Sei1 in Cancer and Diabetes Figure 3. E2f1 activity in Sei1-null cells. (A) Endogenous E2f1 activity was measured by transfecting at least three independent preparations of primary MEFs per genotype with different amounts of a plasmid carrying the luciferase reporter under the regulation of the E2f1-responsive E2f1 promoter. Luciferase activity was measured two days after the transfection. Values represent the average and s.d. (B) Ectopic E2f1 activity was measured as before after co-transfection with different amounts of a plasmid expressing E2f1. (C) Levels of protein p19Arf, a bona-fide E2f1 target, were assessed by immunoblotting of extracts from MEFs of the indicated genotypes transduced with E2f1. These results were confirmed in another independent assay. Actin was used as a loading control. doi:10.1371/journal.pone.0008744.g003 tion by HRasG12V or by the combination of HRasG12V and E1A. We measured the transformation efficiency by plating infected cells at confluent density and counting the emergence of foci after two weeks in culture. We could not observe differences between Sei1-null and wt MEFs when subjected to these oncogenic transformation assays (Fig. 4D). From these data we conclude that the absence of Sei1 has no significant impact on the requirement for external growth factors, ability to immortalize spontaneously, or susceptibility to be oncogenically transformed. Normal Apoptosis of Sei1-Deficient Cells Based on the above negative data, we decided to expand our studies to stress responses and apoptosis. First, we subjected wt and Sei1-null MEFs to different doses of ultraviolet radiation, which triggered the same levels of apoptosis in both genotypes (Fig. 5A). Then, we infected Sei1-null MEFs with the oncoprotein E1A. This oncoprotein sequesters and inhibits Rb [22] and induces a p19Arf/ p53-dependent apoptotic response [23]. As shown in Fig. 5B, expression of E1A induced apoptosis in wt and Sei1-null MEFs to similar levels, while p53-null MEFs were not affected. E1A sensitizes MEFs to other apoptotic stimuli, such as serum withdrawal or adriamycin [23]. However, Sei1-null MEFs infected with E1A were as sensitive to serum withdrawal and adriamycin as were wt MEFs (Fig. 5B). To extend our study to other cell types, we irradiated mice with ionizing radiation and measured apoptosis in thymocytes, however, there were no significant differences between Sei1-null and wt thymocytes (Fig. 5C). Finally, we analyzed the response of p53 and its target p21Cip1 to ionizing radiation in the spleen and the lung. As observed in Fig. 5D, both proteins, p53 and p21, were clearly induced upon irradiation but this induction was not affected by the status of Sei1. Also, basal levels of p21Cip1 and p53 were not detectably higher in Sei1-null tissues compared to wt ones. We conclude that Sei1 does not have a significant impact on the response of p53 to DNA damage and the susceptibility to undergo apoptosis. Sei1-Deficiency Does Not Impact on Tumor Development in Mice To address the impact of Sei1 on tumorigenesis, we first focused on the emergence of spontaneous malignant tumors with aging. Sei1-null mice developed spontaneous malignant tumors with the same incidence and spectrum as wt mice (Fig. 6A). To further explore this issue, we decided to subject our mice to carcinogenic treatments. For this, we performed a carcinogenic assay that consisted in intramuscular injection of 3-methyl-cholanthrene (3MC), which results in the development of fibrosarcomas. We could not observe significant differences between the onset of fibrosarcomas in wt and Sei1-null mice (Fig. 6B). We then PLoS ONE | www.plosone.org 4 January 2010 | Volume 5 | Issue 1 | e8744