ICN1-dependent lymphoma. Therefore, malignant transfor- mation causing independent tumors in different mice must have taken place before the completion of TCR- rearrange- ment because of the expression of a single TCR- chain paired with diverse TCR- chains in different cells from the same tu- mor. This fits well with the finding that TCR- CD4 8+ cells, i.e., immature cells that have undergone pre-TCR sig- naling are tumorigenic and exhibit a significantly higher pro- liferation than their normal counterparts (Fig. 1 d). Because the ICN1-overexpressing CD 4+8+ cells that appear early in blood are not tumorigenic, the data indicate that malignant transformation must occur in precursors of CD4+8+ cells and does take place within a few weeks after BMT with ICN1- overexpressing lineage-negative cells. In contrast to the nonmalignant (nontumorigenic) CD4+8+ TCR- + cells, the malignant CD4+8+ TCR- + or CD4 8+ TCR- + cells derived from the immature CD4 8+ TCR- cells continued to grow, and each phenotypically distinct subset was able to independently cause tumors in nu/ nu recipient mice with very similar kinetics; this is incompat- ible with the notion that the “early” CD4 8+ cells represent tumor stem cells that generate nonmalignant TCR- + prog- eny that constitutes the tumor mass. The tumorigenic poten- tial was mirrored by the up-regulation of several oncogenes, most prominently the c-Myc oncogene that represents an ICN1 target, but also others, such as Akt and the oncogenes of the Ras family, the latter being considered targets of c- Myc. Furthermore, transformed cells exhibited reduced lev- els of cyclin-dependent kinase inhibitors p18 and p19, as well as elevated expression of cyclin-dependent kinase 4 (another c-Myc target) compared with their nonmalignant CD4+8+ counterparts. Increased c-Myc RNA levels were also re- flected in increased levels of RNAs transcribed from genes involved in transcription and translation such as RNA poly- merases, as well as initiation and elongation factors, which are likewise considered to represent targets of c-Myc. However, c-Myc overexpression by itself is not sufficient to cause mono- clonal tumors (7, 8), and needs to be accompanied by further genetic changes to result in outgrowth of monoclonal tumors. Apart from being an ICN1 target, c-Myc expression can be dramatically increased by chromosomal translocation (38). However, SKY analysis of independent tumors did not reveal any translocations, indicating that overexpression was depen- dent on transcriptional regulation of c-Myc by ICN1 (5, 6). Furthermore, analysis by high-resolution oligo array–based comparative genomic hybridization (array CGH) did not ex- hibit any tumor-specific amplifications or deletions, suggesting that genomic instability does not contribute to the generation of tumors initiated by ICN1 overexpression, in spite of the ob- servation that c-Myc overexpression can be associated with genomic instability. Thus, mechanisms other than genomic instability must account for the observation that highly ele- vated levels of c-Myc in tumors did not result in stabilization of apoptosis-inducing p53. Normally this occurs via up-regu- lation of p19Arf, which then sequesters Mdm2 that can then no longer degrade p53. In fact, p53 levels in tumor cells were JEM VOL. 205, November 24, 2008 A RT I C L E much reduced compared with those in the abnormal nonma- lignant or normal CD4+8+ cells, as were levels of p19Arf both at the level of RNA and protein. Down-regulation of the p19Arf protein explains the lack of compensatory p53 up-regu- lation that could have prevented tumor outgrowth and probably did so in the nonmalignant CD4+8+ cells. However, it does not explain the up-regulation of Mdm2 and down-regulation of p53 at the transcriptional level. The reduced expression of p19Arf is at least in part dependent on its transcriptional repres- sor Bmi-1, which can be up-regulated by c-Myc (29) and was specifically up-regulated in T-ALL. Thus, the reduced levels of p19Arf protein in the absence of any mutation are best ex- plained by Bmi-1 up-regulation. It is important to stress how- ever, that the dysregulation of the c-myc-p53 axis in T-ALL does not only involve the usually suspected posttranscriptional mechanisms, but also results from the unexplained dysregula- tion at the transcriptional level of Mdm2 as well as p53. Because of the monoclonal nature of the tumors that even- tually develop, it is likely that another thus far unknown ge- netic event is required for malignant transformation that does not depend on genomic instability, as evident from the CGH data. The cooperating mutation(s) require further scrutiny, and could conceivably involve mutations caused by genetic instability. However, because the tumors develop relatively early, and we did not find mutation by sequencing of genes in the c-Myc-p53 axis, we consider it more likely that the coop- erating event is caused by insertional mutagenesis of the ret- roviral vector itself. This hypothesis will be examined by determining the retroviral integration sites in tumors versus nontumorigenic ICN1-overexpressing cells. MATERIALS AND METHODS Mice. BALB/c WT and BALB/c nu/nu mice were purchased from Taconic. C57BL/6 and Rag-2 / c / mice were purchased from The Jackson Lab- oratory. All mice were kept in specific pathogen–free animal facilities at the Dana-Farber Cancer Institute. All animal procedures were performed in com- pliance with the guidelines of the Dana-Farber Cancer Institute Animal Re- sources Facility, which operates under regulatory requirements of the U.S. Department of Agriculture and Association for Assessment and Accreditation of Laboratory Animal Care, and experimental protocols were approved by the Animal Care and Use Committee of the Dana-Farber Cancer Institute. Tumor transplantation. Nude mice were injected with various numbers of tumor cells (1 × 104 to 2 × 106) and tumor development was subse- quently monitored. Recombinant DNA constructs. The intracellular domain of the murine Notch1 (ICN1) retroviral vector was created by cloning a PCR-amplified ICN1 full-length sequence into a MSCV-IRES-DsRed vector, Mutant E47 cDNA was cloned into a retroviral vector MSCV-IRES-GFP. The MSCV- IRES-DsRed vector and MSCV-IRES-GFP vectors were provided by D. Vignali (St. Jude Children’s Research Hospital, Memphis, TN). Mutant E47 cDNA was provided by X.H. Sun (Oklahoma Medical Research Center, Oklahoma City, OK) and has been previously described (19). The murine ICN1 retroviral plasmid CMMP-ICN1-IRES-EGFP and its parent vector CMMP-IRES-EGFP were used in most infection experiments with ICN1. The retroviral construct encoding a truncated N-terminal fragment of mas- termind-like 1 fused to EGFP (DN-MAML), and the control vector MigR1 was provided by J.C. Aster (Brigham & Women’s Hospital, Boston, MA) and has been previously described (25, 39). 2859