Notch ligands and Hes1 induce Notch-mediated growth arrest/cell death in human B-cell leukemia lines. (A) RNA expression levels of the 4 Notch homologs were measured in 4 pre-B ALL cell lines via RT-PCR. GAPDH was used as a control. The data for Notch1 and Notch2 are in triplicate with standard deviation. Data for Notch3 and Notch4 are averages of duplicate samples. (B) Growth inhibition and cell death in pre-B ALL cell lines during coculture with Notch ligand-expressing fibroblasts (Jagged1 or Jagged2 NIH3T3s) was measured by staining for DNA content (PI) and analysis by flow cytometry. Comparison was made to coculture with untransduced NIH3T3 controls. The top graph represents the percentage of non-G₀/G₁ cells versus days of coculture for 2 different pre-B ALL cell lines (JIM, Nalm-6) (duplicate experiments gave similar results). Bottom graph represents the cell count versus days of coculture (counted in triplicate). Up-regulation of Hes1 by RT-PCR was measured at 24 hours after initiation of coculture with either Jagged1- or Jagged2-expressing fibroblasts. GAPDH was used as the cDNA control. (C) Hes1 expression leads to growth arrest/cell death that is equivalent to intracellular Notch1. The graph shows the percentage of GFP⁺ (transduced) cells versus days from transduction (normalized to day 2) for the 697 pre-B ALL cell line. The second graph shows similar data from 2 other cell lines, JM-1 and Nalm-6. The P values are from repeated-measures ANOVA. In panels B and C, means and standard deviation are shown.