Validating rnai targets

Posted by / 21-Jun-2017 01:49

Validating rnai targets

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.

When cells are explanted from an organism and placed in culture, they have to adapt to an artificial environment, characterized by abnormal concentrations of nutrients and growth factors and the presence of ambient O levels, as well as the absence of surrounding cell types and extracellular matrix components. One of the hallmarks shared by cells undergoing replicative senescence and OIS is the critical involvement of the p53 and p16 is an exquisite sensor that is activated by oncogenic signals and mediates senescence in cultured murine cells, in human cells it does not seem to play a similarly dominant role (Wei et al. This is also exemplified by the signaling routes relaying OIS by RAS (Haferkamp et al. Recent evidence suggests the relevance of OIS also in the context of induced pluripotency in vitro. Concomitant loss of p53 allows cells to override the cytostatic effects of deletions (Chen et al. Similarly, depletion of NF1 causes senescence in vitro, which is eventually accompanied by decreases in ERK and AKT activities (Courtois-Cox et al. An elegant model was proposed in which the increase in RAS activity following NF1 loss is dampened by a negative feedback loop.

Communication between DDR-associated factors and the cell cycle machinery is brought about by phosphorylation and activation of several cell cycle proteins, including CDC25 (a family of phosphatases) and p53.

In addition, differential expression of p53 isoforms has been linked to replicative senescence (Fujita et al. Together, these changes can induce a transient proliferation arrest, allowing cells to repair their damage.

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.

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In recent years, evidence for the existence of premature senescence in vivo has been accumulating rapidly and points to a critical role in tumor suppression. Alternatively, increased proliferation rates associated with p53 loss may result in accelerated kinetics of i PS formation (Hanna et al. To the extent that this can be extrapolated to an in vivo setting, one could imagine that cancer stem cells arise from a similar reprogramming process (Krizhanovsky and Lowe 2009).