2A gene were specially designed and manufactured by Qiagen (Hilden, Germany). The MT-2A siRNA sequences used were as follows: siMT2A_1 (S, 50 -CCG GUU CCU GCA AAU GCA A dTdT-30 ; AS, 50 -UUG CAU UUG CAG GAA CCG G dCdG-30 ) and siMT2A_2 (S, 50 -CGC UCC CAG AUG UAA AGA A dTdT-30 ; AS, 50 -UUC UUU ACA UCU GGG AGC G dGdG-30 ). A negative siRNA sequence (S, 50 -UUC UCC GAA CGU GUC ACG U dTdT-30 ; AS, 50 -ACG UGA CAC GUU CGG AGA A dTdT-30 ) that does not target any human genes was used as a negative control.
post-transfection while protein expression was examined at 72 h post-transfection after optimization. 2.4. Real-time RT-PCR The cells were homogenized using the QiaShredder (Qiagen) and the total RNA was extracted from the cells using RNEasy Micro kit (Qiagen) for 24-well plate samples and RNeasy Mini kit (Qiagen, Hilden, Germany) for other larger samples. cDNA was synthesized from 2 lg of RNA using SuperScript III 1st-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) with random hexamer primers. Each cDNA sample equivalent to 10 ng of total RNA was then used in each real-time RT-PCR reaction. The primers specific for the individual MT isoforms were adapted from Mididoddi et al. [17]. Real-time RT-PCR conditions were optimized by adjusting the annealing temperature to 60 °C. PCR was performed using a Light Cycler (Roche Applied Science, Indianapolis, IN, USA) and a program with the following parameters: initial denaturation at 95 °C for 15 min, 45 cycles performed at 94 °C for 15 s, 60 °C for 25 s and 72 °C for 18 s. Melting curve analysis was carried out at 65 °C for 15 s to verify the specificity of the amplification reaction. The amplified products of MT isoforms and G3PDH were removed from the LightCycler reaction capillaries by short spin and analyzed by agarose gel electrophoresis. Relative quantification was calculated using the DDCT and 2DDCT method, where DCT refers to the difference between the CT values of the target gene and the housekeeping gene, G3PDH. 2.5. Immunocytochemistry
2.2. Cell culture MCF-7 breast cancer cells and MCF-12A normal breast epithelial cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). MCF-7 breast cancer cells were maintained in DMEM supplemented with 10% FBS without any antibiotics. MCF-12A cells were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium, supplemented with 20 ng/ml human epidermal growth factor, 100 ng/ml cholera toxin, 0.01 mg/ml bovine insulin, 500 ng/ml hydrocortisone and 5% FBS. Cell cultures were passaged at subconfluency and maintained at 5% carbon dioxide at 37 °C.
Cells grown on coverslips were fixed in 4% paraformaldehyde. Cells were incubated with primary mouse monoclonal anti-MT antibody at 1:200 dilution, 4 °C overnight. Secondary anti-mouse IgG was then added at a dilution of 1:200 and incubated at room temperature for 1 h before incubation with Avidin Biotin Complex (ABC) solution. The cells were then treated with 3,30 -diaminobenzidine (DAB) in Tris buffered saline (TBS) and 1% hydrogen peroxide for 10 min. Subsequently, cells were washed with TBS before rinsing with 0.1 M of acetate buffer pH 4.8, followed by counterstaining with hematoxylin. After dehydrating the cells with increasing concentration of ethanol and Histoclear, the coverslips were mounted on glass slides.
2.3. Silencing of the MT-2A genes in MCF-7 breast cancer cells
2.6. Growth curve analysis
One microgram of siRNA was diluted to a final volume of 100 ll in opti-MEM I Reduced Serum Medium (Gibco, Auckland, New Zealand) in a 24-well plate. Six microliters of RNAiFect Transfection Reagent (Qiagen) was added to the prediluted siRNA and mixed by pipetting. The samples were incubated at room temperature for 15 min before replacing the culture medium with 300 ll of cell culture medium and addition of the transfection complexes drop wise. The plate was incubated at 37 °C in a humidified 5% CO2 atmosphere. The silencing efficiency in MCF-7 cells was optimized using a positive siRNA (siAClamin). Gene expression in the transfected cells was examined at 48 h
Cells were cultured for up to 72 h post-siRNA transfection and analysis was conducted using alamarBlue (Invitrogen, CA, USA) with continuous monitoring of the treated cells. AlamarBlue was added to a concentration of 10% (v/v) of the total volume of the medium bathing the cells 20 h after seeding. The samples were then incubated for 4 h at 37 °C in a 5% CO2 humidified incubator and absorbance was read at 570 nm with 600 nm as the reference wavelength using the Tecan2000 plate reader. Following which, the medium with alamarBlue was removed and fresh medium was added to the cells. The above procedure with alamarBlue was repeated 4 h before
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the 24, 48 and 72 h time points. The experiments were conducted in triplicates. 2.7. Cell proliferation assay Cell proliferation was determined with the CellTiter 96Ò Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI). At 72 h post-siRNA transfection, 150 ll of dye solution was added to the siRNA transfected cells in a 24-well plate, containing 1000 ll of fresh culture medium. The plate was then incubated for 4 h at 37 °C in a humidified, 5% CO2 atmosphere. After incubation, 1000 ll of Solubilization Solution/Stop Mix was added to each well. The plate was then allowed to stand overnight in a humidified incubator at 37 °C. The absorbance was read at 570 nm with 650 nm as the reference wavelength using the Tecan2000 plate reader.
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2.11. Immunoblot analysis Cell lysate was harvested from the monolayer using MPER (Pierce Biotechnology, Rockford, IL, USA) in the presence of EDTA, phosphatase inhibitor cocktail and protease inhibitor cocktail (Pierce Biotechnology) with the use of a cell scrapper. Cell debris was removed by centrifuging the cell lysate at 13,000 rpm at 4 °C for 5 min. Protein concentration was examined using Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). Twenty micrograms of cell extracts were subjected to separation using 10% sodium dodecyl sulfate– polyacrylamide gel by electrophoresis. The separated proteins were then transferred to PVDF membrane
2.8. Flow cytometry analysis of cell cycle Cells were harvested and washed with PBS before fixing in 70% ethanol. The fixed cells were washed once with PBS before suspending in propidium iodide/RNase solution consisting of a final concentration of 20 lg/ml of propidium iodide (Sigma–Aldrich, Saint Louis, MI, USA), 200 lg/ml of RNase A (Roche Applied Science, Indianapolis, IN, USA) solution in 0.1% Triton-X in PBS. The cells were incubated for 30 min at room temperature before analysis. 2.9. Scanning electron microscopy (SEM) MCF-7 cells grown on glass slides were treated with siMT2A_1 or siNeg for 48 h. Cells were then fixed in 3% glutaraldehyde in phosphate buffer (PB) at pH 7.4 for 1 h. After washing with PB, cells were post-fixed with 1% osmium tetraoxide for 30 min. The samples were subsequently dehydrated with increasing concentrations of ethanol followed by drying in a Critical Point Dryer (Baltec CPD 030, Liechtenstein). The cells were mounted on SEM and sputter-coated with gold before viewing in a Philips XL30 SEM (FEI, Holland). 2.10. Superarray analysis cDNA obtained from siMT2A_1 silenced cells at 48 h were used to examine the expression of the various genes related to different cancer pathways using Superarray technology with cell cycle and metastatic genes (Superarray Bioscience, Frederick, MD, USA) according to the manufacturer’s protocol. cDNA of MCF-7 cells treated with siNegative at 48 h was used as a negative control in this experiment. The quality of the RNA was verified by Nanodrop to have a A260:A280 ratio above 2.0 and A260:A230 ratio above 1.7 before further use. The quality of the cDNA of treated and control cells were verified with RT2 RNA QC PCR array (Superarray Bioscience, Frederick, MD, USA) and silencing efficiency by real-time RT-PCR before Superarray analysis. The normalizer used was the average of five housekeeping genes, viz., 18SrRNA, HPRT1, RPL13A, G3PDH and ACTB, for each cDNA sample.
Fig. 1. Differential expression levels of MT isoforms in breast cell lines. (a) MT isoforms in MCF-7 breast cancer cells. A lower DCT value implies a higher gene expression. (b) MT isoforms in MCF-12A normal breast epithelial cells. (c) Gel electrophoresis of the real-time PCR amplification products of MT-2A isoform and housekeeping gene G3PDH from MCF-7 cells. Ten microliters of the PCR product was electrophoresed on 1.2% agarose gel in the presence of ethidium bromide. Lane designation: lane M, molecular weight marker; lane 1, MT-2A; lane 2, G3PDH.
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(Bio-Rad, Hercules, CA, USA) for Western blotting analysis. The membranes were blocked in 5% non-fat milk (in Trisbuffered saline Tween 20 (50 mM Tris, pH 7.6, 150 mM NaCl and 0.1% Tween 20) for 1 h at room temperature before overnight incubation (at 4 °C) with ATM and cdc25A antibodies at 1:200 and 1:100 dilutions, respectively. b-Actin (BioRad, Hercules, CA, USA) was used as the loading control. HRP conjugated goat-anti-mouse secondary antibodies (Amersham Biosciences, Piscataway, NJ) were used. The membranes were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology). Immunoblot bands were analyzed and quantified by densitometer GS-710 (Bio-Rad, Hercules, CA, USA). 2.12. Statistical analysis Values are presented as means ± SEM. An unpaired twotailed t-test or one-way ANOVA followed by a post hoc Tukey test was performed using the GraphPad Prism version 4.00 for Windows. For comparing differences between growth curves, the two-way ANOVA was used. p < 0.05 was considered significant. 3. Results 3.1. Expression of MT isoforms in breast cell lines Of the eight known functional MT-1 and MT-2 mRNA isoforms, MT1A, 1F, 1H, 1X and 2A isoforms were expressed in MCF-7 breast cancer
cells (Fig. 1a). MT-1A and 1H isoforms were minimally expressed in the breast cancer cells with 1F, 1X and 2A being the main isoforms expressed. MCF-12A normal breast epithelial cells additionally expressed the MT-1E isoform which is similar to that previously reported in primary breast myoepithelial cells [15]. The specificity of the MT and G3PDH primers was verified by melting curve analyses (not shown) and running the amplicons on a 1% gel electrophoresis (Fig. 1b). 3.2. Down-regulation of MT-2A at mRNA and protein levels The relative expression of the MT-2A gene following successful downregulation was examined at 48 h and 72 h post-transfection (Fig. 2a). The MT-2A gene was found to be successfully down-regulated at 94% and 96% with siMT2A_1 and siMT2A_2, respectively, at 48 h. The silencing efficiency decreased to 76% and 78% for siMT2A_1 and siMT2A_2, respectively at 72 h. Down-regulation of the MT-2A gene was verified at the protein level with the use of immunohistochemistry (Fig. 2b). The intensity of MT-immunopositive staining in siMT-2A samples was significantly lesser as compared to siNegative or control samples. Although the E9 antibody detects all MT-1 and MT-2 isoforms, MT-2A is known to be the most abundant isoform in MCF-7 cells [9]. Western blotting was not used for the detection of MT protein levels as to-date there is no well-developed protocol for MT due to the small size (6–7 kDa) of this protein. 3.3. Silencing of MT-2A gene inhibited cell proliferation Cell growth was significantly slower in siMT2A_1 cells (p = 0.0056) and siMT2A_2 cells (p = 0.0147) as compared to siNegative treated cells when analyzed with two-way ANOVA (Fig. 3a). There was no significant difference in growth between siMT2A_1 and siMT2A_2 (p = 0.2812). Furthermore, cell proliferation analysis showed reduced cell proliferation at 72 h post-siRNA transfection in siMT2A_1 cells (p < 0.001) and siMT2A_2 cells (p < 0.05) as compared to cells treated with the siNegative control
Fig. 2. Silencing the MT-2A gene with siRNA down-regulates MT-2A expression at mRNA and protein levels. (a) Silencing efficiency of two independent siRNA, namely siMT2A_1 and siMT2A_2 at 48 h and 72 h post-transfection in MCF-7 breast cancer cells. Total RNA was extracted and converted to cDNA before analysis by quantitative real-time RT-PCR. (b) Immunocytochemistry with primary monoclonal anti-MT E9 antibody and DAB staining in control cells and after treatment with siNegative and siMT2A_1. More intense MT-immunopositive staining was observed in the cytoplasm and nuclei of untreated and siNegative-treated cells as compared to cells treated with siMT2A_1. The nuclear staining observed in the siMT2A-1 treated cells was mainly due to the counterstaining by hematoxylin and not DAB staining. Bar = 48 lm.
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4. Discussion
Fig. 3. Silencing of MT-2A gene inhibits cell proliferation in MCF-7 cells. (a) Growth curve analysis up to 72 h post-siRNA transfection using nontoxic alamarBlue. Relative absorbance was determined by the difference between the absorbance of the cells at 570 nm and the absorbance of reference wavelength at 600 nm. (b) Cell proliferation assay at 72 h postsiRNA transfection examined with CellTiter 96Ò Non-Radioactive Cell Proliferation Assay. Relative absorbance was determined by the difference between the absorbance of the cells at 570 nm and the absorbance of reference wavelength at 650 nm. Results are presented as means ± SEM of triplicate experiments.
(Fig. 3b). Both siRNAs against MT-2A demonstrated an increase in the number of cells in the sub-G1- and G1-phase and decrease in the S and G2/M-phases (Figs. 4a and b). There was a slight increase in the percentage of cells in the sub-G1-phase in the siNegative treated cells as compared to the siMT2A_1 (p = 0.0067) and siMT2A_2 (p = 0.0198) treated cells. There was also a significant increase in the percentage of cells in the G1-phase from 71.3% in the siNegative treated cells to 83.7% in siMT2A_1 (p < 0.0001) and 74.4% in the siMT2A_2 (p = 0.0215) treated cells. A decrease in the percentage of cells in the S-phase was observed in the siMT2A_1 (p = 0.0002) and siMT2A_2 (p = 0.012) treated cells as compared to the siNegative treated cells. In the G2/M-phase, siMT2A_1 treated cells showed a significant decrease in the percentage of cells as compared to siNegative treated cells (7.3% compared with 15.0%, p = 0.0008). There was no significant difference between the untreated cells and siNegative treated cells (p > 0.05) for all phases of the cell cycle. SEM micrographs show a marginal increase in the number of apoptotic cells in siMT2A_1 treated cells as compared to siNegative treated cells (Fig. 4c). 3.4. Differential expression of cell cycle genes We also compared the expression of 16 cell cycle related genes in MT-2A down-regulated MCF-7 cells with siNegative treated MCF-7 cells (Table 1). The genes that were differentially regulated include up-regulation of the ATM gene by 1.31-fold (p < 0.05), down-regulation of cdc25A by 2.28-fold (p < 0.05) and Checkpoint homolog 2 (chk2) by 1.35-fold (p < 0.05). The differential regulation of ATM and cdc25A at protein level was verified by immunoblotting and densitometric analysis (Fig. 5).
MTs have been implicated in breast cancer progression as oncogenic proteins, promoting cell proliferation in several types of cancers [18–21]. Clinical studies have documented a relationship between higher aggressiveness and poorer prognosis of breast cancer with increased MT protein expression [10,22,23]. There is still paucity of information with regard to the role of MT isoforms in tumorigenesis. Although down-regulation of MT-2A by anti-sense oligonucleotides in breast cancer cells have revealed the induction of growth arrest and apoptosis [9,10], the exact mechanism by which MT-2A affects cancer progression in breast cancer has not been well elucidated. Our current study shows a significant reduction in the number of cells with an obvious G1-phase cell cycle arrest when the breast cancer cells were treated with MT-2A siRNA. There was a significant increase in the number of cells in the G1-phase of the siMT-2A cells as compared to the siNegative treated cells. In order to better understand the molecular mechanism(s) that may result in a block at the G1-stage of the cell cycle, expression analysis of cell cycle related genes were conducted using the commercially available Superarray kit. From the gene expression study, cdc25A was found to be significantly down-regulated while ATM was found to be significantly up-regulated at both mRNA and protein levels. Both genes encode proteins belonging to the ATM/Chk2/cdc25A pathway which is known to influence the cell cycle. In the early stages of checkpoint activation, DNA damage sensors are known to relay information to a family of phosphoinositide 3-kinase related kinases (PIKKs) [24] which are essentially serine/threonine kinases. These proteins play essential roles in relaying early signals in cell cycle checkpoints. The two PIKK family members in mammalian cells are ATM and ATR (ATM and Rad3-related). One of the relevant substrate in response to DNA damage for both of these kinases is p53 [25–27]. In mammalian cells, ATM controls cell cycle arrest in G1- and G2-phases in response to DNA damage [28]. In p53-dependant G1 checkpoint, phosphorylation of Ser 15 residue on p53 by ATM will eventually lead to its accumulation by the release of p53 from its substrate [29,30]. Activation of p53 will induce transcription of cyclin-dependent kinase (cdk) inhibitor p21 CIP1/WAF1, leading to a G1-growth arrest [31]. Since ATM was found to be up-regulated in siMT-2A cells in our present study, MT-2A could possibly act upstream of ATM and its substrate p53 to induce G1-growth arrest. This is supported by a previous study where MT-2A was reported to bind to p53 and activate its downstream target gene, p21 [11]. However, our results also indicate that MT-2A can mediate G1-growth arrest via ATM/chk2/cdc25A pathway. In human cells, three cdc25 members, viz., cdc25A, cdc25B and cdc25 C have been characterized [32–34]. Cdc25B and cdc25C mainly regulate the transition from G2 to mitosis phase through cdk1 dephosphorylation [35,36] whereas cdc25A is required for progression from G1- to S-phase by phosphorylation of its primary substrate cdk2, which
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Fig. 4. Silencing of MT-2A gene induces G1-phase cell cycle arrest and a marginal rise in apoptosis. (a) Representative cell cycle profile of MCF-7 cells (control) and MCF-7 cells transiently transfected with siNegative, siMT2 A_1 and siMT2A_2. Cells were stained with propidium iodide and analyzed with FACS flow cytometry at 72 h post-transfection. (b) Proportion of cells in various phases of cell cycle for siMT2A_1, siMT2A_2, siNegative and control treated cells. Results are presented as means ± SEM of triplicate experiments. (c) Scanning electron micrographs of MCF-7 cells treated with siMT2A and siNegative at 48 h post-transfection. Bar = 50 lm. Inset shows an apoptotic cell with a shrunken morphology and the presence of cell blebs. Bar = 5 lm.
Table 1 Expression of cell cycle related genes induced and repressed in MCF-7 cells after siMT-2A treatment. Gene name
Description
Cell cycle related genes ATM Ataxia telangiectasia mutated (includes complementation groups A, C and D) BRCA1 Breast cancer 1, early onset CCNE1 Cyclin E1 CDC25A Cell division cycle 25A CDK2 Cyclin-dependent kinase 2 CDK4 Cyclin-dependent kinase 4 CDKN1A Cyclin-dependent kinase inhibitor 1A (p21, Cip1) CDKN2A Cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) CHEK2 CHK2 checkpoint homolog (S. pombe) E2F1 E2F transcription factor 1 MDM2 Mdm2, transformed 3T3 cell double minute 2, p53 binding protein (mouse) RB1 Retinoblastoma 1 (including osteosarcoma) S100A4 S100 calcium binding protein A4 (calcium protein, calvasculin, metastasin, murine placental homolog) TP53 Tumor protein p53 (Li-Fraumeni syndrome)
GenBank Accession No.
Fold change
NM_000051
1.31*
NM_007294 NM_001238 NM_001789 NM_001798 NM_000075 NM_000389 NM_000077 NM_007194 NM_005225 NM_002392 NM_000321 NM_002961
1.46 1.19 2.28* 1.04 1.08 1.38 1.00 1.35* 1.17 1.08 1.08 1.20
NM_000546
1.07
A list of cell-cycle related genes were subjected to real-time RT-PCR analysis by Superarray technology (n = 3). The fold changes of the respective genes in the siMT2A treated sample with respect to siNegative treated sample are shown above. A positive value indicates a positive up-regulation of the gene while a negative value indicates down-regulation of the gene. * p < 0.05.
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Fig. 5. Silencing of MT-2A gene induces an up-regulation in ATM protein expression with a concomitant decrease in cdc25A protein expression. (a) MCF7 cells were treated with siMT-2A and siNegative. Cell lysates were examined by Western blot analysis with antibodies against ATM and the housekeeping protein b-actin. A representative blot is shown along with adjacent densitometric quantitation of ATM and b-actin band intensities. (b) siMT-2A and siNegative-treated MCF7 cell lysates were examined by Western blot analysis with antibodies against cdc25A and b-actin. A representative blot is shown along with adjacent densitometric quantitation of cdc25A and b-actin band intensities. Results are presented as means ± SEM of triplicate experiments.
Fig. 6. Schematic diagram showing that inhibition of MT-2A expression results in G1-cell cycle block mediated via the ATM/Chk2/cdc25A pathway.
is essential for the DNA replication process [37,38]. The abundance of cdc25A is regulated by the ubiquitin–proteasome degradation pathway in response to inhibition of DNA replication or induction of DNA damage [39,40] which is activated by ATM and ataxia telangiectasia-mutated and Rad3-related (ATR) proteins. ATM activates its downstream effector Chk2 by phosphorylation at Thr68 [41–43] while ATR phosphorylates Chk1 at Ser317 and Ser345 [44]. Activated Chk2 phosphorylates downstream substrates, which include p53 tumor suppressor at Ser20 [45], BRCA1 at Ser988 [46] and Cdc25 family of phosphatases which are involved in cell cycle arrest. Chk2 phosphorylates Cdc25A at Ser123 [47] and Cdc25C at Ser216 [48]. Chk2 autophosphorylation has also been reported to
occur both in cis and in trans [49]. We posit that downregulation of MT-2A may lead to increased expression of ATM which in turn regulate phosphorylation of Chk2 and trigger the ubiquitin–proteasome degradation pathway for cdc25A, thereby leading to p53-independent G1-arrest (Fig. 6). In summary, we have shown that MT-2A affects cell proliferation in breast cancer cells. Moreover, cell cycle gene analysis shows that MT-2A regulates the cell cycle through the ATM/p53 and/or ATM/Chk2/cdc25A pathway. One of the recent focuses in cancer therapeutic strategy is to develop molecular cancer therapeutics. To this end, MT-2A appears to be a promising target molecule for the molecular therapy of breast cancer.
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