Synopsis on cellular senescence and apoptosis☆☆☆★

Celluar senescence 

The hallmark of cellular or replicative senescence is an irreversible arrest of cell proliferation with maintenance of cell functions. At the turn of the 20th century, Alexis Carrel, a distinguished Nobel laureate, surgeon, and cell culturist, demonstrated that fibroblasts from the chick heart were immortal in tissue culture. Because of this finding and the duplication of these results by other laboratories, the prevailing dogma until the 1960s was the belief that all cultured cells were immortal. Because it was recognized that on occasion and at exceedingly low frequencies immortal cells can arise spontaneously from normal cell cultures and that external stimuli such as carcinogens, radiation, and oncogenic viruses are required for cell transformation and immortality, the hypothesis of Carrel was challenged. In 1961, the extensive tissue culture studies of Hayflick and Moorhead described the isolation and characterization of diploid human fibroblasts derived from fetal tissues. Their study results demonstrated unequivocally that cells in culture had a finite number of subcultivations (also referred to as cell passage) of approximately 50 before achieving growth arrest. These findings were not caused by medium components or culture conditions, but instead were an inherent property of the cells themselves in culture. This phenomenon was interpreted as aging at the cell level, therefore refuting the concept of cell immortality, and was established as an in vitro model for which exhaustion of the doubling capacity in human diploid fibroblasts is recognized as cellular senescence. In the last 35 years, many cell types from various animal species have been studied and shown to have a finite replicative life span. By definition, postmitotic cells, which do not replicate, do not undergo cellular senescence. Instead, cellular senescence affects the phenotype of dividing cells only.

In the last decade, the model of cellular senescence has been applied in vitro to human cells comprising living tissues, with the implication of possibly having an important role in the function of tissue integrity. The role of cellular senescence has been extended to explain the aging process, tumor suppression, and impaired tissue function resulting in poor wound healing. In the aforementioned normal process of aging and disease states related to neoplastic transformation and wound impairment, it is worthwhile to review the expected replicative capacity of cells as they undergo sequential population doublings. As seen in Fig 1, the proliferative capacity of the cells decreases with increasing population doublings.

As fibroblasts approach senescence, each cell at a given passage will have undergone a finite number of divisions, and the population of fibroblasts as a whole will be at varying stages toward senescence, depending on the number of population doublings. The entire population of fibroblasts can be said to have reached cellular senescence when every cell in the culture has completed its replicative life span. At this stage, growth arrest is irreversible, and a senescent cell cannot be stimulated by any physiologic mitogens to initiate DNA replication.

Molecular characteristics of cellular senescence 

The hallmark of cellular senescence is the irreversible growth arrest of the cell, with preservation of metabolic function. Morphologically, senescent cells are larger in size and can have polygonal shapes. This appearance may be caused in part by a number of growth regulatory dependent genes being repressed. Although these aspects are the primary characteristics of a senescent cell, the molecular events involving alterations in signal transduction and the regulation of DNA replication are fundamental in distinguishing senescent cells from replicating cells and cells undergoing apoptosis. Fig 2 is a schematic representation of a senescent cell, demonstrating the effect of physiologic mitogens or environmental stimuli on the molecular pathways involving the interaction of cell receptors, secondary messenger moieties, and regulatory proteins leading to the inhibition of DNA replication.

The loss of proliferative capacity in senescent cells is partially explained by the profound attenuated response to growth factors, such as basic fibroblast growth factor, epidermal growth factor, transforming growth factor, and platelet-derived growth factor. Studies in normal human diploid fibroblasts from the WI-38 cell line have demonstrated that the ligand-receptor binding, receptor density (number of receptors per cell surface area), and the affinity of the receptor for the ligand are unchanged in early passage (presenescent) versus late passage (senescent) fibroblasts. The mechanism for the attenuated response of the cell receptor to the mitogenic stimuli appears to be a function of both alterations in the receptor structural moiety and in the postreceptor secondary messenger pathways. The changes in secondary messenger signal transduction involve different molecules consisting of phospholipid metabolite arachidonic acid and the production of prostaglandins, diacylglycerol, protein kinase A and C, cyclic adenosine monophosphate, and intracellular calcium.

There are a number of normally expressed genes in the cell cycle that are not expressed in senescent cells. These genes, such as cyclin A, proliferating cell nuclear antigen, thymidine kinase, and DNA polymerase α, are regulated by the transcription factor E2F. The inability of senescent cells to synthesize and replicate DNA during the cell cycle involves in part the suppression of at least three positive-acting genes. In senescent fibroblasts, the expression of c-fos proto-oncogene, helix-loop-helix id-1 and id-2 genes, and the components of the E2F transcriptional factor is inhibited, triggering the inability of the cell for DNA synthesis. The alterations of the growth-regulating genes in senescent fibroblasts are intimately coupled to various cell-cycle dependent proteins. The expression of cell-cycle regulator proteins tumor suppressor p53, cyclin-dependent kinase inhibitor p21 (cip1/sdi1/waf1) and p16 (cdkn2/ink4a), and the tumor suppressor retinoblastoma susceptibility protein pRb is involved directly or indirectly in the regulation of cell proliferation. The overexpression of p21 is an important regulator of cell proliferation in senescent cells. Because p21 (and also p16) inhibits cyclin-dependent protein kinases, which leads to the constitutive underphosphorylation of pRb and E2F suppression, this protein plays a major role in inhibiting cell proliferation in senescent cells. It is suggested by evidence that inactivation of p21 by genetic modulation can bypass the events of senescence in human diploid fibroblasts and that oncogenic ras (a proliferation cell-regulating protein) can lead to accumulation of p16 and p53, leading to premature cellular senescence.

Senescent cells display altered differentiated functions, which can impart changes on the extracellular environment. Senescent cells display resistance to apoptosis probably through the overexpression of Bcl-2 protein. Because senescent cells would resist programmed cell death, invariably this would lead to the accumulation of these cells within tissues. The accumulation of senescent cells in vivo that have achieved growth arrest has been postulated to have consequences affecting aging, neoplastic differentiation, and impaired tissue integrity and healing. A recent senescent marker called senescent-associated β galactosidase (SA β-gal) persists in aged tissues and increases with increasing population doublings. SA β-gal staining of cultured human fibroblasts, which is a marker for cellular senescence, results in a perinuclear blue staining that characterizes these fibroblasts as senescent. Further changes in senescent cells involve the overproduction of matrix metalloproteinases, collagenases, and gelatinases and decreased amounts of metalloproteinase inhibitors. The imbalance of elevated proteinase activity compared with antiproteinase has been implicated not only in the degradation of important extracellular structural proteins like fibronectin, but also in degrading growth factors and their receptors. It appears that the accumulation of senescent cells in tissues can have profound effects on neighboring cells and ultimately on the normal aging process and in disease states.

Apoptosis 

Apoptosis, a word derived from the Greek word “falling leaves,” is defined as a programmed cell death. Apoptosis has recently received a lot of attention in medicine and science because of its importance during organism development, pathogenesis, and cell transformation. Apoptosis is a fundamental biologic process required to maintain the integrity and homeostasis of a multicellular organism. The etiology of many intractable human diseases, including neurodegenerative disorders such as Alzheimer's and Parkinson's diseases and cancer, is associated with alterations in cell apoptosis. Furthermore, apoptosis is implicated in the progression of cell death during reperfusion injury of ischemic tissue and in brain and myocardial tissues at risk from further insult during a stroke and myocardial infarction, respectively.

Apoptosis should be distinguished from necrosis. Necrotic cell death is pathologic and results from acute cellular injury, leading to rapid cellular edema and lysis. In contrast, cell death by apoptosis has the hallmark of controlled autodigestion of the cell. This process leads to the formation of apoptotic bodies within the intact cell plasma membrane. Unlike cellular necrosis, which is accompanied by an inflammatory response because of the loss of membrane integrity, an important feature of apoptosis is that elimination of apoptotic cells by mononuclear cell phagocytosis occurs without the induction of an inflammatory response. Apoptosis occurs when cells commit suicide in response to exogenous stimuli or intrinsic signals to maintain homeostasis of the organism. The events that commit a cell to either a path of apoptosis or necrotic cell death after a specific stimulus are dictated in the former and not the latter by the activation of the central cell death signal either through a specific set of surface death receptors or directly within the cell by drugs, toxins, or radiation (Fig 3).

The signals for apoptosis induction can be intrinsic or extrinsic. There are many inducers of apoptosis, such as physiologic activators (tumor necrosis factor [TNF], neurotransmitters, growth factor withdrawal), oxygen reactive metabolites, viral infection, chemotherapeutic drugs, radiation (UV and gamma), and toxins. These stimuli can direct their effect by activating specific death receptors, causing perturbations of the cell membrane or metabolic functions, or by causing direct DNA damage. Once a cell has been induced to go through apoptosis, a cascade of events occurs. The first event is provocation of the apoptotic response. For activation of the cell surface receptors, ligands, which are structurally related to the TNF gene superfamily (with the exception of nerve growth factor), bind and stimulate a complex family of death receptors that also belong to the TNF gene superfamily. These death receptors share a similarity in possessing a cysteine-rich extracellular domain and a cytoplasmic homologous sequence named the death domain. There are a number of death receptors that have been characterized, such as TNF receptor family, nerve growth factor receptor, CD95 receptor (also called Fas or Apo1), death receptor 3 (also called DR3, Apo3, LARD, or TRAMP), and DR5 (also called Apo2, TRAIL-R2, TRICK 2, or KILLER). After the activation of the death receptors by the specific stimuli, a central cell death signal is transduced, triggering apoptosis by activation of a group of cysteine proteases. These proteases are called caspases (c ysteinyl asp artatespecific proteinases ), which are inactive precursor polypeptides, and in turn are cleaved at specific aspartate residues and assembled into active heterotetramer proteases that ultimately begin to digest DNA in a defined and specific fashion. One of the signature changes associated with apoptosis is DNA fragmentation. Whereas necrosis-induced death causes nonspecific degradation of chromosomal material, in programmed cell death specific sites of DNA are rapidly restricted or cut such that it creates predictable DNA fragments measuring in multiples of 180 base pairs. In human physiology, apoptosis plays a very important role. Cells that are constantly exposed to UV radiation, such as skin cells, or cells that have a limited life span tend to be resistant to apoptosis. Resistance to apoptosis for these cells is developed for long-term survival. In contrast, immune cells are very susceptible to apoptosis. This is a physiologic attempt to regulate autoantibody production. Pathogenesis occurs when a cell type loses its original susceptibility to apoptosis. When immune cells are activated, they produce antibodies against foreign proteins; however, as soon as antibodies are produced, apoptosis is induced, so that cells producing particular antibodies are quickly destroyed. Apoptosis associated with the vascular diseases is not well known, and this is an area of research that could be further explored.

As described, the hallmark of cellular senescence is the loss of proliferative capacity, whereas the hallmark of apoptosis is sequential cellular events that lead to programmed cell death. These two events are not related and have distinctive biological pathways. Senescent cells are shown to be resistant to apoptosis. However, resistance to apoptosis does not equate with loss of proliferative capacity. Cells such as keratinocytes, which are constantly exposed to UV radiation, display a high degree of resistance to apoptosis. Especially those keratinocytes thought to be stem cells are protected from apoptosis, and they are resistant to apoptosis induced by UV radiation. However, by definition, stem cells have a high proliferative capacity and, when required, divide. Thus senescence and apoptosis are two distinctive biological pathways, and each contributes separately to the development and pathogenesis of an organism.