Senescence-associated β-galactosidase—A biomarker of aging,DNA damage,or cell proliferation restriction?

The most popular biomarker of cellular senescence (BCS) is the activity of senescence-associated β-galactosidase (SA-β-Gal). Today, this is the prevailing BCS in the studies based on the definition of cell senescence (which we do not accept) understood primarily as accumulation in the cells (most often—those not prone to replicative senescence) of certain BCS under the impact of various external factors causing DNA damage. However, some papers provide evidence that SA-β-Gal activity in the cells is not a good BCS, because it often depends not so much on age (in vitro or in vivo) as on the method of research, the presence of certain pathologies, and, what is most important, on the proliferative status of the cells studied. Apparently, the restriction of cell proliferation under certain conditions (due to differentiation, contact inhibition, DNA damage, some diseases, etc.) is itself the factor that stimulates SA-β-Gal expression. In other words, SA-β-Gal appears even in “young” cells if their proliferation is suppressed. Such data, in our opinion, are additional evidence for the validity of our concept of aging, which postulates the leading role of cell proliferation restriction in the age-related accumulation of various macromolecular defects (primarily DNA damage) in cells.     

Does Aging Have a Purpose?

Ideas of proponents and opponents of programmed aging concerning the expediency of this phenomenon for the evolution of living organisms are briefly considered. We think that evolution has no “gerontological” purpose, because the obligate restriction of cell proliferation during the development of multicellular organisms is a factor that “automatically” triggers aging due to the accumulation of various macromolecular lesions in cells as a result of the suppression, or even complete cessation of emergence of new, intact cells. This leads to the “dilution” of stochastic damage (the most important of which is DNA damage) at the level of the entire cellular population. Some additional arguments in favor of the inexpediency of aging for both species and individuals are also listed.     

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

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

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

In the review, the main types of autophagy (macroautophagy, microautophagy, and chaperonemediated autophagy) are shortly described. Data about the character of the influence of autophagy on the aging process and on the development of some neurodegenerative diseases in various organisms are analyzed. It is noted that this effect is usually (though not always) beneficial. Results of investigations of the phenomenon in experiments on mice, nematodes, fruit flies, bacteria, yeast, and cell cultures of higher organisms are considered. Obvious relationship between autophagy activation and cell proliferation restriction is emphasized. The latter, in our opinion, is the main cause of age-related accumulation of various defects (the most important of them is DNA damage) in cells and tissues, which leads to an increase in the death probability (i.e., to aging). It is concluded that studies of the role of autophagy in the aging process on the models of chronological aging in yeast or stationary phase aging of cell cultures could be considered as the most appropriate approach to the problem solution.     

Crystal structure of lumazine synthase from Mycobacterium tuberculosis as a target for rational drug design: binding mode of a new class of purinetrione inhibitors

The enzymes involved in the biosynthesis of riboflavin represent attractive targets for the development of drugs against bacterial pathogens, because the inhibitors of these enzymes are not likely to interfere with enzymes of the mammalian metabolism. Lumazine synthase catalyzes the penultimate step in the riboflavin biosynthesis pathway. A number of substituted purinetrione compounds represent a new class of highly specific inhibitors of lumazine synthase from Mycobacterium tuberculosis. To develop potent antibiotics for the treatment of tuberculosis, we have determined the structure of lumazine synthase from M. tuberculosis in complex with two purinetrione inhibitors and have studied binding via isothermal titration calorimetry. The structures were determined by molecular replacement using lumazine synthase from Saccharomyces cerevisiae as a search model and refined at 2 and 2.3 A resolution. The R-factors were 14.7 and 17.4%, respectively, and the R(free) values were 19.3 and 26.3%, respectively. The enzyme was found to be a pentamer consisting of five subunits related by 5-fold local symmetry. The comparison of the active site architecture with the active site of previously determined lumazine synthase structures reveals a largely conserved topology with the exception of residues Gln141 and Glu136, which participate in different charge-charge interactions in the core space of the active site. The impact of structural changes in the active site on the altered binding and catalytic properties of the enzyme is discussed. Isothermal titration calorimetry measurements indicate highly specific binding of the purinetrione inhibitors to the M. tuberculosis enzyme with dissociation constants in micromolar range.     

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

We believe that cytogerontological models, such as the Hayflick model, though very useful for experimental gerontology, are based only on certain correlations and do not directly apply to the gist of the aging process. Thus, the Hayflick limit concept cannot explain why we age, whereas our “stationary phase aging” model appears to be a “gist model,” since it is based on the hypothesis that the main cause of both various “age-related” changes in stationary cell cultures and similar changes in the cells of aging multicellular organism is the restriction of cell proliferation. The model is applicable to experiments on a wide variety of cultured cells, including normal and transformed animal and human cells, plant cells, bacteria, yeasts, mycoplasmas, etc. The results of relevant studies show that cells in this model die out in accordance with the Gompertz law, which describes exponential increase of the death probability with time. Therefore, the “stationary phase aging” model may prove effective in testing of various geroprotectors (anti-aging factors) and geropromoters (pro-aging factors) in cytogerontological experiments. It should be emphasized, however, that even the results of such experiments do not always agree with the data obtained in vivo and therefore cannot be regarded as final but should be verified in studies on laboratory animals and in clinical trials (provided this complies with ethical principles of human subject research).     

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

There is an opinion that the chronological aging (ChA) of yeast and the stationary phase aging (SPA) of cultured animal and human cells are a consequence of growth medium acidification. However, a number of recent publications indicate that, although this process has a certain influence on the rate of “aging” of cells in the stationary growth phase, it does not determine it completely. Apparently, the key factor in this case is the restriction of cell proliferation, which leads to cell “aging” even under physiologically optimal conditions. During yeast ChA and mammalian cell SPA, the medium is getting acidified to pH ≤ 4. Prevention of acidification can prolong the culture life span, but the cells will still die, although at a slower rate. Effects of medium acidification during ChA and SPA can be explained by activation of highly conserved growth signaling pathways leading to oxidative stress, and these processes, in turn, can play a role in aging of multicellular organisms and development of age-related diseases. Our previous experiments on the effect of buffer capacity of growth medium on SPA of transformed Chinese hamster cells showed that 20 mM HEPES had no effect on cell growth rate; in addition, the growth curves of experimental and control cells reached a plateau on the same day. However, the cell saturation density in the medium with HEPES was lower (i.e., the cells were “older” in terms of the gerontological cell kinetics model); on the other hand, the rate of SPA was markedly reduced, compared to the control, although the cells were still “getting older.” It can be assumed that extracellular pH (by the way, well correlated with intracellular pH) is an important factor (I.A. Arshavsky’s concept of the role of acidic alteration in aging) but not the key factor determining the survival of cells in a stationary culture.     

Protein poly(ADP-ribosyl)ation system: Changes in development and aging as well as due to restriction of cell proliferation

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.