Cytogerontology at the beginning of the third millenium: from "correlative" to "gist" models

For the most part, research in the area of cytogerontology, i.e., investigation of the mechanisms of aging in the experiments on cultured cells, is carried out using the "Hayflick's model". More than forty years have passed since the appearance of that model, and during this period of time, very much data were obtained on its basis. These data contributed significantly to our knowledge of the behavior of both animal and human cultured cells. Specifically, we already know of the mechanisms underlying the aging in vitro. On the other hand, in my opinion, little has changed in our knowledge of the aging of the whole organism. In all likelihood, this can be explained by that the Hayflick's model is, like many others used in the experimental gerontology, correlative, i.e. based on a number of detected correlations. In the case of Hayflick's model, these are correlations between the mitotic potential of cells (cell population doubling potential) and some "gerontological" parameters and indices: species life-span, donor age, evidence of progeroid syndromes, etc., as well as various changes of normal (diploid) cells during long-term cultivation and during aging of the organism. It is, however, well known that very frequently a good correlation has nothing to do with the essence (gist) of the phenomenon. For example, we do know that the amount of gray hair correlates quite well with the age of an individual but is in no way related to the mechanisms of his/her aging and probability of death. In this case, the absence of cause-effect relationships is evident, which are, at the same time, indispensable for the development of gist models. These models, as distinct from the correlative ones, are based on a certain concept of aging. In the case of Hayflick's model, such a concept is absent: we cannot explain, using the "Hayflick's limit", why our organism ages. This conclusion was convincingly confirmed by the discovery of telomere mechanism which determines the aging of cells in vitro. That discovery initiated the appearance of theories attempting to explain the process of aging in vivo also on its basis. However, it has become clear that the mechanisms of aging of the entire organism, located, apparently, in its postmitotic cells, such as neurons or cardiomyocytes, cannot be explained in the framework of this approach. Hence, we believe that it is essential to develop "gist" models of aging using cultured cells. The mechanisms of cell aging in such models should be similar to the mechanisms of cell aging in the entire organism. Our "stationary phase aging" model could be one of such models, which is based on the assumption of the leading role of cell proliferation restriction in the processes of aging. We assume that the accumulation of "senile" damage is caused by the restriction of cell proliferation either due to the formation of differentiated cell populations during development (in vivo) or to the existence of saturation density phenomenon (in vitro). Cell proliferation changes themselves do not induce aging, they only lead to the accumulation of macromolecular defects, which, in turn, lead to the deterioration of tissues, organs, and, eventually, of the entire organism, increasing the probability of its death. Within the framework of our model, we define cell aging as the accumulation in a cell population of various types of damage identical to the damage arising in senescing multicellular organism. And, finally, it is essential to determine how the cell is dying and what the death of the cell is. These definitions will help to draw real parallels between the "genuine" aging of cells (i.e., increasing probability of their death with "age") and the aging of multicellular organisms.     

Cytogerontology at the Beginning of the Third Millennium: From “Correlative” to “Gist” Models

For the most part, research in the area of cytogerontology, i.e., investigation of the mechanisms of aging in the experiments on cultured cells, is carried out using the Hayflick's model. More than forty years have passed since the appearance of that model, and during this period of time, very much data were obtained on its basis. These data contributed significantly to our knowledge of the behavior of both animal and human cultured cells. Specifically, we already know of the mechanisms underlying the aging in vitro. On the other hand, in my opinion, little has changed in our knowledge of the aging of the whole organism. In all likelihood, this can be explained by that the Hayflick's model is, like many others used in the experimental gerontology, correlative, i.e. based on a number of detected correlations. In the case of Hayflick's model, these are correlations between the mitotic potential of cells (cell population doubling potential) and some gerontological parameters and indices: species life-span, donor age, evidence of progeroid syndromes, etc., as well as various changes of normal (diploid) cells during long-term cultivation and during aging of the organism. It is, however, well known that very frequently a good correlation has nothing to do with the essence (gist) of the phenomenon. For example, we do know that the amount of gray hair correlates quite well with the age of an individual but is in no way related to the mechanisms of his/her aging and probability of death. In this case, the absence of cause-effect relationships is evident, which are, at the same time, indispensable for the development of gist models. These models, as distinct from the correlative ones, are based on a certain concept of aging. In the case of Hayflick's model, such a concept is absent: we cannot explain, using the Hayflick's limit, why our organism ages. This conclusion was convincingly confirmed by the discovery of telomere mechanism which determines the aging of cellsin vitro. That discovery initiated the appearance of theories attempting to explain the process of aging in vivo also on its basis. However, it has become clear that the mechanisms of aging of the entire organism, located, apparently, in its postmitotic cells, such as neurons or cardiomyocytes, cannot be explained in the framework of this approach. Hence, we believe that it is essential to develop gist models of aging using cultured cells. The mechanisms of cell aging in such models should be similar to the mechanisms of cell aging in the entire organism. Our stationary phase aging model could be one of such models, which is based on the assumption of the leading role of cell proliferation restriction in the processes of aging. We assume that the accumulation of senile damage is caused by the restriction of cell proliferation either due to the formation of differentiated cell populations during development (in vivo) or to the existence of saturation density phenomenon (in vitro). Cell proliferation changes themselves do not induce aging, they only lead to the accumulation of macromolecular defects, which, in turn, lead to the deterioration of tissues, organs, and, eventually, of the entire organism, increasing the probability of its death. Within the framework of our model, we define cell aging as the accumulation in a cell population of various types of damage identical to the damage arising in senescing multicellular organism. And, finally, it is essential to determine how the cell is dying and what the death of the cell is. These definitions will help to draw real parallels between the genuine aging of cells (i.e., increasing probability of their death with age) and the aging of multicellular organisms.     

Testing of geroprotectors in experiments on cell cultures: Choosing the correct model system

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).     

Decline in regeneration during aging: Appropriateness or stochastics?

There is a standpoint according to which the suppression of the ability of cells in a multicellular organism to proliferate, taking place during aging, as well as the corresponding decline in the regenerative capacities of tissues and organs, is caused by the specialized mechanisms having emerged in the evolution that decrease the risk of malignant transformation and, thereby, provide for protection against cancer. At the same time, various macromolecular defects start to accumulate in senescent cells of the body, which, on the contrary, elevate the probability for malignant transformation of these cells. Thus, according to the mentioned concept, the restriction of cell proliferation is a double-edged sword, which, on the one hand, decreases the probability for malignant tumor development in young age and, on the other hand, limits the lifespan due to accumulation of “spoiled” cells in old age. However, it remains unclear why normal human cells placed under in vitro conditions and thus having no mentioned “anticancer” barriers, which function at the body level only, NEVER undergo spontaneous malignant transformation. In addition, it is unclear how the freshwater hydra escapes both aging and cancer, as it under certain conditions contains no postmitotic and senescent cells at all and under these conditions (excluding the need for sexual reproduction) can live almost indefinitely, possessing a tremendous regenerative potential (a new organism can emerge from even 1/100 part of the old one). Presumably, the restriction of cell proliferation in an aging multicellular organism is not the result of a certain special program. Apparently, there is no program of aging at all, the aging being a “byproduct” of the program of development, whose implementation in higher organisms necessarily requires emergence of cell populations with a very low and even zero proliferative activity, which actually determines the limited ability of the corresponding organs and tissues to regenerate. On the other hand, the populations of highly differentiated cells incapable or poorly capable of reproduction (e.g., neurons, cardiomyocytes, and hepatocytes) are the particular factor that determines the normal functioning of higher animals and humans. Even regeneration of such organs with the help of stem cells may interfere with the necessary links in elaborate systems. The reductionism (“everything is determined by adverse changes in individual cells”), which has recently become widespread in experimental gerontological research, has brought about several model systems for studying the aging mechanisms in isolated cells (Hayflick phenomenon, stationary phase aging model, cellular kinetic model for testing of geroprotectors and geropromoters, etc.). However, it currently seems that data obtained using such models are inappropriate for an automatic extrapolation to the situation in the whole body. Presumably, impairments in regulatory processes functioning at the neurohumoral level are the major players in the mechanisms underlying aging of multicellular organisms rather than a mere accumulation of macromolecular damage in individual cells. It cannot be excluded that a disturbance of such regulation is the particular reason for the abnormal INCREASE in proliferation intensity of some cell populations that are frequently observed in old age and that lead to senile acromegaly and development of numerous benign tumors. It looks like the quality of CONTROL over cells, organs, and tissues becomes poorer with age rather than the quality of the cells themselves, which leads to an increase in the death rate.     

Which aging in yeast is “true”?

Two model systems, “replicative aging” and “chronological aging” (CA), which are used for gerontological research on the yeast Saccharomyces cerevisiae, are compared. In the first case, the number of daughter cells generated by an individual mother cell before cell propagation irreversibly stops is analyzed. This makes the model very similar to the well-known Hayflick model. In the case of CA, the survival of yeast cell population in the stationary phase of growth is studied. It is noted that the second model is similar to the “stationary phase aging” model, which is used in the author’s laboratory for cytogerontological studies on animal and human cells. It is assumed that the concept of cell proliferation restriction as the main cause of age-related accumulation in the cells of multicellular organisms of macromolecular defects (primarily DNA damage) leading to deterioration of tissue and organ functioning and, as a result, to an increase in the death probability allows explaining how the aging process proceeds in almost any living organisms. Apparently, in all cases, this process is initiated by the appearance of slow propagating (or not propagating at all) cells, which leads to the termination of “dilution,” with the help of new cells, of macromolecular defects accumulating at the level of whole cell population. It is concluded that data on the geropromoter or geroprotector activity of various factors obtained in tests on the yeast CA model can be used with a high reliability to understand the mechanisms of human aging and longevity.     

Interpretation of data about the impact of biologically active compounds on viability of cultured cells of various origin from a gerontological point of view

Problems related to the interpretation of data obtained during testing of potential geroprotectors in cytogerontological experiments are considered. It is emphasized that such compounds/physical factors should influence the processes leading to the age-related increase of death probability of multicellular organisms (primarily human, in whose aging gerontologists are mainly interested). However, in the authors’ opinion, compounds that can be used to treat age-related diseases can hardly be classified as geroprotectors. It is noted that, in the model systems using cultured cells, researchers usually evaluate their viability, the criteria of which strongly depend on the aging theory that is shared by the experimenters. In addition, it is very important what cells are used in the studies—normal or transformed cells of multicellular organisms, unicellular eukaryotic or prokaryotic organisms, etc. In particular, the biologically active compounds that decrease the viability of cultured cancer cells, similarly to the compounds that increase the viability of normal cultured cells, may increase the life span of experimental animals and humans. Various problems with interpretation of data obtained with the Hayflick model, the stationary phase aging model, and the cell kinetics model, as well as in experiments on evaluation of cell colony-forming efficiency, are analyzed. The approaches discussed are illustrated on the example of the results of gerontological studies of rapamycin, a well-known mTOR inhibitor. It is assumed that factors retarding the stationary phase aging (chronological aging) of cultured cells are, apparently, the most promising geroprotectors, although the specific mechanisms of their action may vary considerably.     

Evolution of the term “cellular senescence” and its impact on the current cytogerontological research

The term “cellular/cell senescence” was first introduced by Leonard Hayflick to describe the “age-related” changes in normal eukaryotic cells during aging in vitro, i.e., over the exhaustion of their mitotic potential. In the “classic” variant, it was assumed that cells “grow old” with the help of some internal mechanism, which leads to accumulation of various macromolecular defects (DNA damage in the first place). Currently, as a rule, “cellular senescence” means accumulation/appearance of particular “biomarkers of aging” in cells (they are most often transformed cells that do not demonstrate any replicative senescence) under the influence of various external factors (oxidative stress, H₂O₂, mitomycin C, ethanol, ionizing radiation, doxorubicin, etc.) that cause DNA damage. This phenomenon has been called DDR (DNA Damage Response). Among the said biomarkers, there are senescence-associated beta-galactosidase activity, expression of p53 and p21 proteins as well as of proteins involved in the regulation of inflammation, such as IL-6 or IL-8, activation of oncogenes, etc. Thus, “aging/senescence” of cells does not occur simply by itself—it takes place because of the influence of DNA-damaging agents. This approach, in my opinion, despite being very important to define a strategy to fight cancer, distracts us, yet again, from the study of the real mechanisms of aging. It should be emphasized that the “stationary phase aging” model developed in my laboratory also allows registering the occurrence of certain biomarkers of aging in cultured cells, but in this case they arise due to the restriction of their proliferation by contact inhibition, i.e., due to a rather physiological impact, which does not cause any damage to cells by itself (the situation is similar to what we observe in a whole multicellular organism).     

Self-organization in the ontogeny of multicellular organisms: a computer simulation

The progress in understanding the patterns of evolution of ontogeny is hindered by the fact that many features of ontogeny are counterintuitive (as well as the features of other processes related to self-organization, self-assembly, and spontaneous increase in complexity). The basic principle of ontogeny of multicellular organisms is that it is the process of self-assembly of ordered multicellular structures by means of coordinated behavior of many individual modules (cells), each of which follows the same set of"rules" encoded in the genome. These rules are based on the genetic regulatory networks. We hypothesize that many specific features of ontogeny that seem nontrivial or enigmatic are, in fact, the inevitable consequences of this basic principle. If so, they do not need special explanations. In order to verify this hypothesis, we developed the computer program "Evo-Devo" based on the above principle. The program is designed to model the self-assembly of ordered multicellular structures from an aggregation of dividing cells that originate from a single original cell (zygote). Each cell follows a set of rules of behavior ("genotype") that can be specified arbitrarily by the experimenter, and is the same for all cells in the embryo (each cell is programmed in exactly the same way as all other cells). It is not allowed to specify rules for groups of cells or for the whole embryo: only local rules that should be followed at the level of a single cell are possible. The analysis of phenotypic implementation of different genotypes revealed several features which are present in the ontogeny of real organisms and are regularly reproduced in the model. These include: inherent stochasticity; inescapable necessity of development of stabilizing adaptations based on negative feedback in order to decrease this stochasticity; equifinality (noise resistance) resulting from these adaptations; the ability of ontogeny to respond to major perturbations by generating new morphological structures that differ from the "normal" ones, but have similar level of complexity; the similarity of phenotypic manifestations of different mutations; channeling of possible evolutionary transformations of ontogeny; Waddington's creodes; high probability of destabilization of ontogeny (e.g., because of mutations); the possibility of a new morphological character to appear initially as a rare anomaly (low penetrance of many mutations); pleiotropy of mutations affecting ontogeny; spontaneous emergence of morphogenetic correlations; integrity of the developing organism. The fact that these features are regularly reproduced in the model implies that they are probably the inevitable consequences of the basic principle of ontogeny of multicellular organisms formulated above.     

Some remarks on the relationship between autophagy,cell aging,and cell proliferation restriction

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.     

Theoretical prerequisites for an experimental corroboration of the existence of a specialized cellular system in control of multicellular organism tissue proliferation

A theoretical analysis of cell proliferation as a selflimiting process designed to maintain the integrity of an entire multicellular organism and based on the principles of a "hypercycle" suggests the need for the existence, starting at a certain level of multicellular organization, of a specialized system in control of tissue proliferation, a system represented by a body of cells capable of both stimulating and inhibiting the proliferation of a variety of cell types. An analysis of experimental data in different fields of the biological science points to certain T-cell populations as probable candidate for the role of cellular regulators of tissue proliferation. Using as an example the induction of murine liver regeneration by the administration of CCL4, the author demonstrates the dynamics of the formation of cells stimulating and inhibiting regeneration, which conforms well to theoretical considerations.     

Derivation of rigorous conditions for high cell-type diversity by algebraic approach

The development of a multicellular organism is a dynamic process. Starting with one or a few cells, the organism develops into different types of cells with distinct functions. We have constructed a simple model by considering the cell number increase and the cell-type order conservation, and have assessed conditions for cell-type diversity. This model is based on a stochastic Lindenmayer system with cell-to-cell interactions for three types of cells. In the present model, we have successfully derived complex but rigorous algebraic relations between the proliferation and transition rates for cell-type diversity by using a symbolic method: quantifier elimination (QE). Surprisingly, three modes for the proliferation and transition rates have emerged for large ratios of the initial cells to the developed cells. The three modes have revealed that the equality between the development rates for the highest cell-type diversity is reduced during the development process of multicellular organisms. Furthermore, we have found that the highest cell-type diversity originates from order conservation.     

Pathways regulating apoptosis during patterning and development

The patterning and development of multicellular organisms require a precisely controlled balance between cell proliferation, differentiation and death. The regulation of apoptosis is an important aspect to achieve this balance, by eliminating unnecessary or mis-specified cells which, otherwise, may have harmful effects on the whole organism. Apoptosis is also important for the morphogenetic processes that occur during development and that lead to the sculpting of organs and other body structures. Here, we review recent progress in understanding how apoptosis is regulated during development, focusing on studies using Drosophila or Caenorhabditis elegans as model organisms.     

3D printing to construct in vitro multicellular models of melanoma

Currently, there is a lack of suitable models for in-vitro studies of malignant melanoma and traditional single cell culture models no longer reproduce tumor structure and physiological complexity well. The tumor microenvironment is closely related to carcinogenesis and it is particularly important to understand how tumor cells interact and communicate with surrounding nonmalignant cells. Three-dimensional (3D) in vitro multicellular culture models can better simulate the tumor microenvironment due to their excellent physicochemical properties. In this study, 3D composite hydrogel scaffolds were prepared from gelatin methacrylate and polyethylene glycol diacrylate hydrogels by 3D printing and light curing techniques, and 3D multicellular in vitro tumor culture models were established by inoculating human melanoma cells (A375) and human fibroblasts cells on them. The cell proliferation, migration, invasion, and drug resistance of the 3D multicellular in vitro model was evaluated. Compared with the single-cell model, the cells in the multicellular model had higher proliferation activity and migration ability, and were easy to form dense structures. Several tumor cell markers, such as matrix metalloproteinase-9 (MMP-9), MMP-2, and vascular endothelial growth factor, were highly expressed in the multicellular culture model, which were more favorable for tumor development. In addition, higher cell survival rate was observed after exposure to luteolin. The anticancer drug resistance result of the malignant melanoma cells in the 3D bioprinted construct demonstrated physiological properties, suggesting the promising potential of current 3D printed tumor model in the development of personalized therapy, especially for discovery of more conducive targeted drugs.     

Does Aging Have a Purpose?

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.     

IgH-2 cells: a reptilian model for apoptotic studies

Regulation of proper cell number in tissues depends upon a balance between cell proliferation and cell death. The process of apoptosis has thus far been studied in a variety of multicellular organisms from humans to higher plants. In order to broaden our perspective and identify another metazoan system with which to deepen our understanding of the function and evolution of the apoptotic machinery, we have characterized cell death in a reptilian cell line. We show that the death of IgH-2 Iguana (Iguana iguana) heart cells [Clark, H.F., Cohen, M.M., Karzon, D.T., 1970. Characterization of reptilian cell lines established at incubation temperatures of 23 to 36 degrees. Proc. Soc. Exp. Biol. Med. 133, 1039-1047.] is, in response to DNA damaging agents, accompanied by classic morphological changes of apoptosis including detachment from the substrate, cell shrinkage, nuclear pyknosis and externalization of the plasma membrane phospholipid phosphatidylserine. Our biochemical studies show that the death of IgH-2 cells is accompanied by internucleosomal DNA fragmentation and activation of caspases. Our studies with the pan-caspase inhibitor zVAD.fmk implicate caspases in the apoptotic process we observe. This work represents the first detailed molecular and biochemical analysis of apoptosis in cells of an organism of class Reptilia and establishes IgH-2 cells as a suitable model system with which to investigate the phenomenon of caspase dependent apoptosis and the apoptotic machinery in a reptilian model.     

The evolution of robust development and homeostasis in artificial organisms

During embryogenesis, multicellular animals are shaped via cell proliferation, cell rearrangement, and apoptosis. At the end of development, tissue architecture is then maintained through balanced rates of cell proliferation and loss. Here, we take an in silico approach to look for generic systems features of morphogenesis in multicellular animals that arise as a consequence of the evolution of development. Using artificial evolution, we evolved cellular automata-based digital organisms that have distinct embryonic and homeostatic phases of development. Although these evolved organisms use a variety of strategies to maintain their form over time, organisms of different types were all found to rapidly recover from environmental damage in the form of wounds. This regenerative response was most robust in an organism with a stratified tissue-like architecture. An evolutionary analysis revealed that evolution itself contributed to the ability of this organism to maintain its form in the face of genetic and environmental perturbation, confirming the results of previous studies. In addition, the exceptional robustness of this organism to surface injury was found to result from an upward flux of cells, driven in part by cell divisions with a stable niche at the tissue base. Given the general nature of the model, our results lead us to suggest that many of the robust systems properties observed in real organisms, including scar-free wound-healing in well-protected embryos and the layered tissue architecture of regenerating epithelial tissues, may be by-products of the evolution of morphogenesis, rather than the direct result of selection.     

Age‐related AMP‐activated protein kinase alterations: From cellular energetics to longevity

5’ adenosine monophosphate‐activated protein kinase (AMPK) is a key regulator of energy in the cell, which allows the cell/organism to survive with deficit of ATP. Since AMPK is involved in the adaptation to caloric restriction, the role of age‐related changes in AMPK activity in both the aging organism and the aging cell is actively investigated in gerontology. Studies on yeast, worms, flies, rodents, and primates have demonstrated an important effect of this regulator on key signalling pathways involved in the aging process. In some cases, researchers conclude that AMPK promotes aging. However, in our opinion, in such cases, we observe a disturbance in the adaptive ability because of the prolonged cell/organism presence in stressful conditions because the functional capacity of any adaptation system is limited. Interestingly, AMPK can regulate metabolic processes in noncell‐autonomous manner. The main effects of AMPK activation in the cell are realized in restriction of proliferation and launching autophagy. In tissues of an aging organism, the ability of AMPK to respond to energy deficit decreases; this fact is especially critical for organs that contain postmitotic cells. In this review, we have tried to consider the involvement of AMPK in age‐related changes in the cell and in the organism.