Regions precipitated with the TEL-AML1 antibody from NALM-6 cells stably expressing the fusion transcript were mapped to human chromosomal regions (black bars) and compared with regions immunoprecipitated from the induced mouse Ba/F3 cell line (grey bars). were specific for the fusion protein and not bound by native AML1 or TEL. Comparison with gene expression profiles from TEL-AML1-positive patients identified 56 concordantly misregulated genes with negative effects on proliferation and cellular transport mechanisms and positive effects on cellular migration, and stress responses including immunological responses. In summary, this work for the Bambuterol HCl first time gives a comprehensive insight into how TEL-AML1 expression may directly and indirectly contribute to alter cells to become prone for leukemic transformation. in an early B-cell progenitor cell and leads to establishment of a pre-leukemic clone persisting in the bone marrow for several years, insufficient to generate an overt leukaemia.3, 9, 10 It is unclear if the translocation inevitably leads to the disease or if only a small portion progress, as conflicting reports about the incidence of this translocation in healthy newborns exist.3, 11 The global binding pattern of the TEL-AML1 fusion protein on promoter regions in precursor B-cells is not known and several mechanisms of action have been proposed so far. The runt-homology DNA-binding domain of AML1 retained in the TEL-AML1 fusion protein has been shown to be essential for DNA binding.12 Transiently transfected TEL-AML1 blocks AML1-dependent transcription of several promoters with requirement of both, the TEL and AML1 part of the fusion protein.13, 14, 15 These studies proposed that the TEL moiety of the chimeric protein converts AML1 from an activator to a transcriptional repressor. However, alternative mechanisms of TEL-AML1 activity have also been suggested like sequestration of transcriptional cofactors to the cytoplasm16 or dimerization with wild-type protein.17, 18 Studies Bambuterol HCl comparing either patients with and without the TEL-AML1 fusion19 or TEL-AML1-positive cell lines with small hairpin RNA-mediated knock down of the fusion protein indicated expression differences for genes involved in differentiation, apoptosis and immune responses.20, 21 The two latter studies did not find any enrichment for the canonical AML1-binding motif in regulated genes in the acquired mRNA data. In this study, we sought to globally identify promoter regions targeted and regulated by the TEL-AML1 fusion protein. We wanted to differentiate between direct and indirect regulatory effects of the TEL-AML1 fusion protein in a cell system void of secondary aberrations as seen in patients and patient-derived cell lines by using chromatin immunoprecipitation (ChIP)-on-chip to identify promoter-binding sites in combination with mRNA microarray analysis to assess the gene regulatory effect of TEL-AML1. Furthermore, we analyzed the effect of TEL-AML1 expression on the protein output using stable isotope labelling by amino acids in cell culture (SILAC) to deduce indirect regulatory effects of TEL-AML1 independent of promoter binding. Materials and methods Patient samples, control samples and cell lines Four bone marrow samples of patients with TEL-AML1-positive precursor B-cell leukaemia at the time of diagnosis and CD19+ MACS (Miltenyi Biotech, Bergisch Gladbach, Germany)-sorted cells of two healthy donors were obtained after informed consent. No cytogenetic aberration other than t(12;21) was detected in those patient samples. The BA/F3-derived inducible murine cell system was kindly provided by Anthony Ford and has been described elsewhere.22 BA/F3TA+ bearing the inducible TEL-AML1 plasmid as well as the empty vector control cells (BA/F3TA?) Bambuterol HCl were Rabbit Polyclonal to FRS3 induced by 32.5?pM mifepristone treatment for 16?h. NALM-6 (DSMZ ACC 128) and REH (DSMZ Bambuterol HCl ACC 22) cells were cultured according to the provider. TEL-AML1 cDNA was generated with overlap extension PCR from TEL (primers: 5-GGCGCTCGCGAATGTCTGAGACTCCTGCTCAG-3, 5-GGATTCATTCCAAGTATGCATTCTGCTATTCTCCCAATGGGCATGG-3) and AML1 (primers: 5-CCATGCCCATTGGGAGAATAGCAGAATGCATAC TTGGAATGAATCC-3, 5-CCGCGACTAGTTCAGTAGGGCCTCCACACGGCCTC-3),23 cloned into the expression vector pMC324 and transfected into NALM-6 cells using DMRIE-C (Invitrogen, Darmstadt, Germany). Stable cell clones were selected using 400?g/ml hygromycin. Generation and testing of monoclonal antibody.
Regions precipitated with the TEL-AML1 antibody from NALM-6 cells stably expressing the fusion transcript were mapped to human chromosomal regions (black bars) and compared with regions immunoprecipitated from the induced mouse Ba/F3 cell line (grey bars)
