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Because cells are the fundamental building blocks of our bodies, it is logical to assume that cellular changes contribute to the aging process. In this essay I review the methods used to study cellular aging in vitro, in particular replicative senescence, and debate whether these findings could be related to organismal aging.
Inand in contradiction to what was thought limute the time, Leonard Hayflick and Paul Moorhead discovered that human cells derived from hayclick tissues could only divide a finite number of times in culture Hayflick and Moorhead, Phase I is the primary culture, when cells from the explant multiply to cover the surface of the culture flask–most cell types grow in the lab attached to a solid surface.
Cellular Senescence: The Hayflick Limit and Senescent and Aging Cells
Phase II represents the period when cells divide in culture. Briefly, once cells cover a flask’s surface, they stop multiplying.
For cell growth to continue, the cells must be subcultivated. To do so, one removes the hayvlick medium and adds a digestive enzyme called trypsin that dissolves the substances binding cells together. After adding growth medium and pipetting one obtains the cells in a homogeneous suspension that are then divided by two–or more–new flasks. Cells then attach to the new flasks’ surface and start dividing once again until a new subcultivation is required.
Most cells divide vigorously and can often be subcultivated dw a matter of a few days. Oimite several months, however, cells start dividing slower, which marks the beginning of Phase III.
Hayflick and Moorhead noticed that cultures stopped dividing after an average of 50 cumulative population doublings CPDs –splitting one flask of cells into two new flasks of the same size increases the CPDs by one, splitting by four flasks increases the CPDs by two and so on. This phenomenon of growth arrest after a period of apparently normal cell proliferation is known as the Hayflick limit, Phase III phenomenon, or, as it will be called herein, replicative senescence RS.
Part 4: Towards a metrology of aging with telomeres – Work for human longevity
Hayflick and Moorhead worked with fibroblasts, a cell type found in connective tissue widely used in research, but RS has been found in other cell types: In addition, RS is observed in cells derived from embryonic tissues, in cells from adults of all ages, and in cells taken from many animals: The number of CPDs cells undergo in culture varies considerably between cell types and species.
Early results suggested a relation between CPDs cells could endure and the longevity of the species from which the cells were derived. For example, cells from the Galapagos tortoise, which–as described –can live over a century, divide about times Goldstein,while mouse cells divide roughly 15 times Stanley et al.
In addition, cells taken from patients with progeroid syndromes such as Werner syndrome WS — described elsewhere –endure far fewer CPDs than normal cells Salk et al. Exceptions exist and certain cell lines can divide indefinitely without reaching RS.
These are said to be “immortal” and include embryonic germ cells and most cell lines derived from tumors, such as HeLa cells Brunmark et al.
Some types of rat cells have also been claimed as capable of evading RS Mathon et al. The discovery of RS sparked considerable interest and the phenotype of cell senescence in human fibroblasts has been characterized by a series of features, termed biomarkers d’Adda di Fagagna, The most obvious biomarker is growth arrest, i.
Even vigorously dividing cultures are heterogeneous and contain a percentage of growth-arrested cells; this percentage progressively increases until all cells in the population are quiescent, that is, they have stopped dividing Cristofalo and Sharf, ; Smith and Whitney, ; interestingly, the percentage of growth-arrested cells is higher in cells from patients with progeroid syndromes, such as WS cells, when compared with normal cells at the same CPD Kill et al.
Senescent cells are growth arrested in the transition from phase G1 to phase S of the cell cycle Sherwood et al. The growth arrest in RS is irreversible in the sense that growth factors cannot stimulate the cells to divide reviewed in Cristofalo and Pignolo,even though senescent cells can remain metabolically active for long periods of time reviewed in Goldstein, Another important biomarker is cellular morphology Fig.
While cells age in vitro they endure progressive morphological changes e. Briefly, senescent cells are bigger and a senescent population has more diverse morphotypes than cells at earlier CPDs. In fact, a confluent senescent culture has a smaller cellular density than a confluent young culture, though this also occurs because senescent cells are more sensitive to cell-cell contact inhibition. Normal human fibroblasts left and fibroblasts showing a senescent morphology three cells on the right.
Notice the common elongated morphology of senescent cells. Lysosomes are organelles that break down cellular junk. Early reports showed that lysosomes increase in number and size in senescent cells Robbins et al.
Other results also suggest that during in vitro aging increased autophagy–i. Normal human cells are diploid, which means they have two copies of each chromosome.
With each subcultivation the percentage of polyploid cells–i. The expression levels of several genes change during in vitro cellular aging reviewed in Cristofalo et al. One important type of gene overexpressed in senescent cells are inflammatory regulators like interleukin 6 IL6 Shelton et al. Senescent cells also display an increased activity of metalloproteinases which degrade the extracellular matrix reviewed in Campisi, On the other hand, senescent cells have a decreased ability to express heat shock proteins Choi et al.
Telomeres are non-coding regions at the tips of chromosomes. During in vitro aging, the telomeres shorten gradually in each subcultivation Harley et al. The same process might occur in vivo too Hastie et al. Telomere shortening is the primary cause of RS in human fibroblasts, and given their importance, telomeres and their role in aging are discussed in detail in another essay.
hayrlick Assuming human fibroblasts endure 50 CPDs, 2 50 is more than enough cells for several lifetimes Hayflick, The way subcytotoxic stress can accelerate the appearance of the senescent phenotype in cells has been deemed as another form of cellular senescence called stress-induced premature senescence SIPS; see Fig. Schematic drawing of SIPS.
Not surprisingly, haydlick on the dose of stressor used, a cell population will react in different ways. For instance, a high cytotoxic dosage limiye cause such an amount of damage that cellular biochemical activities decrease leading to cellular death by necrosis.
The level of damage sustained by cells determines whether programmed cell death–apoptosis–can unfold or, if the damage is lower, senescence. Since a cellular population is not homogeneous, the dosage of the stressor will shift the percentage of cells executing each of the possible programs depending on the amount of stress, respectively, from no stress to high stress: In addition to Hyflick 2other sources of oxidative damage, such as H 2 O 2 and tert-butylhydroperoxide, and other stressors–e.
The list of stressors that can cause SIPS is constantly growing. Instead of chronic stress, SIPS can be induced based on a single or repeated short exposure s to stressors. Oncogenes such as ras can also induce senescence Serrano et al. As discussed below, because organisms and cells are constantly being exposed to stressors, senescent cells in vivo may derive not only from cell divisions but from cells being exposed to stress.
The connection between organismal aging and cell senescence remains a subject of controversy, in spite of decades of study reviewed in Hayflick, ; de Magalhaes, ; Campisi, ; Campisi and d’Adda di Fagagna, ; Jeyapalan and Sedivy, ; de Magalhaes and Passos, Below I present and discuss some of the key arguments for and against a role of RS and senescent cells in human aging.
At least post partumthere is no relation between the number of CPDs cells can endure haflick the age of the donor Cristofalo et al. Chances are previous studies showing otherwise were biased Cristofalo, Likewise, one study in centenarians failed to find differences in the CPDs cells taken from centenarians could endure when compared to cells from young donors Tesco et al.
As mentioned above, cells at birth from patients with certain progeroid syndromes have fewer divisions than cells from healthy controls. This, however, might be a result of increased cell death or exit from the cell cycle for reasons unrelated to RS Johnson et al. In fact, senescent cells from patients with Werner’s syndrome have different patterns of gene expression Oshima et al.
I should also point out that a caveat of comparing CPDs is that when cell lines are derived from people, the selected cells are those that grow because people, even very old people, never run out hayfflick proliferating cells Tesco et al. As such, these studies would not detect, say, differences in the proportion of proliferating cells.
Although a relation between a species’ longevity and the CPDs its cells can endure in vitro exists, it is debatable if this is related to aging. For one, optimal culture conditions vary from species to species.
As an example, O 2 partial pressure can affect cellular proliferation and there is evidence that O 2 limits the replicative capacity of mouse fibroblasts as these are immortal under low O 2 Xe et al. As such, comparisons between different species may be biased due to intra-species differences in O 2 sensitivity Toussaint et al. In addition, due to the positive correlation between body size and longevity–mentioned before — perhaps cells taken from long-lived animals endure more CPDs because of differences in size, not due to differences in longevity, as supported by results using more sophisticated methods Lorenzini et al.
Senescent cells and senescence-associated biomarkers can be found in various human tissues in vivo associated with both aging and pathology Paradis et al.
Interestingly, stress-prone tissues appear to be the most affected.
For example, fibroblasts cultured hauflick distal lower extremities of patients with venous reflux, which precedes the development of venous ulcers, display characteristics of senescent cells Mendez et al. Similar results also relate cellular senescence to atherosclerosis Minamino et al.
Senescent cells have been found in other mouse tissues too, though possibly through telomere-independent mechanisms Wang et al. Markers of cell senescence, such as p16 INK4a which is discussed elsewherehave been found in the mouse brain Molofsky et al. A gene expression meta-analysis across mammalian tissues and species found signatures of senescent cells in aged tissues de Magalhaes et al.
However, one study monitored p16 INK4a expression with age in mice and found that, while its expression increases with age, total body p16 INK4a expression does not predict overall mortality, raising questions about the role of senescent cells in aging Burd et al. Because senescent cells can secrete proinflammatory cytokines and other factors that disrupt the tissue microenvironment, they may contribute to disruption of cell and tissue function.
Even a small percentage of senescent cells, in fact, may interfere with tissue homeostasis and function Shay and Wright, Indeed, some evidence exist that senescent cells contribute to age-related pathologies such as osteoarthritis Martin and Buckwalter, ; Price et al.
These cytokines’ circulating levels increase in vivo reviewed limitw Lio et al.
Studies using genetically-modified mice found that genetic clearance of senescent cells delays aging-associated disorders in old mice Baker et al. Initially this was observed in progeroid mice that exhibit accelerated aging and accumulate more senescent cells than normally Baker et al. Clearance of senescent cells also did not extend lifespan in progeroid mice, which Baker et al. More recently, using the same genetic approach, the same group found that removing senescent cells in mice preserves health in some tissues, though not in others, protects from cancer and extends median but not maximum lifespan Baker et al.
Therefore, these landmark studies provide evidence that senescent cells can promote age-related phenotypes, at least to a subset of organs.
Clearly, senescent cells can be found in vivo without telomere shortening Melk et al. Since cells taken from old donors do not endure fewer CPDs, one hypothesis is that senescent cells in vivo are not widely caused by shortening telomeres but instead by various stressors and insults.
Exemplifying, studies in centenarians have raised doubts on whether telomere shortening occurs in vivo and whether senescence-associated genes in vitro are also differentially expressed in vivo Mondello et al. Besides, some data indicate that chronic stressors may accelerate risk of a host of age-related diseases by prematurely aging the immune response Kiecolt-Glaser et al.
Lastly, as hinted by the above mentioned results on the impact of O 2 in cell proliferation, RS for many cell lines in vitro and in vivo might instead be better defined as SIPS resulting from oxidative stress. A relationship appears to exist between stress resistance and aging. In model organismsextended longevity is often associated with increased stress resistance reviewed in Longo, Concisely, manipulations in C.