Reinitiation of DNA Synthesis and Cell Division in Senescent Human Fibroblasts by Microinjection of Anti-p53 Antibodies

Abstract

In human fibroblasts, growth arrest at the end of the normal proliferative life span (induction of senescence) is dependent on the activity of the tumor suppressor protein p53. In contrast, once senescence has been established, it is generally accepted that reinitiation of DNA synthesis requires loss of multiple suppressor pathways, for example, by expression of Simian virus 40 (SV40) large T antigen, and that even this will not induce complete cell cycle traverse. Here we have used microinjection of monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, to reinvestigate the effect of blocking p53 function in senescent human fibroblasts. Unexpectedly, we found that both antibodies induce senescent cells to reenter S phase almost as efficiently as SV40, accompanied by a reversion to the "young" morphology. Furthermore, this is followed by completion of the cell division cycle, as shown by the appearance of mitoses, and by a four-to fivefold increase in cell number 9 days after injection. Immunofluorescence analysis showed that expression of the p53-inducible cyclin/kinase inhibitor p21 sdi1/WAF1 was greatly diminished by targeting p53 with either PAb1801 or DO-1 but remained high and, moreover, still p53 dependent in cells expressing SV40 T antigen. As previously observed for induction, the maintenance of fibroblast senescence therefore appears to be critically dependent on functional p53. We suggest that the previous failure to observe this by using SV40 T-antigen mutants to target p53 was most probably due to incomplete abrogation of p53 function.Normal human fibroblasts are capable of only a finite number of cell divisions even under optimum culture conditions, after which they enter a state of viable but permanent growth arrest (29). This phenomenon of cellular senescence has been observed in many other normal cell types (15,23,54) and represents a natural obstacle to clonal expansion (14, 58) which is thought to be an important restriction on the progression of many (although probably not all) human cancers (19).One currently popular model for senescence proposes that an intrinsic cell division clock, possibly based on telomere erosion (4), triggers one or more signal pathways which inhibit key components of the cell cycle regulatory machinery. Two candidate inhibitors whose levels increase with proliferative life span are p16 (2, 25) and p21 sdi1/WAF1 (46); these proteins inhibit the cyclin-dependent kinases CDK4/6 and CDK2, which are required for passage through and exit from the G 1 phase of the cell cycle (51). A related protein, p24 (40), may represent a third inhibitor. One consequence (55) of this inhibition is the failure of senescent cells to phosphorylate a major downstream target of these enzymes-the product of the retinoblastoma (Rb) sensitivity gene, pRb-which in its unphosphorylated form sequesters transcription factors needed for G 1 -S progression (61). Not surprisingly, therefore, escape from senescence is often associated with deregulation of the Rb pathway, either directly throu...

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

“…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.…”

“…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.…”

The centrosomal protein TACC3 controls paclitaxel sensitivity by modulating a premature senescence program

“…RNA interference-mediated suppression of CKIs efficiently triggered mitosis in terminally differentiated skeletal muscle cells, quiescent fibroblasts and senescent embryo kidney cells. Similarly, it has been shown that injection of anti-p53 antibodies or the acute removal of pRb can reactivate the cell cycle in senescent cells (Gire and Wynford-Thomas, 1998;Sage et al, 2003). Thus, these findings implicate that senescent growth arrest can not be reversed by known physiological signals, but upon restoration of TACC3 expression or depletion of mediators involved in checkpoint control, although Aurora-A kinase inhibition was reported to cause irreversible cell cycle arrest (Huck et al, 2010).…”

Aging, tumor suppression and cancer: high wire-act!

“…We do know it is possible to reactivate the growth of some types of senescent cells by inactivating p53 (Gire and Wynford-Thomas, 1998;Beausejour et al, 2003). Cells that express the p16 tumor suppressor protein, which activates the pRB tumor suppressor, are refractory to reversal by p53 inactivation .…”