Cellular senescence

Cellular senescence is a phenomenon characterized by the cessation of cell division. In their experiments during the early 1960s, Leonard Hayflick and Paul Moorhead found that normal human fetal fibroblasts in culture reach a maximum of approximately 50 cell population doublings before becoming senescent. This process called the Hayflick limit is also known as "replicative senescence", since it is brought about through replication. Hayflick's discovery of mortal cells paved the path for the discovery and understanding of cellular aging molecular pathways. Cellular senescence can be initiated by a wide variety of stress-inducing factors. These stress factors include both environmental and internal damaging events, abnormal cellular growth, oxidative stress, autophagy factors, among many other things.
The physiological importance of cell senescence has been attributed to prevention of carcinogenesis, and more recently, aging, development, and tissue repair. Senescent cells contribute to the aging phenotype, including frailty syndrome, sarcopenia, and aging-associated diseases. Senescent astrocytes and microglia contribute to neurodegeneration.

Cellular mechanisms

Stress response and DNA damage

Mechanistically, replicative senescence can be triggered by a DNA damage response due to the shortening of telomeres. Cells can also be induced to senesce by DNA damage in response to elevated reactive oxygen species, activation of oncogenes, and cell-cell fusion. Normally, cell senescence is reached through a combination of a variety of factors. The DNA damage response arrests cell cycle progression until DNA damage, such as double-strand breaks, is repaired. Senescent cells display persistent DDR that appears to be resistant to endogenous DNA repair activities. The prolonged DDR activates both ATM and ATR DNA damage kinases. The phosphorylation cascade initiated by these two kinases causes the eventual arrest of the cell cycle. Depending on the severity of the DNA damage, the cells may no longer be able to undergo repair and either go through apoptosis or cell senescence. Such senescent cells in mammalian culture and tissues retain DSBs and DDR markers. It has been proposed that retained DSBs are major drivers of the aging process. Mutations in genes relating to genome maintenance have been linked with premature aging diseases, supporting the role of cell senescence in aging.
Depletion of NAD+ can lead to DNA damage and cellular senescence in vascular smooth muscle cells.
Although senescent cells can no longer replicate, they remain metabolically active and commonly adopt an immunogenic phenotype that enables them to be eliminated by the immune system. The phenotype consists of a pro-inflammatory secretome, the up-regulation of immune ligands, a pro-survival response, promiscuous gene expression, and stain positive for senescence-associated β-galactosidase activity. Two proteins, senescence-associated beta-galactosidase and p16^Ink4A, are regarded as biomarkers of cellular senescence. However, this results in a false positive for cells that naturally have these two proteins, such as maturing tissue macrophages with senescence-associated beta-galactosidase and T-cells with p16^Ink4A.
The nucleus of senescent cells is characterized by senescence-associated heterochromatin foci and DNA segments with chromatin alterations reinforcing senescence. Senescent cells affect tumour suppression, wound healing, and possibly embryonic/placental development, and a pathological role in age-related diseases.

Cell growth and size

Cell growth plays a crucial role in cell proliferation, regulating cellular homeostasis and cell cycle progression through dynamic changes in cell size. And like DNA damage, it can promote senescence by triggering a prolonged cell cycle arrest. While a typical increase in cell size controls for concentrations of cell cycle activators, an excess of growth can drive a permanent halt on cell proliferation as a result of various mechanistic interactions with cell-cycle signaling pathways and thresholds present.
As the cell increases in size without sufficient proliferation, cellular homeostasis becomes more and more difficult to achieve; the cell experiences cytoplasmic dilution and succumbs to a permanent cell cycle arrest. More particularly, the osmotic stress caused by this overgrowth is linked to an accumulation of p21 during G0/G1 arrest, consequently preventing re-entry into S phase. Additionally, the persistent growth of the cell during this arrest, as driven by mTOR signaling, causes phenotypes characteristic of senescent cells such as cellular hypertrophy, SASP and lysosomal hyperfunctions. The enlarged cells that are able to re-enter the cell cycle are prone to DNA damage and experience abnormalities in signaling for repair, eventually leading to a replication failure and a permanent cell-cycle exit.
Overall, a consistent correlation between larger cell size and senescence has been established. Understanding this mechanistic relationship is useful for addressing different treatment sensitivities in clinical contexts. For tumors presenting growth signal mutations, cell cycle inhibitors hold potential to be a more useful therapeutic, given this cell-size dependency of cellular senescence.

Role of telomeres

s are DNA tandem repeats at the end of chromosomes that shorten during each cycle of cell division. Recently, the role of telomeres in cellular senescence has aroused general interest, especially with a view to the possible genetically adverse effects of cloning. The successive shortening of the chromosomal telomeres with each cell cycle is also believed to limit the number of divisions of the cell, contributing to aging. After sufficient shortening, proteins responsible for maintaining telomere structure, such as TRF2, are displaced, resulting in the telomere being recognized as a site of a double-strand break. This induces replicative senescence. Theoretically, it is possible, upon the discovery of the exact mechanism of biological immortality, to genetically engineer cells with the same capability. The length of the telomere strand has senescent effects; telomere shortening activates extensive alterations in alternative RNA splicing that produce senescent toxins such as progerin, which degrades tissue and makes it more prone to failure.

Role of oncogenes

BRAF^V600E and Ras are two oncogenes implicated in cellular senescence. BRAF^V600E induces senescence through synthesis and secretion of IGFBP7. Ras activates the MAPK cascade which results in increased p53 activation and p16^INK4a upregulation. The transition to a state of senescence due to oncogene mutations are irreversible and have been termed oncogene-induced senescence.
Interestingly, even after oncogenic activation of a tissue, several researchers have identified a senescent phenotype. Researchers have identified a senescent phenotype in benign lesions of the skin carrying oncogenic mutations in neurofibroma patients with a defect that specifically causes an increase in Ras. This finding has been highly reproducible in benign prostate lesions, in melanocytic lesions of UV-irradiated HGF/SF-transgenic mice, in lymphocytes and in the mammary gland from N-Ras transgenic mice, and in hyperplasias of the pituitary gland of mice with deregulated E2F activity. The key to these findings is that genetic manipulations that abrogated the senescence response led to full-blown malignancy in those carcinomas. As such, the evidence suggests senescent cells can be associated with pre-malignant stages of the tumor. Further, it has been speculated that a senescent phenotype might serve as a promising marker for staging. There are two types of senescence in vitro. The irreversible senescence, which is mediated by INK4a/Rb and p53 pathways, and the reversible senescent phenotype, which is mediated by p53. This suggests that the p53 pathway could be effectively harnessed as a therapeutic intervention to trigger senescence and ultimately mitigate tumorigenesis.
p53 has been shown to have promising therapeutic relevance in an oncological context. In the 2007 Nature paper by Xue et al., RNAi was used to regulate endogenous p53 in a liver carcinoma model. Xue et al. utilized a chimaeric liver cancer mouse model and transduced this model with the ras oncogene. They took embryonic progenitor cells, transduced those cells with oncogenic ras, along with the tetracycline transactivator protein to control p53 expression using doxycycline, a tetracycline analog, and tetracycline-responsive short hairpin RNA. In the absence of Dox, p53 was actively suppressed as the microRNA levels increased, so as Dox was administered, p53 microRNA was turned off to facilitate the expression of p53. The liver cancers that expressed Ras showed signs of senescence following p53 reactivation, including an increase in senescence-associated β-galactosidase protein. Even if the expression of p53 was transiently activated or deactivated, senescence via SA β-gal was observed. Xue et al. show that by briefly reactivating p53 in tumors without functional p53 activity, tumor regression is observed. The induction of cellular senescence was associated with an increase in inflammatory cytokines, as is expected based on the SASP. The presence of both senescence and an increase in immune activity can regress and limit liver carcinoma growth in this mouse model.

Signaling pathways

There are several reported signaling pathways that lead to cellular senescence, including the p53 and p16^Ink4a pathways. Both of these pathways are activated in response to cellular stressors and lead to cell cycle inhibition. p53 activates p21 which deactivates cyclin-dependent kinase 2. Without Cdk 2, retinoblastoma protein remains in its active, hypophosphorylated form and binds to the transcription factor E2F1, an important cell cycle regulator. This represses the transcriptional targets of E2F1, leading to cell cycle arrest after the G1 phase.
p16^Ink4a also activates pRB, but through inactivation of cyclin-dependent kinase 4 and cyclin-dependent kinase 6. p16^Ink4a is responsible for the induction of premature, stress-induced senescence. This is not irreversible; silencing of p16^Ink4a through promotor methylation or deletion of the p16^Ink4a locus allows the cell to resume the cell cycle if senescence was initiated by p16^Ink4a activation.
Senescence-associated secretory phenotype gene expression is induced by several transcription factors, including C/EBPβ, of which the most important is NF-κB. Aberrant oncogenes, DNA damage, and oxidative stress induce mitogen-activated protein kinases, which are the upstream regulators of NF-κB.
Inhibition of the mechanistic target of rapamycin suppresses cellular senescence, hence cellular senescence is inhibited by rapamycin.