Western blot, it became apparent that tumor cells exhibited higher levels of these proteins compared with their nontu- morigenic but abnormal or normal counterparts (Fig. 7, a and b). ICN1 has been described to bind specifically in a transcrip- tional complex to c-Myc (5, 6), and we found c-Myc to be up-regulated in nontumorigenic ICN1-overexpressing cells, but further up-regulated in tumor cells at the level of both RNA and protein (Fig. 7 c). It is interesting to note that in studies looking at cyclin D3 deficiency, it was concluded that cyclin D3 was essential for growth of T-ALL (20). These Figure 4. Overexpressed ICN1 inhibits E2A activity by promoting protein degradation at different stages of disease development. (a) E2A E-box motif binding activities were analyzed by electrophoretic mobility shift assay (EMSA) using nuclear extracts of GFP+CD4+8+ (GFP+/ DP) cells derived from various mice. (b) E2A and phosphorylated ERK1/2 protein expression levels were analyzed by Western blot in whole-cell lysates from various cells. (c) Quantitative analysis of E47 mRNA expres- sion in various cell types. GAPDH mRNA was used as control. **, P < 0.01, compared with E2A+/ group. Data are reported as mean ± SEM for tripli- cate experiments. (d) Ubiquitination assay: whole-cell lysates were immuno- precipitated by E2A antibody. Immunoprecipitates were blotted with antiubiquitin antibody. (e) ICN1-induced degradation and phosphorylation of E2A by ERK1/2 MAP kinases. Whole-cell lysates were from various cells that have incubated in the absence or presence of 50 μM PD98059 for 90 min; PD98059 is a specific inhibitor of MEK1 and MEK2. Western blot analysis was performed to evaluate E2A protein expression levels. -Actin is shown as loading control. JEM VOL. 205, November 24, 2008 A RT I C L E data, however, only addressed human T-ALL and murine non- tumorigenic cells. Our results showing that murine T-ALL overexpress cyclin D1 are, in fact, not easily compatible with the notion that cyclin D3 is essential for growth of murine T-ALL. To obtain further insight into differential gene expres- sion, comparative analysis of global gene expression was con- ducted after isolation of RNA from normal CD4+8+ DP thymocytes, nontumorigenic ICN1-overexpressing CD4+8+ cells from peripheral lymphoid tissue, and CD4+8+ tumor cells by Affymetrix chip analysis. Direct targets of Notch, such as Hes1, Hes5, and pT were elevated in nontumorigenic ver- sus normal cells and either maintained or down-regulated in malignant (tumor) cells, in contrast to c-Myc that was further up-regulated in tumor cells (Fig. S1, a and b, available at http://www.jem.org/cgi/content/full/jem.20081561/DC1), consistent with quantitative RT-PCR data and Western blot analysis (Fig. 7, b and c). In regard to cell cycle control and death pathways, both nontumorigenic and tumor cells exhibited reduced transcription of the death-associated protein kinase 1 (DAPK1). Reduced levels of DAPK1 are often found in tu- mors because of the hypermethylation of its promoter (21). Fas was down-regulated in both nontumorigenic and tumor cells versus normal controls. Casting a wider net, the data showed that tumor cells versus nontumorigenic cells were especially deficient in tumor suppressors such as the large tu- mor suppressor 2 (LATS2), often found down-regulated in human lymphoma, and inhibitors of cyclin-dependent ki- nases, such as p19 and p18 (Fig. S1 a). On the other hand, sev- eral oncogenes, including several Ras family oncogenes, the Figure 5. Nondegradable mutant E47 is overexpressed in ICN1- DsRed tumor cells. Western blot analysis of mutant E47 protein expres- sion in whole-cell lysates from ICN1 tumor cells transduced with the empty retroviral vector MSCV-IRES-GFP or retroviral construct MSCV- IRES-GFP-MmE47. -Actin is shown as loading sample. 2855