of tumor cells (T-ALL) with extensively dysregulated gene ex- pression in spite of a normal karyotype and genomic stability. RESULTS Abnormal lymphoid development from ICN1-overexpressing hematopoietic precursors ICN1 overexpression in murine hematopoietic precursor cells from a retroviral IRES vector containing ICN1 and EGFP results in a shutdown of B cell development and ectopic T cell development (Fig. 1) (10). Early after BM transfer (BMT) of retrovirally transduced cells into sublethally irradiated re- cipients, most EGFP+ cells resemble CD4+8+ thymocytes, with the exception that they can be found in lymphoid organs outside of the thymus (Fig. 1 a). Such cells become appar- ent 2 wk after BMT and do not cause tumors when trans- planted into nu/nu recipient mice (Table S1, available at http://www.jem.org/cgi/content/full/jem.20081561/DC1). Consistent with their nonmalignant and nontumorigenic phenotype, these ICN1-overexpressing cells are polyclonal in regard to TCR- rearrangement that is completed before the CD4+8+ stage of T cell development (11, 12). As deter- mined by BrdU labeling, the nontumorigenic but abnormal cells have a rapid turnover, much like CD4+8+ thymocytes, which contain 15% of dividing cells that go through one or two divisions before becoming small cells that subsequently die by apoptosis (13). Accordingly, normal CD4+8+ thymo- cytes and the abnormal ICN1-overexpressing CD4+8+ lym- phoid cells incorporate BrdU quickly. After pulse labeling, the label of normal CD4+8+ thymocytes and the abnormal ICN1-overexpressing CD4+8+ cells is also quickly lost, in- dicating that both cell types do not have a long intermitotic lifespan (Fig. 1 b). However, CFSE labeling of these cell types followed by injection into nu/nu mice (in this case, the CFSE-labeled ICN1-overexpressing cells express a retroviral vector containing DsRed) revealed a clear difference in that control CD4+8+ thymocytes did not divide measurably and disappeared 2 d after transplantation, whereas some of the abnormal, nontumorigenic cells went through three cell divi- sions before their disappearance by day six. Thus, abnormal ICN1-overexpressing CD4+8+ cells early after BMT transfer can be distinguished from normal CD4+8+ thymocytes by their greater, but still limited, proliferation/survival potential (Fig. 1 c). Thus, these early appearing polyclonal cells repre- sent neither tumor cells nor their precursors because they do not generate tumors upon transfer into nude mice. At 2 wk, a minor subset of CD4 8+ EGFP+ cells could be observed in peripheral lymphoid tissue, which, unlike the abnormal CD4+8+ cells, did not express an TCR, and thus corresponded in surface phenotype to CD4 8+ imma- ture thymocytes that represent precursors of CD4+8+ DP cells (Fig. 1, d and e). When these cells were transplanted into nu/nu mice, they generated tumors comprised of CD4+8+ and CD4 8+ cells with TCRs on the cell surface. These data suggested that cells with malignant potential are gener- ated after pre-TCR signaling–dependent TCR- selection (14) and before TCR- rearrangement. This was supported 2852 by the notion that the resulting monoclonal T-ALL carry a sin- gle TCR- rearrangement (Fig. 2 a) but express diverse TCR- chains, as indicated by the partial staining of TCRs with antibodies specific for one particular V subgroup only (Fig. 2 b). Thus, malignant transformation of ICN1-overexpress- ing cells is an event that occurs after pre-TCR signaling, but before the acquisition of the CD4+8+ TCR- + phenotype. In fact, some of the monoclonal tumor cells do not express an TCR on the cell surface, which is likely caused by the observation that they did not succeed in generating produc- tive TCR- rearrangements. The malignant potential of the phenotypically distinct tumor cells is reflected in their DNA labeling kinetics that are faster than those of their normal counterparts with the same surface phenotype. This is true for the “early” ICN1-overexpressing CD4 8+ TCR- cells that cause malignancy after transfer into nu/nu mice and also for the CD4+8+ and CD4 8+ TCR- + tumor cells that each can cause malignant tumors on their own after transfer into nu/nu recipients even when injected in low cell num- bers. The analysis showing that each phenotypically distinct subset causes tumors with identical kinetics when injected in different doses refutes the possible notion that the subset with the most immature phenotype, i.e., the CD4 8+ TCR- subset features tumor stem cells, whereas the more mature subsets feature tumor mass but not necessarily highly tumori- genic cells (Fig. 1 d and Fig. 3). On the basis of these data, it appears useful to distinguish between normal CD4+8+ TCR- + thymocytes, abnormal nonmalignant and nontumori- genic ICN1-overexpressing CD4+8+ TCR- + cells, and tumorigenic CD4 8+ TCR- , as well as CD4+8+ TCR- + and CD4 8+ TCR- + tumor cells when further ana- lyzing tumor development at the subcellular level. We assume that the nontumorigenic but abnormal CD4+8+ cells are de- rived from ICN1-overexpressing CD4 8+ TCR- pre- cursors that have failed to undergo malignant transformation because of a genetic status not suited to cooperate with Notch1 overexpression to generate malignant transformation (see Discussion). Because this sequence of events after ICN1 overexpression has not been elaborated previously, it seems important to remember the contribution of the different sub- sets to malignancy when further analyzing gene expression. Posttranslational elimination of E2A-encoded tumor suppressor proteins Because ICN1 overexpression results in ablation of B cell de- velopment (Fig. 1 a), we investigated its impact on the basic helix-loop-helix proteins encoded by the E2A gene. These proteins bind as homo- or heterodimers with other basic he- lix-loop-helix proteins as transcription factors to ubiquitous E-box motifs and have an essential function in B cell devel- opment (15). In fact, E2A-encoded proteins have an impor- tant role in T cell development as well (14), and E2A-deficient mice regularly develop T-ALL (16). When analyzing binding of E2A protein to E-box motifs by EMSA, it became clear that nuclear extracts of both ICN1-overexpressing polyclonal, nontumorigenic, and tumor cells exhibited strongly reduced NOTCH-INITIATED T-ALL | Li et al.