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A N Khokhlov

Publications and source records attributed to A N Khokhlov.

At least 19 recordsLinked to original sources
PubMed2006

Poly(ADP-ribose)-polymerase-1 and aging: experimental study of possible relationship on stationary cell cultures.

Experiments on "stationary aging" cultures of B11dii-FAF28 Chinese hamster cells showed that contact inhibition of cell growth and further culturing of cells in the stationary phase led to continuous inhibition of enzymes realizing poly-ADP-ribosylation of chromatin proteins. Cell density in the monolayer and percentage of damaged cells detected by trypan blue staining decreases during this process.

Animals↗
PubMed2005

["Stationary phase aging" of cell culture: an attempt of evaluation of growth medium "age" effect].

Cell proliferation rate and 3H-thymidine labeling index of "young" (i. e. harvested in 3 days after subcultivation) cultured Chinese hamster cells (B11 dii-FAF28 line) have been determined in growth medium conditioned by the same cells for various periods of time during their growth and subsequent "stationary phase aging" (medium of different "age"). Cells were serially cultured in Eagle's medium with 10 % bovine serum. The experiment was conducted as follows. The "young" cells were seeded in Carrel's flasks (4500 cells/cm2) with fresh growth medium and placed at 37 degreesC. At definite time intervals, media from 3 randomly selected flasks were filtrated and stored in small glass flasks at 4 degreesC. The cells from all 3 flasks were collected by trypsin treatment and counted with hemocytometer. During the period of 26 day cultivation we collected a set of media of different "age" corresponding to certain points of the growth and "stationary phase aging" curve of the culture. Then, the "young" cells in fresh medium were seeded into tissue culture plates with cover slips placed into wells of the plates (26,600 cells/cm2) and grown at 37degreesC, 5 % CO2 for 2 h. At this point, the medium was replaced with media of different "age". 22 h later (i. e. on the first day after seeding) cell density was evaluated microscopically in all the wells. On the next day (i. e. in 2 days after seeding) 3H-thymidine was added to every well to final concentration 1.85 x 10(4) Bq/ml. After next 24 h (i. e. in 3 days after seeding) cell density was counted again, and the medium was removed. The cover slips were rinsed with Hank's solution and air-dried. Autoradiography was performed in standard manner by photoemulsion exposing for 5 days and subsequent developing in amidol developer. The relative number of nuclei with 10 and more "grains" was revealed microscopically. Based on the obtained results, two basic parameters were evaluated for every "age" medium: 1) cell proliferation activity index calculated as log2 (N3/N1), where N1 - cell density on the first day after seeding, and N3 - the same parameter on the third day after seeding; 2) cell labeling index calculated as percentage of cells with nuclei labeled by 3H-thymidine during incubation from 2nd to 3rd day of cultivation. These two indexes for cell growth in different "age" media appeared to be highly correlating (R = 0.85). Besides, it was found that the observed "age-related" diminishing of ability of the growth media of different "age" to stimulate proliferation of "young" cells cannot completely explain the "stationary phase aging" phenomenon (in particular, even for the "oldest" medium cell labeling index was 65 %). We conclude that the phenomenon is based on exactly intrinsic changes of cells, most likely on molecular level, though environmental effects cannot be entirely excluded. The authors are grateful to the Russian Basic Research Foundation for support (grants 03-04-49030 and 00-04-48049).

Animals↗
PubMed2002

[Results and perspectives of cytogerontologic studies in modern time].

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

Aging↗
PubMed1991

Effects of cholesterol- or 7-ketocholesterol-containing liposomes on colony-forming ability of cultured cells.

Experiments with cultured Chinese hamster cells showed that incubation of the cells with (phosphatidylcholine + cholesterol + 7-ketocholesterol)-containing liposomes (4:3:1 by weight) during two hours led to a decrease in the colony-forming ability of cells down to zero, while (phosphatidylcholine + cholesterol)-containing liposomes (1:1 by weight) reduce this parameter by 90%. Furthermore, the cholesterol-containing liposomes (without 7-ketocholesterol) induce a decrease in the number of the maximal-site colonies accompanied by the corresponding increase in the number of the middle-size colonies.

Animals↗
PubMed1989

[The effect of liposomes containing cholesterol or 7-ketocholesterol on the capacity of cultured cells to form colonies].

The experiments with the cultivated Chinese hamster cells showed that the incubation of cells with (phosphatidylcholine+cholesterol+7-keto-cholesterol)--containing liposomes (4:3:1 by weight) during two hours leads to a decrease in the effectiveness of colony-forming up to zero, while the (phosphatidylcholine+cholesterol-containing liposomes (1:1 by weight) reduce the effectiveness by 90%. Furthermore, the cholesterol-containing liposomes cause the decrease of the number of colonies with maximal size, accompanied by the increase of the number of colonies with middle size.

Animals↗
PubMed1987

[Analysis of the kinetic growth patterns of cultured cells. III. The effect of inoculation density, of a geroprotector-antioxidant and of stationary-phase aging].

With the help of a previously devised model of cultured cell growth kinetics it was shown that the "plateau" level on the growth curve of human embryo diploid fibroblasts increased, if the cells were grown with Epigid geroprotector-antioxidant (equal results with two drug concentrations: 10(-5) and 10(-7) M, resp.). It was also found that the "plateau" level on the growth curve of cultured "3 days aged" Chinese hamster cells (subcultivated to a fresh medium 3 days after a 1:6 subcultivation) lays higher than the "plateau" level of "14 days aged" cells but lower than that of "7 day aged" cells. Furthermore it was shown that an increase in inoculation density of Chinese hamster cells increased proportionately the rate of cell population growth but had a little effect on the "plateau" level of the growth curve. The data obtained are discussed in terms of some "proliferative" theories of cellular ageing.

Animals↗
PubMed1986

[Strengthening of the DNA-protein complex during the stationary aging of cultured cells].

Accumulation of DNA-protein crosslinks during "stationary phase ageing" (i.e. with the increase of non-proliferating period) of cultured transformed Chinese hamster cells and normal embryonic diploid human fibroblasts was determined by the improved method of column chromatography using hydroxylapatite of 14C-valine, 14C-leucine and 3H-thymidine-labelled cell homogenates. Such an accumulation, however, was not observed during "stationary phase ageing" of xeroderma pigmentosum human fibroblasts. Possible mechanisms of the above phenomenon are discussed.

Animals↗
PubMed1985

[Analysis of the kinetic growth patterns of cultured cells. I. The model].

On the basis of the Verhulst-Pearl equation, a model of cultured cell growth kinetics is devised. Analysing both authors' and literature data, a possibility was shown to use the model for description of growth kinetics of different cultured cell types. The proposed method of analysis of cell growth kinetic characteristics also provides a rapid evaluation of the cell population doubling time and of the relative number of non-dividing cells in the population.

Animals↗
PubMed1985

[Analysis of the kinetic growth patterns of cultured cells. II. The action of ionizing radiation, an alkylating agent and a low-frequency electromagnetic field].

With the help of the earlier devised model of cultured cell growth kinetics it is shown that the "plateau" level on the growth curve of Chinese hamster cells decreased proportionally to a dose of the agent applied after a short-term treatment by gamma-radiation or by alkylating agent thiophosphamide. The analysis of our own and literature data enabled us to propose that the lowering of the growth curve may testify to the geropromoter character (i.e. manifesting in ageing promoting) of the investigated factor action. As the low-frequency electromagnetic field induces similar changes in the growth curve, it is related (together with both the above factors investigated) to geropromoters.

Alkylating Agents↗
PubMed1984

[DNA degradation in resting cultured Chinese hamster cells].

A gradual accumulation of the alkali-labile sites in DNA of cultured Chinese hamster cells during "stationary phase ageing" was shown by ultracentrifugation in alkaline sucrose gradients. A possibility is discussed of using the stationary phase mammalian cell cultures as a model for studies of ageing mechanisms.

Animals↗
PubMedDate not supplied

[Enzymatic synthesis of beta-NAD+ selectively marked with tritium at adenine and its use for determining the activity of poly(ADP-ribose) polymerase].

A method of preparation of tritiated beta-HA [symbol: see text]+ from (adenine-2,8-(3)H)ATP and nicotinamide mononucleotide is suggested. This method is based on the baker's yeast (Saccharomyces cerevisiae) enzyme nicotinamide nucleotide adenylyltransferase. Experimental conditions for a nearly complete conversion of labeled ATP to NAD were determined. The tritiated NAD prepared by this method can be used in the quantitative assay of the chromatin enzyme poly(ADP-ribose) polymerase.

Adenine↗
PubMedDate not supplied

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

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

Cell Biology↗