p53 Response to Senescence Figure 6. PPP1CA and pRB co-localize during p53/ras-induced senescence. P53ts or p53ts-Ras were grown at 39uC or incubated at 32uC for 24 hrs. Cells were fixed and labeled with DAPI to identify the nuclei, as well as antibodies against PPP1CA (red) or pRb (green). doi:10.1371/journal.pone.0003230.g006 Since senescence is a safeguard mechanism that may prevent pretumoral cells from further expansion, many studies have recently emphasized the relevance of this possible new therapeutic tool against cancer (reviewed in [41–43]). Our work has identified a set of p53 target genes that affect growth arrest in response to p53 activation. Although our work only identify these 4 genes as the minimal footprint to differentiate growing from p53-arrested cells, these 4 genes have been broadly studied and its relevance in growth arrest and senescence has been established. Tumor suppressor Lats2 has been shown to be necessary for culture-induced replicative senescence in MEFs, since Lats22/2 MEFs bypass this process [48]. Furthermore, cells lacking Lats2 showed increased prevalence of micronuclei, chromosomal defects and aneuploidy [48,49]. Lats2 and p53 establish a positive feedback loop that prevents tetraploidization of cells treated with the microtubule poison nocodazole [49]. Most important, miRNA- 372 and miRNA373 microRNAs directly target Lats2 expression and have been shown to cooperate with oncogenic Val12-Ras in a way that resembles p53 inactivation, acting as oncogenes in testicular germ cell tumors [50]. Finally, Lats2 has been shown down-regulated through promoter hypermethylation [51,52], in association with poor prognosis human breast cancers and acute lymphoblastic leukemia. Lats2 might have a role against cancer development, probably through the induction of senescence, and this could explain the link between its down-regulation and tumoral progression. The tumor-suppressor gene RB1 can suppress S phase entry and cause a transient G1 arrest following DNA damage [53– 55] and the mutations in Rb1 pathway-related genes are associated with poor prognosis in many tumor types. The PTEN/PI3K pathway is also regarded as an effector of cellular senescence [56] through p27kip1 cell cycle inhibitor activation. The key findings obtained in this study may contribute to the current understanding of the molecular basis of senescence and should be of great interest in future senescence studies. Materials and Methods Cell Culture Primary MEFs from p532/2 mice were derived from day 13.5 embryos. Cells expressing murine p53val135 were generated by retrovirus-mediated gene transfer of p53val135 into p532/2 MEFs (p532/2 ts). Cells expressing Val12-Ras were generated by retrovirus-mediated gene transfer of pWZLBlast hVal12-Ras into wild-type MEFs at passage 3 (P3-Ras) and p532/2 ts (p532/2 ts Ras cells). Cells were cultured in Dulbecco’s Modified Eagle’s medium (GIBCO) supplemented with 10% fetal bovine serum (Sigma), 1% penicillin G- streptomycin sulfate (GIBCO), 0.5% fungizone-amphotericin B (GIBCO) and 5 mg/ml plasmocin (InvivoGen). P532/2 MEFs, p532/2 MDM22/2 MEFs and p192/2 MEFs were cultured in Dulbeccos Modified Eagles medium (GIBCO). All media was supplemented with 10% fetal bovine serum (Sigma), 1% penicillin G-streptomycin sulphate (GIBCO), and 0.5% fungizone-amphotericin B (GIBCO) in a humidified CO2 incubator at 37uC. Retroviral Vectors and Gene Transfer The following retroviral vectors were used: p53val135 mutant cDNA in pWZLHygro and pWZLBlast hVal12-Ras. Retrovirus- mediated gene transfer was performed as previously described [44]. Briefly, 56106 LinXE retrovirus producer cells were plated in a 10 cm dish, incubated for 24 h and then transfected by calcium-phosphate precipitation with 20 mg of retroviral plasmid (16 h at 37uC). The medium was changed and the plates were maintained at 32uC for 48 h to increase viral stability. Virus- containing supernatant was filtered through a 0.45 mm filter and supplemented with 8 mg/ml polybrene (Sigma) and an equal volume of fresh medium. Prior to infection, 86105 target fibroblasts were plated per 10 cm dish and incubated for 24 h. PLoS ONE | www.plosone.org 10 September 2008 | Volume 3 | Issue 9 | e3230