Culture medium pH and stationary phase/chronological aging of different cells

Morgunova G.V., Klebanov A.A., Khokhlov A.N.

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.

Morgunova G.V., Klebanov A.A., Khokhlov A.N.

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.

Khokhlov A.N.

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.

Yucel E.B., Eraslan S., Ulgen K.O.

Because of its multifactorial nature, aging is one of the most complicated cell phenomena known. A systems biology approach, which aims to understand the organism as a whole rather than concentrate on the behaviors of individual genes, thus comprises a seamless tool for investigating the aging machinery, which arises mainly as a result of degeneration of the collaboration between signaling and regulatory pathways. In the present study, the effects of medium buffering on the chronological life span are investigated via transcriptome analyses and subsequent integration of the data obtained with the chronological aging network of yeast. The comparative inquiry of transcriptome data of young and old cells grown in buffered and unbuffered media reveals new roles for pH control (e.g. the re‐organization of lipid metabolism and intracellular signaling cascades) that have beneficial consequences on chronological longevity. Integration of the transcriptome data onto the aging network, as well as validation experiments, suggest that Snf1p is a possible intermediate player in the interjunction of sphingolipid and ergosterol metabolisms with extracellular pH control with respect to regulation of the chronological life span. Consequently, a more detailed insight of the chronological aging mechanism of yeast is obtained. The results of the present study provide a solid basis for further research focusing on uncovering the agents that affect aging and age‐related diseases in humans.

Khokhlov A.N., Klebanov A.A., Karmushakov A.F., Shilovsky G.A., Nasonov M.M., Morgunova G.V.

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).

Wasko B.M., Carr D.T., Tung H., Doan H., Schurman N., Neault J.R., Feng J., Lee J., Zipkin B., Mouser J., Oudanonh E., Nguyen T., Stetina T., Shemorry A., Lemma M., et. al.

During chronological aging of budding yeast cells, the culture medium can become acidified, and this acidification limits cell survival. As a consequence, buffering the culture medium to pH 6 significantly extends chronological life span under standard conditions in synthetic medium. In this study, we assessed whether a similar process occurs during replicative aging of yeast cells. We find no evidence that buffering the pH of the culture medium to pH levels either higher or lower than the initial pH of the medium is able to significantly extend replicative lifespan. Thus, we conclude that, unlike chronological life span, replicative life span is not limited by acidification of the culture medium or by changes in the pH of the environment.

Khokhlov A.N.

There is a viewpoint that suppression of the proliferative capacity of cells and impairment of the regeneration of tissues and organs in aging are a consequence of specially arisen during evolution mechanisms that reduce the risk of malignant transformation and, thus, protect against cancer. We believe that the restriction of cell proliferation in an aging multicellular organism is not a consequence of implementing a special program of aging. Apparently, such a program does not exist at all and aging is only a "byproduct" of the program of development, implementation of which in higher organisms suggests the need for the emergence of cell populations with very low or even zero proliferative activity, which determines the limited capacity of relevant organs and tissues to regenerate. At the same time, it is the presence of highly differentiated cell populations, barely able or completely unable to reproduce (neurons, cardiomyocytes, hepatocytes), that ensures the normal functioning of the higher animals and humans. Apparently, the impairment of regulatory processes, realized at the neurohumoral level, still plays the main role in the mechanisms of aging of multicellular organisms, not just the accumulation of macromolecular defects in individual cells. It seems that the quality of the cells themselves does not worsen with age as much as reliability of the organism control over cells, organs and tissues, which leads to an increase in the probability of death.

Khokhlov A.

According to our conception, the aging process is caused by cell proliferation restriction-induced accumulation of various macromolecular defects (mainly DNA damage) in cells of a mature organism or in a cell population. In the case of cell cultures, the proliferation restriction is related to so-called contact inhibition and to the Hayflick's limit, while in the case of multicellular organisms, it is related to the appearance, in the process of differentiation, of organs and tissues consisting of postmitotic and very slowly dividing cells. It is assumed that the proliferation of intact cells prevents accumulation of various errors in a cell population. However, the continuous propagation of all the cells in a multicellular organism is absolutely incompatible with its normal functioning. Thus, the program of development, when it generates postmitotic or slowly dividing cells, automatically leads also to the onset of the aging process (mortality increase with age). Therefore, any additional special program for aging simply becomes unnecessary. This, however, doesn't reject, for some organisms, the reasonability of programmed death, which makes possible the elimination of harmful, from the species point of view, individuals. It is also very important to emphasize that increase or decrease of an organism's lifespan under the effects of various external factors is not always necessarily related to modification of the aging process, though the experimental results in the field are usually interpreted in just this way. I called the experimental-gerontological models similar to the Hayflick's model "correlative", since they are based on some correlations only and not related necessarily to the gist of the aging phenomenon. So, for the Hayflick's model, it is the relationship between population doubling level and donor age, between population doubling potential and species lifespan, between some cell changes in vivo and in vitro, and so forth. If the rationale of the "Hayflick phenomenon" is used, we can't explain why we age. Nevertheless, many authors virtually put a sign of equality between aging in vitro and aging in vivo, which generates conclusions that are of quite doubtful accuracy. A classic illustration of this is the telomere concept of aging. Originally, the principle of shortening end-segments of DNA (telomeres) during each cell division was formulated at the beginning of seventies by the Russian scientist Aleksey Olovnikov and used by him to explain the limited "proliferative" lifespan in vitro of normal cells. Subsequently, the existence of this phenomenon was confirmed by the results of many research reports, the culmination of which was a publication in which the authors demonstrated the possibility of increasing the proliferative potential of normal cells by introducing the enzyme telomerase to them, thus restoring the lost telomere segments. At the moment it looks like the telomere shortening contributes to aging in vitro only, but not to aging in vivo because an organism never realizes the full proliferative potential of its cells. Besides, the most "responsive to aging" are the organs and tissues consisting of postmitotic cells, for which the concept of proliferative potential loses any meaning in practical terms. We developed another "correlative" model--a model for testing of geroprotectors and geropromoters--the "cell kinetics model." It is based on the well-known correlation between the "age" of cultured cells (age of their donor) and their saturation density. The model allowed us to perform preliminary testing of a lot of different compounds and factors that are interesting from a gerontological point of view, but it revealed no information about the real mechanisms of aging. However, the second model we use in our studies--the "stationary phase aging" model--obviously, is a "gist" model. It is based on the assumption that in the cells of stationary cultures various intracellular changes similar to those of an aging organism can be observed. The proliferation restriction in the case is provided, as a rule, just by contact inhibition. Many experimental results confirming this assumption were obtained. "Age-related" changes that are well known from organismal studies were shown to really occur in our experimental stationary cell culture model. Besides, such experiments can be carried out on nearly any type of cells from various biological species. Thus, the evolutionary approach to analysis of the data is provided. Moreover, the changes in the stationary cell cultures become detectable very soon--as a rule, in 2 to 3 weeks after beginning the experiment. All this allows us to suppose that the "stationary phase aging" model should provide a very effective approach to testing of different substances and their cocktails on their activities in terms of accelerating or retarding aging--of course, if their effect is realized on the cell level only.

Gonidakis S., Longo V.D.

Bacteria, which are often considered as avid reproductive organisms under constant selective pressure to utilize available nutrients to proliferate, might seem an inappropriate model to study aging. However, environmental conditions are rarely supporting the exponential growth that is most often studied in laboratories. In the wild, Escherichia coli inhabits environments of relative nutritional paucity. Not surprisingly, under such circumstances, members of an E. coli population age and progressively lose the ability to reproduce, even when environmental conditions provide such an opportunity. Here, we review the methods to study chronological aging in bacteria and some of the mechanisms that may contribute to their age-dependent loss of viability.

Mirisola M.G., Longo V.D.

Comment on: Murakami C, et al. Cell Cycle 2012; 11:3087-96.

Polymenis M., Kennedy B.K.

Comment on: Murakami C, et al. Cell Cycle 2012; 11:3087-96.

Murakami C., Delaney J.R., Chou A., Carr D., Schleit J., Sutphin G.L., An E.H., Castanza A.S., Fletcher M., Goswami S., Higgins S., Holmberg M., Hui J., Jelic M., Jeong K., et. al.

Chronological and replicative aging have been studied in yeast as alternative paradigms for post-mitotic and mitotic aging, respectively. It has been known for more than a decade that cells of the S288C background aged chronologically in rich medium have reduced replicative lifespan relative to chronologically young cells. Here we report replication of this observation in the diploid BY4743 strain background. We further show that the reduction in replicative lifespan from chronological aging is accelerated when cells are chronologically aged under standard conditions in synthetic complete medium rather than rich medium. The loss of replicative potential with chronological age is attenuated by buffering the pH of the chronological aging medium to 6.0, an intervention that we have previously shown can extend chronological lifespan. These data demonstrate that extracellular acidification of the culture medium can cause intracellular damage in the chronologically aging population that is asymmetrically segregated by the mother cell to limit subsequent replicative lifespan.

Longo V., Shadel G., Kaeberlein M., Kennedy B.

Saccharomyces cerevisiae has directly or indirectly contributed to the identification of arguably more mammalian genes that affect aging than any other model organism. Aging in yeast is assayed primarily by measurement of replicative or chronological life span. Here, we review the genes and mechanisms implicated in these two aging model systems and key remaining issues that need to be addressed for their optimization. Because of its well-characterized genome that is remarkably amenable to genetic manipulation and high-throughput screening procedures, S. cerevisiae will continue to serve as a leading model organism for studying pathways relevant to human aging and disease.

Kaeberlein M., Kennedy B.K.

The question of whether aging can be modeled in cell culture has been highly debated among specialists in the field ever since the seminal publication that primary fibroblasts undergo a finite number of population doublings before entering a non-dividing state.1 Although unable to divide, senescent cells can be maintained in a metabolically active state indefinitely if handled appropriately. Initial thoughts were that an accumulation of senescent cells in aging individuals, a now generally accepted phenomenon, would deplete the ability of tissues to repopulate; however, more recent findings indicate that senescent cells have altered secretion profiles that may promote inflammation (and aging itself) in an autocrine fashion.2 Further suggesting a role for senescent cells in aging, a recent report finds that eliminating senescent cells in mice enhances survival of a mouse progeria model.3 But are studies of replicative senescence the only way to model aging in cell culture? Motivated by studies of chronological life span (CLS) in yeast showing that survival in the nutrient-depleted non-proliferaive state was limited by acidification of the culture medium resulting from accumulation of acetic acid,4,5 Leontieva and Blagosklonny6 report in a recent issue of Aging that, at least for cancer cells, acid-induced chronological senescence may also exist for mammalian cells in culture. Chronological aging in yeast is typically studied by culturing cells into stationary phase, maintaining the non-dividing cells in the expired culture medium and periodically measuring cell viability.7 When standard 2% glucose synthetic defined medium is used, the pH of the culture drops from around 4 to below 3 within 2–4 d. Buffering the medium to 6.0 extends CLS as robustly as calorie restriction,4 which is accomplished by reducing initial glucose concentration of the medium.8 Interestingly, calorie restriction itself causes the culture pH to become more alkaline, suggesting that the CLS extension from calorie restriction occurs from reduced acetic acid accumulation and toxicity.9 Although it had been previously reported that alkalinization of the culture medium by addition of NaOH could prolong chronological lifespan,10,11 this observation was not widely known outside of the yeast chronological aging community. The mammalian phenotype is brought about by leaving confluent cancer cells in spent media, which, as any scientist with experience in cell culture knows, turn from red to yellow, and can lead to a rapid loss of cell viability as assessed by plating the cells to new media and counting clones. Leontieva and Blagosklonny show that the color change reflects a reduction in pH brought about by an accumulation of lactic acid. As with yeast, buffering pH extends survival and addition of acid (lactic instead of acetic) shortens the survival period of cells in unspent media. The parallels between yeast CLS and mammalian chronological senescence are intriguing. Both acids are produced primarily as a by-product of fermentative metabolism, and both are known to induce an apoptosis and/or necrosis. Notably, Leontieva and Blagosklonny6 showed that inhibition of mTORC1 by treatment with rapamycin is sufficient to protect cancer cells against lactic acid-induced senescence, a finding strikingly similar to the effect of rapamycin on chronological life span in yeast.12,13 In both cases, the positive effect of rapamycin may be mediated by reduced acid accumulation. It will be of interest to learn whether similar metabolic changes mediated by rapamycin underlie its effects in both systems and, perhaps, in whole animals. A key question is whether the effects of acid on cultured cells is relevant for understanding aging in people. Are there environments in the organism that become more acidic with age, perhaps through cellular release of fermentation-derived acids? It has also been suggested that the burst of reactive oxygen species caused by acetic acid in yeast may mimic aspects of normal aging.14 Interestingly, classic scavengers of free radicals, like NAC, also extend survival in the mammalian chronological senescence assay.6 While the relationship to aging organisms remains unclear, findings in the Leontieva and Blagosklonny study indicate that at least at the cellular level, excess fermentative production of organic acid may be a conserved contributor to the aging of post-replicative cells.

Fabrizio P., Wei M.

The yeast chronological life span (CLS) model has led to the identification of the pro-aging effects of the TOR-Sch9 /S6K and Ras-Adenylate cyclase-PKA pathways, components of which play conserved role in nutrient sensing and aging in mammals [1-4]. One of the early changes that occurs in yeast cells grown in media containing 2% glucose and excess amino acids is the production of acetic acid and acidification of the medium to below pH 4. This acidification has been shown to accelerate yeast aging [5-9]. However, it is clear that it does not explain the effect of the TOR-Sch9/S6K and Ras-AC-PKA pathways on aging since their inhibition extends chronological life span in media that is not acidified and that does not contain acetic acid [10]. The assumption that acetic acid is an organic toxin, which is the key mediator of chronological aging under standard conditions, is probably not true for most genetic backgrounds, since under physiological conditions acetic acid is generated at low levels compared to another metabolite, ethanol [6-7, 11]. Additionally, acetic acid, in spite of its potential toxicity, represents one among several carbon sources that can be utilized by Saccharomyces cerevisiae for growth and metabolism [12-15]. In previous issue of Aging, Leontieva and Blagosklonny describe a yeast-like chronological senescence (CS) model in mammalian cells (Leontieva and Blagosklonny). They show that human tumor cells maintained in stationary culture lose their viability (colony forming units) and that this process is accelerated by medium acidification caused in part by lactate accumulation, which mirrors the accumulation of ethanol and some acetic acid, and the acidification of the medium in S. cerevisiae [5-7, 9]. In yeast, the ethanol accumulated during the growth phase can be used as carbon source during the diauxic shift and the post-diauxic phase, when cells stop dividing and switch from a fermentation- to a respiration-based metabolism [5, 16-17]. Long-lived mutants with deficiencies in the TOR- Sch9/S6K and Ras-AC-PKA pathways deplete ethanol, show a reduced accumulation of extracellular acetic acid [6, 11](M. Wei unpublished results) as well as activate glycerol biosynthesis [11]. As opposed to glucose and ethanol and, possibly, acetic acid, glycerol does not elicit adverse effects on cellular protection and life span suggesting that the Tor1/Sch9-regulated glycerol biosynthesis results in the removal of pro-aging carbon sources [11]. Leontieva and Blagosklonny show that the “yeast-like” chronological senescence in mammalian cells is delayed and attenuated by the inhibition of the mTOR and PI3K signaling pathways, both of which have been implicated in longevity regulation in organisms ranging from yeast to mice. Conditioned medium produced by rapamycin-treated cells was less toxic in inducing CS. However, the addition of rapamycin did not protect fibrosarcoma cells from high concentration of lactate suggesting that rapamycin did not protect cells from CS per se. Rather, inhibition of mTOR affected cellular metabolism and inhibited lactate production during the early phase of stationary survival, which led to a reduced initial lactate accumulation and delayed CS (Leontieva and Blagosklonny). Interestingly, mTOR was spontaneously inactivated after one day in culture, possibly a protective response to lactate accumulation and medium acidification. These results suggest that mTOR promotes CS by favoring lactate production and medium acidification in agreement with the role for TOR-Sch9/S6K in promoting ethanol and acetic acid accumulation in yeast [5, 11, 18]. By contrast, the deletion of either TOR1 or SCH9/S6K are known to extend yeast chronological life span in part by depleting ethanol and acetic acid but largely by mechanisms that are cell autonomous [10-11, 19-21]. It has been argued that acidification of the culture medium and the accumulation of non-fermentable carbon sources such as ethanol and acetic acid render the CLS a paradigm for the identification of “private” mechanisms specific for yeast chronological aging [7, 22-23]. However, not only the yeast CLS method has been remarkably effective in discovering genes later shown to promote aging in mammals [4], it has also revealed the multi-factorial nature of yeast chronological senescence and points to the involvement of diverse cellular processes, such as mitochondrial respiration, reactive oxygen species signaling [1, 19, 24-27], stress response [3, 10, 28], autophagy [29-30], and genome maintenance, in the regulation of life span [31-35]. Although, accumulation of toxic metabolic byproducts may not represent a mechanism of aging in yeast [5-8] or mammalian cells (Leontieva and Blagosklonny [36]), chronological senescence provides a simple model for probing the roles of genes and signaling pathways that affect aging and a powerful platform for high-throughput screening of agents that modulate aging and age-related disease progression.

Keshavarz M., Ahmadi Nasab N.

Over the past decades, cell culture has played an essential role in the development of biology and medicine. As a model system, cell culture has had many applications for comprehending the cellular functions as well as mechanisms of disease production and progression in preclinical studies in various biological fields such as tissue engineering, genetic engineering, cancer research, vaccine production, and drug research. For the first time, in 1907, Ross G. Harrison et al. applied the in vitro cell culture method during a research on the origin of nerve fibers in a kind of frog. They successfully could cultivate the fibers in lymph fluid for several weeks. After that, the cell culture methods constantly have undergone changes and improvements over time for monitoring the function of different types of cells. Nowadays, cell provision sources for in vitro culture include primary cells, secondary cell lines, and more recently, personalized patient-tissue-derived cells.

Morgunova G.V., Shilovsky G.A., Khokhlov A.N.

Abstract The review discusses the role of metabolic disorders (in particular, insulin resistance) in the development of age-related diseases and normal aging with special emphasis on the changes in postmitotic cells of higher organisms. Caloric restriction helps to prevent such metabolic disorders, which could probably explain its ability to prolong the lifespan of laboratory animals. Maintaining metabolic homeostasis is especially important for the highly differentiated long-lived body cells, whose lifespan is comparable to the lifespan of the organism itself. Normal functioning of these cells can be ensured only upon correct functioning of the cytoplasm clean-up system and availability of all required nutrients and energy sources. One of the central problems in gerontology is the age-related disruption of glucose metabolism leading to obesity, diabetes, metabolic syndrome, and other related pathologies. Along with the adipose tissue, skeletal muscles are the main consumers of insulin; hence the physical activity of muscles, which supports their energy metabolism, delays the onset of insulin resistance. Insulin resistance disrupts the metabolism of cardiomyocytes, so that they fail to utilize the nutrients to perform their functions even being surrounded by a nutrient-rich environment, which contributes to the development of age-related cardiovascular diseases. Metabolic pathologies also alter the nutrient sensitivity of neurons, thus disrupting the action of insulin in the central nervous system. In addition, there is evidence that neurons can develop insulin resistance as well. It has been suggested that affecting nutritional sensors (e.g., AMPK) in postmitotic cells might improve the state of the entire multicellular organism, slow down its aging, and increase the lifespan.

Моргунова Г.В., Шиловский Г.А., Хохлов А.Н.

Eigenfeld M., Kerpes R., Becker T.

In yeast, aging is widely understood as the decline of physiological function and the decreasing ability to adapt to environmental changes. Saccharomyces cerevisiae has become an important model organism for the investigation of these processes. Yeast is used in industrial processes (beer and wine production), and several stress conditions can influence its intracellular aging processes. The aim of this review is to summarize the current knowledge on applied stress conditions, such as osmotic pressure, primary metabolites (e.g., ethanol), low pH, oxidative stress, heat on aging indicators, age-related physiological changes, and yeast longevity. There is clear evidence that yeast cells are exposed to many stressors influencing viability and vitality, leading to an age-related shift in age distribution. Currently, there is a lack of rapid, non-invasive methods allowing the investigation of aspects of yeast aging in real time on a single-cell basis using the high-throughput approach. Methods such as micromanipulation, centrifugal elutriator, or biotinylation do not provide real-time information on age distributions in industrial processes. In contrast, innovative approaches, such as non-invasive fluorescence coupled flow cytometry intended for high-throughput measurements, could be promising for determining the replicative age of yeast cells in fermentation and its impact on industrial stress conditions.

Morgunova G.V.

One of the most frequently used model objects in gerontology is yeast, primarily Saccharomyces cerevisiae. Ample data indicating that the lesions in yeast undergoing chronological or stationary phase aging are similar to the age-related lesions in metazoan cells have been accumulated. However, yeast, similarly to any other study objects, also has drawbacks; in particular, although yeast cells are eukaryotes, they are evolutionarily far from mammals. This imposes limitations on the studies of non-conserved metabolic pathways in yeast. In some cases, mammalian cells (for example, Chinese hamster cells) are more suitable for chronological model experiments. They are widely used in industry for manufacturing monoclonal antibodies and recombinant proteins. A significant proportion of these products are produced after cessation of proliferation which initiates chronological aging of the culture. The accumulated data on the features of cell metabolism, as well as growth and duration of the functional activity of the cell culture, are extremely valuable for gerontologists. The exchange of information between these two branches—biotechnological and gerontological—will be beneficial to both of them.

Kirpichnikov M.P., Morgunova G.V., Khokhlov A.N.

Abstract This is a brief review of the latest changes in the editorial policy and content of the Vestnik Moskovskogo Universiteta, Seriya 16: Biologiya journal with special emphasis on the situation with its English-language version Moscow University Biological Sciences Bulletin . The current strategy of the editorial board for evaluation of submitted manuscripts, their rejection, peer reviewing and editing, and the distribution of papers among new sections of the journal are described. The article discusses the requirements for the language of articles, compilation of reference lists, and statistical analysis of the data obtained by the authors. Information is provided on the growth of scientometric indicators of the journal in recent years as well as a list of databases in which the periodical is currently indexed. Differences in interest in published articles between foreign and domestic readers are noted. Data on the number of downloads of the most popular articles from the Springer Nature website are provided. The article analyzes the topics of articles published in the journal in 2017–2019. The authors emphasize the priority for the editorial board of reviews affecting both fundamental and applied aspects of research in the field of biology, biomedicine, and biotechnology.

Guo C., Zhang H., Guan X., Zhou Z.

The anti-aging activity of many plant flavonoids, as well as their mechanisms of action, have been explored in the current literature. However, the studies on the synergistic effects between the different flavonoid compounds were quite limited in previous reports. In this study, by using a high throughput assay, we tested the synergistic effects between different citrus flavonoids throughout the yeast’s chronological lifespan (CLS). We studied the effect of four flavonoid compounds including naringin, hesperedin, hesperitin, neohesperidin, as well as their different combinations on the CLS of the yeast strain BY4742. Their ROS scavenging ability, in vitro antioxidant activity and the influence on the extracellular pH were also tested. The results showed that neohesperidin extended the yeast’s CLS in a concentration-dependent manner. Especially, we found that neohesperidin showed great potential in extending CLS of budding yeast individually or synergistically with hesperetin. The neohesperidin exhibited the strongest function in decreasing the reactive oxygen species (ROS) accumulation in yeast. These findings clearly indicated that neohesperidin is potentially an anti-aging citrus flavonoid, and its synergistic effect with other flavonoids on yeast’s CLS will be an interesting subject for future research of the anti-aging function of citrus fruits.

Khokhlov A.N.

This is a short review concerning the problem of germ line “immortality,” which was already formulated by A. Weismann at the end of the 19th century. Over the following years, it attracted the attention of many gerontologists, who tried to understand the mechanisms of infinite transfer of genetic information from generation to generation with the help of germ cells, which, in contrast to somatic cells, avoid aging in this way. However, it remained unclear how the germ cells of women, which are in fact a population of non-dividing cells (it is similar to stationary phase aging non-subcultured cell culture), provide the mentioned immortality of the germ line. Distinguished Russian gerontologist Zh.A. Medvedev, who passed away recently, published in 1981 his brilliant work “On the Immortality of the Germ Line: Genetic and Biochemical Mechanisms. A Review,” the main points of which are relevant up to today. His paper just discusses the possible mechanisms of such “immortality.” They are analyzed in detail in the current article and can be reduced mainly to the existence of a number of barriers that, in most cases, do not allow progeny to emerge from “old” germ cells (although certain “rejuvenating” processes in the gametes still go). Therefore, children are “born young.” Some alternative approaches to explaining the immortality of the germ line are also considered. Special attention is paid to the “parental age effect” and the role of eggs and sperm cells in this phenomenon.

Khokhlov A.N., Morgunova G.V., Klebanov A.A.

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 antiaging 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 geroprotectors 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.

Morgunova G.V., Karmushakov A.F., Klebanov A.A., Khokhlov A.N.

Partial uncoupling of the processes of oxidative phosphorylation and energy storage in the form of ATP (“mild” uncoupling) helps reduce the production of reactive oxygen species and can also mimic the effect of calorie restriction. A number of studies have shown that uncouplers, such as 2,4-dinitrophenol (DNP), affect the lifespan of Drosophila, yeast, mice, and rats as well as the manifestation of “age-related” changes in cultures of mammalian and human cells undergoing replicative senescence. This paper is devoted to studying the effect of DNP on the growth and subsequent dying out of “stationary phase aging” Chinese hamster cells. Using the method for evaluating the colony-forming efficiency of cells, the maximum permissible concentration was selected, 5 ×10–5 M, in which the substance presumably induces “mild” uncoupling and does not inhibit cell proliferation. At higher concentrations, DNP has a cytotoxic effect on the studied cell culture. Under the influence of DNP in the potentially “mild” uncoupling concentration (5.6 × 10–7 M), the kinetics of cell growth and dying out does not change, and the lifespan of the cell culture does not increase. This effect may be due to the type of cells studied. In addition, there is a probability that the optimal concentration lies in the range from 5 ×10–7 to 5 × 10–5 M or even lower than 5 × 10–7 M.

Morgunova G.V., Klebanov A.A.

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.

Khokhlov A.N.

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.

Morgunova G.V., Klebanov A.A.

Despite the fact that oxidation products of nucleotides and nucleosides are markers of oxidative stress, reports of the paradoxical ability of these compounds to protect cells from the harmful effects of reactive oxygen species began to appear more often. Among all nitrogenous bases, guanine is most susceptible to the influence of oxidative stress; therefore, guanosine is oxidized more often than other bases. In the present work, the effect of exogenous 8-oxo-2′-deoxyguanosine on the growth and “stationary phase aging” (accumulation of “age-related” changes in cultured cells during cell proliferation slowing down within a single passage and subsequent “aging” in the stationary growth phase) of nonsubcultured transformed Chinese hamster cells was studied. We showed that the nucleoside is rapidly absorbed by the cells from the medium, but it does not affect the growth of the culture, and impairs the viability of the cells in the late stationary growth phase. Thus, no mitogenic or geroprotective effect of 8-oxo-2′-deoxyguanosine was found.

Khokhlov A.N., Klebanov A.A., Morgunova G.V.

Recently, a large number of papers have appeared that describe the successful use of various biologically active compounds (short peptides, mitochondrial antioxidants, antidiabetic biguanides, mimetics of dietary restriction, autophagy modulators, etc.) as geroprotectors. However, in our opinion, in most cases, the positive results of such studies are determined by a “successful” selection of control objects. Animals with certain abnormalities are often used for this purpose, so that any favorable effect on the corresponding pathological processes leads to an increase in their lifespan. In addition, control animals can be normal (i.e., wildtype) but placed under certain extreme conditions that can be overcome just by using certain biologically active compounds. Thus, in this case, the treatment of pathologies rather than the effect on fundamental processes of aging is observed. There is a point of view that the results of Clive McCay’s well-known experiments, which have significantly prolonged the life of rats by limiting caloric intake, were determined by the facts that, firstly, the control animals fed ad libitum (which is absolutely untypical for animals in the wild) and, secondly, Fisher-344 rats, which were used in these experiments, are short-lived. The above considerations, apparently, also apply to the gerontological experiments on cultured cells. In particular, we sometimes hear remarks from our colleagues regarding the model of “stationary phase aging” of cell cultures, which is used in our laboratory, due to the fact that most of the experiments are performed on transformed rather than normal cells. However, this approach seems to us quite justified, because the phenomenon of “stationary phase”/chronological aging is common to a wide variety of cells, including bacteria, yeasts, cyanobacteria, mycoplasmas, as well as animal and plant cells. Cells with an unlimited mitotic potential do not change either from experiment to experiment or during long-term cultivation both with and without subcultivation (within the framework of the stationary phase aging model), which cannot be said of the normal diploid fibroblasts, whose telomeres are shortened with each division. In the period from seeding to entering the stationary phase of growth, the cells divide up to ten times! We believe that, to search for effective geroprotectors that affect the fundamental mechanisms of aging, it is necessary to perform studies on “maximally healthy” animals or on “maximally stable” model systems.

Shilovsky G.A., Shram S.I., Morgunova G.V., Khokhlov A.N.

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.