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Abstract

We believe that cytogerontological models such as the Hayflick model, though very useful for experimental gerontology, are based only on certain correlations and do not directly apply to the gist of the aging process. Thus, the Hayflick limit concept cannot explain why we age, whereas our "stationary phase aging" model appears to be a "gist model," since it is based on the hypothesis that the main cause of both various "age-related" changes in stationary cell cultures and similar changes in the cells of aging multicellular organism is the restriction of cell proliferation. The model is applicable to experiments on a wide variety of cultured cells, including normal and transformed animal and human cells, plant cells, bacteria, yeasts, mycoplasmas, etc. The results of relevant studies show that cells in this model die out in accordance with the Gompertz law, which describes exponential increase of the death probability with time.. Therefore, the "stationary phase aging" model may prove effective in testing of various geroprotectors (anti-aging factors) and geropromoters (pro-aging factors) in cytogerontological experiments. It should be emphasized, however, that even the results of such experiments do not always agree with the data obtained in vivo and therefore cannot be regarded as final but should be verified in studies on laboratory animals and in clinical trials (provided this complies with ethical principles of human subject research).
ISSN 00963925, Moscow University Biological Sciences Bulletin, 2014, Vol. 69, No. 1, pp. 10–14. © Allerton Press, Inc., 2014.
Original Russian Text © A.N. Khokhlov, A.A. Klebanov, A.F. Karmushakov, G.A. Shilovsky, M.M. Nasonov, G.V. Morgunova, 2014, published in Vestnik Moskovskogo Universiteta.
Biologiya, 2014, No. 1, pp. 13–18.
10
Alive or dead are those cells
We study in our flasks and wells?
Cytogerontology deals with analysis of aging
mechanisms on cultured cells [1–7]. It is the cytoger
ontological approach that is increasingly often used to
test potential geroprotectors (physical or chemical
factors retarding the process of aging, i.e.,
increase in
the probability of death with age). It should be empha
sized that cytogerontology as a branch of gerontology
cannot successfully develop in the absence of correct
general gerontological concepts and definitions.
Unfortunately, the growing interest in experimen
tal gerontological research during recent years has
resulted in a paradoxical situation: although the num
ber of publications in this field is increasing, only a
minor part of them is actually devoted to the mecha
nisms of aging. In our opinion, this is due, among oth
ers, to the following circumstances:
(1) As a rule, the authors ignore the classical defi
nition of aging as a complex of agerelated changes
that increase the probability of death.
(2) The emphasis in the studies is on increase or
decrease in life span, although this often has no rela
tion to modification of the aging process (in particular,
it is possible to prolong the life span of nonaging
organisms, while the fact of aging itself is not necessar
ily indicative of low longevity).
(3) The control group often consists of animals
with certain abnormalities or genetic disorders, so that
any favorable influence on the corresponding patho
logical processes results in life span prolongation.
(4) Too much significance is assigned to increase or
decrease in the average life span, which is largely
determined by factors unrelated to aging.
(5) An increasing number of gerontological exper
iments are performed on model systems providing
only indirect information on the mechanisms of aging,
and its interpretation largely depends on the basic
concept maintained by a given research team. In par
ticular, this concerns the usage of the term “cell/cellu
lar senescence,” which was originally introduced to
designate a complex of various adverse changes occur
ring in normal cells due to the exhaustion of their pro
liferative potential [1, 8–10]. Today, however, many
authors apply it to the phenomenon of suppression of
proliferative activity in cells (including transformed
cells) under the effect of various DNAdamaging fac
tors, which is accompanied by a certain cascade of
intracellular events [11–13].
GERONTOLOGY
Testing of Geroprotectors in Experiments on Cell Cultures:
Choosing the Correct Model System
A. N. Khokhlov, A. A. Klebanov, A. F. Karmushakov, G. A. Shilovsky,
M. M. Nasonov, and G. V. Morgunova
Evolutionary Cytogerontology Sector, School of Biology, Moscow State University, Moscow, 119992 Russia
email: khokhlov@mail.bio.msu.ru
Received September 1, 2013
Abstract
—We believe that cytogerontological models, such as the Hayflick model, though very useful for
experimental gerontology, are based only on certain correlations and do not directly apply to the gist of the
aging process. Thus, the Hayflick limit concept cannot explain why we age, whereas our “stationary phase
aging” model appears to be a “gist model,” since it is based on the hypothesis that the main cause of both
various “agerelated” changes in stationary cell cultures and similar changes in the cells of aging multicellular
organism is the restriction of cell proliferation. The model is applicable to experiments on a wide variety of
cultured cells, including normal and transformed animal and human cells, plant cells, bacteria, yeasts, myco
plasmas, etc. The results of relevant studies show that cells in this model die out in accordance with the
Gompertz law, which describes exponential increase of the death probability with time. Therefore, the “sta
tionary phase aging” model may prove effective in testing of various geroprotectors (antiaging factors) and
geropromoters (proaging factors) in cytogerontological experiments. It should be emphasized, however, that
even the results of such experiments do not always agree with the data obtained in vivo and therefore cannot
be regarded as final but should be verified in studies on laboratory animals and in clinical trials (provided this
complies with ethical principles of human subject research).
Keywords:
cytogerontology, geroprotectors, cultured cells, “stationary phase aging,” Hayflick limit, cell via
bility, Gompertz law.
DOI:
10.3103/S0096392514020035
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TESTING OF GEROPROTECTORS IN EXPERIMENTS ON CELL CULTURES 11
(6) Finally, there is the issue of what we call “the
problem of reductionism.” In the absolute majority of
gerontological theories proposed in the past few
decades, the mechanisms of both “normal” and accel
erated or retarded aging of multicellular organisms are
reduced to certain macromolecular changes (no mat
ter stochastic or programmed) in their constituent
cells. As a consequence, numerous model systems
have been developed to study “agerelated” changes in
the cells relieved from “organismal noise” associated
with the functioning of the neurohumoral system.
Such reductionism in experimental gerontology (“it
all depends on adverse changes in individual cells”)
has played its role, particularly in the development of
the Hayflick model and also of some models used in
our laboratory, such as the “stationary phase aging”
model, the cell kinetic model for testing of geroprotec
tors and geropromoters, and the model based on eval
uation of cell colonyforming capacity.
Unfortunately, the model based on the Hayflick
limit concept (aging in vitro) is apparently not directly
related to the mechanisms of aging, as has been
repeatedly noted previously [7, 14–21]. In other
words, we cannot conclusively explain why we age by
relying solely on the phenomenon of limited mitotic
potential of normal cells, which is practically never
fully utilized in vivo. However, owing to A.M. Olovni
kov’s theory of marginotomy [22–24], we at least
know today how this phenomenon is realized in the
cells.
It is not excluded that, if the human life span were
extended severalfold, some cell populations would
eventually exhaust their mitotic potential (thereby
reaching the Hayflick limit), which could have
resulted in the “second wave” of aging, but this has not
occurred so far. It should be noted, however, that some
researchers still hold to the opinion that the shortening
of telomeres in the cells is the key mechanism of aging
(e.g., see [25]).
Unlike the Hayflick model, which is based on a
series of correlations [4, 18], our model of “stationary
phase aging” (accumulation of “agedependent” inju
ries in cultured cells whose proliferation is restricted in
a certain way, preferably by contact inhibition) is a
“gist” model based on the assumption that processes
taking place in this model system are essentially simi
lar to those in an aging multicellular organism [2, 4,
26–31]. In fact, this assumption directly issues from
our concept that the restriction of cell proliferation is
the main mechanism providing for the accumulation
of macromolecular defects in cells of aging multicellu
lar organisms [2, 7, 17, 18]. Moreover, our recent stud
ies have shown that cultured cells in the stationary
growth phase indeed “senesce by Gompertz” (figure);
i.e., the probability of their death exponentially
increases with time in accordance with the Gompertz
law [7, 18]. Incidentally, similar results were obtained
even with the suspension cultures of
Acholeplasma
laidlawii
[32], and our previous experiments with this
mycoplasma showed that its “stationary phase aging”
could be successfully delayed by treatment with gero
protectorantioxidant 2ethyl6methyl3hydroxy
pyridine chlorohydrate [33]. It should be noted that
the curves shown in the figure were obtained with
transformed cells. Under appropriate conditions,
most cancer cells are capable of proliferating indefi
nitely, with a given cell line (but not individual cells!)
being “immortal.” For example, the wellknown
HeLa cell line has been maintained in hundreds of
laboratories over more than 60 years. However, when
the growth of such a culture is restricted by certain
physiological means (not causing cell death), various
defects at different structural and functional levels
begin to accumulate in the cells, and the probability of
their death increases; i.e., the cells age in true sense
[34].
At the same time, with regard to the reliability the
ory, it should be taken into account that an aging mul
ticellular organism should not necessarily consist of
senescing cells: the cells can simply die out “by expo
nent” (i.e., without senescence), as in the case of
radioactive decay.
50000
3813
Time after
subcultivation
, days
Cell density, cells/cm
2
100000
150000
200000
250000
300000
350000
18 23 28 33
0.001
0.01
0.1
1
10
3
2
1
Mortality rate
Survival curve for a stationary culture of transformed Chinese hamster cells: (
1
) experimental points, (
2
) data approximation by
the Gompertz equation, (
3
) change in the cell death rate with time.
0
12
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KHOKHLOV et al.
Unfortunately, the obtaining of survival curves for
cultured cells is associated with certain technical and
methodological difficulties, which must be noted here.
First, the cells can divide, which disturbs the integrity
of the cohort. Second, it is not a simple task to cor
rectly determine the moment of cell death. A variety of
socalled probes for this purpose are available today,
but they often yield markedly divergent results. In
other words, the same cell is identified as live in one
test and as dead in another test. The method of evalu
ating cell colonyforming capacity [35, 36] is applica
ble only to proliferating cells and, therefore, cannot be
used for evaluating the viability of postmitotic cells
(e.g., neurons or cardiomyocytes). As for cultured “sta
tionary phase aged” cells, many of them can simply fail
to survive the traumatic procedure of their removal from
the growth surface and subsequent cloning.
Our numerous experiments provide evidence that
changes in the cells occurring in our model system are
indeed similar to those in the cells of aging multicellu
lar organisms. They include accumulation of DNA
singlestrand breaks and DNA–protein crosslinks,
DNA demethylation, changes in the level of sponta
neous sister chromatid exchanges, structural defects in
the cell nucleus, alterations in the plasma membrane,
retardation of mitogenstimulated proliferation,
impairment of colonyforming capacity, changes in
dealkylase activity of cytochrome P450, accumula
tion of 8oxo2'deoxyguanosine (a known biomarker
of aging) in the DNA, increase in the number of cells
with senescenceassociated betagalactosidase activity
(the most popular biomarker of cell senescence), inhi
bition of poly(ADPribosyl)ation of chromatin pro
teins, etc. [2, 7, 37–45].
It should be emphasized that such experiments can
be performed with cells of different origin, including
bacteria, yeasts (currently most widely used in experi
ments on “stationary phase aging”), plant cells, myco
plasmas, etc. This provides a basis for the evolutionary
approach to the analysis of experimental results [46].
Moreover, the “agerelated” changes in cells of sta
tionary cultures can be revealed within a relatively
short time: as a rule in 2–3 weeks after the start of the
experiment.
It is also important that in studies on the Hayflick
model, it is fairly difficult to correctly perform
repeated experiments with the same strain, because
the cells continuously change from passage to passage
(“no man ever steps into the same river twice”),
whereas the “stationary phase aging” model allows, as
already mentioned above, of experimentation with
transformed (or normal but immortalized) animal and
human cells with an unlimited mitotic potential, so
that multiple replication of an experiment is no longer
a problem [34].
All the aforesaid suggests that the “stationary phase
aging” model can be effectively used to test various
agents (drugs) or their combinations for their potential
ability to accelerate or retard aging, provided their
effect is realized only at the cell level.
Unfortunately, we have recently got the impression
that even the data obtained with such “gist” cell cul
ture models cannot be directly extrapolated to the sit
uation in the organism as a whole [7, 18, 30, 31]. Our
cytogerontological tests of various geroprotectors on
the models of “stationary phase aging”, cell kinetics
[47], and cell colonyforming capacity [35] have
shown that these factors fairly often have no favorable
effect on the viability of cultured cells, even though
they prolong the life span of experimental animals and
improve the state of human health. This fact suggests
that the effect of a geroprotector in many cases mani
fests itself only at the organismal level (probably due to
activation/suppression of certain biochemical or neu
rophysiological processes) and is not limited to the
improvement of viability of individual cells. Appar
ently, the same is also true of geropromoters. Thus, it
was probably a serious mistake to perform experiments
with cell cultures so as to exclude the influence of the
endocrine and central nervous systems (which actually
was the main purpose of gerontologists, beginning
from studies by Alexis Carrel [48, 49]). By all
accounts, the results of cytogerontological experi
ments should be thoroughly verified in studies on lab
oratory animals and even in clinical trials (provided
this complies with ethical principles of human subject
research). Of course, this will lessen our chance for an
early breakthrough in studies aimed at retarding the
process of aging, but the reliability of the obtained data
will be significantly higher.
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J. Exp. Med.
,
1913, vol. 18, no. 3, pp. 287–289.
Translated by N. Gorgolyuk
... Another model system-the "stationary-phase," or chronological, aging of cell cultures (Fig. 1)-is based on the idea of proliferation restriction as the main trigger of aging [37][38][39][40]. Within the chronological/"stationary-phase" aging, proliferation is restricted when a cell culture or a suspension of microorganisms enters the stationary growth phase, i.e., stops dividing (quiescence). ...
... Aging is promoted by proliferation restriction, which does not need to be caused by exposure to damaging chemical or physical factors. This model system is universal and applicable for cells of various origins, including protozoa, bacteria, fungi, algae, etc. [40]. ...
... In addition, survival curves are constructed in experimental studies of aging of yeast and bacteria in the chronological/"stationary phase" aging model [10][11][12][13][14]. We have previously shown that a mammalian cell culture in the "stationary phase aging" model dies out in accordance with the Gompertz law [15]. The aim of the present study was to attempt to use the procedures that are used for the analysis of the survival curves of experimental animals in experiments with non-subcultured mammalian cell cultures dying out in the course of stationary phase aging. ...
... Gompertz equation, i.e., undergo aging (Fig. 2). This fact suggests that their death probability increases exponentially with time, similarly to that of aging animals or humans [15,16]. ...
Aging organisms die out in accordance with the "Gompertz law," i.e., the probability of their death increases with age. Survival curve construction is the main tool for gerontologists to study aging and test anti-aging drugs. The analysis of survival curves includes obtaining some indices characterizing aging of the population , for example, the average and maximum lifespan, the mortality rate, and the aging rate. Testing gero-protectors can be correctly performed only by obtaining such curves. The dying out of stationary cell populations bacteria , yeast, and mammalian cell cultures-also occurs in accordance with the Gompertz equation. In this regard, it is reasonable to use the construction of survival curves and their analysis to study the "aging" of non-subcultured cell cultures and testing anti-aging drugs on them. We used this approach in our experiments, due to which we were able to detect the positive anti-aging effect of the Quinton Marine Plasma on stationary phase aging culture of Chinese hamster cells.
... Later, however, taking into account the results of the studies of the "chronological aging" of yeast and bacteria [25][26][27][28], we obtained a number of respective survival curves for cells in the stationary phase of growth in a non-subcultured culture. Mathematical analysis of these curves showed that these cells died in a good agreement with the Gompertz formula ( Fig. 2; schematic illustration is based on the results of several of our experiments) [29]. In this regard, we later performed a series of studies aimed at analyzing the influence of certain biologically active compounds particularly on the kinetics of the death of cultured cells in the stationary phase of growth (see., e.g., [7,[29][30][31][32][33]). ...
... Mathematical analysis of these curves showed that these cells died in a good agreement with the Gompertz formula ( Fig. 2; schematic illustration is based on the results of several of our experiments) [29]. In this regard, we later performed a series of studies aimed at analyzing the influence of certain biologically active compounds particularly on the kinetics of the death of cultured cells in the stationary phase of growth (see., e.g., [7,[29][30][31][32][33]). It was assumed that the deceleration of this process can serve as a basis for classifying the agent of interest as a geroprotector [34,35]. ...
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This is a brief overview of the ideas of the possibility of using the cell kinetic model developed by the author in the 1980s to test, in experiments on cell cultures, potential geroprotectors and geropromoters that slow down or accelerate, respectively, the aging process in animals and humans. The history of the evolution of this model—from estimation of only the cell reproduction rate and saturation density in a non-subcultured cell culture to constructing survival curves in the stationary phase of growth and to a further analysis of the possible interrelation between all parts of the curve of cells’ growth and subsequent dying out—is considered. Possible approaches to mathematical and statistical analysis of the data obtained within the framework of this model system are analyzed. It is emphasized that such studies can be carried out on cells of a very different nature (normal and transformed human and animal cells, plant cells, yeast, mycoplasmas, bacteria, etc.), which makes possible an evolutionary approach to the interpretation of the results obtained. At the same time, in the author’s opinion, the most promising experiments are those carried out on immortalized cells of humans and animals, since they are not cancerous on the one hand and have an unlimited mitotic potential on the other hand and, therefore, do not “age” in the process of numerous divisions, as, for example, normal human diploid fibroblasts do. It is assumed that the appropriate mathematical analysis of the entire growth and dying out curve of a non-subcultured cell culture (from seeding into a culture flask to the complete death of all cells) may allow the clarification of certain relationships between the development and aging of a multicellular organism and to increase the reliability of identifying promising geroprotectors.
... It is known that many parameters used in investigations of aging can be in good correlation with the organism's age but be quite unrelated with increase in death probability with age [23,24], which is necessarily included in classical determina tion of aging of living organisms (see for example [25,26]). Therefore, it is recommended to perform geronto logical studies either in longitudinal experiments or in experiments using so called "gist"/essential models [27,28]. These imply model systems that are based not only on correlations identified in gerontological studies (for example, the well known Hayflick model), but on specif ic mechanisms of aging postulated by the authors. ...
... It was supposed that cell culture growth and monolayer formation could be com pared to growth and development of the whole organism [32 34]. In the framework of this concept, it was pro posed to use stationary cell cultures for studies on age related changes that occur in cells of an aging organism [24,26,28,31,35]. ...
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It is well known that the number of dividing cells in an organism decreases with age. The average rate of cell division in tissues and organs of a mature organism sharply decreases, which is probably a trigger for accumulation of damage leading to disturbance of genome integrity. This can be a cause for the development of many age-related diseases and appearance of phenotypic and physiological signs of aging. In this connection, the protein poly(ADP-ribosyl)ation system, which is activated in response to appearance of various DNA damage, attracts great interest. This review summarizes and analyzes data on changes in the poly(ADP-ribosyl)ation system during development and aging in vivo and in vitro, and due to restriction of cell proliferation. Special attention is given to methodological aspects of determination of activity of poly(ADP-ribose) polymerases (PARPs). Analysis of relevant publications and our own data has led us to the conclusion that PARP activity upon the addition of free DNA ends (in this review referred to as stimulated PARP activity) is steadily decreasing with age. However, the dynamics of PARP activity measured without additional activation of the enzyme (in this review referred to as unstimulated activity) does not have such a clear trend: in many studies, the presented differences are statistically non-significant, although it is well known that the number of unrepaired DNA lesions steadily increases with aging. Apparently, the cell has additional regulatory systems that limit its own capability of reacting to DNA damage. Special attention is given to the influence of the cell proliferative status on PARP activity. We have systematized and analyzed data on changes in PARP activity during development and aging of an organism, as well as data on differences in the dynamics of this activity in the presence/absence of additional stimulation and on cellular processes that are associated with activation of these enzymes. Moreover, data obtained in different models of cellular aging are compared.
... The model of stationary phase aging (SPA) of different animal and human cells has long been used in the Evolutionary Cytogerontology Sector (School of Biology, Moscow State University) to analyze the mechanisms of aging and test potential geroprotectors and geropromoters [2]. This model is based on the idea that restriction of cell proliferation leads to the accumulation of macromolecular damage in cultured cells, similar to that in postmitotic cells of an aging multicellular organism [3][4][5]. ...
... In our opinion, the main conclusion that can be drawn from the aforementioned publications is that preventing acidification of the growth medium is a way to prolong the life span of either yeast or mammalian cell culture, but the cells will still die off, although at a lower rate. It is noteworthy that our experiments on the SPA of mammalian cells cultured in hermetically sealed flasks showed that their "aging" proceeded in accordance with the Gompertz law [2]. On the other hand, the curves presented in some papers on the effect of buffer capacity of the growth medium on yeast ChA kinetics [26,39] clearly show that, in the absence of pH-stabilizing agents, the cells simply die off in exponential numbers; i.e., no aging is observed. ...
Article
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There is an opinion that the chronological aging (ChA) of yeast and the stationary phase aging (SPA) of cultured animal and human cells are a consequence of growth medium acidification. However, a number of recent publications indicate that, although this process has a certain influence on the rate of “aging” of cells in the stationary growth phase, it does not determine it completely. Apparently, the key factor in this case is the restriction of cell proliferation, which leads to cell “aging” even under physiologically optimal conditions. During yeast ChA and mammalian cell SPA, the medium is getting acidified to pH ≤ 4. Prevention of acidification can prolong the culture life span, but the cells will still die, although at a slower rate. Effects of medium acidification during ChA and SPA can be explained by activation of highly conserved growth signaling pathways leading to oxidative stress, and these processes, in turn, can play a role in aging of multicellular organisms and development of age-related diseases. Our previous experiments on the effect of buffer capacity of growth medium on SPA of transformed Chinese hamster cells showed that 20 mM HEPES had no effect on cell growth rate; in addition, the growth curves of experimental and control cells reached a plateau on the same day. However, the cell saturation density in the medium with HEPES was lower (i.e., the cells were “older” in terms of the gerontological cell kinetics model); on the other hand, the rate of SPA was markedly reduced, compared to the control, although the cells were still “getting older.” It can be assumed that extracellular pH (by the way, well correlated with intracellular pH) is an important factor (I.A. Arshavsky’s concept of the role of acidic alteration in aging) but not the key factor determining the survival of cells in a stationary culture.
... В наших исследованиях мы уже неоднократно показывали, что клеточная популяция «стационарно стареющих» клеток млекопитающих вымирает в соответствии с уравнением Гомпертца [6][7][8], по-хожие результаты получают исследователи, работающие на бактериях [9] и дрожжах (хронологическая модель старения) [10]. Помимо этого, во всех указанных клетках происходит накопление биомаркеров старения, а также по истечении некоторого времени активируется/подавляется экспрессия важных, с точки зрения геронтологии, генов и белков, как и у старых организмов [9,11,12]. Если экологические, генетические и аккумуляционные механизмы развития «патологий» клетки довольно легко представить (воздействие pH среды, наличие мутаций у популяции клеток, накопление повреждений в ДНК со временем), то с онтогенетическими дело обстоит сложнее. Условными «онтогенетическими» механизмами можно назвать те механизмы, существующие у клеток, которые помогают расти их популяции, но которые в конечном итоге приводят клетку к возникновению «болезней» -накоплению липофусцина и ассоциированной со старением бета-галактозидазы, избыточной активации комплекса TOR (target of rapamycin) и ухудшению регуляторных свойств АМФ-активируемой протекинкиназы (AMPK, AMP-activated protein kinase). ...
Article
Dilman in his book «Four Models of Medicine» (1987) summarizes the ideas about mechanisms of origin of the «main» noninfectious human diseases and the nature of aging. He considers these diseases from the point of four basic models of medicine – environmental, genetic, accumulative and ontogenetic. Obesity, type II diabetes, atherosclerosis, cancer – all these diseases accompany the aging process and are associated with metabolic disorders. Some mechanisms for the development of such pathologies, for example, hormonal dysregulation, are realized only at the level of the body, however, at present we see that metabolic disorders manifest at the cellular level, and the mechanisms of the origin of «diseases» are also characteristic to some extent of cell populations. Modern scientific research confirms many of the assumptions by Dilman. Moreover, they are moving in the same direction, and therefore, despite the fact that more than 30 years have passed since the publication of the book, the ideas remain relevant.
... According to the de nition to which we adhere, aging is the set of age-related changes in the organism leading to an increase in the probability of death (Khokhlov 2010(Khokhlov , 2014bKhokhlov et al. 2012Khokhlov et al. , 2014. Over time, the ability of the organism to withstand environmental impacts decreases, the ability to resist infections is reduced, and the risk of development of age-related diseases increases. ...
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In this chapter, we shortly describe the main types of autophagy (macroautophagy, microautophagy, and chaperone-mediated autophagy). Data about the character of the influence of autophagy on the aging process and on the development of some diseases in various organisms are analyzed. It is noted that this effect is usually beneficial though not always, because cancer cells also use autophagy for survival. Results of investigations of this phenomenon in experiments on mice, nematodes, fruit flies, bacteria, yeast, and cell cultures of higher organisms are considered. Obvious relationship between autophagy activation and cell proliferation restriction is emphasized. The latter, in our opinion, is the main cause of age-related accumulation of various defects (the most important of them is DNA damage) in cells and tissues, which leads to an increase in the death probability (i.e., to aging). Data about some activators (calorie restriction mimetics) and inhibitors of autophagy in terms of their potential effect on aging and longevity are reviewed. It is concluded that studies of the role of autophagy in the aging process on the models of chronological aging in yeast or stationary phase aging of cell cultures could be considered as the most appropriate approach to the problem solution. We believe that it is better to trigger macroautophagy in a natural way rather than by using various calorie restriction mimetics (rapamycin, resveratrol, etc.), because addiction to them may develop over time.
... During many years of research on the stationary phase aging model, our premise was that cultured cells whose proliferation is restricted in some way (preferably by contact inhibition) accumulate "age-related" defects similar to those in cells of aging multicellular organisms (and geroprotectors should postpone/retard the accumulation), with the kinetics of cell death in this model system remaining behind the scene. Our subsequent studies have shown that mammalian cells in this model die out in accordance with the Gompertz law; that is, they age in the true sense (Khokhlov 2010b;Khokhlov et al. 2014;Khokhlov and Morgunova 2017). In other words, the probability of their death increases exponentially with age, as in aging animals and humans. ...
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A critical review of various methods of testing of geroprotectors (anti-aging compounds or physical factors) in experiments on cultured cells is presented. Some cytogerontological models (the Hayflick model, the stationary phase aging model, the cell kinetics model, and the colony-forming ability evaluation) are reviewed with a focus on general gerontological definitions (aging, death probability, survival curve, cell senescence, biomarkers of aging, aging and non-aging organisms, age-related diseases, etc.). The main attention has been paid to methodological aspects of geroprotectors research with these models. Problems arising when constructing survival curves for cultured cells in the stationary phase aging model are analyzed. In particular, consideration is given to some problems encountered when using the most widespread molecular probes designed for live/dead cell viability assays. In addition, the possible role of the growth medium acidification in the stationary phase aging phenomenon is reviewed. It is concluded that extracellular pH which, by the way, is well correlated with intracellular pH, is very important but not the key factor determining survival of cells in a stationary culture. A note is made that the evaluation of colony-forming efficiency, though optimal for cell viability assessment, is unfortunately not applicable to post-mitotic or very slowly propagating cells. Some questions regarding the interpretation of data obtained in such studies in application to humans are also considered. Popular approaches to choosing biomarkers of cell aging/senescence are briefly reviewed. It is assumed that the least number of problems associated with interpreting the results of testing potential geroprotectors in cytogerontological experiments arises when such studies are performed using normal human cells in the model of stationary phase aging, which is based on the concept of cell proliferation restriction as the main cause of accumulation of macromolecular lesions (mainly DNA damage) in cells of multicellular organisms with age that, in turn, leads to the aging (increase in the death probability) of the organisms. It is assumed that this theory answers almost all the listed questions to any universal concept of aging. However, in the authors' opinion, even their approach will not give the final answer to the question of whether or not the studied factor is a geroprotector. The main conclusion is that gerontologists analyzing the possibilities for retarding or even blocking the aging process currently have no fully adequate alternative to the construction of survival curves for the cohorts of animals or humans, even though this approach is highly expensive and requires great labor expenditures. Apparently, all the cytogerontological models reviewed provide only preliminary testing of potential anti-aging factors.
... According to the concept of aging, which has repeatedly been presented in a number of our articles (see, e.g., [7,[11][12][13]), the proliferation of cells that form tissues and organs of the vast majority of multicellular organisms is restricted due to the accumulation in them of various macromolecular defects, the most important of which are DNA lesions (because the lesion of the principal template often cannot be fixed). Later, through a chain of different events, they lead to an increased death probability of an organism (i.e., to aging). ...
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Ideas of proponents and opponents of programmed aging concerning the expediency of this phenomenon for the evolution of living organisms are briefly considered. We think that evolution has no “gerontological” purpose, because the obligate restriction of cell proliferation during the development of multicellular organisms is a factor that “automatically” triggers aging due to the accumulation of various macromolecular lesions in cells as a result of the suppression, or even complete cessation of emergence of new, intact cells. This leads to the “dilution” of stochastic damage (the most important of which is DNA damage) at the level of the entire cellular population. Some additional arguments in favor of the inexpediency of aging for both species and individuals are also listed.
... AUTOPHAGY AND AGING According to the definition to which we adhere, aging is the set of age-related changes in the organism leading to an increase in the death probability [9][10][11][12]. Over time, the ability of the organism to withstand environmental impacts decreases, the ability to resist infections is reduced, and the risk of development of age-related diseases increases. ...
Article
Full-text available
In the review, the main types of autophagy (macroautophagy, microautophagy, and chaperonemediated autophagy) are shortly described. Data about the character of the influence of autophagy on the aging process and on the development of some neurodegenerative diseases in various organisms are analyzed. It is noted that this effect is usually (though not always) beneficial. Results of investigations of the phenomenon in experiments on mice, nematodes, fruit flies, bacteria, yeast, and cell cultures of higher organisms are considered. Obvious relationship between autophagy activation and cell proliferation restriction is emphasized. The latter, in our opinion, is the main cause of age-related accumulation of various defects (the most important of them is DNA damage) in cells and tissues, which leads to an increase in the death probability (i.e., to aging). It is concluded that studies of the role of autophagy in the aging process on the models of chronological aging in yeast or stationary phase aging of cell cultures could be considered as the most appropriate approach to the problem solution.
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The isolation and characterization of 25 strains of human diploid fibroblasts derived from fetuses are described. Routine tissue culture techniques were employed. Other than maintenance of the diploid karyotype, ten other criteria serve to distinguish these strains from heteroploid cell lines. These include retention of sex chromatin, histotypical differentiation, inadaptability to suspended culture, non-malignant characteristics in vivo, finite limit of cultivation, similar virus spectrum to primary tissue, similar cell morphology to primary tissue, increased acid production compared to cell lines, retention of Coxsackie A9 receptor substance, and ease with which strains can be developed. Survival of cell strains at - 70 °C with retention of all characteristics insures an almost unlimited supply of any strain regardless of the fact that they degenerate after about 50 subcultivations and one year in culture. A consideration of the cause of the eventual degeneration of these strains leads to the hypothesis that non-cumulative external factors are excluded and that the phenomenon is attributable to intrinsic factors which are expressed as senescence at the cellular level. With these characteristics and their extremely broad virus spectrum, the use of diploid human cell strains for human virus vaccine production is suggested. In view of these observations a number of terms used by cell culturists are redefined.
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The history of experimental-gerontological studies on cell cultures numbers already more than 100 years. Basics of the investigations were invented by Carrel in the beginning of XX century, from the 60th of the same century the Hayflick's model began very popular, and from the 80th – the models using various stationary cell cultures. It is assumed that in such experiments we can investigate directly molecular-cellular mechanisms of aging avoiding any influence of the factors (central nervous system, hormones, etc.) manifesting their effects at the whole organism level only. Unfortunately, in the last years rather much data indicating that reduction of fundamental mechanisms of aging to the cellular level only could be incorrect appeared. The results of our recent studies of mild uncoupling effect on "stationary phase aging" of cell culture confirm this idea too. It is possible that the multiple age changes in cells just activate some mechanisms realizing at the organism level only and leading, eventually, to the force of mortality increase, i.e. to aging.
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The history of gerontological studies on cell cultures is shortly reviewed. It is emphasized that the main purpose of the studies was to eliminate the central nervous and endocrine systems' influences considered as the side effects and to separate "senile" changes at the cellular-molecular level only. But, actually, all the cytogerontological models used did not take into consideration the basic definition of aging as the complex of age-related organism’s changes leading to increase of death probability (rate of mortality). Being based on some data obtained formerly (including the data from author's lab – in particular, concerning "mild" uncoupling effect on survival curves of cultured cells) a conclusion was made that age changes in macroorganism's cells shouldn't necessarily be harmful, i.e. induce lowering of cell viability. Perhaps, they are being realized at the organism level only (i.e. quite with the help of nervous and endocrine systems) just triggering the aging process and inducing the increase of macroorganism death probability.
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The 8-oxo-dG/dG ratio in DNA of cultured transformed Chinese hamster cells was analyzed during their "stationary phase aging". Amount of 8-oxo-dG and dG in DNA hydrolyzate was evaluated by HPLC-EC. The cells grew and "aged" for 15 days. As expected, the 8-oxo-dG/dG ratio increased with cell "age". It did not change significantly from 4th to 8th day (6.26 x 10(-5) and 4.42 x 10(-5), correspondingly) and then abruptly increased to 15th day of "age" (22.40 x 10(-5)). The results are in accordance with the conception of cell proliferation restriction as the starting mechanism of ageing and the method can be used for evaluation of cell culture biological age when testing new compounds for their geroprotector or geropromoter activity.
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