Aging and carcinogenesis

Balance on the verge of a foul

Konstantin Andreev, Post-scienceYesterday an old woman came up to me on the street and offered to buy an eternal needle for primus.

You know, Adam, I didn't buy it. I don't need an eternal needle, I don't want to live forever.
I. Ilf, E. Petrov "Golden calf"The very concept of life and death, as such, in a single cell and any multicellular structures, whether it is a culture on the bottom of a Petri dish, a section of the epithelium lining the lung, a 959-cell nematode Caenorhabditis elegans or, in extreme cases, a whole person, has a completely different meaning.

For complexly organized systems, to which we belong, the definition of “living-inanimate” is formulated quite simply and is based on half a dozen signs that students of any biological faculties are told about during their first semester of study there. And only the presence of all of them together makes it possible to attribute the studied object to wildlife.

From the point of view of physiology, the key of these signs should be the ability of a living individual to exchange matter and energy with the environment, which includes processes such as nutrition, respiration and excretion. From a biochemical point of view, a living system, perhaps, should be called one within which the reactions of metabolism continue and the constancy of its chemical composition is maintained. In the understanding of biophysicists, life is an eternal struggle with entropy or, more precisely, the ability to maintain the level of disorder in the system below that outside it. Genetics defines a living object as one that has the ability to reproduce itself, so that all molecules, cells, tissues, etc. are similar in structure to their predecessors. From the point of view of embryology, life is meant as the final process of ontogenesis, starting with the state of a single-celled zygote and ending with the moment when all further changes and the acquisition of new properties are completed. Moreover, the quality of these changes, whether it is growth with the accumulation of biomass or, on the contrary, the death of tissues, does not play a role (that is, when you are pointed out to a growing bald spot, you can smartly parry it by saying that you are not aging at all, but, one might say, developing). Ethologists, in turn, prioritize the presence of irritability in a living object, or a selective reaction to external influences. If you poke, say, an ordinary amoeba (Amoeba proteus) with something sharp, then it should quite sensibly crawl away, and if, for example, you add something nutritious to the medium, then, on the contrary, it will immediately begin to move along the concentration gradient of this nutrient towards its source. A similar experiment, by the way, will work perfectly on a sleeping colleague, unless you have to poke something heavier, and the smell of freshly brewed coffee will play the role of an attractive factor instead of a nutrient medium in this case. The presence of such a selective reaction gives grounds to assume that we have a living organism in front of us.

For a single cell, everything looks a little different. To begin with, the entire life cycle for it is just a time interval between two mitotic divisions, and any act of birth of a new cell is inextricably linked with the death of its predecessor. On the other hand, mitosis, which by its very nature presupposes the complete identity of the two resulting daughter cells, de jure provides her with a kind of formal immortality. In reality, of course, everything happens somewhat differently, and there are many factors in nature that disrupt the proper course of mitosis (try, for example, treating a cell with colchicine and see what happens). And under normal conditions, there are also enough varieties of cell division that lead to an uneven redistribution of genetic material between descendants, including crossing over during meiosis, amitosis, poly- and aneuploidy, karyogenesis, leading to the formation of a multinucleated syncytium, etc.

Nevertheless, any of the above variants of cell division for the tissue as a whole will serve as the basis for its further growth. And so that this growth does not get out of control, and there are three types of real cell death, widely described in the scientific literature - autophagy, necrosis and apoptosis. Of all of them, only the last one is a programmed and precisely controlled process and therefore a key player in all those processes that will be discussed below.

I want to emphasize right away that any organism exists in conditions of incessant balancing between dividing and dying cells, in much the same way as accounting reduces debit with credit, and for an adult whose body, on average, consists of ^10-13 cells, their every second turnover is somewhere around 10 ^{6-10 7}. If this rather fragile balance is suddenly disturbed for some reason, then, depending on which side it is shifted, we will have either tumor growth or tissue necrosis at the exit. It is curious, but it is cell tissue, or cell culture as a level of organization of living matter, in the case when the intensity of cell proliferation is not compensated by their death, that is the only example of actual immortality, which is successfully used in modern science. And these only immortal cells are cancer.

The Eternal Life of Henrietta LacksAmong the many immortalized cell lines, the most famous today is the HeLa line.

It was obtained in 1951 from cervical endothelial cells taken from a cancer patient named Henrietta Lacks (HeLa is an acronym for the first and last name Henrietta Lacks) shortly before her death. Initially infected with the papilloma virus, cancer cells were susceptible to chromosomal aberrations and contained 82 human chromosomes instead of the standard 46. However, another, much more curious and significant feature of them turned out to be "eternity" – the ability to endlessly share in laboratory conditions. And when it was discovered that HeLa cells could be sent by mail and even more – stored in a freezer for decades and then continue to be cultivated again on an artificial nutrient medium, this immediately led to a huge stir in the scientific world, and the HeLa line immediately dispersed to laboratories in all corners of the globe. Subsequently, however, there was a serious scandal with their participation. When the analysis of chromosome sets of a number of other cell lines used for research revealed cases of their contamination by HeLa cells, this led to the fact that all the results obtained on the basis of experiments on these lines and already published in the scientific press could not in fact be considered reliable. As a result, there was considerable confusion, which cost someone a career and reputation. However, despite this, interest in HeLa has not cooled down to this day, since cancer cells, with a certain degree of assumption, are a fairly adequate model for finding answers to many biomedical questions. And it was the HeLa cells that made it possible for Jonas Salk to create a vaccine against polio, participated in the cloning project of the famous Dolly sheep (preliminary experiments on nuclear transplantation were conducted just on HeLa), and were also used to compile genetic maps, practice artificial insemination and other scientific tasks. And even flew into space in 1960 as part of the space program of Soviet geneticists.

Weisman's postulate and the Hayflick limitWhat exactly makes cancer cells immortal?

And why are all the other cells susceptible to what we call aging? And, in the end, what is aging itself? The first to approach this issue closely was the famous zoologist and theorist of evolutionary doctrine August Weisman, who proposed back in 1881 his postulate, which later became famous, which stated that in somatic cells "... the ability to grow by division is not eternal, but limited," which causes the aging of the whole organism. In his opinion, only germ cells could undergo division indefinitely. Of course, all these conclusions were purely empirical, since there was no question of any experimental methods of working with living cells at that time. What's there! After all, even the Schleiden-Schwann cellular theory that there is a cell at all was formulated only thirty years earlier.

Later, Weisman's postulate was tested for strength by the experiments of the French surgeon and pathophysiologist A. Carrel, who for the first time put into practice the technique of growing tissue culture isolated from the body. The technique of the experiment was simple and seemed to completely refute the Weismann assumption about the so-called "mortality" of somatic cells. A cut-off piece of the myocardium of a chicken heart, placed in a nutrient medium and incubated in a thermostat, was then divided into two equal parts, which were transplanted into new, separate test tubes and incubated further. Such replanting could be continued for a long time, almost years, and throughout all these passages, myocardial fibroblasts continued and continued to divide. For these studies, Carrel was awarded the Nobel Prize in 1912, although, as it turned out later, his experiment was not performed quite cleanly. The reason for this was the embryonic fluid used as a nutrient medium to maintain the vital activity of the cell culture – the fresh cells contained in it, getting into the sample and actively dividing, thereby created an imaginary effect of immortality of "old" fibroblasts, because what can you see in a test tube!

And the experiment was redone half a century later by the American cytologist L. Heiflik. Unlike Carrel, he placed in the nutrient medium not by itself a piece of tissue extracted from the body, but pretreated with trypsin, so that the tissue disintegrated into individual cells. In addition, the choice of the nutrient medium itself was different – instead of embryonic serum or blood plasma, Hayflick used an artificially selected solution of amino acids, salts and other low-molecular components sufficient to support cellular reproduction. Otherwise, the method was the same – getting into the nutrient medium, fibroblasts immediately began to divide, and as soon as their layer reached a certain size, the sample was divided in half, treated with trypsin once again and transferred to a new test tube/Petri dish. And after a certain number of transplants, approximately corresponding to 50 cell divisions, tissue growth stopped, and the cells that stopped dividing died after a while. This phenomenon occurred regularly, inevitably and was observed in all repeated experiments subsequently conducted by independent groups of researchers, and not only for fibroblasts, but also for any other types of somatic cells. The critical number of divisions allocated to somatic cells for their life was called the "Hayflick limit", and the experiment completely rehabilitated Weisman's theory regarding the mortality of cell lines, in particular, and any organisms in general.

Telomerase theory of aging. How to circumvent the lawLet's return to the peculiarities of cancer cells.

They are the only ones who manage to overcome this barrier. And this happens due to two interrelated factors – activation of the enzyme telomerase in them, on the one hand, and blocking of the mechanisms of apoptosis – programmed cell death, on the other. How does this whole scheme work approximately? Telomeres are non–coding regions of chromosomes localized at their ends (that is, two for each linear chromosome) and consisting of repeatedly repeating (approximately 10-15 thousand) short nucleotide fragments, for example, in most vertebrates it is TTAGGG. Although it is known that telomeric DNA does not carry any genetic information, nevertheless, its role is very significant. Firstly, with unplanned exposure to exonucleases (enzymes that "cleave" the terminal mononucleotides from the polynucleotide chain), telomeres take the hit, the semantic DNA does not suffer, and the negative effect is thus minimized. However, even this is not the main thing. The main function of telomeres is to protect the coding DNA from losses during the shortening of chromosomes during their replication. The problem arises due to the fact that any DNA polymerase synthesizes a daughter chain only in a strictly defined direction - from the 5' end to the 3', moreover, it cannot start it from scratch. To initiate the operation of this macromolecular machine, a seed, or so-called RNA primer, is needed, which exists for a fairly short time and is immediately removed by it when the DNA polymerase moves further along the chain. That is, with each subsequent act of replication, the 5' end of the newly synthesized DNA turns out to be shorter by 10-30 nucleotides, which in the absence of a telomere buffer would inevitably lead to the loss of genes. In the present case, the telomeres themselves are consumed, gradually shortening to a certain limit (usually by 2-3 thousand repetitions), after which the replication of chromosomes stops, and the cell stops dividing. And this moment exactly coincides with the Hayflick limit.

Telomerase is a very interesting and specific enzyme, or rather, a ribozyme, that is, consisting not only of a protein component, but also carrying its own RNA matrix, which means that it is capable of "rewriting" the information contained in it in the form of DNA sequences. About the same as HIV reverse transcriptase does, just a little for other purposes. In this case, in order to complete the telomeric sections of chromosomes. The trick is that the telomerase gene in ordinary somatic cells remains inactive for most of the time and is expressed, as a rule, only in germ cells and cells that have not yet undergone differentiation (stem cells). And in cancer cells (not always, but somewhere in about 85% of cases), this gene is turned on constantly, which helps them bypass the Hayflick limit and become "immortalized", that is, immortal. Telomerase itself, by the way, is used in diagnostics as a tumor process.

Cellular specialization. Choosing a "profession"If we do not go into the details of the mechanism itself, then, it would seem, firstly, what is wrong for an ordinary somatic cell to keep telomerase constantly in the "on" state, and secondly, why is the limit of telomere shortening only about twenty percent of their length?

After all, the stock of terminal repeats is large enough to allow a much larger number of DNA replication cycles before the coding regions of the chromosome are affected.

The answer to the first question is quite simple – for the correct performance of the functions assigned to it, any cell must undergo a process of final differentiation. This is obvious: for example, cardiomyocytes (muscle cells of the myocardium) and beta cells in the islets of Langerhans (insulin-secreting cells of the pancreas), which perform functions completely far from each other, cannot have a similar structural structure. Stem cells, of course, do not count here, since they do not have any other function besides division; just as the task of germ cells is just the preservation and transfer of genetic information to offspring (for spermatozoa, perhaps, you can also add the ability to flagellar movement in order to move, but no more that). However, as the stages of differentiation progress, an absolutely pluripotent stem cell first turns into a semi-stem progenitor cell, the choice of a further fate for which is already very limited (that is, a semi–stem blood cell can still differentiate into different types of cells of the circulatory system, for example, into leukocytes or lymphocytes, but there is no way to give rise to another tissue). And then into the final cell, which is either not capable of division at all (neurons), or is capable only a limited number of times. It goes without saying that the most difficult and difficult work in the differentiation of stem cells is the regulation of the entire process. So complex that its description would probably take up all the remaining pages, and besides, at this stage of the development of science, half of them would be full of gaps. If all the cells of the body, not just stem cells, had the ability to divide endlessly, to control the growth of tissue in the right amount and in the right place, it would most likely be impossible even with 97% of the regulatory DNA in the human genome.

It is quite difficult to answer the question why the cell does not use the entire length of telomeres, but "stops" ahead of time. But it's pretty well known how it happens. Having information about where the gene we are interested in is localized, it is quite easy to remove it or make it incapacitated. This method in molecular biology is called the "gene knockout" method, which allows you to get an organism devoid of one or another trait for which this gene is responsible, and thus its function can be identified by the phenotypic differences between the "knockout" organism and control. Experiments of this kind have demonstrated that normal-length telomeres in cells maintain a number of genes in a blocked state. This is a special class of genes – the so-called anti–oncogenes, or tumor suppressor genes, whose purpose is to be a counterweight to oncogenes and proto-oncogenes, allowing them to work only during strictly regulated periods of the cell cycle (in particular, before the cell enters the mitosis phase). This maintains a balance between the probability for the cell to go into division or remain in a differentiated state. When telomeres are shortened, these genes are immediately activated and start a program of repression of cell proliferation.

The most famous representative of suppressor genes today is the p53 gene, a gene with a very rich, downright action-packed history of his research, about which for a long time it was impossible even to say with certainty which side he was on, and many researchers considered him just the same oncogene. Subsequently, it was proved that the protein encoded by it (with a molecular weight of 53,000 daltons), being a transcription factor, changes the expression of a number of other target genes, whose products, in turn, inactivate the cyclins necessary for the operation of special proteins of cyclin-dependent kinases, which are responsible for the entry of the cell into mitosis. The result of this tricky chain is the stopping of the cell cycle.

Apoptosis is a programmed suicideHowever, inhibition of mitosis is not the only result of the p53 protein.

In order to exclude the possibility of spreading unwanted genetic information throughout the body, it is better not just to sterilize the "wrong" cell, but to remove it itself for reliability. To be sure. In this case, the program of her death, known as apoptosis, is launched in her. The term itself (from Greek. apo – separation + ptosis – fall) initially had nothing to do with cells at all, but was put into circulation back in Ancient Rome by the doctor and naturalist Galen to describe leaf fall. The fact that leaf fall occurs annually, at about the same time and according to a similar scenario (that is, from some point of view, it is like a programmed event) determined the choice of the name, which was then given to the process of genetically controlled cell death. Apoptosis itself was first described by John Kerr in 1972 and since then has come under such close attention of the scientific world and has been studied so thoroughly that today a search query for the keyword "apoptosis" in the database of the National Center for Biotechnological Information of the USA (National Center for Biotechnological Information, NCBI) gives a list of scientific publications in the amount of more than 230 thousands.

Unlike necrosis, apoptosis is an absolutely natural procedure for any multicellular organism aimed at maintaining a constant number of cells in the population and, as a consequence, tissue homeostasis. It occurs imperceptibly to the eye (without the development of the inflammatory process), but at the same time, almost constantly. An unnecessary cell is quietly removed, without any interference to the work of neighboring cells, and replaced with a new one, which allows you to preserve the structure of the organ as a whole. Its residues are absorbed by phagocytic cells and go into business further as a building material. In the early stages of embryogenesis, apoptosis, oddly enough, is also present – in order to remove rudimentary formations without disturbing the normal maturation of the fetus with an inflammatory reaction. A classic example of this is the connective tissue between the phalanges of the fingers (so that we don't walk later with webbed feet, like waterfowl). In certain cases, apoptosis may be intentionally disabled – where intensive cell division and rapid tissue growth are needed. For example, for reparative regeneration in external injuries, such as wounds, burns, etc. However, excessive apoptosis under standard conditions is likely to also lead to pathologies – only different. Most often they will be autoimmune and immunodeficiency diseases, blood supply disorders leading to ischemia and strokes, neurodegenerative processes, starting with Alzheimer's disease, and so on. All this once again confirms the hackneyed theory that everything is good at the right time, in the right place and in moderation.

Accuracy vs. SpeedSo, the main problem lying in the practical plane is how to make the mutually exclusive processes of cell death and cell survival work or turn off exactly when we need it?

And, of course, the most puzzling problem is how to do this selectively only for those cells that interest us, without affecting all the others? By itself, the scheme of blocking / induction of apoptosis at the molecular level has many self-regulating control points, which are well studied, and which can easily be affected artificially. The whole question is how to selectively deliver the substance that will activate apoptosis to the target. In principle, this is quite solvable, and for a long time there has been a whole direction in molecular therapy developing a target-specific transportation of reagent molecules to cancer cells using various kinds of nanoparticles (target-specific nanoparticles drug delivery). But if it is possible to purposefully affect a single cell, then why not just put the same drug for standard chemotherapy into the carrier nanoparticle, which, unlike the cunning strategy of triggering apoptosis, will kill the malignant cell for sure and with a guarantee, and without the side effects characteristic of chemotherapy. And of course, science will always follow the simplest and most logical path.

Blocking apoptosis, where it is excessive, in order to regenerate and, moreover, "rejuvenate" tissue is generally a dead end path, since the risk of getting a cell with fatal errors in the genome increases disproportionately to the benefit received.

With the telomeric theory of aging, the situation is even more ambiguous. In 2009, Elizabeth Blackburn and Carol Grider, who discovered and described this enzyme 24 years earlier, received their Nobel Prize in Physiology and Medicine "for the discovery of the mechanism of chromosome protection by telomeres and the enzyme telomerase". However, despite the fact that at the moment the theory itself is read experimentally proven, a considerable number of other experimental facts still call it into question, take at least the notorious cloned sheep Dolly. The length of telomeres in her cells was much less than that of an ordinary individual of her age (apparently due to the fact that the nucleus transplanted into the cytoplasm of the egg from the "mother" sheep at the time of cloning belonged to an adult organism, which means that all subsequent cells of the embryo automatically carried nuclei, as if "older" than his own age). However, no signs of premature aging were observed either in Dolly or in other cloned animals (including mice, cattle, etc.).

These contradictions have allowed some scientists to put forward a number of alternative theories of cellular aging, in which the role of the molecular switch is assigned not to telomeric sites, but to extra–chromosomal DNA, in particular, mitochondrial. However, these assumptions also do not explain everything and are more reminiscent of catching a black cat in a dark room.

And finally, as they say in English, the last but not the least – or rather, just the most important and key thing, because of which we live, on average, for 70 years, not 700, and somewhere in the fifth decade we personally get acquainted with doctors of all profiles. The reason is as follows. Almost all molecular machines in a cell that directly or indirectly work with information about an organism encoded in DNA, whether it is matrix replication (DNA polymerase), transcription (RNA polymerase) or translation (ribosomes), do not work with impeccable accuracy at all. And even the presence of well-thought-out mechanisms for correcting their errors cannot guarantee that this information will be reproduced without distortion. For example, DNA polymerase makes mistakes with a frequency of 1 time per million nucleotides it reads (¹⁰⁶). It would seem that this is the highest accuracy, but if we estimate the number of nucleotides in the average gene (5x10 ⁴), the number of genes in the genome (3x10 ⁴), the frequency of "use" of the gene during the life cycle of the cell (varies depending on its functions) and the total number of cells in the human body (10 ¹³), then it turns out that mistakes happen much more often than we would like. Therefore, despite the fact that the cell seems to be a self–sustaining system, it wears out over time. But not quite in the sense in which, say, a car tire wears out, but rather in a similar way to the operating system on a computer. Over the course of life, there is a gradual accumulation of errors in the genome, so the cell always faces a choice – either to divide often, quickly, always have a "freshly baked" biosynthetic apparatus and take the amount, but risk getting some lethal (or carcinogenic) mutation for itself earlier. Or, on the contrary, be careful: lengthen, if possible, the intervals between divisions, reduce the activity of metabolism to a minimum, thereby insuring yourself from possible errors during replication, but limiting your functionality and risking not being able to restore your population in case of external damage. This strategy is chosen by neurons that, as is known, are not able to regenerate, are practically incapable of dividing, and can afford it, since they are usually well protected from the outside by the bone skeleton, and energy is provided by adjacent astroglia cells. But, alas, all cells will not be able to do this, otherwise there will simply be no one to work in the body.

Thus, time in any case always works against us, and carcinogenesis is not for nothing called the disease of old age. Theoretically, it is not so difficult to make cells immortal, but until there is a way to reproduce genetic information with absolute accuracy and prevent the accumulation of mutations over the course of life, there will be little sense from this. After all, it is painful to live up to 150 years, being just a bunch of all genetic diseases known to science - not God knows what a rosy prospect, and hardly anyone would want to live so long, but just as bad.

Literature:

• DePinho RA. The age of cancer. Nature. 2000 Nov 9; 408(6809):248-54.
• Mathon NF, Lloyd AC. Cell senescence and cancer, Nat Rev Cancer. 2001 Dec;1(3):203-13.
• Steller H. Mechanisms and genes of cellular suicide. Science 1995; Vol. 267:1445-1449.
• Kerr JF, Wyllie AH, Currie AR. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972; Vol. 26:239-257.
• Collado M, Blasco MA, Serrano M. Cellular senescence in cancer and aging. Cell. 2007 Jul 27;130(2):223-33.
• Campisi J. Cancer and aging: rival demons? Nat Rev Cancer. 2003 May;3(5):339-49.
• Alenzi FQ. Links between apoptosis, proliferation and the cell cycle. Br J Biomed Sci. 2004;61(2):99-102.
• Kong Y, Cui H, Ramkumar C, Zhang H. Regulation of Senescence in Cancer and Aging. J Aging Res. 2011 Mar 8;2011:963172.

The author is a post–graduate student of the Department of Biological and Chemical Sciences, a researcher at the Laboratory of Cell Membrane Biophysics at the Illinois Institute of Technology (Chicago, USA)

Portal "Eternal youth" http://vechnayamolodost.ru06.03.2013