essential role in early tumor growth because it is deleted in CD4-expressing tumor cells, but not their CD4 8+ TCR- precursors. This result is in line with reports on T-ALL cell lines (23). The essential role of ICN1 for tumor growth in vivo was shown by introducing dominant-negative mastermind-like 1 (DNMAML1) (25) through retroviral transduction into pri- mary ICN1-overexpressing tumor cells using an approach similar to that used to test the effect of mutant E47 on tumor development (Fig. 6), i.e., the retroviral vector containing ICN1 and DsRed was used to generate tumors and the ret- roviral vector containing DNMAML1 and EGFP was intro- duced into tumor cells before transplantation into nude mice. Even though 99% pure double-fluorescent tumor cells were sorted and injected into nu/nu recipients, double-fluorescent cells did not form tumors, whereas DsRed, ICN1-expressing cells survived selectively in the injected hosts and generated Figure 7. Specific gene expression. (a) NF- B DNA binding activity was analyzed by electrophoretic mobility shift assay (EMSA) using nuclear extracts of GFP+/CD4+8+ DP cells derived from various mice. (b) c-Myc, cyclin D1, and cyclin D3 protein levels in various cells were analyzed by Western blot in whole-cell lysates. -Actin is shown as loading control. (c) Total RNA isolated from various cells was analyzed by quantitative real-time PCR using specific primers for c-Myc. GAPDH mRNA was used as control. *, P < 0.05; **, P < 0.01. Data are shown as mean ± SEM for triplicate experiments. (d) The c-Myc-p53 axis: Western blot analysis in whole-cell lysates from various cells was performed. -Actin is shown as loading sample. (e) Quantitative analysis of p19Arf, Mdm2, p53, and Bmi-1 mRNA expression in various cells. GAPDH mRNA was used as control. **, P < 0.01; ***, P < 0.001. Data are shown as mean ± SEM for triplicate ex- periments. N, CTRL GFP+/DP thymus; NT, ICN1, GFP+/DP spleen 2 wk; TU, ICN1 GFP+/DP spleen 8 wk. JEM VOL. 205, November 24, 2008 A RT I C L E tumors at a much later stage compared with ICN1-express- ing controls (Fig. 6 c). These results indicate that c-Myc and ICN1 are both required for the maintenance of tumors as previously established for cell lines (5, 25). The c-Myc—p53 pathway in malignant transformation The tumor suppressor p53 can counteract the oncogenic ef- fect of elevated c-Myc by inducing cell death. This can occur through c-Myc–dependent increased production of p19Arf that sequesters the p53-degrading enzyme Mdm2 in the nu- cleolus, preventing it from reaching p53, which is thereby stabilized and induces apoptosis in c-Myc–overexpressing cells (26). Thus, this scenario depicts the regulation of the c- myc-p53 axis mostly by posttranscriptional mechanism. Sev- eral mechanisms can interfere with this protective pathway, in particular mutations in either c-Myc (27) or p53 (28), or the activation of the c-Myc–dependent transcriptional repres- sor Bmi-1 that can down-regulate p19Arf (29). To investigate whether the c-Myc-p53 axis (28) is intact in ICN1-depen- dent tumor cells with elevated levels of c-Myc, we deter- mined c-Myc and p53 levels by Western blotting. As shown in Fig. 7 d, in spite of the highly elevated levels of the c-Myc protein in tumor cells, the level of the p53 protein was down compared with nontumorigenic ICN1-overexpressing cells, indicating that the c-Myc–dependent pathway of p53 stabili- zation was not operative in tumor cells. Furthermore, p19Arf protein was down-regulated and Mdm2 protein was up-reg- ulated in tumor cells compared with nontumorigenic ICN1- overexpressing cells. Curiously, all changes in protein expression were mirrored by changes at the level of RNA, indicating the existence of regulatory mechanisms in addition to posttran- scriptional modifications. Also, the transcriptional repressor Bmi-1 was up-regulated in tumor cells at the level of RNA, providing an explanation for the low-level expression of p19Arf at the level of RNA. Normal karyotype and genomic stability of tumors Recent analysis of human T-ALL lines has indicated consid- erable genomic instability, whereas genomic instability of mu- rine T-ALL was artificially introduced (30). Also, overexpression of c-Myc was reported to result in genomic instability (31). When several independent primary T-ALL samples from ICN1- overexpressing mice samples were subjected to SKY analysis, it became clear that the independent T-ALL samples exhib- ited a normal karyotype (Fig. 8) as has also been observed in subsets of primary human T-ALL samples (32, 33). Further- more, high-resolution oligo array–based comparative genomic hybridization of four independent primary T-ALL samples revealed expected deletions at the TCR- , TCR- , and TCR- loci, as well as the lgH locus; these deletions are also found among the majority of normal CD4+8+ thymocytes. Importantly, however, there were no deletions or amplifica- tions that occurred only in tumor cells, but not in normal cells or nontumorigenic cells, excluding genomic instability as the cause of malignant transformation in this mouse model. These results therefore show that dysregulated gene expression 2857