clinical trials have shown that DS children with AML, and in particular AMkL, have extremely high event-free survival (EFS) rates (,80–100%) when treated with cytosine arabinoside (ara-C)/ anthracycline-based chemotherapy.[2–5,9] This is in marked contrast to the ,50% EFS rates typically seen for non-DS pediatric AML patients, and the ,35% EFS rates for non-DS pediatric AMkL patients.[2–5,9] What are the molecular bases for the increased incidence of leukemia in DS children, and in particular, TMD and AMkL? Acquired somatic mutations of the transcription factor gene GATA1 (localized to Xp11.23) have been consistently detected in nearly all DS TMD and AMkL cases, while mutations have not been detected in DS acute lymphoblastic leukemia and non-DS AML and AMkL except for rare cases.[10–12] GATA1 gene encodes a zinc finger transcription factor that binds to the WGATAR motif and is essential for normal erythroid and megakaryocytic differentiation.[13] The net effect of the mutations is the introduction of stop codons either before or after methionine 84 that results in a 40-kDa truncated GATA1 protein (designated GATA1s), initiated from a downstream translation start site and distinguishable from GATA1 (50-kDa).[12] Both GATA1s and GATA1 show similar DNA binding abilities and interact with partner proteins, such as ‘‘Friend of GATA1’’ (FOG1), though GATA1s exhibits altered transactivation capacity due to the loss of the N-terminal activation domain.[12] This could potentially contribute to the uncontrolled proliferation of poorly differentiated megakaryocytic precursors. In fact, GATA1s has been shown to lead to hyperproliferation of a unique, previously unrecognized yolk sac and fetal liver progenitor in transgenic mice, which may account for the transient nature of TMD in DS.[14] The uniform detection of somatic GATA1 gene mutations in DS AMkL cases suggests that loss of the wild-type GATA1 and/or synthesis of GATA1s in DS AMkL may somehow contribute to the high EFS rates of DS AMkL patients. Indeed, a relationship between GATA1 and AML outcome was suggested by a Japanese clinical study in which non-DS AML patients with lower GATA1 transcript expression experienced the highest complete remission rates.[15] In our previous study, when the wild-type GATA1 was ectopically over-expressed in a DS AMkL cell line, CMK (harbors a mutated GATA1 gene and only expresses GATA1s), it resulted in significantly increased resistance to ara-C, suggesting that loss of GATA1 could be responsible for the enhanced therapeutic responses of DS AMkL patients.[16] However, it is not clear whether the enhanced therapeutic responses of DS AMkL patients are due primarily to loss of the wild-type GATA1 and/or due to unique biological functions of GATA1s in DS AMkL cases. To date, no studies have been reported to determine the role of GATA1s in a human DS AMkL cell line model. In this study, we explored the functional role of GATA1s in DS AMkL biology and therapy using lentivirus shRNA to knockdown GATA1 in the DS AMkL cell line, CMK, which expresses only GATA1s and no wild-type GATA1.[17] Our results suggest that GATA1s has unique functions in facilitating DS leukemogenesis and in modulating therapeutic responses by repressing differentiation towards the megakaryocytic lineage, and by promoting prolifer- ation and survival, potentially through regulating expression of Bcl-2 and other relevant genes. Materials and Methods Cell Culture The DS AMkL cell line, CMK, was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ; Braunschweig, Germany). The parental CMK cells and the The Role of GATA1s in DS AMkL shRNA stable clones were cultured in RPMI 1640 with 10% fetal bovine serum (Hyclone, Logan, UT) and 2 mM L-glutamine plus 100 U/ml penicillin and 100 mg/ml streptomycin, in a 37uC humidified atmosphere containing 5% CO2/95% air. Lentiviral ShRNA Knockdown of GATA1 and Bcl-2 in CMK Cells Knockdown of GATA1 and Bcl-2 genes in the CMK cells was performed using shRNA lentivirus (Sigma-Aldrich, St. Louis, MO), as previously reported.[18,19] The sequences for the negative control, GATA1, and Bcl-2 shRNAs are shown in Figure S1. Two clones for each gene (designated CMK-5a and CMK-5b for GATA1, and CMK-b7 and CMK-b8 for Bcl-2) with decreased expression of GATA1s and Bcl-2, respectively, were selected for further studies. A pool of cells from the negative control infection (lentivirus expressing a shRNA with limited homology to any known human genes) was used as the negative control (designated CMK-neg) for the GATA1 and Bcl-2 shRNA stable clones. Cell Differentiation and Proliferation Assays The effects of GATA1s on megakaryocytic differentiation were assessed by flow cytometry analysis of cell surface markers of the CMK-neg, -5a, and -5b stable clones, as previously described.[20] To determine the effects of GATA1s on cell proliferation, the CMK-neg, -5a, and -5b stable clones were seeded at 2.56104 cells/mL in T-25 flasks and counted every 24 hours to determine doubling times for each stable clone. To verify possible changes in cell cycle progression, CMK-5a, -5b, and –neg cells were harvested and fixed with ice-cold 70% (v/v) ethanol for 24h. After centrifugation at 2006g for 5 min, the cell pellets were washed with phosphate-buffered saline (PBS, pH 7.4) and resuspended in PBS containing propidium iodine (PI) (50 mg/ mL), triton X-100 (0.1%, v/v), and DNase-free RNase (1 mg/mL). The DNA contents were determined by flow cytometry using a FACScan flow cytometer (BD Biosciences, San Jose, CA). Cell cycle analysis was done with the Multicycle software (Phoenix Flow Systems, Inc.). BrdU Labeling and Flow Cytometric Analysis The CMK-neg, -5a, and -5b cells were cultured in flasks (2256105/ml) and incubated for 1 hour with 10 mM BrdU (5- Bromo-29-deoxyuridine, Sigma-Aldrich, St. Louis, MO). Cells were harvested, washed, fixed and treated with 2 N HCl. After washing with PBS, cells were blocked with 0.2% Tween-20 in PBS and incubated with fluorescein isothiocyanate (FITC) conjugated anti-BrdU primary antibody (BD Pharmingen FITC Mouse Anti- BrdU Set, San Diego, CA) in the same buffer for 2 hours at room temperature. Cells were washed and resuspended in PBS with 2 mg/mL 7-AAD (Sigma-Aldrich, St. Louis, MO) and subse- quently submitted to flow cytometry. Flow cytometric analysis was done on at least 10,000 cells from each sample, using a FACS Calibur flow cytometer (BD BioSciences). Three independent experiments were completed. In Vitro Ara-C Cytotoxicity Assays For determinations of in vitro ara-C cytotoxicities, the CMK stable clones were cultured in complete medium with dialyzed fetal bovine serum (FBS) in 96-well plates at a density of 86104 cells/ mL. Cells were cultured for 96 h with a range of concentrations of ara-C at 37uC, and viable cell numbers were determined using the Cell Titer-blue reagent (Promega, Madison, WI) and a fluores- cence microplate reader. The IC50 values were calculated as the concentrations of ara-C necessary to inhibit 50% proliferation PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27486