ANTICANCER RESEARCH 30: 1163-1168 (2010)

ALCAM, Activated Leukocyte Cell Adhesion Molecule, Influences the Aggressive Nature of Breast Cancer Cells, a Potential Connection to Bone Metastasis

SIMON DAVIES and WEN G. JIANG

Metastasis and Angiogenesis Research Group, Cardiff University School of Medicine Heath Park, Cardiff CF14 4XN, United Kingdom

Abstract. Introduction: ALCAM, activated leukocyte cell Activated leukocyte cell adhesion molecule (ALCAM), also adhesion molecule, is connected to the progression of certain known as metastasis clone D (MEMD) or CD166, solid tumours and has been shown to be a prominent feature was first identified on activated leukocytes and in haemopoietic for tumours that subsequently developed bone metastasis. The stem cells and myeloid progenitors (1, 2). The encoding present study investigated the biological influence of ALCAM ALCAM is located on the long arm of human 3 on breast cancer cells in connection with bone biological (3q13.1-q13.2) (1). It is organized into 16 exons spanning environment. Μaterials and Methods: Suitable breast cancer nearly 150 kb of DNA (3). ALCAM is a of the cells were transfected with either the ALCAM expression immunoglobulin superfamily that is involved in homophilic construct or anti-ALCAM transgene, to create sublines that adhesion and in heterotypic binding to the lymphocyte cell had differential expression of ALCAM. The growth, migration surface receptor CD6 (4, 5). ALCAM- mediated cell adhesion and invasion of the cells were evaluated in the presence or is mediated by both homophilic (ALCAM-ALCAM) and absence of matrix prepared from human bones. heterophilic (ALCAM-CD6) (6) interactions between cells, Results: ZR-751ΔALCAM (ALCAM knockdown) and MDA-MB- with the heterophilic interactions being approximately 100 231ALCAMexp (overexpressing ALCAM) were constructed. times stronger than the homophilic interactions (7). MDA MB-231ALCAMexp cells showed a slower rate of growth The influence of ALCAM on cell adhesion has inspired a compared with control cells. However, in the presence of bone number of studies to evaluate its expression in human tumours matrix proteins, MDA MB-231ALCAMexp showed a including melanoma, prostate cancer, breast cancer, colorectal significantly reduced rate of growth, p<0.01 vs. control cells. carcinoma, bladder cancer and oesophageal squamous cell In contrast, ZR-751ΔALCAM cells grew faster compared with carcinoma. ALCAM expression has been shown to have control cells. MDA MB-231ALCAMexp displayed a significantly clinical implications. In malignant melanoma, approximately reduced (p=0.012) and ZR-751ΔALCAM cells significantly half of the cases investigated were found to have ALCAM increased invasiveness (p=0.02) vs. their respective controls positivity and the expression is seen in the vertical growth cells. In an ECIS-based cell migration assay, MDA-MB- phase of melanoma (6). In prostate cancer, colorectal 231ALCAMexp cells showed marked reduction in migration. carcinoma and oesophageal squamous cell carcinoma, Inclusion of bone matrix proteins therefore further reduced ALCAM expression was lost in high-grade tumours (8-10). the migration speed of MDA MB-231ALCAMexp cells. ALCAM has also been shown to be involved in capillary Conclusion: Loss of ALCAM in breast cancer cells facilitates tubule formation (11) and in vessel invasion into cartilage in the invasive behaviour of breast cancer and high levels of vitro (12). Degen et al. were the first to look for the presence ALCAM in the cells have a suppressive role in the aggressive of ALCAM in breast cancer, and performed northern blot nature of breast cancer cells. analysis of MEMD (a partner to ALCAM) mRNA and found expression in the MCF-7 mammary carcinoma cell line. Studies have more recently shown ALCAM in resected breast tumours (13) and King et al. shown a decreased level of Correspondence to: Wen G. Jiang, Metastasis and Angiogenesis ALCAM expression correlating with the nodal involvement, Research Group, Cardiff University School of Medicine, Heath the grade, the TMN stage, the Nottingham Prognostic Index Park, Cardiff CF14 4XN, U.K. Tel: +44 2920742895, Fax: +44 2920761623, e-mail: [email protected] NPI and the clinical outcome (local recurrence and death) (13). These finding have recently been substantiated by Key Words: ALCAM, CD166, breast cancer, bone metastasis, cell Jezierska et al., who similarly concluded that low ALCAM adhesion. concentrations correlated with an aggressive phenotype (14).

0250-7005/2010 $2.00+.40 1163 ANTICANCER RESEARCH 30: 1163-1168 (2010)

Table I. Primers used in the present study.

Primers Sequence (5’-3’) Size (bp)

ALCAMrib1F CTG CAG CAC TTT TCC ATT CCT GTA CCA TGT CTG ATG AGT CCG TGA GGA 48 ALCAMrib1R ACT AGT TAT CCA GTA TGT CAA TTT TCG TCC TCA CGG ACT 39 ALCAMrib2F CTG CAG TAC GTC AAG TCG GAA AGG TCT CTG ATG CGT CCG TGA GGA 45 ALCAMrib2R ACT AGT ATG GAG ATA CCA TTT TTC GTC CTC ACG GAC T 37 ALCAMrib3F CTG CAG CTG ATC CTT GCA TTA CTC TGA TGA GTC CGT GAG GA 41 ALCAMrib3R ACT AGT CAG AAA ACT ACA CTT TGT CTT TTC GTC CTC AGG ACT 42 ALCAMF6 TTA TCA TAC CTT GCC GAC TT 19 ALCAMR6 GGG TGG AAG TCA TGG TAT AG 20 GAPDHF8 GGC TGC TTT TAA CTC TGG TA 20 GAPDHR8 GAC TGT GGT CAT GAG TCC TT 20 ALCAMExF1 AAT ATG GAA TCC AAG GGG G 19 ALCAMExR1 GGC TTC AGT TTT GTG ATT GT 20 ALCAMExF2 AAT ATG GAA TCC AAG GGG 18 ALCAMExR2 GGC TTC AGT TTT GTG ATT G 19

The published data on the function of ALCAM in breast quantitative PCR (Qabsolute) were obtained from AbGene, Surrey, cancer are not wholly consistent, there is however growing England, UK). Mouse monoclonal antibody ALCAM/CD166 (for evidence in support of initial findings showing that a reduced immunohistochemistry) was obtained from Novocastra Laboratories (Newcastle upon Tyne, UK). Peroxidase conjugated anti-mouse level of ALCAM expression is an indicator of poor prognosis antibodies were from Sigma (Poole, Dorset, UK) and a biotin universal in breast cancer (13, 14). It has been reported recently that was from Dako, Ltd. (Carpinteria, CA, USA). Bone matrix proteins ALCAM expression is particularly aberrant in breast tumours were prepared from fresh human bone tissues obtained immediately which subsequently develop bone metastatis, and that after hip replacement, and collected under the ethical committee ALCAM expression level is linked to both the clinical approval. Bones were crushed at ice cold temperature and outcome and the presence of bone metastasis (15). subsequently processed using a BioRuptor instrument (Wolf It is not clear how ALCAM expression may affect the Laboratories, York, UK), in order to extract matrix proteins. cellular behaviour of breast cancer cells, and whether the Construction of hammerhead ribozyme transgenes targeting the expression profile may influence osteotrophy of breast cancer human ALCAM and mammalian expression vector for ALCAM. cells. In the present study, a series of breast cancer sublines Hammerhead ribozymes that specifically target ALCAM , based on with different expression of ALCAM were developed and the secondary structure of human ALCAM, have been generated as their biological behaviour was tested, including growth, previously described (16-18) (primer details shown in Table I). Anti- migration and invasiveness. Furthermore, these parameters ALCAM transgenes were used to transfect ZR-751 cells which were were tested in the presence of bone matrix proteins. strongly positive for ALCAM (Figure 1A). Following selection of transfected cells with blasticidin (used at 5 μg/ml) and verification, the following stably transfected cells were established: ALCAM Materials and Methods knock-down cells (designated here as ZR751ALCAMrib), plasmid-only control cells (ZR751pEFa), and the wild type (ZR751WT). Full length Materials. Human breast cancer cell lines, MDA MB-231 and ZR 7- human ALCAM coding region was amplified from a cDNA library 51, were obtained from ATCC (American Type Culture Collection, of human breast tissues using the primers listed in Table I. Reverse Maryland, USA). Other cells were obtained from ECACC (European transcription was carried out using a reverse transcription(RT) kit Collection of Animal Cell Culture, Salisbury, England, UK). The (Sigma, Poole, Dorset, England, UK) and amplification using an endothelial cell line HECV was from obtained from Interlab Cell line extensor PCR master mix which has an additional proof reading Collection (ICLC, Naples, Italy). Recombinant human hepatocyte polymerase (AbGene Ltd). The ALCAM full length coding product growth factor/scatter factor (rhHGF/SF) was a gift from Dr. T. was similarly cloned into the pEF6 vectors. MDA MB-231 cells Nakamura, Osaka University Medical School, Osaka, Japan. Matrigel which were negative for ALCAM were transfected with either the (reconstituted ) was obtained from Collaborative control vector or ALCAM expression vector. Stably transfected cells Research Products (Bedford, MA, USA). Transwell plates equipped were designated as MDA231pEF/His and MDA231ALCAMexp, for with a porous insert (pore size 8 μm) were obtained from Becton control transfection and ALCAM expression, respectively. Dickinson Labware (Oxford, UK). DNA gel extraction and plasmid extraction kits were obtained from Sigma (Poole, Dorset, England). RNA preparation and RT-PCR. Total RNA was extracted from cells Anti-actin and anti-GAPDH were from Santa Cruz Biotechnologies using an RNA extraction kit (AbGene Ltd) and the concentration Inc., (Santa Cruz, CA, USA). Molecular biology grade agarose and was quantified using a spectrophotometer (Wolf Laboratories, York, DNA ladder were obtained from Invitrogen (Carlsbad, CA, USA). England, UK). cDNA was synthesized using a first strand synthesis Master mix for routine polymerase chain reaction (PCR) and with an oligodt primer (AbGene). PCR was performed using sets of

1164 Davies and Jiang: ALCAM Influences the Aggressive Nature of Breast Cancer Cells

Figure 1. A: Secondary structure of ALCAM mRNA used to identify Figure 2. A: Changes in absorption, indicating growth, of the different targeting site for ribozymes. B: Expression profile of ALCAM transcript MDA MB 231 cell lines after overexpressing ALCAM, in different in human breast cancer cell lines. ZR751 and MDA MB 231 which were concentrations of bone matrix (BMP). B: Changes in absorption, found to be strongly positive and negative, respectively were chosen to indicating growth, of the ZR-751 cell lines after ALCAM expression was be suitable for transfection. C: Creation of sublines with differential knocked down, in different concentrations of growth medium. expression of ALCAM. Left: ZR-751 transfected with anti-ALCAM ribozyme transgenes showing that trangene-1 and -2 were more active than transgene-3; Right: MDA MB-231 cells were transfected with ALCAM expression vector. ALCAMexp4 vector appeared more effective. the array surface with a cysteine solution, the arrays were incubated with complete medium for 1 h. Respective cells (300,000 per well) in the same volume of medium were added to the arrays and incubated overnight to reach confluence. Baseline electric resistance primers (Table I) with the following conditions: 5 min at 95˚C, and (without addition of cancer cells) was first recorded, and then cancer then 20 sec at 94˚C-25 s at 56˚C, 50 s at 72˚C for 36 cycles, and cell or control medium was added carefully to the cancer cells. finally 72˚C for 7 min. β-actin was amplified and used as a house Electrical changes were monitored continuously for up to 24 h. In keeping control. PCR products were then separated on a 0.8% the 9600 system, the monitoring was at fixed 30 Hz. In the 1600R agarose gel, visualised under UV , photographed using a system, two conditions were recorded: 400 Hz, 4,000 Hz and 40,000 tm Unisave camera (Wolf Laboratories) and documented with Hz for screening the nature of endothelial changes and 4,000 Hz for Photoshop-CS4 (San Jose, CA, USA) software. fix frequency for cell modelling. Statistical analysis was carried out using Minitab (version 12) In vitro cell growth assay. This was based on a previously reported (State College, PA, USA). TheAnderson-Darling test was used for method (20) Cells were plated into 96-well plated at 2,000 cells/well normality testing and the Student’s t-test for significance testing. followed by a period of up to to 5 day’s incubation. Cells were fixed in 10% formaldehyde on the day of plating and daily for the subsequent 5 days. 0.5% Crystal violet (w/v) was used to stain cells. Results Following washing, the stained crystal violet was dissolved with 10% (v/v) acetic acid and the absorbance was determined at a Expression of ALCAM in breast cancer cell lines and wavelength of 540 nm using an ELx800 spectrophotometer (Wolf establishing stably transfected sublines. As shown in Figure Laboratories, York, UK). Absorbance represents the cell number. 1B, MDA MB-463, ZR751 and BT474 were positive, MDA MB-436, MCF-7, MDA MB-468 and BT482 were weakly Electric cell-substrate impedance sensing (ECIS)-based cell positive for ALCAM transcripts. In contrast, MDA MB-435, adhesion assay. Two models of ECIS instrument were used: ECIS 9600 for screening and ECIS 1600R for modelling. In both systems, MDA MB-231, BT549 and MDA MB-157 were negative. ZR- 8W10E arrays which have 10 electrodes in each well were used 751 and MDA MB-231 were selected for knocking down (Applied Biophysics Inc, NJ, USA) (21-23). Following treatment of (using ribozyme transgenes) and over-expression, respectively.

1165 ANTICANCER RESEARCH 30: 1163-1168 (2010)

Figure 3. A: Adherence of the different MDA MB-231 cell lines in standard and hepatocyte growth factor (HGF). B: Adherence of the different ZR- 751 cell lines in standard and hepatocyte growth factor (HGF). C: Adherence of the different MDA MB-231 cell lines in bone matrix protein (BMP) and a combination of bone matrix protein (BMP) and hepatocyte growth factor (HGF). D: Attachment of MDA MB cell lines pEF6 and Exp 2 to the ECIS central electrode over a 6-hour period. E: Attachment of each of the MDA MB cell lines to the ECIS central electrode over a 6-hour period in standard medium, bone matrix protein (BMP), hepatocyte growth factor (HGF) or a combination of BMP and HGF.

Figure 1C shows that transgene-1 and 2 and expression cells resulted in significant reduction of cell adhesion to construct-4 were effective in knocking down the ALCAM , Matrigel (Figure 3A). Using hepatocyte transcript. Stable transfectants were established from these growth factor (HGF) as a stimulus, wild-type and control strains and used for subsequent studies. cells showed a significant increase in the adhesion. However, expression of ALCAM also substantially reduced the Levels of ALCAM expression breast cancer cells directly affect adhesion in the cell. In ZR-751 cells, knocking down the rate of cell growth in bone matrix proteins. When ALCAM ALCAM increased the adhesion of the cells to Matrigel was overexpressed MDA MB-231 cells showed substantially (Figure 3B). Using a BMP-coated surface, the same reduction reduced rate of growth in the presence of bone matrix proteins of adhesion is shown as when MDA MB-231 cells (Figure 2A). This is particularly so when the concentration of overexpress ALCAM (Figure 3C). Using the ECIS method, BMP was higher than 15%. In contrast, the change of growth the reduction of adhesion to culture surface (Figure 3D) and pattern was not fully reproduced in ZR-751 cells after BMP-coated surface (Figure 3E) were similarly observed. knocking down ALCAM (Figure 2B), which may suggest that the incomplete knockdown as seen in Figure 1C may have In vitro invasiveness of breast cancer cells is affected by the contributed to this action. expression profile of ALCAM. Two matrix models of the in vitro invasiveness were used: Matrigel and Matrigel mixed ALCAM expression impacts on the matrix adhesion of breast with BMP. As shown in Figure 4A, overexpression of cancer cells. Overexpression of ALCAM in MDA MB-231 ALCAM in MDA MB-231 cells resulted in significant

1166 Davies and Jiang: ALCAM Influences the Aggressive Nature of Breast Cancer Cells

reduction of invasiveness in the Matrigel model. A smaller but nonetheless similar reduction is seen with the Matrigel mixture (Figure 4C). In contrast, knockdown ALCAM from ZR-751 resulted in an increase in invasiveness of invasion into Matrigel (Figure 4B).

Discussion

This study shows that ALCAM expression is different in different breast cancer cell lines. It is further shown that overexpression of ALCAM in breast cancer cells results in marked reduction of cell growth, adhesion to matrix and migration. The opposite has been seen when ALCAM is knocked out of breast cancer cells. Data from the present study support the clinical observation that ALCAM expression in breast cancer may be linked to the likelihood of the tumour developing bone metastasis (15). The growth of both MDA MB-231 and ZR-751 cells was investigated in a variety of different concentrations of both standard medium and BMP. It was clear that high levels of ALCAM expression (in wild-type ZR-751 and in MDA MB- 231ALCAMexp) are associated with cell growth under routine culture conditions and in the presence of BMP. Thus, the presence of ALCAM reduces the growth rate in cancer cells and the absence of ALCAM increases it. This conclusion correlates well with a clinical study where more aggressive fast growing tumors had very low levels of ALCAM (13). The ability of a cancer cell to adhere to the either a basement membrane of standard medium or of BMP is the first stage in the spread of a cancer cell. As described previously, the ability to adhere to the basement membrane of a blood vessel wall allows extravasation and subsequent intravasation at a distant site. An increase in the ability of a cancer cell to adhere would increase the ability of the cancer to metastasize. The presence of ALCAM is predicted to reduce the ability of cancer cells to adhere to a basement membrane, since the tumors with the lowest levels of ALCAM that metastasised in this study. In performing the adhesion experiments, as well as using standard medium and BMP, the same media were run with HGF added, since this has been shown to increase the ability of cancer cells to adhere. The results obtained from the adhesion experiment showed that MDA MB-231 and ZR-751, when manipulated to have different expression pattern of ALCAM, showed a correlation between the levels of ALCAM and adhesiveness. The same inverse correlation has been observed between the level of ALCAM and invasiveness of breast cancer. These results are Figure 4. A: Invasion of the MDA MB 231 cell lines through an artificial in line with observations made in a previous clinical study, in basement membrane in the presence of standard medium and hepatocyte which tumours which subsequently developed bone metastasis growth factor (HGF). B: Invasion of the ZR-751 cell lines through an tend to have low levels of ALCAM (15). artificial basement membrane in the presence of standard medium and hepatocyte growth factor (HGF). C: Invasion of the MDA MB-231 cell The present study thus provides new evidence that breast lines through an artificial basement membrane in the presence of cancer cells expressing low levels of ALCAM are likely to different concentrations of bone matrix protein (BMP). adhere to bone matrix and survive in an environment that also

1167 ANTICANCER RESEARCH 30: 1163-1168 (2010) contain materials obtained from bone matrix. The method by 11 Ohneda O, Ohneda K, Arai F, Lee J, Miyamoto T, Fukushima Y, which ALCAM interacts with specific protein(s) from the Dowbenko D, Lasky LA and Suda T: ALCAM (CD166): its role bone preparation remains to be elucidated further. However, in hematopoietic and endothelial development. Blood 98: 2134- 2142, 2001. these observations, together with recent reports from clinical 12 Arai F, Ohneda O, Miyamoto T, Zhang X and Suda T: studies, point strongly to an inhibitory role of ALCAM in the Mesenchymal stem cells in perichondrium express activated development of bone metastasis from breast cancer. leukocyte cell adhesion molecule and participate in bone marrow formation. J Exp Med 195: 1549-1563, 2002. Acknowledgements 13 King JA, Ofori-Acquah SF, Stevens T, Al-Mehdi AB, Fodstad O and Jiang WG: Activated leukocyte cell adhesion molecule in The authors would like to thank Cancer Research Wales and the breast cancer: prognostic indicator. Breast Cancer Res 6: 478- Albert Hung Foundation for supporting this work. 487, 2004. 14 Jezierska A, Matysiak W and Motyl T: ALCAM/CD166 protects References breast cancer cells against apoptosis and autophagy. Med Sci Monit 12: 263-273, 2006. 15 Davies SR, Dent C, Watkins G, King JA, Mokbel K and Jiang 1 Bowen MA, DD Patel, Li X, Modrell B, Malacko AR, Wang W- WG: Expression of the cell to cell adhesion molecule, ALCAM, C, Marquardt H, Neubauer M, Pesando JM, Francke U, Haynes in breast cancer patients and the potential link with skeletal BF and Aruffo A: Cloning, mapping, and characterization of metastasis. Oncology Rep 19: 555-561, 2008. activated leukocyte-cell adhesion molecule (ALCAM), a CD6 16 Davies G, Jiang WG and Mason MD: Cell–cell adhesion molecules ligand. J Exp Med 181: 2213-2220, 1995. and signaling intermediates and their role in the invasive potential 2 Uchida N, Yang Z, Combs J, Pourquie O, Nguyen M, of prostate cancer cells. J Urol 163: 985-992, 2000. Ramanathan R, Fu J, Welply A, Chen S, Weddell G, Sharma AK, 17 Jiang WG, Davies G, Martin TA, Parcc C, Watkins G, Mason Leiby KR, Karagogeos D, Hill B, Humeau L, Stallcup WB, MD, Mokbel K and Mansel RE: Molecular targeting of Hoffman R, Tsukamoto AS, Gearing DP and Peault B: The matrilysin and its impact on tumour growth in vivo, the potential characterization, molecular cloning, and expression of a novel implications in breast cancer therapy. Clin Cancer Res 11: 6012- hematopoietic cell antigen from CD34_ human bone marrow 6019, 2005. cells. Blood 89: 2706-2716, 1997. 18 Jiang WG, Grimshaw D, Lane J, Martin TA, Parr C, Davies G, 3 Ikeda K and Quertermous T: Molecular isolation and Laterra J and Mansel RE: Retroviral hammerhead transgenes to characterization of a soluble isoform of activated leukocyte cell cMET and HGF/SF inhibited growth of breast tumour, induced adhesion molecule that modulates endothelial cell function. J by . Clin Cancer Res 9: 4274-4281, 2003. Biol Chem 279: 55315-55323, 2004 19 Jiang WG, Ablin RJ, Kynaston HG and Mason MD: The 4 Burns F, von Kannen S, Guy L, Raper J, Kamholz J and Chang Prostate Transglutaminase (TGase-4, TGaseP) regulates the S: DM-GRASP, a novel immunoglobulin superfamily axonal interaction of prostate cancer and vascular endothelial cells, a surface protein that supports neurite extension. Neuron 7: 209- potential role for the ROCK pathway. Microvascular Res 77: 220, 1991. 150-157, 2009. 5 Peduzzi J, Irwin M and Geisert E: Distribution and 20 Jiang WG, Hiscox S, Hallett MB, Horrobin DF, Mansel RE and characteristics of a 90 kDa protein, KG-CAM in the rat CNS. Puntis MCA: Regulation of the expression of E-cadherin on Brain Res 640: 296-307, 1994. human cancer cells by gamma linolenic acid. Cancer Res 55: 6 van Kempen LC, Nelissen JM, Degen WG, Torensma R, Weidle 5043-5048, 1995. UH, Bloemers HP, Figdor CG and Swart GW: Molecular basis 21 Giaever I and Keese CR: Monitoring behavior in tissue for the homophilic activated leukocyte cell adhesion molecule culture with an applied electric field. Proc Natl Acad Sci USA (ALCAM)-ALCAM interaction. J Biol Chem 276: 25783-25790, 81: 3761-3764, 1984. 2001. 22 Giaever I and Keese CR: Micromotion of mammalian cells 7 Hassan, NJ, Barclay AN and Brown MH: Frontline: Optimal T measured electrically. Proc Natl Acad Sci USA 88: 7896-7900, cell activation requires the engagement of CD6 and CD166. Eur 1991. J Immunol 34: 930-940, 2004. 23 Jiang WG, Martin TA, Russell-Lewis J, Ye L, Douglas-Jones A 8 Kristiansen G, Pilarsky C, Wissmann C, Stephan C, Weissbach and Mansel RE: Eplin-alpha expression in human breast cancer, L, Loy V, Loening S, Dietel M and Rosenthal A: ALCAM/CD166 the impact on cellular migration and clinical outcome. Mol is up-regulated in low-grade prostate cancer and progressively Cancer 7: 71, 2008. lost in high-grade lesions. Prostate 54: 34-43, 2003. 9 Weichert W, Knosel T, Bellach, J, Dieal M and Kistianse G: ALCAM/CD166 is overexpressed in colorectal carcinoma and correlates with shortened patient survival. J Clin Pathol 57: 1160-1164, 2004. 10 Verma A, Shukla NK, Deo SVS, Gupta SD and Ralham R: MEMD/ALCAM: A potential marker for tumor invasion and Received February 7, 2010 nodal metastasis in esophageal squamous cell carcinoma. Revised March 26, 2010 Oncology 68: 463-470, 2005. Accepted March 26, 2010

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