30 October 2012

Cancer and aging: a mini-review

Is aging a payment for suppressing cancerous tumors?

Article for the contest "bio/mol/text"

Конкурс научно-популярных статей «био/мол/текст»

More than 50 years have passed since the phenomenon of cell aging was proved on fibroblast culture, but the existence of old cells in the body has long been questioned. There was no evidence that the aging of individual cells plays an important role in the aging of the whole organism. In recent years, molecular mechanisms of cell aging, their connection with oncological diseases and inflammation have been discovered. According to modern concepts, inflammation plays a leading role in the genesis of almost all age-dependent diseases, which ultimately lead the body to death. It turned out that old cells, on the one hand, act as tumor suppressors (since they irreversibly stop dividing themselves and reduce the risk of transformation of surrounding cells), and on the other hand, the specific metabolism of old cells can cause inflammation and degeneration of neighboring precancerous cells into malignant ones. Currently, clinical trials are underway of drugs that selectively eliminate old cells in organs and tissues, thereby preventing degenerative changes in organs and cancer.

Cell division. The cell cycle.There are about 300 types of cells in the human body, and they are all divided into two large groups: some can divide and multiply (that is, they are mitotically competent), and others – postmitotic – do not divide:

these are neurons that have reached the extreme stage of differentiation, cardiomyocytes, granular leukocytes and others.

In our body, there are renewing tissues in which there is a pool of constantly dividing cells that replace spent or dying cells. Such cells exist in the crypts of the intestine, in the basal layer of the skin epithelium, in the bone marrow (hematopoietic cells). Cell renewal can occur quite intensively: for example, connective tissue cells in the pancreas are replaced every 24 hours, gastric mucosa cells – every three days, leukocytes – every 10 days, skin cells – every six weeks, approximately 70 g of proliferating small intestine cells are removed from the body daily [1].

Stem cells that exist in almost all organs and tissues are able to divide indefinitely. Tissue regeneration occurs due to the proliferation of stem cells, which can not only divide, but also differentiate into cells of the tissue whose regeneration is taking place.

Stem cells are present in the myocardium, in the brain (in the hypocampus and in the olfactory bulbs) and in other tissues. This opens up great hopes in terms of the treatment of neurodegenerative diseases and myocardial infarction [2-4].

Constantly renewing tissues contribute to an increase in life expectancy. During cell division, tissue rejuvenation occurs: new cells take the place of damaged ones, while repair (elimination of DNA damage) occurs more intensively and regeneration is possible in case of tissue damage. It is not surprising that vertebrates have significantly higher life expectancy than invertebrates – the same insects whose cells do not divide in adulthood.

But at the same time, renewing tissues are subject to hyperproliferation, which leads to the formation of tumors, including malignant ones. This is due to irregularities in the regulation of cell division and an increased frequency of mutagenesis in actively dividing cells. According to modern concepts, in order for a cell to acquire the property of malignancy, it needs 4-6 mutations*.

It is worth remembering, among other things, that mutation mutations are different, and according to the latest genomic research, in each generation a person acquires about 60 new mutations (which were not in the DNA of his parents). Obviously, most of them are quite neutral (see "Over a thousand: the third phase of human genomics"). – Ed.

Mutations occur rarely, and in order for a cell to become cancerous – this is calculated for human fibroblasts – about 100 divisions must occur (this number of divisions usually occurs in a person at about the age of 40) [5].

In order to protect against itself, special cellular mechanisms of tumor suppression have formed in the body. One of them is the replicative aging of cells (senescence), which consists in the irreversible stopping of cell division at the G1 stage of the cell cycle. With aging, the cell stops dividing: it does not respond to growth factors and becomes resistant to apoptosis.

Hayflick LimitThe phenomenon of cell aging was first discovered in 1961 by Leonard Hayflick and colleagues on fibroblast culture.

It turned out that cells in human fibroblast culture live for a limited time under good conditions and are able to double approximately 50±10 times, and this number was called the Hayflick limit [6, 7]. Before Hayflick's discovery, the prevailing view was that cells are immortal, and aging and death are a property of the organism as a whole.

This concept was considered irrefutable largely due to the experiments of Carrel, who maintained the culture of chicken heart cells for 34 years (it was thrown out only after his death). However, as it turned out later, the immortality of the Carrel culture was an artifact, because along with the embryonic serum, which was added to the culture medium for cell growth, the embryonic cells themselves got there (and, most likely, the Carrel culture was no longer what it was at the beginning).

Cancer cells are truly immortal. Thus, HeLa cells isolated in 1951 from Henrietta Lacks' cervical tumor are still used by cytologists (in particular, a vaccine against polio was developed with the help of HeLa cells). These cells have even been in space.

As it turned out, the Hayflick limit depends on age: the older a person is, the fewer times his cells are doubled in culture. Interestingly, frozen cells during defrosting and subsequent cultivation seem to remember the number of divisions before freezing. In fact, there is a "division counter" inside the cell, and upon reaching a certain limit (the Hayflick limit), the cell stops dividing – it becomes senescent. Senescent (old) cells have a specific morphology – they are large, flattened, with large nuclei, highly vacuolized, their gene expression profile changes. In most cases, they are resistant to apoptosis.

However, the aging of the body cannot be reduced only to the aging of cells. This is a much more complex process. There are old cells in a young body, but there are few of them! When senescent cells accumulate in tissues with age, degenerative processes begin that lead to age-related diseases. One of the factors of these diseases is the so–called senile "sterile" inflammation, which is associated with the expression of pro-inflammatory cytokines by old cells.

Another important factor of biological aging is the structure of chromosomes and their tips – telomeres.

Telomeric theory of agingIn 1971, our compatriot Alexey Matveyevich Olovnikov suggested that the Hayflick limit is associated with the "under-replication" of the end sections of linear chromosomes (they have a special name – telomeres).

The fact is that in each cycle of cell division, telomeres are shortened due to the inability of DNA polymerase to synthesize a copy of DNA from the very tip [8, 9]. In addition, Olovnikov predicted the existence of telomerase (an enzyme that adds repeating DNA sequences to the ends of chromosomes), based on the fact that otherwise DNA in actively dividing cells would quickly be "eaten" and the genetic material would be lost. (The problem is that telomerase activity fades in most differentiated cells.)

Telomeres (Fig. 1) play an important role: they stabilize the tips of chromosomes, which otherwise, as cytogenetics say, would become "sticky", i.e. subject to various chromosomal aberrations, which leads to degradation of genetic material.

Figure 1. Telomeres are the end sections of chromosomes.
Since a person has 23 pairs of chromosomes (that is, 46 pieces), telomeres turn out to be 92.

Telomeres consist of repeating (1000-2000 times) sequences (5’–TTAGGG–3’), which in total gives 10-15 thousand nucleotide pairs for each chromosomal tip. At the 3’ end, telomeres have a rather long single-stranded DNA section (150-200 nucleotides) involved in the formation of a lasso-type loop [10, 11] (Fig. 2).

Figure 2. Composition and structure of telomeres. Multiple cell division in the absence of telomerase activity
leads to telomere shortening and replicative aging.

Several proteins are associated with telomeres, forming a protective "cap" – this complex is called shelterin (Fig. 3). Shelterin protects telomeres from the action of nucleases and adhesion, and, apparently, it is he who preserves the integrity of the chromosome.

Figure 3. The structure of the telomeric complex (shelterin).
Telomeres are located at the ends of chromosomes and consist of tandem repeats TTAGGG,
which end with a 32-member protruding single-stranded fragment.
Telomeric DNA is associated with shelterin, a complex of six proteins: TRF1, TRF2, RAP1, TIN2, TPP1 and POT1.

Unprotected ends of chromosomes are perceived by the cell as damage to genetic material, which activates DNA repair. The telomeric complex, together with shelterin, "stabilizes" the chromosomal tips, protecting the entire chromosome from destruction. In senescent cells, the critical shortening of telomeres disrupts this protective function [12, 13], and therefore chromosomal aberrations begin to form, which often lead to malignancy. To prevent this from happening, special molecular mechanisms block cell division, and the cell goes into a state of senescence – an irreversible stop of the cell cycle. At the same time, the cell is guaranteed not to multiply, which means it will not be able to form a tumor. In cells with impaired senescence ability (which multiply despite telomere dysfunction), chromosomal aberrations are formed.

The length of telomeres and the rate of their shortening depends on age. In humans, the length of telomeres varies from 15 thousand nucleotide pairs (so-called n.p.) at birth to 5 t.n.p. in chronic diseases. Telomere length is maximal in 18-month-old children, and then it quickly decreases to 12 t.n.p. by the age of five. After that, the shortening rate decreases [14].

Telomeres shorten in different people at different rates. E. Blackburn (winner of the 2009 Nobel Prize in Physiology or Medicine) has found that women who are constantly experiencing stress (for example, mothers of chronically ill children) have significantly shorter telomeres compared to their peers (by about ten years!). E. Blackburn's laboratory has developed a commercial test to determine the "biological age" of people based on the length of telomeres.

Interestingly, mice have very long telomeres (50-40 t.n.p., compared with 10-15 t.n.p. in humans). In some lines of laboratory mice, the telomere length reaches 150 t.n. Moreover, in mice, telomerase is always active, which prevents telomeres from shortening. However, this, as everyone knows, does not make mice immortal. Moreover, tumors develop much more often in them than in humans, which suggests that telomere shortening as a mechanism of protection against tumors in mice does not work [15].

When comparing the length of telomeres and telomerase activity in different mammals, it turned out that species characterized by replicative aging of cells have a long lifespan and a large weight. These are, for example, whales, whose life expectancy can reach 200 years. For such organisms, replicative aging is simply necessary, since too many divisions give rise to many mutations that need to be dealt with somehow. Presumably, replicative aging is such a mechanism of struggle, which is also accompanied by telomerase repression [16].

The aging of differentiated cells occurs differently. Both neurons and cardiomyocytes are aging, but they do not divide! For example, they accumulate lipofuscin, an aging pigment that disrupts the functioning of cells and triggers apoptosis. Fat accumulates in the cells of the liver and spleen with age.

The relationship of replicative cell aging with the aging of the body, strictly speaking, has not been proven, but age-related pathology is also accompanied by cell aging (Fig. 4).

Figure 4. Histochemically stained for beta-galactosidase activity
human fibroblasts of the WI-38 line. A: young; B: old (senescent).

Malignant neoplasms of the elderly are mostly associated with renewable tissues. Oncological diseases in developed countries are one of the main causes of morbidity and mortality, and an independent risk factor for cancer is simply... age. The number of deaths from tumor diseases increases exponentially with age, as does the overall mortality. This tells us that there is a fundamental link between aging and carcinogenesis.

Telomerase is an enzyme that has been predictedThere must be a mechanism in the body that compensates for the shortening of telomeres, – this assumption was made by A.M. Olovnikov.

Indeed, in 1984, such an enzyme was discovered by Carol Grader and named telomerase. Telomerase (Fig. 5) is a reverse transcriptase that increases the length of telomeres, compensating for their underreplication. In 2009, E. Blackburn, K. Grader and D. Shostak were awarded the Nobel Prize for the discovery of this enzyme and a series of works on the study of telomeres and telomerase.

Figure 5. Telomerase contains a catalytic component (reverse transcriptase TERT),
telomerase RNA (hTR or TERC) containing two copies of the telomeric repeat
and being a matrix for the synthesis of telomeres, and the protein dyskerin.

According to E. Blackburn, telomerase is involved in regulating the activity of about 70 genes. Telomerase is active in embryonic and embryonic tissues, in stem and proliferating cells. It is found in 90% of cancerous tumors, which ensures the unstoppable proliferation of cancer cells. Currently, among the drugs that are used to treat cancer, there is also a telomerase inhibitor. But in most somatic cells of the adult body, telomerase is not active.

Many stimuli can lead a cell to a state of senescence – telomere dysfunction, DNA damage caused by mutagenic environmental influences, endogenous processes, strong mitogenic signals (overexpression of Ras, Raf, Mek, Mos, E2F-1 oncogenes, etc.), chromatin disorders, stress, etc. In fact, cells stop dividing–become senescent–in response to potentially cancer-causing events.

Guardian of the GenomeTelomere dysfunction, which occurs when they are shortened or when shelterin is disrupted, activates the p53 protein.

This transcription factor leads the cell to a state of senescence, or causes apoptosis. In the absence of p53, chromosome instability develops, characteristic of human carcinomas. Mutations in the p53 protein are found in 50% of breast adenocarcinomas and in 40-60% of cases of colorectal adenocarcinoma. Therefore, p53 is often called the "guardian of the genome".

Telomerase is reactivated in most tumors of epithelial origin, which are characteristic of the elderly. It is believed that telomerase reactivation is an important stage of malignant processes, because it allows cancer cells to "ignore" the Hayflick limit. Telomere dysfunction promotes chromosomal mergers and aberrations, which in the absence of p53 most often leads to malignant neoplasms.

On the molecular mechanisms of cell agingIn order to understand the molecular mechanisms of the cell transition to a state of senescence, I will remind you how cell division occurs.

The process of cell reproduction is called proliferation. The lifetime of a cell from division to division is called the cell cycle. The proliferation process is regulated both by the cell itself – autocrine growth factors – and by its microenvironment – paracrine signals.

The activation of proliferation occurs through the cell membrane, in which there are receptors that perceive mitogenic signals – these are mainly growth factors and intercellular contact signals. Growth factors usually have a peptide nature (about 100 of them are known to date). These are, for example, platelet growth factor, which is involved in thrombosis and wound healing, epithelial growth factor, various cytokines – interleukins, tumor necrosis factor, colony-stimulating factors, etc. After activation of proliferation, the cell leaves the resting phase G0 and the cell cycle begins [19] (see Fig. 6).

Figure 6. Diagram of the cell cycle.

The cell cycle is divided into four stages:
1. G1 (pre–synthetic) is the period when the cell is preparing for DNA replication. At this stage, the cell cycle may stop if DNA damage is detected (at the time of repair). If errors in DNA replication are detected and they cannot be corrected by repair, the cell does not go to stage S.
2. S (synthetic) – when DNA replication occurs.
3. G2 (post-synthetic) – preparation of the cell for mitosis, when the accuracy of DNA replication is checked; if under-replicated fragments or other violations in synthesis are detected, the transition to the next stage (mitosis) does not occur.
4. M (mitosis) – the formation of a cell spindle, segregation (divergence of chromosomes) and the formation of two daughter cells (actually division).

The cell cycle is regulated by cyclin-dependent kinases, different for each stage of the cell cycle. They are activated by cyclins and inactivated by a number of inhibitors. The purpose of such a complex regulation is to ensure DNA synthesis with as few errors as possible, so that the daughter cells have absolutely identical hereditary material. Verification of the correctness of DNA copying is carried out at four "control points" of the cycle: if errors are detected, the cell cycle stops and DNA repair is activated. If violations of the DNA structure can be corrected, the cell cycle continues. If not, it is better for the cell to "commit suicide" (by apoptosis) in order to avoid the possibility of becoming cancerous.

The molecular mechanisms leading to irreversible cell cycle arrest are controlled by tumor suppressor genes, including p53 and pRB associated with cyclin-dependent kinase inhibitors. Suppression of the cell cycle in the G1 phase is carried out by the protein p53, acting through an inhibitor of cyclin-dependent kinase p21. Transcription factor p53 is activated when DNA is damaged, and its function is to remove from the pool of replicating cells those that are potentially oncogenic (hence the nickname p53 – "guardian of the genome"). This idea is confirmed by the fact that p53 mutations are detected in ~50% of cases of malignant tumors. Another manifestation of p53 activity is associated with apoptosis of the most damaged cells.

Cell senescence and age-dependent diseasesSenescent cells accumulate with age and contribute to age-related diseases.

They reduce the proliferative potential of the tissue and deplete the stem cell pool, which leads to degenerative disorders of the tissue and reduces the ability to regenerate and renew.

Senescent cells are characterized by specific gene expression: they secrete inflammatory cytokines and metalloproteinases that destroy the intercellular matrix. It turns out that old cells provide sluggish senile inflammation, and the accumulation of old fibroblasts in the skin causes an age-related decrease in the ability to heal wounds (see Fig. 7).

Figure 7. The relationship between cell aging and aging of the body.

Old cells also stimulate proliferation and malignancy of nearby precancerous cells, due to the secretion of epithelial growth factor [20]. Senescent cells accumulate in many human tissues, are present in atherosclerotic plaques, in skin ulcers, in arthritic joints, as well as in benign and preneoplastic hyperproliferative lesions of the prostate and liver. When irradiated with cancerous tumors, some cells also go into a state of senescence, thereby ensuring relapses of the disease.

Thus, cellular aging demonstrates the effect of negative pleiotropy, the essence of which is that what is good for a young organism can become bad for an old one. The most striking example is the processes of inflammation. A pronounced inflammatory reaction contributes to the rapid recovery of a young organism in infectious diseases. In old age, active inflammatory processes lead to age-related diseases. Now it is considered that inflammation plays a decisive role in almost all age-related diseases, starting with neurodegenerative ones.

It turns out a paradox: the aging of cells in a young body protects against cancer, and in an old one – contributes to it! Currently, in the USA, the Mayo Clinic is conducting research on the effect of "elimination" of old cells from the body. Encouraging results have been obtained on animals on increasing life expectancy and slowing down the clinical manifestations of age–related diseases if senescent cells - good citizens, but bad neighbors - are selectively eliminated from the tissues of old animals.

For a list of the literary sources indicated in the text, see the original article.

Portal "Eternal youth" http://vechnayamolodost.ru30.10.2012

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