Fyn-proto-oncogene or general transcripts of genes enhanc- ing proliferation (CDK4) and antagonizing cell death were specifically up-regulated in tumor cells, as were genes en- hancing transcription and translation and representing c-Myc target genes (Fig. S1 b) (5, 22). These data suggest a scenario in which high levels of ICN1 alone result in elimination or down-regulation of several tumor suppressors, such as E2A- encoded proteins and DAPK1, and in up-regulation of sev- eral Notch targets and survival genes in nontumorigenic cells, but only moderately up-regulated c-Myc levels. These changes, however, are insufficient to cause malignant transformation and lead only to moderately increased survival/proliferation of cells with a CD4+8+ phenotype. The nontumorigenic cells do, in fact, express higher Annexin V levels than tumor cells, indicating their higher susceptibility to apoptosis. Also, non- tumorigenic cells still express appreciable levels of tumor sup- pressors that are down-regulated in malignant tumor cells (Fig. S1, a and b). Of special interest was the observation that the p53 inducible nuclear protein 1 gene was up in nontu- morigenic versus tumor cells, indicating that p53 activity was increased in nontumorigenic versus tumor cells. These obser- vations may explain the nonmalignant phenotype of the ICN1- overexpressing nontumorigenic cells. C-Myc and ICN1 overexpression are essential for tumor development and growth The gene expression data suggest that up-regulation of c- Myc may play an essential role in tumorigenesis, as has been suggested by analysis of select T-ALL cell lines (5). The es- sential role of c-Myc for tumor growth was confirmed in ex- periments where floxed c-Mycfl/fl (23) mice were crossed with CD4-cre (24) mice and BM cells from c-Mycfl/fl CD4- cre+/ mice were infected with the ICN1 and EGFP bicis- tronic retroviral vector. As shown in Fig. 6 d, the deletion of c-Myc at the CD4+8+ stage of T cell development prevented tumor formation, but did not prevent the generation of EGFP+ extrathymic nontumorigenic cells, whereas in CD4- cre mice, tumors developed normally. Thus, c-Myc plays an Figure 6. Essential role of ICN1 and c-Myc, but not E47, down-regulation in tumor growth. Tumor development and growth dependent on c-Myc and ICN1, respectively. The empty GFP-IRES retroviral vector (a), the GFP-IRES retroviral vector containing mutant E47 (b), or the GFP-IRES retrovi- ral vector containing DNMAML1 (c) were introduced into tumors expressing a DsRed-ICN1 retrovirus. Double-fluorescent tumor cells were sorted and 106 cells were injected into nu/nu recipient mice. Tumor cells were analyzed when mice had developed palpable tumors. Nude mice injected with tumors con- taining empty retroviral vectors died of lymphoma 3 wk of after transfer. Mice injected with cells containing MmE47 or DN-MAML1 developed tumors 7 wk after injection. Double-fluorescent cells did not form tumors in c. (d) Analysis of GFP+ and GFP cells in blood of Rag2 / , c / mice that were irradiated and reconstituted with retrovirally transduced BM cells from either floxed c-Myc, CD4-cre, or CD4-cre mice. CD4-cre mice were killed at 8.5 wk when they exhibited large lymphoblastic tumors. In contrast, CD4-cre c-MycFL/FL mice developed no tumors up to 15 wk. 2856 NOTCH-INITIATED T-ALL | Li et al.