to therapy. Our approach involved lentiviral shRNA knockdown of the GATA1 gene in a human DS AMkL cell line, CMK, which harbors a mutated GATA1 gene and expresses only GATA1s. Interestingly, down-regulation of GATA1s in CMK cells resulted in impaired cell proliferation and increased differentiation toward the megakaryocytic lineage to extents that paralleled the decreased levels of GATA1s seen in the stable clones (Fig. 1 and Fig. 2). The increase in megakaryocytic differentiation of the CMK-5a and CMK-5b cells was further supported by the substantially induced expression of proplatelet formation genes, such as PF4 and TUBB1 (Fig. 6). In addition, down-regulation of GATA1s was accompa- nied by both up- and down-regulation of a large number of genes, including genes related to cell proliferation and cell differentiation, such as IL1A. Previous studies have established that interleukin 1 enhances proliferation of megakaryocytic progenitors.[25,37] Our finding that GATA1s promotes cell proliferation and represses differentiation toward megakaryocytic lineage in DS suggests that expression of GATA1s in itself could be a causal factor in leukemogenesis in DS. EFS rates for DS patients with AMkL are significantly higher than non-DS AML patients, and in particular, non-DS AMkL patients,[2–5] suggesting that the presence of GATA1s may contribute to the high EFS rates of DS AMkL patients. Our previous study suggested that loss of GATA1 may contribute to the enhanced chemotherapy responses of DS AMkL compared to non-DS AML.[38] However, it is not clear whether expression of GATA1s itself contributes to the increased chemotherapy sensitivities, as seen in DS AMkL patients, compared to non-DS AML patients. In the present study, we found that down- regulation of GATA1s in CMK cells resulted in significantly increased in vitro sensitivities to ara-C, daunorubicin, and VP-16 compared to that in the negative control, suggesting that expression of GATA1s can confer resistance to chemotherapy in DS AMkL. Accordingly, expression of GATA1s by itself is unlikely to explain the enhanced chemotherapy responses in DS AMkL compared to non-DS AML. Rather, expression levels of GATA1s may represent an important biomarker related to chemotherapy sensitivity or resistance in DS AMkL. Loss of GATA1 may represent a significant contributing factor accounting for the increased chemotherapy sensitivity of DS AMkL. The role of GATA1s in chemosensitivity only applies to the DS AML group itself (based on the lack of expression in non-DS AML) and its relative impact may be modest. Nonetheless, assessing the role of GATA1s expression in DS AMkL may identify a small subset of DS patients with refractory or relapsed disease.[39] A study of DS patients with TMD reported that differences in GATA1s levels were associated with the risk of progression to myeloid leukemias, supporting the notion that GATA1s may impact the clinical features of DS leukemia cases. [40] What are the molecular mechanism(s) responsible for the effects of GATA1s on ara-C sensitivity? Our real-time RT-PCR assays References 1. Taub JW (2001) Relationship of chromosome 21 and acute leukemia in children with Down syndrome. J Pediatr Hematol Oncol 23: 175–178. 2. Ravindranath Y, Abella E, Krischer JP, Wiley J, Inoue S, et al. (1992) Acute myeloid leukemia (AML) in Down’s syndrome is highly responsive to chemotherapy: experience on Pediatric Oncology Group AML Study 8498. Blood 80: 2210–2214. 3. Lange BJ, Kobrinsky N, Barnard DR, Arthur DC, Buckley JD, et al. (1998) Distinctive demography, biology, and outcome of acute myeloid leukemia and myelodysplastic syndrome in children with Down syndrome: Children’s Cancer Group Studies 2861 and 2891. Blood 91: 608–615. 4. Gamis AS, Woods WG, Alonzo TA, Buxton A, Lange B, et al. (2003) Increased age at diagnosis has a significantly negative effect on outcome in children with The Role of GATA1s in DS AMkL showed no significant differences in dCK, CDA, and hENT-1 transcript levels among the stable clones (data not shown). However, significantly increased basal apoptosis in the CMK-5a and -5b clones was detected compared to that in the CMK-neg cells. This was accompanied by decreased levels of Bcl-2 protein, which was certainly responsible for the increased basal apoptosis and increased ara-C sensitivities in the CMK-5a and -5b clones, as demonstrated by our lentiviral shRNA knockdown experiments. Surprisingly, down-regulation of GATA1s resulted in increased Bcl-2 transcripts in the GATA1 shRNA stable clones compared to the CMK-neg cells, suggesting that GATA1s must confer some means of posttranscriptional regulation of Bcl-2 expression in CMK cells. Current studies are underway to determine the mechanisms underlying the regulation of Bcl-2 expression by GATA1s in CMK cells. To summarize, our findings suggest that GATA1s has unique functions in DS AMkL. GATA1s appears likely to facilitate DS leukemogenesis and to confer resistance to chemotherapy by promoting proliferation and survival, and by repressing differen- tiation towards the megakaryocytic lineage, potentially by regulating expression of Bcl-2 and other relevant genes. Supporting Information Figure S1 Sequences for the negative control, GATA1, and Bcl-2 shRNAs. (DOC) Table S1 Summary of primers used for real-time RT- PCR in this study. (XLS) Table S2 GATA1s responsive probes identified by microarray analysis. (XLSX) Table S3 GATA1s responsive probes related to cell differentiation. (XLSX) Table S4 GATA1s responsive probes related to cell proliferation. (XLSX) GATA1s responsive probes related to cell Table S5 death. (XLSX) Author Contributions Conceived and designed the experiments: YG JT. Performed the experiments: AX HE AD TB JB GD CX SB. Analyzed the data: AX HE AD TB JB GD CX SB LM YG JT. Wrote the paper: AX LM YG JT. Down syndrome and acute myeloid leukemia: a report from the Children’s Cancer Group Study 2891. J Clin Oncol 21: 3415–3422. 5. Athale UH, Razzouk BI, Raimondi SC, Tong X, Behm FG, et al. (2001) Biology and outcome of childhood acute megakaryoblastic leukemia: a single institution’s experience. Blood 97: 3727–3732. 6. Zipursky A, Thorner P, De Harven E, Christensen H, Doyle J (1994) Myelodysplasia and acute megakaryoblastic leukemia in Down’s syndrome. Leuk Res 18: 163–171. 7. Zipursky A (2003) Transient leukaemia–a benign form of leukaemia in newborn infants with trisomy 21. Br J Haematol 120: 930–938. 8. Klusmann JH, Creutzig U, Zimmermann M, Dworzak M, Jorch N, et al. (2008) Treatment and prognostic impact of transient leukemia in neonates with Down syndrome. Blood 111: 2991–2998. PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e27486