o sublethally irradiated recipients. All mice became hunched, moribund and had ruffled fur after 20 2901691 21 days. Therefore, spleens and livers from these mice were analysed for expression of CD4, CD8 and TCRb by flow cytometry. All 4 mice demonstrated a similar CD4+8+ population and CD8+ SP population as the primary mouse. These data reveal that the Fli-1-induced pre-T LBL is transplantable. To preclude that site specific integration effects could be responsible for the observed pre-T LBL development, a genomic Southern blot of both primary and secondary pre-T LBLs was undertaken with a GFP specific probe. The results unequivocally demonstrate that each individual pre-T LBL had different multiple integration sites and as such the observed pre-T LBL is due to aberrant FLI-1 expression. Additionally, we sequenced integration sites in 5 tumours and could find no association with known oncogenes. Interestingly, analysis of TCRb rearrangements revealed that most Fli-1 pre-T LBL possessed one dominant clone but were oligoclonal. The exceptions to this were those that did not express TCRb. As expected, identical TCRb rearrangements were found in the thymus 
and spleen of the same Fli-1 transplanted mice and in primary and secondary 17526600 Fli-1 transplanted mice. Discussion Fli-1 was initially identified as an insertion site in erythroleukaemia in BALB/c mice and subsequently shown to be required for the development of megakaryocytes. Fli-1 has also been found to control myeloid and B cell development. However, the data presented here for the first time, demonstrate that Fli-1 also plays a role in T cell development. Fli-1 overexpression leads to a block in the DN3 to DN4 transition with a subsequent inhibition of the DN to DP transition. The Fli-1-induced DN3 arrest is consistent with endogenous Fli-1 downregulation at the DN2 stage as enforced Fli-1 expression in DN3 would increase FLI-1 levels and inhibit progression to DN4. No Increase in Pro-survival Bcl-2 Family mRNAs in Fli1 Pre-T LBL Fli-1 has been demonstrated to upregulate Bcl-2 in erythroleukaemia. Therefore, we analysed primary and secondary Fli-1 leukaemias and MigR1 controls for Bcl-2, Bcl-xL and Mcl1 mRNA expression by Q-PCR. Surprisingly, all Fli1 pre-T LBLs, whether primary, secondary or grown in vitro had lower levels of Bcl-2 family members, Bcl-2, Bcl-xL and Mcl-1. These data show that overexpression of the pro-survival Bcl-2 family members are unlikely to be involved in the induction of Fli-1 pre-T LBL. Fli-1 Overexpression Induces T Cell Vatalanib web Leukemia control 6 weeks post transplant. Data is representative of three independent experiments. C. Fli-1 pre-T LBL possess 59 Notch1 deletions detectable by PCR. Primers flanking the 59 Notch1 deletion site generate a small 485 bp PCR fragment of genomic DNA in Fli-1 tumours, which is absent in control cells where the same primers span a 12,251 bp region of undeleted WT DNA. Lanes 1, 8 & 21:2-Log DNA ladder, 27 and 917: MigR1 control or primary and secondary Fli-1 spleen cells, bone marrow or thymocytes as indicated. 17; radiationinduced thymic lymphoma, 18: Fli-1 transduced T cell precursors grown on OP9-DL1 cells, 19: Fli-1-transduced FDC-P1 cells, 20: Mixl1transduced leukaemic BM cells. G3PDH: positive gDNA PCR control. D. Notch1 mRNA upregulation in leukaemic Fli-1 mice. Q-PCR of MigR1 control, Fli-1 primary, secondary and in vitro Fli-1 pre-T LBL cells from the thymus, spleen or liver. Samples and analysis as in Fig. 5. E. Notch1 mRNA upregul
Posted inUncategorized