p53 Is Preferentially Recruited to the Promoters of Growth Arrest Genes p21 and GADD45 during Replicative Senescence of Normal Human Fibroblasts

Abstract

Replicative senescence is the terminal growth arrest that most normal human cells enter into after a fixed number of divisions in vitro, limiting the proliferative potential of a cell and preventing genomic instability caused by critically short telomeres. Thus, senescence presents a tumor-suppressive mechanism and a barrier to tumor formation. However, senescent cells are inherently resistant to apoptosis and, as they accumulate in aging tissues, may contribute to organ dysfunction and promote tumor progression as part of the stromal environment. Replicative life span in normal human cells can be extended by inactivation of the tumor suppressor gene p53 or its direct target, the cyclin-dependent kinase inhibitor p21, suggesting a direct role for this pathway in senescence. However, p53 recruitment to promoters of target genes during replicative senescence has not been shown in live cells. In this study, we used chromatin immunoprecipitation to determine that p53 preferentially occupied the promoters of growth arrest genes p21 and GADD45 in senescent normal human diploid fibroblasts but not the promoters of other target genes that recruited p53 following doxorubicin-induced DNA damage, such as apoptosis regulators TNFRSF10b, TNFRSF6, and PUMA. This differential recruitment of p53 in senescent versus doxorubicin-treated fibroblasts was accompanied by differences in post-translational modification of p53. These data provide mechanisms for both the growth arrest mediated by p53 and the resistant nature of senescent cells to apoptosis despite p53 activity.

Loss of p21Sdi1 expression in senescent cells after DNA damage accompanied with increase of miR-93 expression and reduced p53 interaction with p21Sdi1 gene promoter

“…Increased p53 in response to genotoxic damage temporally regulates selective binding to p21 and MDM2 promoters; p21 induces growth arrest with DNA damage repair, whereas MDM2 degrades p53 in human cells. In contrast to the genotoxic damage, replicatively senescent cells reveal failure of p53 activation by phosphorylation on serine residues and by acetylation on lysine residues, therefore, p53 binding to p21 and GADD45 is specifically higher than the target genes regulating apoptosis [13]. On the other hand, critically short human telomeres induce senescence either by activating p53 or p16 INK4a /pRB pathway, and suppression of both pathways is required to suppress senescence of aged human cells.…”

Expression of an IFN-Inducible Cellular Senescence Gene, IFI16, Is Up-Regulated by p53

“…p53 protein level increases in certain normal old human diploid fibroblasts (HDF), such as IMR90 and MRC5, during replicative senescence (8)(9)(10)(11)(12). Likewise, the p53 sequencespecific DNA-binding activity and transcriptional activity increase during replicative senescence (9).…”

“…Moreover, functional inactivation of p53 using viral oncoproteins, such as SV40 large T antigen, or knockdown of p53 expression allows cells to bypass the onset of replicative senescence, resulting in an extension of proliferation potential of cells (13). These studies (8)(9)(10)(11)(12)(13) have established a role for p53 in cellular senescence.…”

“…p53 is a transcription factor, which is activated in cells in response to certain stimuli, such as DNA damage, hypoxia, oxidative stress, or other cellular stress (16)(17)(18). Moreover, telomere shortening in normal human cells triggers cellular senescence through activation of ataxia-telangiectasia mutated kinase, which phosphorylates p53 on Ser 15 , resulting in activation of p53 (10)(11)(12). The activated p53 binds to its DNA-binding consensus sequence that is present in its target genes (12)(13)(14)(15).…”

“…Moreover, telomere shortening in normal human cells triggers cellular senescence through activation of ataxia-telangiectasia mutated kinase, which phosphorylates p53 on Ser 15 , resulting in activation of p53 (10)(11)(12). The activated p53 binds to its DNA-binding consensus sequence that is present in its target genes (12)(13)(14)(15). The binding of p53 to its target genes is known to result in transcriptional activation of genes, such as p21 and Gadd45, or transcriptional repression of genes, such Bcl2 and MAP4 (12,13).…”