myc transgene for E2f4-null mice (E2f42/2 compared to E2f42/2 Em-myc+: p = 0.0002) (Figure 2B). It appears that the acceleration of cell cycle progression driven by Myc in B lineage cells is not significantly affected by the loss of E2F2 or E2F4 activities. An alteration in the apoptotic potential of Myc could account for the differences in Myc-initiated tumor onset among wild type, E2f2-null and E2f4-null animals. Possibly in E2f22/2 mice apoptosis is reduced whereas in E2f42/2 mice apoptosis is potentiated. Freshly isolated bone marrow B lineage cells (B220+) from E2f22/2 Em-myc transgenics and E2f42/2 Em-myc transgenics were found to have comparable percentages of activated caspase-3 positive cells as their wild type and heterozygous Em-myc siblings, around 0.6%, and all the non- transgenic siblings had less than half this percentage of apoptotic cells (data not shown). Since divergent viability may be masked by clearance in vivo, the survival of B lineage cells under culture conditions where deregulated Myc induces apoptosis was assessed [30,31]. The bulk of B lineage cells, excepting progenitors upstream of small pre-B cells, was enriched by negative selection from the bone marrow, spleen and mesenteric lymph node. The resulting population of small pre-B cells, immature B cells and mature B cells from each mouse was cultured for eight hours in medium lacking cytokines. The cultured cells were sampled at two- hour intervals, and B220+ cells assessed for viability based on activated caspase 3 and 7-AAD staining (Figure S4). As expected, the decline in viability was faster for cells from Em-myc transgenics than from non-transgenics (Figure S4 and Figure 3A and 3B). Notably, when mice in the E2F2 cohort were compared, cells from E2f22/2 Em-myc mice lost viability to a similar extent over eight hours as cells from wild type and heterozygous siblings (Figure 3A). Likewise, in an experiment assessing E2F4 cohort mice, cells from E2f42/2 Em-myc mice declined in viability similarly to those from their wild type and heterozygous siblings (Figure 3B). Thus, the faster tumor onset for E2f2-deficient mice appears not attributable to a general apoptotic deficiency and the slower tumor onset for E2f4-null mice unlikely the result of increased apoptosis, at least in the small pre-B cells and more mature stages assessed here that constitute the large majority of B lineage cells. Myc-mediated tumor emergence is almost invariably associated with a disabling of the ARF-p53 tumor suppressor pathway [8,14]. Mutations that are indicative of pathway disruption include: p53 deletion, ARF deletion, or overexpression of ARF, Mdm2 or mutant p53. Examples of these disruptions in a sampling of tumors are shown in Figures S5, S6, and S7. Consistent with the earlier studies, tumors from E2F wild type Em-myc mice showed evidence of disruption of the ARF-Mdm2-p53 pathway (Figure 3C). The largely late-onset lymphomas from E2f4-null mice also demon- strated disruptions in this pathway. Importantly, the spectrum and overall incidence of defects in the E2f2+/2 and E2f22/2 lymphomas were very similar to that shown by the E2f2+/+ lymphomas. In contrast, other modifiers of Myc-induced lympho- magenesis such as Bim and Bax relieve or modify the strong selective pressure for functional inactivation of this pathway [32,33]. That loss of ARF-p53 function was still associated with development of tumors in the E2f22/2 mice further supports the evidence that E2F2 deficiency does not compromise Myc-induced apoptosis (Figure 3A). Expansion of B lineage progenitor populations in E2f22/2 mice Given that E2F2 deficiency does not alter the proliferation or apoptosis of pretumorous B lineage cells in response to Myc, we focused on the possibility that there may be a different underlying mechanism driving the accelerated tumor emergence, one E2F and Myc-Induced Lymphoma involving development of the B lineage and the response to Myc. As noted in one of the earliest descriptions of the Em-myc model, it is possible that different onsets could reflect different extents of lineage expansion in response to Myc and therefore numbers of vulnerable cells [24]. Overall viable white blood cell number in the bone marrow did not change with E2F2 status while Em-myc positive mice had modestly higher counts (data not shown). As expected, the B cell lineage expanded as a proportion of the bone marrow in response to the Em-myc transgene in E2f2+/+ mice (Figure 4A) and the expansion favored less mature over more mature B cells across all three genotypes (Figure 4B). Importantly, there was a similar degree of expansion for the E2f2+/2 and E2f22/2 mice. Recent studies have suggested, however, that lymphomagenesis likely initiates in B lineage progenitor cells making the effects of various mutations on progenitor populations particularly relevant [34]. For instance, the Em-myc bcl22/2 mice develop tumors at the same rate as Em-myc bcl2+/+ mice despite decreased pre-B, immature B and mature B lymphocytes; significantly, they do have similar numbers of pro-B cells [34]. Similarly, Em-myc/max41 mice develop lymphoma almost as quickly at Em-myc mice despite a severe deficit in more mature, peripheral B cells [35]. Conversely, the increased progenitor population of early/large pre-B stage cells exhibited by Phospholipase Cc2-deficient mice is associated with accelerated lymphomagenesis [36]. As shown in Figure 4C, CFU pre-B colony assays using bone marrow from non-transgenic E2F2 cohort mice indicate that there were more B lineage progenitors in E2f22/2 marrow (P,0.0001) and E2f2+/2 marrow (P = 0.0406) than wild type marrow. The proportion of early B lineage cells (B220+ CD43+) identified by flow cytometry was also greater in non-transgenic E2f22/2 marrow than in non- transgenic E2f2+/+ marrow (P = 0.0008; Figure 4D). The increased proportions of progenitors in E2f2-deficient mice extended into pre-tumorous Em-myc positive mice: there was the trend, although not statistically significant, for more immature B lineage cells as a proportion of the total bone marrow in E2f22/2 (P = 0.0769) and E2f2+/2 Em-myc transgenics (P = 0.1393) compared to E2f2+/+ Em- myc transgenic mice (Figure 4D). Additional analysis revealed that a significant proportion of the E2f22/2 Em-myc lymphomas were not monoclonal. Assessment of Igh locus rearrangement patterns by Southern analysis indicated that almost 40% of the E2f22/2 tumors were biclonal or oligoclonal whereas tumors from other genotypes, whether in the E2F2 cohort or in other cohorts, were predominantly monoclonal, in agreement with past studies [5] (Figure 5A and Figure S8A and S8B). This degree of oligoclonality was, however, less extensive than that which occurs with the homozygous Em- myc/Em-myc mice [37] and when retrovirally-expressed myc was expressed in mice reconstituted with p532/2 hematopoietic stem cells [38]. In addition, several tumors were analyzed by flow cytometry for isotypic surface marker expression (Figure 5B). Seven out of ten tumors emerging from E2f22/2 mice displayed a complex pattern of isotypic surface markers. Such complexity, while not uncommon for Em-myc lymphomas in general [22], is consistent with multiple clones. Of note, two out of eight tumors from E2f2+/2 mice were similarly complex. In contrast, the majority of tumors from other Em-myc mice displayed single, homogeneous patterns of surface markers and could be clearly classified as being either pro/pre-B or immature B lymphomas. Taken together, these findings support the conclusion that in the E2f22/2 Em-myc mice there is an increased population of B lineage cells susceptible to lymphomagenesis resulting in the occasional emergence of more than one independent tumor. Using a different Myc transgenic system, Cory and colleagues noted the emergence PLoS Genetics | www.plosgenetics.org 5 September 2009 | Volume 5 | Issue 9 | e1000640