P1A as an Oncogene Figure 1. Expression of P1A. A. Diagram of Emmb-P1A transgene. P1A open reading frame (P1A-ORF) is under controlled by enhancer of immunoglobulin heavy chain (Em) and mb-1 promoter (Pmb-1), followed by ID2 gene and an SV40 polyadenylation signal sequence. B. Western blot analysis of P1A protein expression in J558 plasmacytoma cells, R1 murine ES cells, thymus, spleen, testis from P1A Tg mice and BALB/c WT mice; actin as internal control. doi:10.1371/journal.pone.0013439.g001 P1A in lymphoid cells. P1A protein was detectable by Western blot in P1A Tg thymus by the anti-P1A antibody against the C- terminus of the P1A protein, however it was not detected in P1A Tg spleen even with much more protein loaded (Figure 1B). As shown in Figure 1B, P1A protein level was highest in J558 plasmacytoma cells followed by mouse R1 embryonic stem cells and then the testis, according to the actin loading amount. P1A Tg mice developed thymic tumors To increase tumor susceptibility, we crossed the P1A transgene to Rag22/2 background which is immune-deficient due to lack of T and B lymphocytes. We observed that 19 out of 49 P1A Tg mice in Rag2-deficient background developed thymic lymphoma after 7 months of age. The Rag22/2P1ATg mice had enlarged thymuses compared to control mice (Figure 2A). In addition to the thymus, the spleen was also enlarged (data not shown). Moreover, tumor infiltration into target organs, including liver and lung, were also widespread (data not shown). In addition, when using tumor incidence as a survival end point, P1A Tg mice in immunodefi- cient background (Rag22/2) had significant shorter survival rates due to the tumor formation compared to control groups (p = 0.0004, Figure 2B). A trend of increased tumor was also noted in WT background, although the increase was not statistically significant (p = 0.1157, Figure 2C). In order to determine the lineages of the tumors, we analyzed the cell surface markers of the thymic tumors. As shown in Figure 3, only 1 of the 7 tumors analyzed expressed significant levels of CD4 and CD8. In contrast the majority of tumors expression markers of non-T lineages, including that of B cells (B220+), plasmacytoid dendritic cells (B220+CD11c+) , myeloid cells (B220+Gr1+CD11b+). The result is consistent with the fact that Rag2 deficiency results in the thymocyte differentiation being blocked before they can reach the CD4+CD8+ stage [13]. It is noteworthy that all but one of tumors expresses B220, a marker for the B cell lineage. P1A Tg bone marrow (BM) cells exhibit higher proliferation and more hematopoietic colony forming units We determined P1A Tg BM cell proliferation ability by BrdU incorporation assay. BrdU is an analog of the DNA precursor thymidine. The proliferating cells will incorporate BrdU into their DNA. The amount of BrdU in the DNA of cells can be detected with specific anti-BrdU fluorescent antibodies followed by flow cytometry. After 24 hours of labeling BrdU in vivo, we analyzed the BrdU incorporation rate by flow cytometry. Forty-six percent of BM cells from wild-type (WT) control mice were BrdU positive. However, the P1A Tg mice had a significant increase in the proportion of BrdU-positive BM cells both in immunocompro- mised (P1A+Rag2+/+) and immunodeficient (P1A+Rag22/2) backgrounds compared to WT mice (p = 0.02 and 0.03, respec- tively, Figure 4 AB). We also cultured P1A Tg BM cells and control cells in methylcellulose based medium to quantify potential progenitors in vitro. The hematopoietic progenitors were able to proliferate and differentiate, resulting in the generation of mature cells. Colony- forming units (CFUs) were measured in semi-solid media with appropriate cytokines. We found P1A Tg BM had much higher CFU compared to control BM. The increase was more obvious in the second round culture (Figure 4C). According to their morphology, the increases of colony number in all colony types, including BFU-E (burst-forming unit-erythroid), CFU-G (colony- forming unit granulocyte), CFU-M (colony-forming unit macro- phage) and CFU-GM (colony-forming unit granulocyte macro- phage), were seen in P1A Tg BM by inverted microscopy (Figure 4D). PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13439