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 is known as "replicative senescence", or the Hayflick limit. 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 for 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 (ROS), activation of oncogenes, and cell- cell fusion. Normally, cell senescence is reached through a combination of a variety of factors (i.e., both telomere shortening and oxidative stress). The DNA damage response (DDR) arrests cell cycle progression until DNA damage, such as double-strand breaks (DSBs), are 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 has been linked with premature aging diseases, supporting the role of cell senescence in aging (see DNA damage theory of 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 consisting of a pro-inflammatory secretome, the up-regulation of immune ligands, a pro-survival response, promiscuous gene expression (pGE), 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.

Senescent cells can undergo conversion to an immunogenic phenotype that enables them to be eliminated by the immune system. This phenotype consists of a pro-inflammatory secretome, the up-regulation of immune ligands, a pro-survival response, promiscuous gene expression (pGE) and stain positive for senescence-associated β-galactosidase activity. The nucleus of senescent cells is characterized by senescence-associated heterochromatin foci (SAHF) and DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS). Senescent cells affect tumour suppression, wound healing and possibly embryonic/placental development and a pathological role in age-related diseases.

Role of telomeres

Telomeres 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 (OIS).

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 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 (tta) protein to control p53 expression using doxycycline, a tetracycline analog and tetracycline responsive short hairpin RNA (shRNA). 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 B-galactosidase protein. Even if the expression of p53 was transiently activated or deactivated, senescence via SA B-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 is able to 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(Cdk 2). Without Cdk 2, retinoblastoma protein (pRB) 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 (Cdk 4) and cyclin-dependent kinase 6 (Cdk 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 (SASP) gene expression is induced by a number of 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.

Characteristics of senescent cells

Senescent cells are especially common in skin and adipose tissue. Senescent cells are usually larger than non-senescent cells. Transformation of a dividing cell into a non-dividing senescent cell is a slow process that can take up to six weeks.

Senescent cells affect tumor suppression, wound healing and possibly embryonic/placental development, and play a pathological role in age-related diseases. There are two primary tumor suppressor pathways known to mediate senescence: p14arf/p53 and INK4A/RB. More specifically p16INK4a-pRb tumor suppressor and p53 are known effectors of senescence. Most cancer cells have a mutated p53 and p16INK4a-pRb, which allows the cancer cells to escape a senescent fate. The p16 protein is a cyclin dependent kinase inhibitor (CDK) inhibitor and it activates Rb tumor suppressor. p16 binds to CDK 4/6 to inhibit the kinase activity and inhibit Rb tumor suppressor via phosphorylation. The Rb tumor suppressor has been shown to associate with E2F1 (a protein necessary for transcription) in its monophosphorylated form, which inhibits transcription of downstream target genes involved in the G1/S transition. As part of a feedback loop, increased phosphorylation of Rb increases p16 expression that inhibits Cdk4/6. Reduced Cdk4/6 kinase activity results in higher levels of the hypo-phosphorylated (monophosphorylated) form of Rb, which subsequently leads to reduced levels of p16 expression.

The removal of aggregated p16 INK 4A positive senescent cells can delay tissue dysfunction and ultimately extend life. In the 2011 ''Nature'' paper by Baker et al. a novel transgene, INK-ATTAC, was used to inducibly eliminate p16 INK4A-positive senescent cells by action of a small molecule-induced activation of caspase 8, resulting in apoptosis. A BubR1 H/H mouse model known to experience the clinicopathological characteristics of aging-infertility, abnormal curvature to the spine, sarcopenia, cataracts, fat loss, dermal thinning, arrhythmias, etc. was used to test the consequences of p16INK4a removal. In these mice p16 INK4a aggregates in aging tissues including the skeletal and eye muscle, and adipose tissues. Baker et al. found that if the senescent cells are removed, it is possible to delay age-associated disorders. Not only does p16 play an important role in aging, but also in auto-immune diseases like rheumatoid arthritis that progressively lead to mobility impairment in advanced disease.

In the nervous system, senescence has been described in astrocytes and microglia, but is less understood in neurons. Because senescence arrests cell division, studies of senescence in the brain were focused mainly on glial cells and less studies were focused on nondividing neurons. Analyzing single nucleus RNA-Seq data from human brains suggested p19 as a marker for senescent neurons, which are strongly associated with neurons containing neurofibrillary tangle.

SASP

The secretome of senescent cells is very complex. The products are mainly associated with inflammation, proliferation, and changes in the extracellular matrix. A Senescence Associated Secretory Phenotype (SASP) consisting of inflammatory cytokines, growth factors, and proteases is another characteristic feature of senescent cells. There are many SASP effector mechanisms that utilize autocrine or paracrine signalling. SASP induces an unfolded protein response in the endoplasmic reticulum because of an accumulation of unfolded proteins, resulting in proteotoxic impairment of cell function. Autophagy is upregulated to promote survival.

Considering cytokines, SASP molecules IL-6 and IL-8 are likely to cause senescence without affecting healthy neighbor cells. IL-1beta, unlike IL-6 or IL-8, is able to induce senescence in normal cells with paracrine signaling. IL-1beta is also dependent on cleavage of IL-1 by caspase-1, causing a pro-inflammatory response. Growth factors, GM-CSF and VEGF also serve as SASP molecules. From the cellular perspective, cooperation of transcriptional factors NF-κB and C/EBPβ increase the level of SASP expression. Regulation of the SASP is managed through a transcription level autocrine feedback loop, but most importantly by a continuous DDR. Proteins p53, p21, p16ink4a, and Bmi-1 have been termed as major senescence signalling factors, allowing them to serve as markers. Other markers register morphology changes, reorganization of chromatin, apoptosis resistance, altered metabolism, enlarged cytoplasm or abnormal shape of the nucleus. SASPs have distinct effects depending on the cellular context, including inflammatory or anti-inflammatory and tumor or anti-tumor effects. While considered a pro-tumorogenic effect, they likely support already tumor-primed cells instead of shifting healthy cells into transformation. Likewise, they operate as anti-tumor protectors by facilitating the elimination of damaged cells by phagocytes. The SASP is associated with many age-related diseases, including type 2 diabetes and atherosclerosis. This has motivated researchers to develop senolytic drugs to kill and eliminate senescent cells to improve health in the elderly. The nucleus of senescent cells is characterized by senescence-associated heterochromatin foci (SAHF) and DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS).

Clearance of senescent cells by the immune system

Due to the heterogeneous nature of senescent cells, different immune system cells eliminate different senescent cells. Specific components of the senescence-associated secretory phenotype (SASP) factors secreted by senescent cells attract and activate different components of both the innate