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

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.

Campisi J.

For most species, aging promotes a host of degenerative pathologies that are characterized by debilitating losses of tissue or cellular function. However, especially among vertebrates, aging also promotes hyperplastic pathologies, the most deadly of which is cancer. In contrast to the loss of function that characterizes degenerating cells and tissues, malignant (cancerous) cells must acquire new (albeit aberrant) functions that allow them to develop into a lethal tumor. This review discusses the idea that, despite seemingly opposite characteristics, the degenerative and hyperplastic pathologies of aging are at least partly linked by a common biological phenomenon: a cellular stress response known as cellular senescence. The senescence response is widely recognized as a potent tumor suppressive mechanism. However, recent evidence strengthens the idea that it also drives both degenerative and hyperplastic pathologies, most likely by promoting chronic inflammation. Thus, the senescence response may be the result of antagonistically pleiotropic gene action.

Alinkina E.S., Vorobyova A.K., Misharina T.A., Fatkullina L.D., Burlakova E.B., Khokhlov A.N.

In order to clarify possible cytological mechanisms that underlie the beneficial effects of carvacrol-bearing essential oils on health and mental abilities, we studied one of them (oregano essential oil) in experiments on transformed cultured Chinese hamster cells. Possible cytotoxic or mitogenic effects of the preparation at various concentrations were preliminarily estimated by analyzing the cell culture density after 4 days of cultivation. The preparation concentration in the growth medium (on carvacrol basis) varied from 1 × 10−15 up to 5 × 10−4 M (on carvacrol basis). As a result, two concentrations were selected for further experiments, including 2.5 × 10−5 M as the maximal absolutely non-toxic concentration and 2.5 × 10−4 M as the concentration at which the oregano essential oil decreased approximately 2-fold the final cell density of the grown culture. It was found that the preparation at 2.5 × 10−5 M had no effect on either the colony-forming ability of the cells or the saturation density of the culture (which is a marker of its “biological age”) or kinetics of its “stationary phase aging” (degradation of cultured cells in the stationary phase of growth, similar to age-related changes of the cells in aging organism). On the contrary, the oregano essential oil at 2.5 × 10−4 M abruptly diminished colony-forming ability of the cells and influenced as a “pro-aging” factor on the saturation density of the cell culture and kinetics of the cell death induced by “stationary phase aging.” Based on our own concept of aging and the data obtained, we assumed that detected increase in the life span of mice under the influence of the oregano essential oil could be determined by certain functional changes at the organismal level only, but is not associated with any geroprotective (anti-aging) activity of the preparation, which is manifested at the cellular level and improves the cell viability.

Sikora E., Arendt T., Bennett M., Narita M.

Cellular senescence as the state of permanent inhibition of cell proliferation is a tumour-suppressive mechanism. However, due to the associated secretory phenotype senescence can also contribute to cancer and possibly other age-related diseases, such as obesity, diabetes, atherosclerosis and Alzheimer's disease. There are two major mechanisms of cellular senescence; replicative senescence depends on telomere erosion or dysfunction whilst stress-induced premature senescence (SIPS) is telomere-independent and also includes oncogene-induced senescence (OIS). The senescence phenotype is characterised by altered cellular morphology, increased activity for senescence-associated-β-galactosidase (SA-β-GAL), increased formation of senescence-associated heterochromatin foci (SAHF) and promyelocytic leukemia protein nuclear bodies (PML NBs), permanent DNA damage, chromosomal instability and an inflammatory secretome. Some of these markers have been identified in cells from age-related pathologies. However, to improve our understanding of the contribution of cellular senescence to organismal ageing and age-related disease, it is imperative to define an unequivocal signature of cellular senescence that is functionally connected with normal and pathological ageing. Herein, we describe the processes leading to senescence, and the current biomarkers of cellular senescence, with particular emphasis on the causal role of DNA damage responses involved in the process. We highlight the gaps in our knowledge both of the processes leading to senescence, and the signature of cellular senescence both in vitro and in vivo. A well-defined set of senescence biomarkers for ageing and age-related disease would have a strong impact on the diagnosis, staging and predicted outcomes of age-related disease, providing the basis for a pharmacological intervention to postpone ageing and age-related disease.

Lawless C., Wang C., Jurk D., Merz A., Zglinicki T.V., Passos J.F.

Cellular senescence, the irreversible loss of replicative capacity, might be a tumour suppressor and a contributor to age-related loss of tissue function. The absence of quantitative tests for reliability of candidate markers for senescent cells is a major drawback in cell population studies. Fibroblasts in culture constitute mixed populations of proliferation-competent and senescent cells, with transition between these with increasing population doublings (PD). We estimated senescent fraction in human and mouse fibroblasts with high precision from easily observed growth curves using a dynamic simulation model. We also determined senescent fractions, at various PD (over a wide range of senescent cell frequencies) using candidate senescence markers: Ki67, p21 (CDKN1A), γH2AX, SAHF and Sen-β-Gal either alone or in combination, and compared with those derived from growth curves. This comparison allowed ranking of candidate markers. High rankings were obtained for Sen-β-Gal, SAHFs and the combination of Ki67 negativity with high (>5 per nucleus) γH2A.X foci density in MRC5 fibroblasts. We demonstrate that this latter marker combination, which can easily be performed in paraffin-embedded tissue, gives quantitative senescent cell frequency estimates in mouse embryonic fibroblast cultures and in mouse intestinal sections. The technique presented is a framework for quantitative assessment of markers for senescence.

Khokhlov A.N.

The history of gerontological experiments on cell cultures is reviewed. Cytogerontological studies and aging theories by Weismann, Carrel, Hayflick, and the author are compared. It is emphasized that the basic notion of aging mechanisms was deeply revised several times within the 20th century. It is concluded that at present the aging of multicellular organisms cannot be satisfactorily explained with the help of cytogerontological studie’s data. Experiments on cell cultures need to be combined with fundamental gerontological studies, including survival curve analysis for humans or experimental animals.

Khokhlov A.N.

According to author’s concept, the aging process is caused by cell proliferation restriction-induced accumulation of various macromolecular defects (mainly DNA damage) in cells of an organism or cell population. In case of cell cultures, this proliferation restriction is related to either so-called contact inhibition or Hayflick’s limit, and in case of multicellular organisms, to the appearance, in the process of differentiation, of organs and tissues consisting of postmitotic or very slowly dividing cells. Cell proliferation is absolutely incompatible with normal functioning of a macroorganism. Thus, the development program automatically leads to a situation inducing the aging process (martaliry rates increase with age). Therefore, any special program of aging simply becomes senseless. This, however, does not reject for some organisms the reasonability of programmed death, which makes possible the elimination of harmful, from the species’s point of view, individuals. It is also very important to understand that increase or decrease of organism’s life span under the action of various factors are not necessarily related to a modification of the aging process, even though the experimental results in this field are interpreted just in this way.

Harman D.

My short paper, ‘‘Origin and evolution of the free radical theory of aging: a brief personal history, 1954–2009’’ by myself was recently published in Biogerontology. In this paper I inadvertently failed to make clear the difference between the research fields of oxygen toxicity and aging. I indicated that Drs. Rebeca Gershman and Daniel Gilbert were reported to apparently equate ‘‘oxygen toxicity’’ with ‘‘aging.’’ Although free radicals are involved in both fields (see Harman, D., Ann. N.Y. Acad. Sci., 1067: 10–21, 2006; pages 16–17), time is required to manifest the complex product attributed to aging. Further, the research fields of aging and oxygen toxicity differ clinically. Oxygen toxicity is of relatively little importance to ‘‘man,’’ whereas aging is of major importance. Judging from the literature concerned with both aging and oxygen toxicity, this difference may not be appreciated by some.

CRISTOFALO V.

Kang H.T., Lee C.J., Seo E.J., Bahn Y.J., Kim H.J., Hwang E.S.

Inhibition of human papillomavirus (HPV) E6 and E7 transcription by means of the E2 protein of bovine papillomavirus 1 (BPV1) has been shown to induce acute growth arrest in HPV-positive cervical carcinoma cells. This state of arrest is marked by the expression of senescence phenotypes including SA beta-Gal activity and lipofuscin accumulation. In this study, we examined the reversibility of these phenotypes by exogenously expressing the E6 and E7 genes into HeLa cells growth-arrested by the depletion of E6/E7. Re-expression of E7 (but not E6) in 2 days following E2 transduction induced the cells to resume growth. The proliferating cells manifested the phenotype of untreated HeLa cells, suggesting that E7 is the major factor responsible for the continued proliferation and the suppression of the senescence phenotype in cervical carcinoma cells. However, E7 in 5 days following E2 transduction did not prevent HeLa cells from entering the senescent state, indicating that the arrested state becomes irreversible. Our results suggest that, upon depletion of the viral oncoproteins, a senescent state is irreversibly induced in HeLa cells after a period of commitment. The status and cellular location of certain factors involved in signal transduction and cell cycle control was altered as well along with this irreversibility transition.

Khokhlov A.N.

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.

Untergasser G., Gander R., Rumpold H., Heinrich E., Plas E., Berger P.

The family of transforming growth factors betas (TGF-betas) comprises molecules involved in growth inhibition, stress-induced premature senescence, epithelial mesenchymal transition and differentiation processes. The aim of this study was to clarify the effect of long term exposure of human prostate basal cells to TGF-betas, which are found in high concentrations in prostatic fluid and areas of benign prostatic hyperplasia (BPH). Basal cell cultures established from prostate explants (n=3) were either grown into cellular senescence, or stimulated with TGF-beta1, beta2 and beta3. Similar to cellular senescence, TGF-beta stimulation resulted in an increase of SA-beta galactosidase (SA-beta-gal) activity, flattened and enlarged cell morphology, and down-regulation of the inhibitor of differentiation Id-1. TGF-beta-treated prostate epithelial cells neither showed terminal growth arrest nor induction of important senescence-relevant genes, such as p16(INK4A), IFI-6-16, IGFBP-3 or Dkk-3. Cells stained positive for cytokeratins 8/18, but did not express other lumenal markers, such as prostate-specific antigen and androgen-receptors. TGF-betas increased also the expression of the mesenchymal marker vimentin, indicating that basal epithelial cells underwent differentiation with lumenal and mesenchymal features. In contrast, in vitro-differentiated neuroendocrine-like cells from prostate organoide cultures, expressing chromogranin A and cytokeratin 18, strongly stained positive for SA-beta-gal. Thus, SA-beta-gal activity is not only a marker for senescence, but also for differentiation of human prostate epithelial cells. With regard to the in vivo situation, in addition to cellular senescence, TGF-beta could contribute to the increased number of SA-beta-gal positive epithelial cells in BPH.

Choi J., Shendrik I., Peacocke M., Peehl D., Buttyan R., Ikeguchi E.F., Katz A.E., Benson M.C.

Cellular senescence is a unique cellular response pathway thought to be closely associated with the aging process. The senescent phenotype is characterized by the loss of a cell's ability to respond to proliferative and apoptotic stimuli even while normal metabolic activity and vitality is maintained. Recently, a novel biomarker, senescent-associated beta-galactosidase (SA-beta-gal), was found to identify cells with the senescent phenotype. In the present study, we examined whether human prostatic epithelial cells adopt a senescence-associated phenotype after prolonged culture and analyzed a series of human benign prostatic hyperplasia (BPH) specimens to determine whether the cellular senescence process might be a factor in the development of BPH.A primary culture of epithelial cells was established from the normal tissue of the peripheral zone of a radical prostatectomy specimen and was serially passaged until senescence. Forty-three human prostate specimens were obtained subsequent to radical prostatectomy or transrectal ultrasound-guided biopsy. The cultured cells and tissue specimens were histochemically stained to reveal the expression of SA-beta-gal, the cellular senescence biomarker.As has been reported for other types of cultured cells, human prostatic epithelial cells demonstrated widespread expression of the cellular senescence marker, SA-beta-gal, on prolonged culture. In our survey of hypertrophied human prostate tissues, 17 specimens (40%) of the 43 analyzed demonstrated positive staining for SA-beta-gal. In these tissues, SA-beta-gal expression was noted only in the epithelial cells. No statistical correlation (P = 0.42) between the chronologic age of the patient donor and SA-beta-gal expression was found. However, a high prostate weight (greater than 55 g) was found to correlate strongly with the expression of the SA-beta-gal biomarker (P = 0. 0001).Cultured prostatic epithelial cells expressed SA-beta-gal on reaching replicative senescence in vitro. The survey of human BPH specimens for the senescent marker showed that prostatic epithelial cells in patients with BPH with more advanced enlargement of the prostate (greater than 55 g prostate weight) expressed SA-beta-gal, and the prostates from patients with BPH that weighed less than 55 g tended to lack senescent epithelial cells. On the basis of these results, we propose that the accumulation of senescent epithelial cells may play a role in the development of the prostatic enlargement associated with BPH.

Severino J., Allen R.G., Balin S., Balin A., Cristofalo V.J.

Cytochemically detectable beta-galactosidase (beta-gal) at pH 6.0 has been reported to increase during the replicative senescence of fibroblast cultures and has been used widely as a marker of cellular senescence in vivo and in vitro. In this study, we have characterized changes in senescence-associated (SA) beta-gal staining in early and late passage cultures, cultures established from donors of different ages, virally immortalized cells, and tissue slices obtained from donors of different ages. The effects of different culture conditions were also examined. While we confirm the previous report that SA beta-gal staining increased in low-density cultures of proliferatively senescent cells, we were unable to demonstrate that it is a specific marker for aging in vitro. Cultures established from donors of different ages stained for SA beta-gal activity as a function of in vitro replicative age, not donor age. We also failed to observe any differences in SA beta-gal staining in skin cells in situ as a marker of aging in vivo. The level of cytochemically detectable SA beta-gal was elevated in confluent nontransformed fibroblast cultures, in immortal fibroblast cultures that had reached a high cell density, and in low-density, young, normal cultures oxidatively challenged by treatment with H2O2. Although we clearly demonstrate that SA beta-gal staining in cells is increased under a variety of different conditions, the interpretation of increased staining remains unclear, as does the question of whether the same mechanisms are responsible for the increased SA beta-gal staining observed in senescent cells and changes observed in cells under other conditions.

Krishna D.R., Sperker B., Fritz P., Klotz U.

Apparently two forms of beta-galactosidase (beta-GAL) in cells or tissue sections can be detected by enzyme histochemical staining (X-GAL). Using a sensitive and specific HPLC method we have determined the pH dependent activity of beta-GAL in cell lines of lung carcinoma (A549), colon carcinoma (Caco2-TC7), promyelocytic leukemia (HL60), hepatoma (HepG2) and human liver homogenates. The HPLC method has been validated and the influence of pH and substrate concentration was studied. There was a good linear correlation between HPLC and quantitative enzyme histochemistry (pH 4.5: r = 0.985; pH 6.0: r = 0.967). Both, pH 4.5 beta-GAL and pH 6 beta-GAL could be demonstrated in all biological material tested and pH 6 beta-GAL activity was always lower (25-50%) than pH 4.5 activity. In Caco2-TC7 cells both activities increased by a factor of 10 from day 3 to day 17 after seeding. In addition, since the beta-GAL activity decreased with increase in pH both in human liver homogenates (independent of the age of the donor) as well as in tumor cell lysates in a similar fashion we believe that the activity at pH 6 can hardly be considered as an exclusive 'senescence marker'. In addition, the more sensitive HPLC method could demonstrate activity in cells that showed negative reaction with X-GAL.

MORGUNOVA G.V., KHOKHLOV A.N.

The search and testing of drugs with senolytic activity is one of the new directions in gerontology. The number of “senescent” cells that increases with age contributes to the development of age-related diseases and chronic non-infectious inflammation. Removing “senescent” cells or suppressing their influence on surrounding tissues seems a logical step to improve the quality of life and, possibly, prolong lifespan. However, drugs that have senolytic and senomorphic activity in model systems cause the development of a number of side effects in clinical trials. In this review, we consider the main advances in the field of senotherapy, the prospects for the use of senotherapy drugs, and the limitations that researchers and clinicians may encounter.

Morgunova G.V., Khokhlov A.N.

The search for and testing of drugs with senolytic activity is a new direction in gerontology. The increasing number of “senescent” cells with age contributes to the development of age-related diseases and chronic non-infectious inflammation. Removing “senescent” cells or suppressing their influence on surrounding tissues seems like a logical step to improve quality of life and possibly prolong it. However, drugs with senolytic and senomorphic activity in model systems induce the development of a number of side effects in clinical trials. In this review, we discuss the main advances in senotherapy, the prospects for the use of senotherapeutics, and the limitations that researchers and clinicians may encounter.

Cooper I.D., Kyriakidou Y., Petagine L., Edwards K., Elliott B.T.

In the pursuit of longevity and healthspan, we are challenged with first overcoming chronic diseases of ageing: cardiovascular disease, hypertension, cancer, dementias, type 2 diabetes mellitus. These are hyperinsulinaemia diseases presented in different tissue types. Hyperinsulinaemia reduces endogenous antioxidants, via increased consumption and reduced synthesis. Hyperinsulinaemia enforces glucose fuelling, consuming 4 NAD+ to produce 2 acetyl moieties; beta-oxidation, ketolysis and acetoacetate consume 2, 1 and 0, respectively. This decreases sirtuin, PARPs and oxidative management capacity, leaving reactive oxygen species to diffuse to the cytosol, upregulating aerobic glycolysis, NF-kB and cell division signalling. Also, oxidising cardiolipin, reducing oxidative phosphorylation (OXPHOS) and apoptosis ability; driving a tumourigenic phenotype. Over time, increasing senescent/pathological cell populations occurs, increasing morbidity and mortality. Beta-hydroxybutyrate, an antioxidant, metabolite and signalling molecule, increases synthesis of antioxidants via preserving NAD+ availability and enhancing OXPHOS capacity. Fasting and ketogenic diets increase ketogenesis concurrently decreasing insulin secretion and demand; hyperinsulinaemia inhibits ketogenesis. Lifestyles that maintain lower insulin levels decrease antioxidant catabolism, additionally increasing their synthesis, improving oxidative stress management and mitochondrial function and, subsequently, producing healthier cells. This supports tissue and organ health, leading to a better healthspan, the first challenge that must be overcome in the pursuit of youthful longevity.

Khokhlov A.N.

The author’s view on the current state of gerontological research is presented. He believes that the widespread departure from the principles of classical gerontology, formulated back in the 20th century, has not been reflected in the works in the field of biology of aging (both theoretical and experimental) in the best way. The neglect of the fundamental principles of gerontological research has led to the fact that, in most works, the classical definition of aging as a set of age-related changes in practically healthy individuals leading to an increase in the rate of mortality is ignored. The emphasis is on assessing the average and maximum life span of the studied organisms, even if they are ageless. Extending the lifetime of such objects cannot be considered a modification of the rate of their aging. It is emphasized that special attention is now being paid to molecular age-related changes, which some gerontologists consider aging, although this is just its possible mechanism or consequence. However, geroprotectors are very often studied exactly by assessing the modification of the rate of such age-related changes. At the same time, as classical gerontology rightly believes, the principles of which the author urges to adhere to, without taking the survival curves of the control and experimental cohorts, it is impossible to draw a correct conclusion about whether the studied compound is a geroprotector. At the same time, an approach to the formation of such cohorts, including an assessment of the minimum required number of organisms in them, as well as the “quality” of their health, is very important. Several gerontological articles that have been published in the most highly ranked scientific journals and, therefore, have attracted much attention of relevant specialists are considered. This attention was expressed, among other things, in the high citation rate of these works, although they were performed with significant violations of the principles of classical gerontology, which were subsequently identified by other researchers. It is also emphasized that, at present, the rating of a scientific journal for many gerontological readers has become much more important than the correctness of the results and ideas presented in the article. A list of methodological problems is given, which, according to the author, not only complicate the situation with modern gerontological research but also make tangible progress in this area practically unattainable.

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.

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.

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.

Khokhlov A.N., Morgunova G.V.

A brief comparative analysis of different approaches to cell viability testing in cytogerontological experiments is performed with a focus on problems in constructing survival curves for cultured cells in the “stationary phase aging” model. It is emphasized that the choice of methods to this end depends mainly on the researchers’ ideas about molecular and cellular mechanisms of aging. A note is made that the evaluation of colonyforming efficiency, though optimal for cell viability assessment, is unfortunately not applicable to postmitotic or very slowly propagating cells. Consideration is also given to some problems encountered when using the most popular molecular probes designed for live/dead cell viability assays.

Khokhlov A.N., Morgunova G.V., Ryndina T.S., Coll F.

The effect of isotonic Quinton Marine Plasma (QMP) solution 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 phase of growth) of transformed Chinese hamster cells was studied. No positive effects of QMP on the studied viability indexes of the cultured cells were found in any of the experiments. It is assumed that QMP, like many other potential anti-aging agents the authors studied recently (2,4-dinitrophenol in concentrations that provide mild uncoupling, the essential oil of oregano, hydrated C60-fullerene, etc.), can demonstrate its beneficial effect only at the level of the whole organism, triggering neurohumoral mechanisms that are not present in cytological model systems.

Khokhlov A.N.

The long history of ideas about the most famous “immortal” (non-aging) organism, freshwater hydra, is shortly reviewed. Over the years this polyp has attracted the attention of naturalists interested in problems of aging and longevity. In recent years, this interest has abruptly increased with the accent on fine mechanisms providing an almost complete lack of aging in hydra. It is emphasized that hydra immortality is based on indefinite self-renewal capacity of its stem cells. It is this fact that allows the polyp to continuously replace the “outworn” cells of the organism, keeping all its characteristics unchanged for an almost unlimited time. It is concluded that the applicability of the data obtained in gerontological experiments on hydra to human being is, unfortunately, very limited because normal functioning of many important organs and tissues in highly developed organisms is determined by the presence of postmitotic cells (neurons, cardiomyocytes, etc.), which actually cannot be replaced.

Kirpichnikov M.P., Khokhlov A.N.

The paper considers the history of how the scientific journal Moscow University Biological Sciences Bulletin (MUBSB) evolved during the last 7 years. It is the English edition of the Russian scientific peer-reviewed journal of the School of Biology of Lomonosov Moscow State University MSU Vestnik (Herald). Series 16. Biology. MUBSB is published by Allerton Press, a member of the Nauka/Interperiodica International Academic Publishing Company since 2007. The rapid progress of MUBSB in recent years is apparently due to the journal having been distributed since 2007 by the internationally renowned Springer publishing consortium that places electronic versions of all articles on its website, which has, to all appearances, led to a manifold increase in the number of journal subscribers. As a result, the number of downloads of MUBSB papers from the publishing company website also raised by an order of magnitude from 2007 to 2013. The growing popularity of the journal is noted to have lead to its inclusion in a number of international databases, and this, in turn, has increased its attractiveness for a large number of authors, including Russian nonmembers of Moscow State University, as well as scientists from research institutes and universities of other countries. The main features of the spectrum of the papers published in MUBSB are briefly considered.