Figure 8. Genomic analysis. (left) SKY analysis of ICN1-induced tumor cells. SKY table of stained ICN1-induced tumor chromosomes using SKY paint shows the spectral, classified, and banded-inverted chromosomes. (right) Array CGH. Genomic DNAs extracted from ICN1-induced malignant tumor GFP+CD4+8+ (Tumor XLT7), abnormal nonmalignant ICN1-overexpressing cells GFP+CD4+8+, and control GFP+CD4+8+ thymocytes were subjected to high- resolution oligo array–based comparative genomic hybridization. Tail DNA from the same mouse was used as reference for the hybridization. Malignant tumor GFP+CD4+8+ cells from four independent tumor mice were analyzed by Array CGH. in murine T-ALL initiated by ICN1 overexpression does not result from genomic instability. DISCUSSION Cellular and molecular pathways involved in tumorigenesis initiated by a single aberrantly expressed gene are difficult to study retrospectively in human tumor cell lines because of the unknown impact of unrelated genetic changes that may have independently contributed to tumor formation, as well as further genetic changes that are required for in vitro growth (30). For this reason, disease progression was explored in a murine model of T-ALL initiated exclusively by retrovirus- mediated overexpression of ICN1 in lin BM cells. In fact, mutations resulting in overexpression of ICN1 are frequently observed in human T-ALL samples (2–4). However, in all cases, it is unknown whether such mutations initiated tumor development or just contributed later to the progression of disease. In a mouse model of ICN1-initiated T-ALL, it was shown by Aster et al. that retrovirally induced overexpression of ICN1 in lineage-negative BM cells resulted in extrathymic T cell development and T-ALL (10). In this study, we have characterized various stages during tumor development with the aim to define cellular and molecular pathways in ICN1- dependent tumorigenesis. BMT with cells retrovirally transduced with a bicistronic retroviral vector containing ICN1 and a fluorescence marker resulted after 2 wk in accumulation of CD4+8+ cells outside of the thymus. Such ICN1-overexpressing cells were not only abnormal in regard to their anatomical location but also exhibited an extended proliferation/survival potential com- pared with their intrathymic counterparts. These cells, how- ever, were nontumorigenic after transplantation into nude 2858 mice, and hence do not qualify as tumor progenitors. Molec- ular analysis of these early appearing nontumorigenic CD4+8+ cells revealed the ICN1-dependent, degradation of E2A tu- mor suppressor that normally binds to ubiquitous E-box mo- tifs and in the thymus reduces proliferation while promoting differentiation of pre-TCR–expressing thymocytes (16).The ubiquitin-dependent degradation of E2A-encoded proteins in ICN1-overexpressing cells explains the lack of B cell de- velopment of ICN1-overexpressing BM cells (15). Nontumorigenic CD4+8+ cells exhibit multiple TCR- rearrangements consistent with their polyclonal nonmalig- nant and nontumorigenic phenotype. Global gene expression analysis confirmed that these cells differ extensively from normal CD4+8+ thymocytes; expression of several Notch tar- get genes is increased, as is the expression of genes involved in proliferation/survival, whereas RNAs coding for cell cycle inhibitors and death-promoting proteins are reduced. These data fit well with the functional studies showing increased survival and proliferation of ICN1 overexpression among nontumorigenic CD4+8+ cells. The proliferative capacity, TCR- rearrangement status, and gene expression profile of these abnormal but nontu- morigenic cells are distinct from those of monoclonal, malig- nant CD4+8+ tumor cells that express a single TCR- chain but diverse TCR- chains, implying that pre-TCR signaling (the pre-TCR consisting of a TCR- chain that is disulfide linked to the invariable pre-TCR- chain) (34) makes a ma- jor contribution to malignancy, being absolutely essential for T-ALL generation in some (10) but not all studies (35, 36). In fact, Notch1 signaling and pre-TCR signaling do cooper- ate in normal T cell development to induce survival and proliferation (37), as they do cooperate in the initiation of NOTCH-INITIATED T-ALL | Li et al.