Figure 2.

Function of the Ink4a/Arf transgene in MEFs. (A) Activation of the transgenic Ink4a/Arf allele in response to in vitro culture stress. MEFs of the indicated genotype were serially passaged according to the 3T3 protocol. In the case of Ink4a/Arf (–/–;tg/·) MEFs (right panels), transgene-produced p16^Ink4a and p19^Arf reached maximal levels at around passage 35. In the case of Ink4a/Arf (+/–) MEFs (left panels), maximal levels were reached at around passage 4. In both cases, cells expressing p16^Ink4a and p19^Arf, either from the transgene or from the endogenous allele, were overgrown by cells that had lost expression of p16^Ink4a and p19^Arf. This overall pattern was reproducibly observed in four independent (–/–;tg/·) MEF cultures, each from a different embryo (see Supplementary Fig. S1). (B) Activation of the transgenic Ink4a/Arf allele in response to Ras-induced oncogenic stress. MEFs of the indicated genotype and passage were retrovirally transduced with an empty vector or with a vector expressing oncogenic H-RasV12, and the levels of p16^Ink4a and p19^Arf were determined at day 2 post-selection (see Supplemental Material). Immunoblots are representative of three assays with independent MEF cultures. (C) The transgenic Ink4a/Arf allele provides neoplastic resistance to oncogenic Ras. (Black bars) MEFs of the indicated genotype and passage were infected with oncogenic Ras or with empty vector, and their proliferation was measured at day 0 and day 4 post-selection. The ratio between day 4 and day 0 in Ras-infected cells was normalized as the percentage of the corresponding ratio in vector-infected cells. (Gray bars) MEFs (10⁶ cells) were transfected with a plasmid encoding oncogenic Ras, and 3 wk later the total number of neoplastic foci was scored. Data for proliferation and neoplastic foci correspond to the average and standard deviation of three and five assays, respectively, with independent MEF cultures. (D) Activation of the transgenic Ink4a/Arf allele in response to E1a-induced oncogenic stress. MEFs were retrovirally transduced with an empty vector or with a vector expressing E1a, and the levels of p16^Ink4a and p19^Arf were determined by immunoblotting. Data are representative of three assays with independent MEF cultures. (E) The transgenic Ink4a/Arf allele provides increased sensitivity to E1a-mediated apoptosis. MEFs retrovirally transduced with E1a (+) or empty vector (–) were treated or not with doxorubicin, as indicated, and apoptotic cells were quantified 24 h later by cytometry. Data correspond to a representative assay from a total of three assays with independent MEF cultures.