Reversal of Senescence in Mouse Fibroblasts through Lentiviral Suppression of p53

Abstract

Senescence is generally defined as an irreversible state of G 1 cell cycle arrest in which cells are refractory to growth factor stimulation. In mouse embryo fibroblasts (MEFs), induction of senescence requires the presence of p19 ARF and p53, as genetic ablation of either of these genes allows escape from senescence and leads to immortalization. We have developed a lentiviral vector that directs the synthesis of a p53-specific short hairpin transcript, which mediates stable suppression of p53 expression through RNA interference. We show that suppression of p53 expression in senescent MEFs leads to rapid cell cycle re-entry, is associated with loss of expression of senescence-associated genes, and leads to immortalization. These data indicate that senescence in MEFs is reversible and demonstrate that both initiation and maintenance of senescence is p53-dependent.

Cooperation between p53 and p130(Rb2) in induction of cellular senescence

“…This is consistent with observations of others on senescent embryonic mouse fibroblasts (MEFs) treated with p53-siRNA. 35 Only 0.5-1% of the senescent MEFs divided successfully. The data shown here suggest that not the growth-suppressive functions of p130(Rb2) and p53 per se, but secondary changes were responsible for the change to the irreversible status.…”

“…Thus, in agreement with the observations of others, senescent cells have the potential to escape the arrest and may even resume proliferation when any of the major control proteins such as p53 or pRb is inactivated. 1,2,35,37 Cells which lack p16INK4a, a frequent feature of tumor cells, seem to be prone to escape the arrest more efficiently. 38 The silent accumulation of DNA damage may increase the risk to select for spontaneous cell mt's, which can overcome the arrest.…”

“…In contrast to other cell systems, p53 was constitutively present in the nucleus both at the restrictive (391C) as well as at the permissive temperatures (301C) Figure 1 Induction of a senescence phenotype by p53 . (a) SDS-PAGE and autoradiography of 35 S-methionine-labeled p53 , immunoprecipitated (IP: p53) from C6D10-p53 Val-135 cell lysates with antibodies detecting the mt (pAb240), the wt (pAb246) or both conformations (pAb248) of p53; p53 changes from the mt (a, lane 1) to the wt conformation (a, lane 2) when shifted to 301C; the wt conformation of p53 is degraded rapidly and reaches a low level, as shown by immunofluorescence (b) and WB (c). (d) C6D10-p53 and control cultures (C6D10-neo) assayed for BrdU incorporation by immunofluorescence microscopy; the percentage of BrdU-incorporating C6D10-p53 Val-135 cells decreases to less than 5% after the shift to the permissive temperature; shown are the mean values and SDs of a representative experiment; at each time point, 10 microscopic areas containing at least 50 cells per area were selected randomly and assayed for BrdU incorporation.…”

“…The following antibodies were used: mouse monoclonal antibodies (mAbs) to p53 pAb240, pAb246, pAb248 and pAb421; [35][36][37] antibodies to cyclin D (M-20), cyclin E (M-20), cyclin A (H432), CDK2(D-12), p21(H164 and Sx118), p107 (sc-318), p130(Rb2) (sc-317), GFP (sc-8334 and sc-9996) from Santa Cruz Biotechnology (Santa Cruz, USA); CDK4 (H22) (Biomol, Hamburg, Germany); pRb(p105) (G3-245) (BD Biosciences, San Diego, USA), cleaved caspase 3 (Cell Signaling Technology), tubulin (Oncogene Research, San Diego, USA), cyclin A (C-4710) (Sigma, Munich, Germany), rabbit anti-lamin B. Incorporation of BrdU was monitored with a mouse mAb to BrdU (Boehringer Mannheim, Mannheim, Germany); normal goat serum and normal donkey serum were purchased from DAKO (Glostrup, Denmark); FITC-and Texas Red-conjugated secondary antibodies were from Jackson Immuno Research Laboratories (West Grove, USA). The plasmids used were: pcDNA3E1A12S (T Dobner, Regensburg, Germany) and pIRES2-AcGFP1-cycA.…”

siRNA-based approaches in cancer therapy

“…[79][80][81][82] In general, biosafety is the major concern in the production of retroviral and lentiviral vectors, and currently they are in use in HIV patients only. Adenoviral vectors are widely used both in cells and in vivo; however, their inability to integrate into the genome has proved advantageous in terms of safety but at the cost of inefficient long-term gene silencing.…”