Validating rnai targets
In addition to p53, replicative senescence is linked to the RB tumor suppressor and its signaling partners, including p16–RB pathways is essential for induction of senescence in a variety of human cell strains.
The first evidence for this came from experiments employing the viral oncoprotein SV40 large T antigen (LT) and mutants thereof lacking the ability to inhibit p53 or RB.
Moreover, the DNA damage kinases ATM and ATR are activated in senescent cells (D'Adda di Fagagna et al. After amplification of the DDR signal, these kinases activate CHK1 and CHK2 kinases.
Consistent with Hayflick's proposal, we now know that, with the propagation of human cells in culture, telomeres (the protective chromosomal termini) are progressively shortened, ultimately causing cells to reach their “Hayflick limit.” This barrier has been termed replicative (cellular) senescence, since it is brought about by replication.
Telomeres are subject to attrition due to the fact that DNA polymerase fails to completely replicate the lagging strands.
Elongation of their life span can be achieved by culturing in serum-free medium supplemented with a number of defined growth factors (Loo et al. Long-term exit from the cell cycle is the central and, in our view, only indispensable marker for the identification of all types of cellular senescence both in vitro and in vivo. At least in some in vitro settings, this can be bypassed by altering the cell culture conditions (Ramirez et al. This notwithstanding, at least in melanomagenesis, genetic and immunohistochemical evidence in mice and humans suggests that p16 plays a redundant role in senescence (Gruis et al. In certain settings, pro-oncogenic effects result: The proliferative rate, migration, and invasion of premalignant cells are enhanced when they are cocultured with, or grown in medium conditioned by, senescent fibroblasts (Krtolica et al. Similar findings have been reported for several additional weakly oncogenic epithelial cell lines that show increased tumorigenic potential upon exposure to senescent fibroblasts (Krtolica et al. Intriguingly, in addition to influencing their microenvironment, some inflammatory cytokines have an indispensable role in the establishment and maintenance of the senescence arrest. This is relayed by the C/EBPβ and NF-κB transcription factors and is associated with the activation of an inflammatory transcriptome.
1987) or by culturing under physiological oxygen conditions (Parrinello et al. Consistent with this, oxidative stress induces cessation of replication in cultured human cells (Packer and Fuehr 1977; Chen et al. 1995), while the replicative potential of human melanocytes and epithelial cells depends largely on the composition of the culture medium used, as well as on the use of feeder layers (Ramirez et al. Senescence of MEFs can be bypassed also by inactivation of family genes (Tanaka et al. However, it is important to realize that, although senescent cells by definition withdraw from the cell cycle, their inability to replicate is far from unique. 2009), while p53 and p21–RB pathway collaborates with ectopic h TERT expression in the immortalization of both primary human epithelial cells (Kiyono et al. 2001), consistent with the view discussed above that inadequate circumstances can launch the senescence program (Sherr and De Pinho 2000). For example, signaling through the IL-6 and IL-8 (CXCR2) receptors is essential, in a cell-autonomous fashion, for cells to enter senescence in response to oncogenic BRAF or replicative exhaustion, respectively (Acosta et al. Correspondingly, elevated levels of cytokines are found in senescent human neoplasms.