Mutational activation of Notch1 and deletion of Tcra are etiologically associated with the development of T-cell leukemia/lymphoma. (A) Array CGH analysis of CEP2A (top) and CEP5A (bottom) cells shows almost identical chromosome changes in the 2 cell lines, including 14qC2 segmental loss as well as gain of chr7, chr10, and chr15. (B) Detailed view of murine Tcra deletion in both CEP2A and CEP5A cell lines. (C) Genomic PCR confirms loss of Tcra in the CEP2A and CEP5A cells. (D) Flow cytometric analysis reveals that both CEP2A and CEP5A cells are negative for cell-surface Tcra. (E) Western blot analysis, using antibodies against the activated Notch1 (Val 1774 antibody, Cell Signaling Technologies), showing that activated Notch1 (different bands resulting from different truncating mutations) is present in LN isolated from the primary (1°) and secondary (2°) T-LBL mice compared with 3 murine FGFR1-related neoplasm cell lines. A spleen sample from #3 AML mouse is used as a negative control. (F) Schematic of the Notch1 gene showing the relative locations of the primers used to analyze the deletion mutants (top). A 5′ deletion (brackets) creates a 500-base pair fragment using the P1/P2 primers, as shown in LN from different T-LBL mice. In normal cells, the wild-type 11.5-kb fragment cannot be amplified.12 (G) γ-Secretase inhibitors DAPT and Comp E significantly inhibit CEP2A and CEP5A cell growth in vitro at micromolar concentrations.
