Results and perspectives of cytogerontologic studies in modern time

The overwhelming majority of research in the field of cytogerontology (i.e. investigating mechanisms of aging in experiments with cultured cells) has been done using the widely applied Hayflick's model. More than 40 years have passed since the appearance of the model, and during this time numerous data were obtained on its basis. The data significantly contributed to our knowledge of the behavior of cultured animal and human cells. In particular, we know enough about the in vitro aging phenomenon. But in my opinion, little has changed in our knowledge of aging in the whole organism. This may be, presumably, because Hayflich's model, like many other models used in experimental gerontology, is correlative, i.e. based on a great variety of detected correlations. In Hayflick's model these are correlations between the cell mitotic potential (cell population doubling potential) and the number of "gerontological" parameters and indices, such as the species life span, donor's age, evidence of progeroid syndromes, etc, and also correlations between various changes of normal (diploid) cells during a long-term cultivation and in the course of organismal aging. However, it is well known that a good correlation does not frequently have anything in common with the essence (gist) of the phenomenon under investigation. For example, the amount of grey hair in the individual is known to excellently correlate with his or her age, being, however, in no way associated with mechanisms of aging or probability of death. In this case, the absence of cause-effect relationships is evident. But it is these particular relationships that are totally indispensable for gist models developing. Such models, different from the correlative ones, are based on a definite concept of aging phenomenon. With the Hayflick's model, such a concept is absent, since using "Hayflick's limit" one cannot explain why the human organism is aging eventually. This can be exemplified by a discovery of a telomere mechanism, which is claimed to determine cell aging in vitro. This discovery triggered an outburst of theories aimed to explain on its basis as well the process of aging in vivo. However, now it is clear that mechanisms of the whole organism aging, hidden, presumably, in its postmitotic cells (neurons or cardiomyocytes) cannot be accounted for by this approach. In view of all stated above, we consider as indispensable the elaboration of "gist" models of aging using cultured cells. Mechanism of cell aging in these models must be similar to those in the whole organism. We believe that one of such models may be our "stationary phase aging" model, based on an assumption of the leading role of cell proliferation restriction in aging. We assume that accumulation of "senile" damage may by caused by the restriction of cell proliferation due to both the formation of differentiated cell populations in the course of development, and the existence of saturation density phenomenon (in vitro). Cell proliferation changes by themselves do not induce any aging processes, but lead only to accumulating macromolecular defects, which in their turn generate deterioration of tissues, organs, and eventually of the whole organism, thus increasing the probability of its death. Within the framework of our model, we define cell aging as the accumulation in a cell population of different types of damage identical to the damage arising in senscencing multicellular organism. And finally, we consider as very important the future studies aimed to determine the process of cell dying and cell death in general. Availability of such definitions would help to draw real parallels between the "genuine" cell aging (i.e. the increased probability of cell destruction with "age") and aging of the multicellular organism.     

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).     

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.     

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.     

Constitutive and induced functions of the p53 gene

The p53 tumor suppressor serves to secure genetic stability of multicellular organisms. It suppresses the accumulation of mutations in somatic cells and substantially decreases the probability of malignant diseases. The p53 gene acts highly selectively through multiple mechanisms. Under relatively favorable conditions, p53 helps to maintain intracellular homeostasis by balancing anabolic and catabolic processes and by timely elimination of reactive oxygen species. These functions of p53 facilitate maximal efficiency and survival of cells under conditions of physiological stresses. In the case of grave molecular damage caused by severe stress, a significant amount of highly active p53 is induced leading to irreversible growth arrest and programmed cell death. The induced functions of p53 secure the timely elimination from the organism of damaged and potentially dangerous cells. Collectively, the functions of p53 contribute to the prevention of malignant and other diseases and decelerate the aging process.     

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.     

Pathways of apoptosis and importance in development

The elimination of cells by programmed cell death is a fundamental event in development where multicellular organisms regulate cell numbers or eliminate cells that are functionally redundant or potentially detrimental to the organism. The evolutionary conservation of the biochemical and genetic regulation of programmed cell death across species has allowed the genetic pathways of programmed cell death determined in lower species, such as the nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster to act as models to delineate the genetics and regulation of cell death in mammalian cells. These studies have identified cell autonomous and non-autonomous mechanisms that regulate of cell death and reveal that developmental cell death can either be a pre-determined cell fate or the consequence of insufficient cell interactions that normally promote cell survival.     

Autophagy, mitochondria and cell death in lysosomal storage diseases

Lysosomal storage diseases (LSDs) are debilitating genetic conditions that frequently manifest as neurodegenerative disorders. They severely affect eye, motor and cognitive functions and, in most cases, abbreviate the lifespan. Postmitotic cells such as neurons and mononuclear phagocytes rich in lysosomes are most often affected by the accumulation of undegraded material. Cell death is well documented in parts of the brain and in other cells of LSD patients and animal models, although little is known about mechanisms by which death pathways are activated in these diseases, and not all cells exhibiting increased storage material are affected by cell death. Lysosomes are essential for maturation and completion of autophagy-initiated protein and organelle degradation. Moreover, accumulation of effete mitochondria has been documented in postmitotic cells whose lysosomal function is suppressed or in aging cells with lipofuscin accumulation. Based upon observations in the literature and our own data showing similar mitochondrial abnormalities in several LSDs, we propose a new model of cell death in LSDs. We suggest that the lysosomal deficiencies in LSDs inhibit autophagic maturation, leading to a condition of autophagic stress. The resulting accumulation of dysfunctional mitochondria showing impaired Ca2+ buffering increases the vulnerability of the cells to pro-apoptotic signals.     

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).     

Is There a Molecular Logic That Sustains Neuronal Functional Integrity and Survival? Lipid Signaling Is Necessary for Neuroprotective Neuronal Transcriptional Programs

A challenge to civilization is the growing incidence in the loss of sight and cognition due to increased life expectancy. Therefore, we are confronted with a rise in the occurrence of photoreceptor- and neuronal-survival failure, as reflected mainly by age-related macular degeneration (AMD) and Alzheimer’s disease (AD). Nervous system development is driven by neuronal apoptotic cell death, and thereafter, for the entire lifespan of an organism, neurons are postmitotic cells. In neurodegenerative diseases, apoptosis and other forms of cells death lead to selective neuronal loss. Although age is the main risk factor, not everyone develops these diseases during aging. Despite decades of important findings about neuronal cell death, the specific mechanisms that regulate neuronal survival remain incompletely understood.     

Die for the community: an overview of programmed cell death in bacteria

Programmed cell death is a process known to have a crucial role in many aspects of eukaryotes physiology and is clearly essential to their life. As a consequence, the underlying molecular mechanisms have been extensively studied in eukaryotes and we now know that different signalling pathways leading to functionally and morphologically different forms of death exist in these organisms. Similarly, mono-cellular organism can activate signalling pathways leading to death of a number of cells within a colony. The reason why a single-cell organism would activate a program leading to its death is apparently counterintuitive and probably for this reason cell death in prokaryotes has received a lot less attention in the past years. However, as summarized in this review there are many reasons leading to prokaryotic cell death, for the benefit of the colony. Indeed, single-celled organism can greatly benefit from multicellular organization. Within this forms of organization, regulation of death becomes an important issue, contributing to important processes such as: stress response, development, genetic transformation, and biofilm formation.     

Informational hypothesis of aging: How does the germ line “avoid” aging?

An informational hypothesis of aging is formulated, and a model is proposed for the survival of a population of multicellular organisms under conditions of informational degradation of the cell genetic material (accumulation of random errors in the genome). The germline cells are not anyhow privileged in this respect, but the genetic material undergoes “rejuvenation” (decrease in the number of errors) during its transition from parents to progeny, owing to meiotic crossover (which changes the error density in individual gametes) and subsequent selection of the most functional (i.e. most error-free) gametes to form the progeny genomes. With a simulation-type model, it is shown that such a mechanism ensures stability of the population gene pool in many generations, though in any individual organism the amount of genome errors in all cells increases with aging.

     

The evolution of cell death programs as prerequisites of multicellularity

One of the hallmarks of multicellularity is that the individual cellular fate is sacrificed for the benefit of a higher order of life-the organism. The accidental death of cells in a multicellular organism results in swelling and membrane-rupture and inevitably spills cell contents into the surrounding tissue with deleterious effects for the organism. To avoid this form of necrotic death the cells of metazoans have developed complex self-destruction mechanisms, collectively called programmed cell death, which see to an orderly removal of superfluous cells. Since evolution never invents new genes but plays variations on old themes by DNA mutations, it is not surprising, that some of the genes involved in metazoan death pathways apparently have evolved from homologues in unicellular organisms, where they originally had different functions. Interestingly some unicellular protozoans have developed a primitive form of non-necrotic cell death themselves, which could mean that the idea of an altruistic death for the benefit of genetically identical cells predated the invention of multicellularity. The cell death pathways of protozoans, however, show no homology to those in metazoans, where several death pathways seem to have evolved in parallel. Mitochondria stands at the beginning of several death pathways and also determines, whether a cell has sufficient energy to complete a death program. However, the endosymbiotic bacterial ancestors of mitochondria are unlikely to have contributed to the recent mitochondrial death machinery and therefore, these components may derive from mutated eukaryotic precursors and might have invaded the respective mitochondrial compartments. Although there is no direct evidence, it seems that the prokaryotic-eukaryotic symbiosis created the space necessary for sophisticated death mechanisms on command, which in their distinct forms are major factors for the evolution of multicellular organisms.     

Modelling the formation of necrotic regions in avascular tumours

The mechanisms underlying the formation of necrotic regions within avascular tumours are not well understood. In this paper, we examine the relative roles of nutrient deprivation and of cell death, from both the proliferating phase of the cell cycle via apoptosis and from the quiescent phase via necrosis, in changing the structure within multicellular tumour spheroids and particularly the accumulation of dead cell material in the centre. A mathematical model is presented and studied that accounts for nutrient diffusion, changes in cell cycling rates, the two different routes to cell death as well as active motion of cells and passive motion of the dead cell material. In studying the accumulation of dead cell matter we do not distinguish between the route by which each was formed. The resulting mathematical model is examined for a number of scenarios. Results show that in many cases the size of the necrotic core is closely correlated with low levels in nutrient concentration. However, in certain cases, particularly where the rate of necrosis is large, the resulting necrotic core can lead to regions of non-negligible nutrient concentration-dependent upon the mode of cell death.     

Systems biology of yeast cell death

Programmed cell death (PCD) (including apoptosis) is an essential process, and many human diseases of high prevalence such as neurodegenerative diseases and cancer are associated with deregulations in the cell death pathways. Yeast Saccharomyces cerevisiae, a unicellular eukaryotic organism, shares with multicellular organisms (including humans) key components and regulators of the PCD machinery. In this article, we review the current state of knowledge about cell death networks, including the modeling approaches and experimental strategies commonly used to study yeast cell death. We argue that the systems biology approach will bring valuable contributions to our understanding of regulations and mechanisms of the complex cell death pathways.     

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.     

Phenoptosis: programmed death of an organism

Programmed cell death (apoptosis) is well-established in many multicellular organisms. Apoptosis purifies a tissue from cells that became useless or even harmful for the organism. Similar phenomena are already described also at subcellular level (suicide of mitochondria, i.e., mitoptosis) as well as at supracellular level (degradation of some organs temporarily appearing in the course of ontogenesis and then disappearing by means of apoptosis of the organ-composing cells). Following the same logic, one may put a question about programmed death of an organism as a mechanism of purification of a kin, community of organisms, or population from individuals who became unwanted for this kin, etc. A putative mechanism of such kind is proposed to be coined "phenoptosis" by analogy with apoptosis and mitoptosis. In a unicellular organism (the bacterium Escherichia coli), three different biochemical mechanisms of programmed death are identified. All of them are actuated by the appearance of phages inside the bacterial cell. This may be regarded as a precedent of phenoptosis which prevents expansion of the phage infection among E. coli cells. Purification of a population from infected individuals looks like an evolutionary invention useful for a species. Such an invention has high chances to be also employed by multicellular organisms. Most probably, septic shock in animals and humans serves as an analog of the phage-induced bacterial phenoptosis. It is hypothesized that the stress-induced ischemic diseases of brain and heart as well as carcinogenesis if they are induced by repeated stresses also represent phenoptoses that, in contrast to sepsis, are age-dependent. There are interrelations of programmed death phenomena at various levels of complexity of the living systems. Thus, extensive mitoptosis in a cell leads to apoptotic death of this cell and extensive apoptosis in an organ of vital importance results in phenoptotic death of an individual. In line with this logic, some cases are already described when inhibition of apoptosis strongly improves the postischemic state of the organism.     

"Candidatus Midichloria" endosymbionts bloom after the blood meal of the host, the hard tick Ixodes ricinus

"Candidatus Midichloria mitochondrii," an intracellular symbiont of the tick Ixodes ricinus, is the only described organism able to invade the mitochondria of any multicellular organism. We used quantitative PCR to examine cycles of bacterial growth and death throughout the host's development and found that they correspond with the phases of engorgement and molt, respectively.