activity (Fig.4 a). According to the literature, the results could have been caused by distinct mechanisms; the reduced activ- ity of E2A in EMSA could have been caused by either up- regulation of the pre-TCR by overexpressed ICN1 or by increased pre-TCR signaling resulting in increased Id1 or Id3 levels that prevent binding of E2A-encoded proteins to E- box motifs (17, 18). Alternatively, overexpressed ICN1 could have resulted in E2A protein phosphorylation and degrada- tion (19). Apparently, the second mechanism was responsible because there were reduced levels of E2A protein as deter- mined by Western blotting (Fig. 4 b), but not reduced RNA levels as analyzed by quantitative RT-PCR analysis (Fig. 4 c). The reduced levels of E2A-encoded proteins were caused by ubiquitination-dependent degradation of E2A depending on phosphorylation by activated ERK kinase (Fig. 4 d). In fact, inhibition of ERK phosphorylation (19) by the specific MEK1 inhibitor PD98059 restored the levels of E2A-encoded pro- Figure 2. TCR expression by malignant and nonmalignant cells. (a) TCR- rearrangement in various cell types. Analysis of TCR- rear- rangement by Southern blot analysis in GFP+CD4+8+(GFP+/DP) and GFP+CD4 8+(GFP+/CD8+) thymocytes derived from retroviral GFP-empty vector–transplanted mice, as well as GFP+CD4+8+(GFP+/DP) and GFP+CD4 8+(GFP+/CD8+) splenocytes derived from retroviral GFP-ICN1– transplanted mice at 2 and 8 wk after BMT. Molecular markers are shown on the left. (b) TCR- and TCRV- 2 expression by various lym- phocyte subsets. (left) Staining with the pan TCR- antibody H57. (right) Staining with V 2 TCR antibody. 2854 teins (Fig. 4 e). Similarly, reduced levels of E2A proteins were observed in both nontumorigenic and tumor cells. Thus, as observed in ICN1-overexpressing fibroblasts (19), the E2A- encoded tumor suppressors were eliminated in ICN1-over- expressing lymphoid cells by posttranslational modifications. The ICN1-dependent reduction of E2A-encoded tumor sup- pressors by itself was insufficient to cause tumors within the first few months of observation. To evaluate the role of E2A-encoded E47 protein as a po- tential tumor suppressor in T-ALL, nondegradable mutant E47 (19) was introduced into ICN1-overexpressing tumors. Dou- ble fluorescence–labeled tumor cells were sorted and injected into nu/nu recipient mice, whereas control mice received tu- mor cells transduced with an empty EGFP vector. Cells over- expressing E47 (Fig. 5) had a clear growth disadvantage because they generated tumors more slowly than ICN1-overexpressing cells transduced with an empty vector. Also, after injection of 98% pure double-labeled tumor cells expressing the two retro- viral vectors, there was some selection favoring cells no longer expressing mutant E47. This selection resulted in tumors in which half of the cells no longer expressed mutant E47, indi- cating that in ICN1-overexpressing tumor cells, E47 can act as a tumor-suppressor, albeit not one that is sufficiently strong to completely prevent the outgrowth of E47+ tumors (Fig. 6). Thus, the posttranslational elimination of the E2A-encoded tumor suppressor by overexpressed ICN1 contributes to Notch1-induced malignant growth, which is consistent with the observation that this tumor suppressor has a role in pre- venting proliferation and facilitating differentiation in normal T cell development. On the other hand, it appears that E2A reduction may not be essential for tumor formation after ICNI overexpression because E47-overexpressing tumors could still be detected, albeit at a later stage. Tumor-specific gene expression When analyzing the expression of a few specific candidate genes such as c-Myc, NF- B, cyclin D1, and cyclin D3 by Figure 3. Kinetic analysis of tumor development. Various cell num- bers of ICN1-overexpressing cells with a distinct surface phenotype (as indicated) were injected into nu/nu mice and the kinetics of tumor devel- opment were monitored. NOTCH-INITIATED T-ALL | Li et al.