FIG. 4.
ΔNp73 rescues Ras-induced senescence in primary MEFs. (A) Primary MEFs coexpressing ΔNp73 and oncogenic Ras exhibit a transformed phenotype in vitro. 129 MEFs coexpressing GFP and H-RasV12, ΔNp73 and H-RasV12, ΔNp73 L322P and H-RasV12, or p53 R175H and RasV12 were evaluated. Morphologically, the ΔNp73+H-RasV12 cells were highly refractile, were no longer contact inhibited, and resembled cells transformed by mutant p53+RasV12. This effect was not seen with tetramerization-defective ΔNp73 L322P+H-RasV12. (B) ΔNp73 rescues Ras-induced senescence in primary MEFs. The growth properties of 129 primary MEFs that were infected with retroviruses expressing GFP or ΔNp73, followed by infection with H-RasV12-, K-RasV12-, or N-RasD12-expressing retroviruses 2 days later, were evaluated. Cells were plated at equal densities and then counted every 3 days. Whereas Ras-expressing MEFs ceased to grow after 12 days and slowly lost cells due to concomitant cell death, ΔNp73+Ras-expressing MEFs continue to proliferate. MEFs were at passage 5 at the time of infection; 20 days later corresponds to six additional passages. After further passage in culture, fully transformed cells arose in the ΔNp73 cultures. Two such pooled populations were injected into nude mice, leading to the development of high-penetrance, short-latency tumors (see Table 1). (C) ΔNp73 and Ras cooperate in promoting focus formation. 129 MEFs were transduced with retroviral vectors expressing GFP+Ras, ΔNp73+Ras, mutant ΔNp73+Ras, and p53 R175H+Ras. Infected cells were selected with puromycin for 48 h. A total of 3 × 103 cells were mixed with 105 uninfected MEFs and plated onto 6-cm dishes in triplicate. After 14 days, plates were stained with Giemsa and photographed. K-RasV12 was used on the left, and H-RasV12 was used on the right. Loss of contact inhibition was also seen with N-RasD12 (not shown). (D) Expression analysis of MEFs from the focus formation assays in panel C 4 days after transduction with retroviruses encoding RasV12+GFP (lane 1), RasV12+ΔNp73 (lane 2), RasV12+p53 R175H (lane 3), and RasV12+ΔNp73 L322P (lane 4). Immunoblot analyses of 50-μg portions of total cell extracts with the indicated antibodies were done. DO-1 was specific for human p53, whereas CM-5 was raised against mouse p53, although it cross-reacts with human p53. (E) Expression analysis of MEFs from focus formation assays in panel C 4 days after transduction with retroviruses encoding RasV12+GFP (lane 1), RasV12+ΔNp73 (lane 2), RasV12+p53 R175H (lane 3), and RasV12+ΔNp73 L322P (lane 4). Immunoblot analyses of 50-μg portions of the total cell extracts with the indicated antibodies were carried out. For panels D and E, K-Ras was used on the left and H-Ras was used on the right.