title: A unique role of GATA1s in Down syndrome acute megakaryocytic leukemia biology and therapy.
abstract: BACKGROUND
  Acute megakaryocytic leukemia (AMkL) in Down syndrome (DS) children is uniformly associated with somatic GATA1 mutations, which result in the synthesis of a shorter protein (GATA1s) with altered transactivation activity compared to the wild-type GATA1. It is not fully established whether leukemogenesis and therapeutic responses in DS AMkL patients are due to loss of the wild-type GATA1 or due to a unique function of GATA1s.
  
  
  METHODS
  Stable clones of CMK cells with decreased GATA1s or Bcl-2 levels were generated by using GATA1- or BCL-2-specific lentivirus shRNAs. In vitro ara-C, daunorubicin, and VP-16 cytotoxicities of the shRNA stable clones were determined by using the Cell Titer-blue reagent. Apoptosis and cell cycle distribution were determined by flow cytometry analysis. Changes in gene transcript levels were determined by gene expression microarray and/or real-time RT-PCR. Changes in protein levels were measured by Western blotting. In vivo binding of GATA1s to IL1A promoter was determined by chromatin immunoprecipitation assays.
  
  
  RESULTS
  Lentivirus shRNA knockdown of the GATA1 gene in the DS AMkL cell line, CMK (harbors a mutated GATA1 gene and only expresses GATA1s), resulting in lower GATA1s protein levels, promoted cell differentiation towards the megakaryocytic lineage and repressed cell proliferation. Increased basal apoptosis and sensitivities to ara-C, daunorubicin, and VP-16 accompanied by down-regulated Bcl-2 were also detected in the CMK GATA1 shRNA knockdown clones. Essentially the same results were obtained when Bcl-2 was knocked down with lentivirus shRNA in CMK cells. Besides Bcl-2, down-regulation of GATA1s also resulted in altered expression of genes (e.g., IL1A, PF4, and TUBB1) related to cell death, proliferation, and differentiation.
  
  
  CONCLUSIONS
  Our results suggest that GATA1s may facilitate leukemogenesis and potentially impact therapeutic responses in DS AMkL by promoting proliferation and survival, and by repressing megakaryocytic lineage differentiation, potentially by regulating expression of Bcl-2 protein and other relevant genes.
authors: Division of Pediatric Hematology/Oncology, Children's Hospital of Michigan, Detroit, Michigan, United States of America.
authors: Ana C
authors: AC
authors: Xavier
authors: Holly
authors: H
authors: Edwards
authors: Alan A
authors: AA
authors: Dombkowski
authors: Tugce B
authors: TB
authors: Balci
authors: Jason N
authors: JN
authors: Berman
authors: Graham
authors: G
authors: Dellaire
authors: Chengzhi
authors: C
authors: Xie
authors: Steven A
authors: SA
authors: Buck
authors: Larry H
authors: LH
authors: Matherly
authors: Yubin
authors: Y
authors: Ge
authors: Jeffrey W
authors: JW
authors: Taub
mesh_terms: D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D003561:Cytarabine / Q000494:pharmacology / Q000627:therapeutic use; D003630:Daunorubicin / Q000494:pharmacology / Q000627:therapeutic use; D004314:Down Syndrome / Q000150:complications*; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D005047:Etoposide / Q000494:pharmacology / Q000627:therapeutic use; D050982:GATA1 Transcription Factor / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics; D055785:Gene Knockdown Techniques; D006801:Humans; D053582:Interleukin-1alpha / Q000235:genetics; D016086:Lentivirus / Q000235:genetics; D007947:Leukemia, Megakaryoblastic, Acute / Q000150:complications / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology*; D008533:Megakaryocytes / Q000187:drug effects / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; C508584:IL1A protein, human; D053582:Interleukin-1alpha; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D003561:Cytarabine; D005047:Etoposide; D003630:Daunorubicin