Genetic regulation of cell proliferation. Mechanisms of cell division
Proliferative processes in acute inflammation begin soon after the influence of the phlogogenic factor on the tissue and are more pronounced along the periphery of the inflammation zone. One of the conditions for the optimal course of proliferation is the attenuation of the processes of alteration and exudation.
Proliferation
Phagocytes also produce and release into the intercellular fluid a number of biologically active substances that regulate the development of either immunity, allergies, or a state of tolerance. Thus, inflammation is directly related to the formation of immunity or immunopathological reactions in the body.
Proliferation, a component of the inflammatory process and its final stage, is characterized by an increase in the number of stromal and, as a rule, parenchymal cells, as well as the formation of intercellular substance at the site of inflammation. These processes are aimed at the regeneration of altered and/or replacement of destroyed tissue elements. Various biologically active substances, especially those stimulating cell proliferation (mitogens), are essential at this stage of inflammation.
The forms and degree of proliferation of organ-specific cells are different and are determined by the nature of the cell populations (see the article “Cell Population” in the Appendix “Reference of Terms”).
In some organs and tissues (for example, liver, skin, gastrointestinal tract, respiratory tract), the cells have a high proliferative ability, sufficient to eliminate structural defects at the site of inflammation.
In other organs and tissues this ability is very limited (for example, in the tissues of tendons, cartilage, ligaments, kidneys, etc.).
In a number of organs and tissues, parenchymal cells have virtually no proliferative activity (for example, cardiac muscle myocytes, neurons). In this regard, upon completion of the inflammatory process in the tissues of the myocardium and nervous system, stromal cells, mainly fibroblasts, which also form non-cellular structures, proliferate at the site of inflammation. As a result, a connective tissue scar is formed. At the same time, it is known that parenchymal cells of these tissues have a high ability for hypertrophy and hyperplasia of subcellular structures.
Activation of proliferative processes correlates with the formation of biologically active substances that have an anti-inflammatory effect (a kind of anti-inflammatory mediators). The most effective among them include:
Inhibitors of hydrolases, in particular proteases (for example, antitrypsin), microglobulin, plasmin or complement factors;
Antioxidants (eg, ceruloplasmin, haptoglobin, peroxidases, SOD);
Polyamines (eg putrescine, spermine, cadaverine);
Glucocorticoids;
Heparin (suppresses adhesion and aggregation of leukocytes, activity of kinins, biogenic amines, complement factors).
Replacement of dead and damaged tissue elements during inflammation is noted after their destruction and elimination (this process is called wound cleansing).
The proliferation reactions of both stromal and parenchymal cells are regulated by various factors. The most significant among them include:
Many inflammatory mediators (for example, TNF, which suppresses proliferation; leukotrienes, kinins, biogenic amines, which stimulate cell division).
Specific metabolic products of leukocytes (for example, monokines, lymphokines, ILs, growth factors), as well as platelets, that can activate cell proliferation.
Low molecular weight peptides released during tissue destruction, polyamines (putrescine, spermidine, spermine), as well as nucleic acid breakdown products that activate cell reproduction.
Hormones (GH, insulin, T4, corticoids, glucagon), many of them capable of both activating and suppressing proliferation depending on their concentration, activity, synergistic and antagonistic interactions; for example, glucocorticoids in low doses inhibit, and mineralocorticoids activate regeneration reactions.
Proliferation processes are also influenced by a number of other factors, for example, enzymes (collagenase, hyaluronidase), ions, neurotransmitters and others.
. Chapter II
Cell reproduction. Problems of cell proliferation in medicine.
2.1. Life cycle of a cell.
The cellular theory states that cells arise from cells by dividing the original. This position excludes the formation of cells from non-cellular matter. Cell division is preceded by reduplication of their chromosomal apparatus, DNA synthesis in both eukaryotic and prokaryotic organisms.
The time a cell exists from division to division is called the cell or life cycle. Its magnitude varies significantly: for bacteria it is 20-30 minutes, for a shoe 1-2 times a day, for an amoeba about 1.5 days. Multicellular cells also have different abilities to divide. In early embryogenesis they divide frequently, and in the adult body they mostly lose this ability, as they become specialized. But even in an organism that has reached full development, many cells must divide to replace worn-out cells that are constantly sloughed off and, finally, new cells are needed to heal wounds.
Therefore, in some populations of cells, divisions must occur throughout life. Taking this into account, all cells can be divided into three categories:
1. By the time a child is born, nerve cells reach a highly specialized state, losing the ability to reproduce. During ontogenesis, their number continuously decreases. This circumstance also has one good side; if nerve cells divided, then higher nervous functions (memory, thinking) would be disrupted.
2. Another category of cells is also highly specialized, but due to their constant exfoliation, they are replaced by new ones and this function is performed by cells of the same line, but not yet specialized and have not lost the ability to divide. These cells are called renewing cells. An example is the constantly renewed cells of the intestinal epithelium, hematopoietic cells. Even bone tissue cells can be formed from unspecialized ones (this can be observed during the reparative regeneration of bone fractures). Populations of unspecialized cells that retain the ability to divide are usually called stem cells.
3. The third category of cells is an exception, when highly specialized cells under certain conditions can enter the mitotic cycle. We are talking about cells that have a long lifespan and where, after complete growth, cell division occurs rarely. An example is hepatocytes. But if 2/3 of the liver is removed from an experimental animal, then in less than two weeks it is restored to its previous size. The same are the cells of the glands that produce hormones: under normal conditions, only a few of them are able to reproduce, and under altered conditions, most of them can begin to divide.
The cell cycle means the repeated repetition of sequential events over a certain period of time. Typically, cyclic processes are graphically depicted as circles.
The cell cycle is divided into two parts: mitosis and the interval between the end of one mitosis and the beginning of the next - interphase. The autoradiography method made it possible to establish that in interphase the cell not only performs its specialized functions, but also synthesizes DNA. This period of interphase is called synthetic (S). It begins approximately 8 hours after mitosis and ends after 7-8 hours. The interval between the S-period and mitosis was called presynthetic (G1 - 4 hours) after the synthetic period, before mitosis itself - postsynthetic (G2). happening over the course of about an hour.
Thus, there are four stages in the steel cell cycle; mitosis, G1 period, S period, G2 period.
Establishing the fact of DNA duplication in interphase means that during interphase the cell cannot perform specialized functions; it is busy building cellular structures, synthesizing building materials that ensure the growth of daughter cells, accumulating energy expended during mitosis itself, and synthesizing specific enzymes for DNA replication . Therefore, interphase cells, in order to fulfill their functions prescribed by the genetic program (become highly specialized), must temporarily or permanently leave the cycle during the G0 period, or remain in an extended G1 (no significant differences in the state of cells of the G0 and G1 periods were noted, since it is possible to return from G0 cells in a cycle). It should be especially noted that in multicellular mature organisms, the majority of cells are in the G0 period.
As already mentioned, the increase in the number of cells occurs only due to the division of the original cell, which is preceded by a phase of accurate reproduction of genetic material, DNA molecules, chromosomes.
Mitotic division includes new cell states: interphase, decondensed and already reduplicated chromosomes pass into the compact form of mitotic chromosomes, an achromatic mitotic apparatus is formed, which is involved in chromosome transfer, chromosomes diverge to opposite poles and cytokinesis occurs. The process of indirect division is usually divided into the following main phases: prophase, metaphase, anaphase and telophase. The division is conditional, since mitosis is a continuous process and the change of phases occurs gradually. The only phase that has a real beginning is anaphase, in which
chromosomes begin to separate. The duration of individual phases is different (on average, prophase and telophase - 30-40", anaphase and metaphase - 7-15"). At the beginning of mitosis, a human cell contains 46 chromosomes, each of which consists of 2 identical halves - chromatids (a chromatid is also called the S-chromosome, and a chromosome consisting of 2 chromatids is called the d-chromosome).
One of the most remarkable phenomena observed in mitosis is the formation of the spindle. It ensures the alignment of d-chromosomes in one plane, in the middle of the cell, and the movement of S-chromosomes to the poles. The spindle is formed by the centrioles of the cell center. Microtubules are formed in the cytoplasm from the protein tubulin.
In the G1 period, each cell contains two centrioles; by the time of the transition to the G2 period, a daughter centriole is formed near each centriole and a total of two pairs are formed.
In prophase, one pair of centrioles begins to move to one pole, the other to the other.
Between pairs of centrioles, a set of interpolar and chromosomal microtubules begins to form towards each other.
At the end of prophase, the nuclear membrane disintegrates, the nucleolus ceases to exist, chromosomes (d) spiral, the spindle moves to the middle of the cell and d-chromosomes find themselves in the spaces between the microtubules of the spindle.
During prophase, D chromosomes undergo a path of condensation from thread-like structures to rod-shaped ones. The shortening and thickening of (d-chromosomes continues for some time in metaphase, as a result of which metaphase d-chromosomes have sufficient density. A centromere is clearly visible in the chromosomes, dividing them into equal or unequal arms, consisting of 2 adjacent S- chromosomes (chromatids). At the beginning of anaphase, the S chromosomes (chromatids) begin to move from the equatorial plane to the poles. Anaphase begins with the splitting of the centromeric region of each chromosome, as a result of which the two S chromosomes of each d chromosome are completely separated from each other. This means that each daughter cell receives an identical set of 46 S chromosomes. After centromere separation, one half of the 92 S chromosomes begins to move to one pole, the other half to the other.
To this day, it has not been established precisely under what forces the movement of chromosomes to the poles occurs. There are several versions:
1. The spindle contains actin-containing filaments (as well as other muscle proteins), it is possible that this force is generated in the same way as in muscle cells.
2. The movement of chromosomes is caused by the sliding of chromosomal microtubules along continuous (interpolar) microtubules with opposite polarity (McItosh, 1969, Margolis, 1978).
3. The speed of chromosome movement is regulated by kinetochore microtubules to ensure orderly segregation of chromatids. Most likely, all of the listed mechanisms for achieving a mathematically precise distribution of hereditary substance to daughter cells cooperate.
Towards the end of anaphase and the beginning of telophase, a constriction begins to form in the middle of the elongated cell; it forms the so-called cleavage furrow, which, going deeper, divides the cell into two daughter cells. Actin filaments take part in the formation of the furrow. But as the furrow deepens, the cells are connected to each other by a bundle of microtubules called the median body, the remainder of which is present for some time in interphase. Parallel to cytokinesis, chromosome decoiling occurs at each pole in the reverse order from the chromosomal to the nucleosomal level. Finally, the hereditary substance takes the form of clumps of chromatin, either tightly packed or decondensed. The nucleolus, nuclear envelope, surrounding chromatin and karyoplasm are formed again. Thus, as a result of mitotic cell division, the newly formed daughter cells are identical to each other and are a copy of the mother cell, which is important for the subsequent growth, development and differentiation of cells and tissues.
2.2. Mechanism of regulation of mitotic activity
Maintaining the number of cells at a certain, constant level ensures overall homeostasis. For example, the number of red and white blood cells in a healthy body is relatively stable, although these cells die, they are constantly replenished. Therefore, the rate at which new cells are formed must be regulated to match the rate at which they die.
To maintain homeostasis, it is necessary that the number of different specialized cells in the body and the functions they must perform be under the control of various regulatory mechanisms that maintain all this in a stable state.
In many cases, the cells are given a signal that they need to increase their functional activity, and this may require an increase in the number of cells. For example, if the Ca content in the blood drops, then the cells of the parathyroid gland increase the secretion of the hormone, and the calcium level reaches normal. But if the animal’s diet does not have enough calcium, then additional production of the hormone will not increase the content of this element in the blood. In this case, the cells of the thyroid gland begin to rapidly divide, so that an increase in their number leads to a further increase in the synthesis of the hormone. Thus, a decrease in a particular function can lead to an increase in the population of cells providing these functions.
In people who find themselves in high mountains, the number of red blood cells sharply increases (at an altitude of less than 02) in order to provide the body with the necessary amount of oxygen. Kidney cells react to a decrease in oxygen and increase the secretion of erythropoietin, which enhances hematopoiesis. After the formation of a sufficient number of additional red blood cells, hypoxia disappears and the cells producing this hormone reduce its secretion to normal levels.
Cells that are fully differentiated cannot divide, but their numbers can still be increased by the stem cells from which they originate. Nerve cells cannot divide under any circumstances, but they can increase their function by increasing their processes and multiplying the connections between them.
It should be noted that in adult individuals the ratio of the overall sizes of various organs remains more or less constant. When the existing ratio of organ sizes is artificially disrupted, it tends to normal (removal of one kidney leads to an increase in the other).
One of the concepts that explains this phenomenon is that cell proliferation is regulated by special substances called kelons. It is assumed that they have specificity for different types of cells and organ tissues. It is believed that a decrease in the number of kelons stimulates cell proliferation, for example, during regeneration. Currently, this problem is being carefully studied by various specialists. Evidence has been obtained that keylons are glycoproteins with a molecular weight of 30,000 – 50,000.
2.3. Irregular types of cell reproduction
Amitosis. Direct division, or amitosis, is described earlier than mitotic division, but is much less common. Amitosis is the division of a cell in which the nucleus is in an interphase state. In this case, chromosome condensation and spindle formation do not occur. Formally, amitosis should lead to the appearance of two cells, but most often it leads to the division of the nucleus and the appearance of bi- or multinucleated cells.
Amitotic division begins with fragmentation of the nucleoli, followed by division of the nucleus by constriction (or invagination). There may be multiple divisions of the nucleus, usually of unequal size (in pathological processes). Numerous observations have shown that amitosis almost always occurs in cells that are obsolete, degenerating and unable to produce full-fledged elements in the future. So, normally, amitotic division occurs in the embryonic membranes of animals, in the follicular cells of the ovary, and in giant trophoblast cells. Amitosis has a positive meaning in the process of tissue or organ regeneration (regenerative amitosis). Amitosis in aging cells is accompanied by disturbances in biosynthetic processes, including replication, DNA repair, as well as transcription and translation. The physicochemical properties of chromatin proteins in cell nuclei, the composition of the cytoplasm, the structure and functions of organelles change, which entails functional disorders at all subsequent levels - cellular, tissue, organ and organismal. As destruction increases and restoration fades, natural cell death occurs. Amitosis often occurs during inflammatory processes and malignant neoplasms (induced amitosis).
Endomitosis. When cells are exposed to substances that destroy spindle microtubules, division stops, and chromosomes will continue the cycle of their transformations: replicate, which will lead to the gradual formation of polyploid cells - 4 p. 8 p., etc. This transformation process is otherwise called endoreproduction. The ability of cells to undergo endomitosis is used in plant breeding to obtain cells with a multiple set of chromosomes. For this purpose, colchicine and vinblastine are used, which destroy the filaments of the achromatin spindle. Polyploid cells (and then adult plants) are large in size; the vegetative organs from such cells are large, with a large supply of nutrients. In humans, endoreproduction occurs in some hepatocytes and cardiomyocytes.
Another, rarer result of endomitosis is polytene cells. During polyteny in the S-period, as a result of replication and non-disjunction of chromosomal strands, a multi-stranded, polytene structure is formed. They differ from mitotic chromosomes in their larger size (200 times longer). Such cells are found in the salivary glands of dipteran insects and in the macronuclei of ciliates. On polytene chromosomes, swellings and puffs (transcription sites) are visible - an expression of gene activity. These chromosomes are the most important object of genetic research.
2.4. Problems of cell proliferation in medicine.
As is known, tissues with a high rate of cell turnover are more sensitive to the effects of various mutagens than tissues in which cells are renewed slowly. However, for example, radiation damage may not appear immediately and does not necessarily weaken with depth; sometimes it even damages deep-lying tissues much more than superficial ones. When cells are irradiated with X-rays or gamma rays, gross disturbances occur in the cell life cycle: mitotic chromosomes change shape, they break, followed by incorrect joining of fragments, and sometimes individual parts of chromosomes disappear altogether. Spindle anomalies may occur (not two poles in the cell, but three will form), which will lead to uneven divergence of chromatids. Sometimes cell damage (large doses of radiation) is so significant that all attempts by the cell to begin mitosis are unsuccessful and division stops.
This effect of radiation partly explains its use in tumor therapy. The goal of radiation is not to kill tumor cells in interphase, but to cause them to lose their ability to undergo mitosis, which will slow or stop tumor growth. Radiation in doses that are not lethal to the cell can cause mutations leading to increased proliferation of altered cells and give rise to malignant growth, as often happened to those who worked with X-rays, not knowing about their danger.
Cell proliferation is affected by many chemicals, including drugs. For example, the alkaloid colchicine (contained in colchicum corms) was the first drug that relieved joint pain due to gout. It turned out that it also has another effect - stopping division by binding to tubulin proteins from which microtubules are formed. Thus, colchicine, like many other drugs, blocks the formation of the spindle.
On this basis, alkaloids such as vinblastine and vincristine are used to treat certain types of malignant neoplasms, becoming part of the arsenal of modern chemotherapeutic anticancer drugs. It should be noted that the ability of substances such as colchicine to stop mitosis is used as a method for the subsequent identification of chromosomes in medical genetics.
Of great importance for medicine is the ability of differentiated (and germ) cells to maintain their potential for proliferation, which sometimes leads to the development of tumors in the ovaries, in the section of which cell layers, tissues, and organs are visible as a “mush”. Scraps of skin, hair follicles, hair, ugly teeth, pieces of bones, cartilage, nervous tissue, fragments of the eye, etc. are revealed, which requires urgent surgical intervention.
2.5. Pathology of cell reproduction
Mitotic cycle abnormalities.. The mitotic rhythm, usually adequate to the need for restoration of aging, dead cells, can be changed under pathological conditions. A slowdown of the rhythm is observed in aging or poorly vascularized tissues, an increase in the rhythm is observed in tissues under various types of inflammation, hormonal influences, in tumors, etc.
REGULATION OF THE CELL CYCLE
Introduction
Activation of proliferation
Cell cycle
Cell cycle regulation
Exogenous regulators of proliferation
Endogenous regulators of the cell cycle
Pathways of CDK regulation
G1 phase regulation
S phase regulation
G2 phase regulation
Regulation of mitosis
DNA damage
Ways to repair DNA double-strand breaks
Cellular response to DNA damage and its regulation
Tissue regeneration
Regulation of tissue regeneration
Conclusion
Bibliography
Introduction
The cell is the elementary unit of all living things. There is no life outside the cell. Cell reproduction occurs only through division of the original cell, which is preceded by the reproduction of its genetic material. Activation of cell division occurs due to the influence of external or internal factors on it. The process of cell division from the moment of its activation is called proliferation. In other words, proliferation is the multiplication of cells, i.e. an increase in the number of cells (in culture or tissue) that occurs through mitotic divisions. The duration of the cell's existence as such, from division to division, is usually called the cell cycle.
In the adult human body, cells of different tissues and organs have different abilities to divide. In addition, with aging, the intensity of cell proliferation decreases (i.e., the interval between mitoses increases). There are populations of cells that have completely lost the ability to divide. These are, as a rule, cells at the terminal stage of differentiation, for example, mature neurons, granular blood leukocytes, cardiomyocytes. In this regard, the exception is the immune B- and T-memory cells, which, being in the final stage of differentiation, are able to begin to proliferate when a certain stimulus appears in the body in the form of a previously encountered antigen. The body has constantly renewing tissues - various types of epithelium, hematopoietic tissues. In such tissues there are cells that constantly divide, replacing spent or dying cell types (for example, intestinal crypt cells, cells of the basal layer of the integumentary epithelium, hematopoietic cells of the bone marrow). There are also cells in the body that do not reproduce under normal conditions, but again acquire this property under certain conditions, in particular when it is necessary to regenerate tissues and organs. The process of cell proliferation is tightly regulated both by the cell itself (regulation of the cell cycle, cessation or slowdown of the synthesis of autocrine growth factors and their receptors) and its microenvironment (lack of stimulating contacts with neighboring cells and matrix, cessation of secretion and/or synthesis of paracrine growth factors). Dysregulation of proliferation leads to unlimited cell division, which in turn initiates the development of the oncological process in the body.
Activation of proliferation
The main function associated with the initiation of proliferation is assumed by the plasma membrane of the cell. It is on its surface that events occur that are associated with the transition of resting cells to an activated state that precedes division. The plasma membrane of cells, due to the receptor molecules located in it, perceives various extracellular mitogenic signals and ensures the transport into the cell of the necessary substances that take part in the initiation of the proliferative response. Mitogenic signals can be contacts between cells, between a cell and a matrix, as well as the interaction of cells with various compounds that stimulate their entry into the cell cycle, which are called growth factors. A cell that has received a mitogenic signal to proliferate starts the process of division.
CELL CYCLE
The entire cell cycle consists of 4 stages: presynthetic (G1), synthetic (S), postsynthetic (G2) and mitosis itself (M). In addition, there is a so-called G0 period, which characterizes the resting state of the cell. In the G1 period, cells have diploid DNA content per nucleus. During this period, cell growth begins, mainly due to the accumulation of cellular proteins, which is caused by an increase in the amount of RNA per cell. In addition, preparations for DNA synthesis begin. In the next S-period, the amount of DNA doubles and the number of chromosomes accordingly doubles. The post-synthetic G2 phase is also called premitotic. During this phase, active synthesis of mRNA (messenger RNA) occurs. This stage is followed by the cell division itself, or mitosis.
The division of all eukaryotic cells is associated with the condensation of duplicated (replicated) chromosomes. As a result of division, these chromosomes are transferred to daughter cells. This type of division of eukaryotic cells - mitosis (from the Greek mitos - threads) - is the only complete way to increase the number of cells. The process of mitotic division is divided into several stages: prophase, prometaphase, metaphase, anaphase, telophase.
REGULATION OF THE CELL CYCLE
The purpose of the regulatory mechanisms of the cell cycle is not to regulate the passage of the cell cycle as such, but to ensure, ultimately, the error-free distribution of hereditary material during the process of cell reproduction. The regulation of cell reproduction is based on the change in states of active proliferation and proliferative organ. Regulatory factors that control cell reproduction can be divided into two groups: extracellular (or exogenous) or intracellular (or endogenous). Exogenous factors are found in the cell microenvironment and interact with the cell surface. Factors that are synthesized by the cell itself and act inside it are classified as endogenous factors. This division is very arbitrary, since some factors, being endogenous in relation to the cell producing them, can leave it and act as exogenous regulators on other cells. If regulatory factors interact with the same cells that produce them, then this type of control is called autocrine. With paracrine control, the synthesis of regulators is carried out by other cells.
EXOGENOUS REGULATORS OF PROLIFERATION
In multicellular organisms, the regulation of the proliferation of various cell types occurs due to the action of not one growth factor, but a combination of them. In addition, some growth factors, being stimulators for some types of cells, behave as inhibitors in relation to others. Classic growth factors are polypeptides with a molecular weight of 7-70 kDa. To date, more than a hundred such growth factors are known. However, only a few of them will be discussed here.
Perhaps the largest body of literature is devoted to platelet-derived growth factor (PDGF). Released upon destruction of the vascular wall, PDGF is involved in the processes of thrombus formation and wound healing. PDGF is a potent growth factor for quiescent fibroblasts. Along with PDGF, epidermal growth factor (EGF), which is also capable of stimulating the proliferation of fibroblasts, has been studied no less thoroughly. But, besides this, it also has a stimulating effect on other types of cells, in particular on chondrocytes.
A large group of growth factors are cytokines (interleukins, tumor necrosis factors, colony-stimulating factors, etc.). All cytokines are multifunctional. They can either enhance or inhibit proliferative responses. For example, different subpopulations of CD4+ T lymphocytes, Th1 and Th2, producing a different spectrum of cytokines, are antagonists towards each other. That is, Th1 cytokines stimulate the proliferation of cells that produce them, but at the same time suppress the division of Th2 cells, and vice versa. Thus, normally the body maintains a constant balance of these two types of T-lymphocytes. The interaction of growth factors with their receptors on the cell surface leads to the launch of a whole cascade of events inside the cell. As a result, transcription factors are activated and proliferative response genes are expressed, which ultimately initiates DNA replication and the cell enters mitosis.
ENDOGENOUS REGULATORS OF THE CELL CYCLE
In normal eukaryotic cells, progression through the cell cycle is tightly regulated. The cause of cancer is cell transformation, usually associated with violations of the regulatory mechanisms of the cell cycle. One of the main results of cell cycle defects is genetic instability, since cells with defective cell cycle control lose the ability to correctly duplicate and distribute their genome between daughter cells. Genetic instability leads to the acquisition of new features that are responsible for tumor progression. Cyclin-dependent kinases (CDKs) and their regulatory subunits (cyclins) are major regulators of the cell cycle. Cell cycle progression is achieved through the sequential activation and deactivation of different cyclin-CDK complexes. The action of cyclin-CDK complexes is to phosphorylate a number of target proteins in accordance with the phase of the cell cycle in which a particular cyclin-CDK complex is active. For example, cyclin E-CDK2 is active in late G1 phase and phosphorylates proteins required for progression through late G1 phase and entry into S phase. Cyclin A-CDK2 is active in the S and G2 phases, it ensures the passage of the S phase and entry into mitosis. Cyclin A and cyclin E are central regulators of DNA replication. Therefore, misregulation of the expression of any of these cyclins leads to genetic instability. It has been shown that the accumulation of nuclear cyclin A occurs exclusively at the moment when the cell enters the S phase, i.e. at the moment of G1/S transition. On the other hand, it was shown that the level of cyclin E increased after passing the so-called restriction point (R-point) in late G1 phase, and then decreased significantly when the cell entered S phase.
WAYS OF REGULATION CDK
The activity of cyclin-dependent kinases (CDKs) is tightly regulated by at least four mechanisms:
1) The main way CDK is regulated is by binding to cyclin, i.e. In its free form, the kinase is not active, and only the complex with the corresponding cyclin has the necessary activities.
2) The activity of the cyclin-CDK complex is also regulated by reversible phosphorylation. In order to acquire activity, phosphorylation of CDK is necessary, which is carried out with the participation of the CDK activating complex (CAC), consisting of cyclin H, CDK7 and Mat1.
3) On the other hand, in the CDK molecule, in the region responsible for substrate binding, there are sites whose phosphorylation leads to inhibition of the activity of the cyclin-CDK complex. These sites are phosphorylated by a group of kinases, including Wee1 kinase, and dephosphorylated by Cdc25 phosphatases. The activity of these enzymes (Wee1 and Cdc25) varies significantly in response to various intracellular events, such as DNA damage.
4) Finally, some cyclin-CDK complexes may be inhibited due to binding to CDK inhibitors (CKIs). CDK inhibitors consist of two groups of proteins, INK4 and CIP/KIP. INK4 inhibitors (p15, p16, p18, p19) bind to and inactivate CDK4 and CDK6, preventing interaction with cyclin D. CIP/KIP inhibitors (p21, p27, p57) can bind to cyclin-CDK complexes containing CDK1, CDK2, CDK4 and CDK6. It is noteworthy that under certain conditions, CIP/KIP inhibitors can enhance the kinase activity of cyclin D-CDK4/6 complexes
REGULATION G 1 PHASE
In the G1 phase, at the so-called restriction point (restriction point, R-point), the cell decides whether to divide or not. The restriction point is the point in the cell cycle after which the cell becomes unresponsive to external signals until the completion of the entire cell cycle. The restriction point divides the G1 phase into two functionally distinct stages: G1pm (postmitotic stage) and G1ps (presynthetic stage). During G1pm, the cell evaluates the growth factors present in its environment. If the necessary growth factors are present in sufficient quantities, the cell enters G1ps. Cells that have entered the G1ps period continue to progress through the entire cell cycle normally, even in the absence of growth factors. If the necessary growth factors are absent in the G1pm period, then the cell enters a state of proliferative dormancy (G0 phase).
The main result of the cascade of signaling events that occurs due to the binding of growth factor to the receptor on the cell surface is the activation of the cyclin D-CDK4/6 complex. The activity of this complex increases significantly already in the early G1 period. This complex phosphorylates targets necessary for progression into S phase. The main substrate of the cyclin D-CDK4/6 complex is the retinoblastoma gene product (pRb). Unphosphorylated pRb binds and thereby inactivates transcription factors of the E2F group. Phosphorylation of pRb by cyclin D-CDK4/6 complexes leads to the release of E2F, which enters the nucleus and initiates the translation of protein genes necessary for DNA replication, in particular the cyclin E and cyclin A genes. At the end of the G1 phase, there is a short-term increase in the amount of cyclin E, which portends the accumulation of cyclin A and the transition to S phase.
The following factors can cause cell cycle arrest in the G1 phase: increased levels of CDK inhibitors, deprivation of growth factors, DNA damage, external influences, oncogenic activation
REGULATION S PHASES
S phase is the stage of the cell cycle when DNA synthesis occurs. Each of the two daughter cells that are formed at the end of the cell cycle must receive an exact copy of the DNA of the mother cell. Each base of the DNA molecules that make up the 46 chromosomes of a human cell must be copied only once. That is why DNA synthesis is extremely tightly regulated.
It has been shown that only DNA from cells in G1 or S phase can replicate. This suggests that DNA must be<лицензирована>for replication and that the piece of DNA that has been duplicated loses this<лицензию>. DNA replication begins at the binding site of proteins called ORC (Origin of replicating complex). Several components required for DNA synthesis bind to ORC in late M or early G1 phase, forming a prereplicative complex, which actually gives<лицензию>DNA for replication. At the G1/S transition stage, more proteins necessary for DNA replication are added to the prereplicative complex, thus forming an initiation complex. When the replication process begins and a replication fork is formed, many components are separated from the initiation complex, and only the components of the post-replication complex remain at the replication initiation site.
Many studies have shown that the normal functioning of the initiation complex requires the activity of cyclin A-CDK2. In addition, for the successful completion of the S phase, the activity of the cyclin A-CDK2 complex is also required, which, in fact, is the main regulatory mechanism that ensures the successful completion of DNA synthesis. Arrest in S phase can be induced by DNA damage.
REGULATION G 2 PHASES
G2 phase is a stage of the cell cycle that begins after DNA synthesis is complete but before condensation begins. The main regulator of the G2 phase is the cyclin B-CDK2 complex. Cell cycle arrest in the G2 phase occurs due to inactivation of the cyclin B-CDK2 complex. The regulator of the G2/M transition is the cyclin B-CDK1 complex; its phosphorylation/dephosphorylation regulates entry into the M phase. DNA damage or the presence of unreplicated regions prevents the transition to M phase.
Cellular proliferation- increase in the number of cells through mitosis,
leading to tissue growth, as opposed to another method of increasing it
masses (for example, edema). There is no proliferation in nerve cells.
In the adult body, developmental processes related to
with cell division and specialization. These processes can be either normal
normal physiological, and aimed at restoring the
organism due to a violation of its integrity.
The importance of proliferation in medicine is determined by the ability to cle-
current of different tissues to division. The healing process is associated with cell division
healing of wounds and tissue restoration after surgical operations.
Cell proliferation underlies regeneration (recovery)
lost parts. The problem of regeneration is of interest to me-
dicines, for reconstructive surgery. There are physiological,
reparative and pathological regeneration.
Physiological- natural restoration of cells and tissues in
ontogeny. For example, the change of red blood cells, skin epithelial cells.
Reparative- restoration after damage or death of the adhesive
current and fabrics.
Pathological- proliferation of tissues not identical to healthy tissues;
Yum.
For example, the growth of scar tissue at the site of a burn, cartilage at
fracture site, proliferation of connective tissue cells at the site of our
cervical tissue of the heart, cancerous tumor.
Recently, it has been customary to separate animal tissue cells according to their properties.
ability to divide into 3 groups: labile, stable and static. TO labile
in the process of vital activity of the body (blood cells, epithelium, mucus)
gastrointestinal tract, epidermis, etc.).
ability to divide into 3 groups: labile, stable and static. stable include cells of organs such as the liver, pancreas,
ductal gland, salivary glands, etc., which exhibit limited
new ability to divide.
TO static include cells of the myocardium and nervous tissue, which
Some, according to most researchers, do not share.
The study of cell physiology is important for understanding it
togenetic level of organization of living things and mechanisms of self-regulation
cells that ensure the integral functioning of the entire organism.
Chapter 6
GENETICS HOW THE SCIENCE. REGULARITIES
INHERITANCE SIGNS
6.1 Subject, tasks and methods of genetics
Heredity and variability are fundamental properties
properties of living things, since they are characteristic of living beings of any level of organiza-
nization.
The science that studies the patterns of heredity and variability news is called
genetics.
Genetics as a science studies heredity and hereditary variability, namely, it deals with next:
problems
1) storage of genetic information;
2) transfer of genetic information;
3) implementation of genetic information (use of it in specific
specific signs of a developing organism under the influence of the external environment);
4) changes in genetic information (types and causes of changes,
mechanisms).
The first stage of development of genetics - 1900–1912. Since 1900 - redesigned
Covering of G. Mendel's laws by scientists H. De Vries, K. Correns, E. Cher-
poppy seed Recognition of G. Mendel's laws.
Second stage 1912–1925 - creation of the chromosome theory of T. Mor-
Ghana. Third stage 1925–1940 - discovery of artificial mutagenesis and
genetic processes of evolution.
Fourth stage 1940–1953 - research on gene control
physiological and biochemical processes.
The fifth stage from 1953 to the present - the development of molecular
biology.
Some information on the inheritance of traits was known
a very long time ago, but the scientific basis for the transmission of traits was first
set out by G. Mendel in 1865 in the work: “Experiments on plant
hybrids."
These were advanced thoughts, but contemporaries did not give
the significance of his discovery. The concept of “gene” did not exist at that time and G. Men-
del spoke about the “hereditary inclinations” contained in the reproductive cells
kah, but their nature was unknown.
development of genetics as a science. In 1902, T. Boveri, E. Wilson and D. Setton made
They made an assumption about the connection of hereditary factors with chromosomes.
In 1906, W. Betson introduced the term “genetics”, and in 1909, V. Johansen -
"gene". In 1911, T. Morgan and collaborators formulated the main principles
Zheniya chromosomal theory of heredity.
They proved that genes
located at certain chromosomal loci in a linear order, according to
tion of a certain feature.
Basic methods of genetics: hybridological, cytological and
mathematical. Genetics actively uses methods of other related
sciences: chemistry, biochemistry, immunology, physics, microbiology, etc.
The cell cycle is the period of a cell's life from one division to another or from division to death. The cell cycle consists of interphase (the period outside of division) and cell division itself.
At the end of the G1 period, it is customary to distinguish a special moment called the R-point (restriction point, R-point), after which the cell necessarily enters the S period within several hours (usually 1-2). The time period between the R-point and the beginning of the S period can be considered as preparatory to the transition to the S period.
The most important process that occurs in the S period is the doubling or reduplication of DNA. All other reactions occurring in the cell at this time are aimed at ensuring DNA synthesis. Such auxiliary processes include the synthesis of histone proteins, the synthesis of enzymes that regulate and ensure the synthesis of nucleotides and the formation of new DNA strands.
The general situation goes like this. The cell constantly contains special enzyme proteins that, by phosphorylating other proteins (at serine, tyrosine or threonine residues in the polypeptide chain), regulate the activity of genes responsible for the passage of the cell through one or another period of the cell cycle. These enzyme proteins are called cyclin-dependent protein kinases (cdc). There are several varieties, but they all have similar properties. Although the amount of these cyclin-dependent protein kinases may vary at different periods of the cell cycle, they are constantly present in the cell, regardless of the period of the cell cycle, that is, they are abundant. In other words, their synthesis or quantity does not limit or regulate the passage of cells through the cell cycle. However, in pathology, if their synthesis is impaired, their number is reduced, or there are mutant forms with altered properties, then this, of course, can affect the course of the cell cycle.
Why can’t such cyclin-dependent protein kinases themselves regulate the passage of cells through periods of the cell cycle? It turns out that they are in an inactive state in cells, and in order for them to be activated and start working, special activators are needed. They are cyclins. There are also many different types of them, but they are not constantly present in cells: they appear and then disappear. At different phases of the cell cycle, different cyclins are formed, which bind to Cdk to form different Cdk‑cyclin complexes. These complexes regulate different phases of the cell cycle and are therefore called G1-, G1/S-, S- and M-Cdk (Fig. from my Fig. cyclins). For example, the passage of a cell through the G1 period of the cell cycle is ensured by a complex of cyclin-dependent protein kinase-2 (cdk2) and cyclin D1, cyclin-dependent protein kinase-5 (cdk5) and cyclin D3. Passage through a special restriction point (R-point) of the G1 period is controlled by the complex of cdc2 and cyclin C. The transition of a cell from the G1 period of the cell cycle to the S period is controlled by the complex of cdk2 and cyclin E. For the transition of a cell from the S period to the G2 period, the cdk2 complex and cyclin are required A. Cyclin-dependent protein kinase 2 (cdc2) and cyclin B are involved in the transition of the cell from the G2 period to mitosis (M period). Cyclin H in association with cdk7 is required for the phosphorylation and activation of cdc2 in complex with cyclin B.
Cyclins are a new class of proteins discovered by Tim Hunt that play a key role in controlling cell division. The name “cyclins” comes from the fact that the concentration of proteins of this class changes periodically in accordance with the stages of the cell cycle (for example, it falls before the start of cell division).
The first cyclin was discovered by Hunt in the early 1980s, during experiments with frog and sea urchin eggs. Later, cyclins were found in other living beings.
It turned out that these proteins changed little during evolution, as did the cell cycle control mechanism, which came from simple yeast cells to humans in a “conserved” form.
Timothy Hunt (R. Timothy Hunt), together with fellow Englishman Paul M. Nurse and American Leland H. Hartwell, received the Nobel Prize in Physiology or Medicine in 2001 for the discovery of genetic and molecular mechanisms of cell cycle regulation – a process that is essential for the growth, development and very existence of living organisms
Cell cycle checkpoints
1. The point of exit from the G1 phase, called Start - in mammals and the restriction point in yeast. After passing through the restriction point R at the end of G1, the onset of S becomes irreversible, i.e. processes leading to the next cell division are started.
2. Point S – checking the accuracy of replication.
3. G2/M transition point – checking the completion of replication.
4. Transition from metaphase to anaphase of mitosis.
Regulation of replication
Before replication begins, the Sc ORC complex (origin recognition complex) sits on ori, the replication origin point. Cdc6 is present throughout the cell cycle, but its concentration increases early in G1, where it binds to the ORC complex, to which Mcm proteins then join to form the pre-replicative complex (pre-RC). Once the pre-RC is assembled, the cell is ready to replicate.
To initiate replication, S-Cdk binds to protein kinase (?), which phosphorylates pre-RC. In this case, Cdc6 dissociates from the ORC after the start of replication and is phosphorylated, after which it is ubiquitinated by SCF and degraded. Changes in the pre-RC prevent replication from starting again. S-Cdk also phosphorylates some Mcm protein complexes, which triggers their export from the nucleus. Subsequent protein dephosphorylation will restart the process of pre-RC formation.
Cyclins are Cdk activators. Cyclins, like Cdks, are involved in various processes besides cell cycle control. Cyclins are divided into 4 classes depending on the time of action in the cell cycle: G1/S, S, M and G1 cyclins.
G1/S cyclins (Cln1 and Cln2 in S. cerevisiae, cyclin E in vertebrates) reach their maximum concentration in the late G1 phase and decrease in the S phase.
The G1/S cyclin–Cdk complex triggers the onset of DNA replication by turning off various systems that suppress the S-phase Cdk in the G1 phase. G1/S cyclins also initiate centrosome duplication in vertebrates and the formation of a spindle body in yeast. A decrease in the G1/S level is accompanied by an increase in the concentration of S cyclins (Clb5, Clb6 in Sc and cyclin A in vertebrates), which forms an S cyclin-Cdk complex that directly stimulates DNA replication. S cyclin levels remain high throughout the S, G2 phases and the onset of mitosis, where it helps initiate mitosis in some cells.
M-cyclins (Clb1,2,3 and 4 in Sc, cyclin B in vertebrates) appear last. Its concentration increases as the cell enters mitosis and reaches a maximum in metaphase. The M-cyclin-Cdk complex involves spindle assembly and sister chromatid alignment. Its destruction in anaphase leads to exit from mitosis and cytokinesis. G1 cyclins (Cln3 in Sc and cyclin D in vertebrates) help coordinate cell growth with entry into a new cell cycle. They are unusual because their concentration does not vary with cell cycle phase, but changes in response to external growth regulatory signals.
Programmed cell death
In 1972, Kerr et al. published an article in which the authors presented morphological evidence of the existence of a special type of cell death, different from necrosis, which they called “apoptosis.” The authors reported that structural changes in cells during apoptosis go through two stages:
1st – formation of apoptotic bodies,
2nd – their phagocytosis and destruction by other cells.
The causes of death, the morphological and biochemical processes of the development of cell death can be different. But still they can be clearly divided into two categories:
1. Necrosis (from the Greek nekrosis - necrosis) and
2. Apoptosis (from Greek roots meaning “falling away” or “disintegration”), which is often called programmed cell death (PCD) or even cell suicide (Fig. 354).
Two pathways of cell death
a – apoptosis (promoted cell death): / – specific compression of the cell and condensation of chromatin, 2 – fragmentation of the nucleus, 3 – fragmentation of the cell body into a series of apoptotic bodies; b – necrosis: / – swelling of the cell, vacuolar components, chromatin condensation (karyorrhexis), 2 – further swelling of membrane organelles, lysis of nuclear chromatin (karyolysis), 3 – rupture of membrane components of the cell – cell lysis
N. is the most common nonspecific form of cell death. It can be caused by severe cell damage as a result of direct trauma, radiation, toxic agents, hypoxia, complement-mediated cell lysis, etc.
The necrotic process goes through a number of stages:
1) paranecrosis - similar to necrotic, but reversible changes;
2) necrobiosis – irreversible dystrophic changes, characterized by a predominance of catabolic reactions over anabolic ones;
3) cell death, the time of which is difficult to determine;
4) autolysis - decomposition of a dead substrate under the action of hydrolytic enzymes of dead cells and macrophages. In morphological terms, necrosis is equivalent to autolysis.
Despite the huge amount of work, there is no agreed upon and precise definition of the concept of “apoptosis”.
Aloptosis was usually characterized as a special form of cell death, different from necrosis in morphological, biochemical, molecular genetic and other characteristics.
A. is cell death caused by internal or external signals that in themselves are not toxic or destructive. A. is an active process requiring energy, gene transcription and denovo protein synthesis.
A significant number of agents have been discovered that cause apoptosis of these cells, in addition to radiation and glucocorticoids:
Ca2+ ionophores
Adenosine
Cyclic AMP
Tributyltin
Hyperthermia
A study of the kinetics of DNA degradation in lymphoid cells in vivo and in vitro showed:
The first clear signs of decay appear, as a rule, more than 1 hour after exposure, more often by the end of the 2nd hour.
Internucleosomal fragmentation continues for several hours and ends mainly 6, less often 12 hours after exposure.
Immediately from the moment of degradation, the analysis reveals a large number of small DNA fragments, and the ratio between large and small fragments does not change significantly during apoptosis.
The use of inhibitors of ATP synthesis, protein synthesis and gene transcription slows down the process of apoptosis. There is no such dependence in the case of N.
As can be seen from a comparison of the definitions of necrosis and apoptosis, there are both similarities and significant differences between the two types of cell death.
| Characteristic | Necrosis | Apoptosis |
| functional | irreversible cessation of her life activity; | |
| morphologically | violation of the integrity of membranes, changes in the nucleus (pyknosis, rhexis, lysis), cytoplasm (edema), cell destruction; | loss of microvilli and intercellular contacts, condensation of chromatin and cytoplasm, reduction in cell volume (shrinkage), formation of vesicles from the plasma membrane, cell fragmentation and the formation of apoptotic bodies; |
| biochemically | impaired energy production, coagulation, hydrolytic breakdown of proteins, nucleic acids, lipids; | hydrolysis of cytoplasmic proteins and internucleosomal DNA decay; |
| genetically | – loss of genetic information; and ending with autolysis or heterolysis with an inflammatory reaction. | structural and functional restructuring of the genetic apparatus and culminating in its absorption by macrophages and (or) other cells without an inflammatory reaction. |
Cell death is regulated by cell-cell interactions in various ways. Many cells in a multicellular organism require signals in order to stay alive. In the absence of such signals or trophic factors, a program of “suicide” or programmed death develops in cells. For example, neuronal culture cells die in the absence of neuronal growth factor (NGF), prostate cells die in the absence of testicular androgens, breast cells die when the level of the hormone progesterone drops, etc. At the same time, cells can receive signals that trigger processes in target cells that lead to death such as apoptosis. Thus, hydrocortisone causes the death of lymphocytes, and glutamate causes the death of nerve cells in tissue culture; tumor necrosis factor (TNF) causes the death of a wide variety of cells. Thyroxine (thyroid hormone) causes apoptosis of tadpole tail cells. In addition, there are situations when apoptotic cell death is caused by external factors, such as radiation.
The concept of “apoptosis” was introduced when studying the death of some liver cells during incomplete ligation of the portal vein. In this case, a peculiar picture of cell death is observed, which affects only individual cells in the liver parenchyma.
The process begins with the fact that neighboring cells lose contact, they seem to shrink (the original name for this form of death is shrinkagenecrosis - necrosis by cell compression), specific chromatin condensation occurs in the nuclei along their periphery, then the nucleus fragments into separate parts, followed by the cell itself fragments into individual bodies delimited by the plasma membrane - apoptotic bodies.
Apoptosis is a process that leads not to lysis or dissolution of the cell, but to its fragmentation and disintegration. The fate of apoptotic bodies is also unusual: they are phagocytosed by macrophages or even normal neighboring cells. In this case, an inflammatory reaction does not develop.
It is important to note that in all cases of apoptosis - whether during embryonic development, in an adult organism, normally or during pathological processes - the morphology of the cell death process is very similar. This may indicate the commonality of apoptosis processes in different organisms and in different organs.
Studies on various objects have shown that apoptosis is the result of genetically programmed cell death. The first evidence of the presence of a genetic program of cell death (PCD) was obtained by studying the development of the nematode Caenorhabditiselegans. This worm develops in just three days, and its small size makes it possible to follow the fate of all its cells, starting from the early stages of fragmentation to a sexually mature organism.
It turned out that during the development of Caenorhabditiselegans, only 1090 cells are formed, of which some 131 nerve cells spontaneously die by apoptosis, leaving 959 cells in the body. Mutants were discovered in which the process of elimination of 131 cells was disrupted. Two genes, sed-3 and sed-4, were identified, the products of which cause apoptosis of 131 cells. If these genes are absent or altered in mutant Caenorhabditiselegans, then apoptosis does not occur and the adult organism consists of 1090 cells. Another gene was also found - sed-9, which is a suppressor of apoptosis: with the mutation of sed-9, all 1090 cells die. An analogue of this gene was discovered in humans: the bcl‑2 gene is also a suppressor of apoptosis in various cells. It turned out that both proteins encoded by these genes, Ced-9 and Bc1-2, have one transmembrane domain and are localized in the outer membrane of mitochondria, nuclei and endoplasmic reticulum.
The system of development of apoptosis turned out to be very similar in nematodes and vertebrates; it consists of three parts: a regulator, an adapter and an effector. In Caenorhabditiselegans, the regulator is Ced-9, which blocks the adapter protein Ced-4, which in turn does not activate the effector protein Ced-3, a protease that acts on cytoskeletal and nuclear proteins (Table 16).
Table 16. Development of programmed cell death (apoptosis)
Sign ──┤ – inhibition of the process, sign ─→ – stimulation of the process
In vertebrates, the ACL system is more complex. Here the regulator is the Bc1-2 protein, which inhibits the adapter protein Apaf-1, which stimulates the cascade of activation of special proteinases - caspases.
Enzymes – participants in the process of apoptosis
Thus,
Once it begins in the cell, such degradation quickly proceeds “to the end”;
Not all cells enter into apoptosis immediately or in a short period of time, but gradually;
DNA breaks occur along linker (internucleosomal) DNA;
Degradation is carried out by endo-, but not exonucleases, and these endonucleases are activated or gain access to DNA not as a result of direct interaction with the agent that causes apoptosis, but indirectly, since quite a significant time passes from the moment the cells come into contact with such an agent until the onset of degradation, and, therefore, DNA fragmentation is not the first characteristic “apoptotic” reaction of a cell at the molecular level. In fact, if degradation were triggered as a result of direct interaction of endonucleases or chromatin with an agent, then in the case of, for example, the action of ionizing radiation, apoptosis would occur quickly and simultaneously in almost all cells.
Based on these conclusions, deciphering the molecular mechanism of apoptosis development “focused” on identifying the endonucleases(s) that carry out DNA fragmentation and the mechanisms that activate endonucleases.
Endonucleases
1. Degradation is carried out by DNase I. The process is activated by Ca2+ and Mg2+ and suppressed by Zn2+.
However, there are facts that argue against the participation of DNase I in the process of DNA fragmentation. It is known that this enzyme is absent in the nucleus, however, this argument is not very weighty, since the relatively small size of its molecules, 31 kDa, in the case of disruption of the permeability of the nuclear membrane makes the participation of DNase I in DNA degradation quite real. Another thing is that when chromatin is processed in vitro, DNase I causes breaks not only in the linker part, but also in nucleosomal DNA.
2. Another endonuclease considered as the main enzyme for DNA degradation is endonuclease II [Barry 1993]. This nuclease, when processing nuclei and chromatin, carries out internucleosomal fragmentation of DNA. Despite the fact that its activity does not depend on divalent metal ions, the question of the participation of endonuclease II in DNA degradation has not yet been resolved, since the enzyme is not only located in lysosomes, but is also released from cell nuclei.
3. endonuclease with a molecular weight of 18 kDa. This enzyme was isolated from the nuclei of rat thymocytes dying by apoptosis [Gaido, 1991]. It was absent in normal thymocytes. The activity of the enzyme manifests itself in a neutral environment and depends on Ca2+ and Mg2+.
4. γ-nuclease with a molecular weight of 31 kDa, which has a “classical” dependence on Ca, Mg and Zn ions. The activity of this enzyme was increased in the nuclei of rat thymocytes treated with glucocorticoids.
5. endonuclease with a molecular weight of 22.7 kDa, an enzyme whose activity appears in the nuclei of rat thymocytes only after the action of glucocorticoids and is suppressed by the same inhibitors as internucleosomal DNA degradation.
Caspases are cysteine proteases that cleave proteins at aspartic acid. In the cell, caspases are synthesized in the form of latent precursors, procaspases. There are initiator and effector caspases. Initiator caspases activate latent forms of effector caspases. More than 60 different proteins serve as substrates for the action of activated caspases. This is, for example, focal adhesion structure kinase, the inactivation of which leads to the separation of apoptotic cells from their neighbors; these are lamins that are disassembled by the action of caspases; these are cytoskeletal proteins (intermediate filaments, actin, gelsolin), the inactivation of which leads to a change in the shape of the cell and the appearance of bubbles on its surface, which give rise to apoptotic bodies; this is an activated CAD protease that cleaves DNA into oligonucleotide nucleosomal fragments; these are DNA repair enzymes, the suppression of which prevents the restoration of DNA structure, and many others.
One example of the unfolding of an apoptotic response may be the reaction of a cell to the absence of a signal from a necessary trophic factor, such as nerve growth factor (NGF), or androgen.
In the cytoplasm of cells in the presence of trophic factors, another participant in the reaction is in an inactive form - the phosphorylated protein Bad. In the absence of a trophic factor, this protein is dephosphorylated and binds to the Bc1–2 protein on the outer mitochondrial membrane and thereby inhibits its antiapoptotic properties. After this, the membrane proapoptotic protein Bax is activated, opening the way for ions entering the mitochondrion. At the same time, cytochrome c is released from the mitochondria through the pores formed in the membrane into the cytoplasm, which binds to the adapter protein Araf-1, which in turn activates procaspase 9. Activated caspase 9 triggers a cascade of other procaspases, including caspase 3, which, being proteinases, they begin to digest mixed proteins (lamins, cytoskeletal proteins, etc.), which causes apoptotic cell death, its disintegration into parts, into apoptotic bodies.
Apoptotic bodies, surrounded by the plasma membrane of the destroyed cell, attract individual macrophages, which engulf and digest them using their lysosomes. Macrophages do not respond to neighboring normal cells, but recognize apoptotic ones. This is due to the fact that during apoptosis, the asymmetry of the plasma membrane is disrupted and phosphatidylserine, a negatively charged phospholipid, which is normally located in the cytosolic part of the bilipid plasma membrane, appears on its surface. Thus, through selective phagocytosis, tissues are cleared of dead apoptotic cells.
As mentioned above, apoptosis can be caused by a number of external factors, such as radiation, the action of certain toxins, and inhibitors of cellular metabolism. Irreversible DNA damage causes apoptosis. This is due to the fact that the accumulating transcription factor, the p53 protein, not only activates the p21 protein, which inhibits cyclin-dependent kinase and stops the cell cycle in the G1 or G2 phase, but also activates the expression of the bax gene, the product of which triggers apoptosis.
The presence of checkpoints in the cell cycle is necessary to determine the completion of each phase. Cell cycle arrest occurs when DNA is damaged in the G1 period, when DNA replication is incomplete in the S phase, when DNA is damaged in the G2 period, and when the connection between the spindle and chromosomes is disrupted.
One of the control points in the cell cycle is mitosis itself, which does not enter anaphase if the spindle is not assembled correctly and in the absence of complete connections of microtubules with kinetochores. In this case, there is no activation of the APC complex, no degradation of cohesins connecting sister chromatids, and no degradation of mitotic cyclins, which is necessary for the transition to anaphase.
DNA damage prevents cells from entering the S period or mitosis. If these damages are not catastrophic and can be restored through reparative DNA synthesis, then the cell cycle block is removed and the cycle reaches its completion. If the DNA damage is significant, then somehow stabilization and accumulation of the p53 protein occurs, the concentration of which is normally very low due to its instability. The p53 protein is one of the transcription factors that stimulates the synthesis of the p21 protein, which is an inhibitor of the CDC-cyclin complex. This causes the cell cycle to arrest at the G1 or G2 stage. During a block in the G1 period, a cell with DNA damage does not enter the S phase, as this could lead to the appearance of mutant cells, which may include tumor cells. Blockade in the G2 period also prevents the process of mitosis of cells with DNA damage. Such cells, with a blocked cell cycle, subsequently die by apoptosis, programmed cell death (Fig. 353).
With mutations leading to the loss of p53 protein genes, or with their changes, blockade of the cell cycle does not occur, the cells enter mitosis, which leads to the appearance of mutant cells, most of which are non-viable, others give rise to malignant cells.
Selective damage to mitochondria, in which cytochrome c is released into the cytoplasm, is also a common cause of apoptosis. Mitochondria and other cellular components are especially affected by the formation of toxic reactive oxygen species (ATS), under the influence of which nonspecific channels with high permeability for ions are formed in the inner mitochondrial membrane, as a result of which the mitochondrial matrix swells and the outer membrane ruptures. In this case, proteins dissolved in the intermembrane space, together with cytochrome c, enter the cytoplasm. Among the released proteins are factors that activate apoptosis and procaspase 9.
Many toxins (ricin, diphtheria toxin, etc.), as well as antimetabolites, can cause cell death through apoptosis. When protein synthesis in the endoplasmic reticulum is disrupted, procaspase 12, localized there, participates in the development of apoptosis, which activates a number of other caspases, including caspase 3.
Elimination is the removal of individual cells by apoptosis and is also observed in plants. Here, apoptosis includes, as in animal cells, an induction phase, an effector phase and a degradation phase. The morphology of plant cell death is similar to changes in animal cells: chromatin condensation and nuclear fragmentation, oligonucleotide degradation of DNA, compression of the protoplast, its fragmentation into vesicles, rupture of plasmodesmata, etc. However, protoplast vesicles are destroyed by hydrolases of the vesicles themselves, since plants do not have cells similar to phagocytes. Thus, PCD occurs during the growth of root cap cells, during the formation of perforations in leaves, and during the formation of xylem and phloem. Leaf fall is associated with selective death of cells in a certain zone of the cutting.
The biological role of apoptosis, or programmed cell death, is very large: it is the removal of cells that have spent their time or are unnecessary at a given stage of development, as well as the removal of altered or pathological cells, especially mutant or infected with viruses.
So, in order for cells to exist in a multicellular organism, signals for their survival are needed - trophic factors, signaling molecules. These signals can be transmitted over a distance and captured by the corresponding receptor molecules on target cells (hormonal, endocrine signaling), this can be paracrine communication when the signal is transmitted to a neighboring cell (for example, neurotransmitter transmission). In the absence of such trophic factors, the apoptosis program is implemented. At the same time, apoptosis can be caused by signaling molecules, for example, during resorption of the tail of tadpoles under the influence of thyroxine. In addition, the action of a number of toxins that affect individual parts of cell metabolism can also cause cell death through apoptosis.
Apoptosis in the pathogenesis of diseases
1. In the immune system
2. ONCOLOGICAL DISEASES
3. VIRAL INFECTION (apoptosis-inducing: human immunodeficiency, chicken anemia; apoptosis inhibitors: cytomegalovirus, Epstein-Barr, herpes)
4. A. and NEURONS OF THE CEREBRAL CORTEX
PRINCIPLES OF CORRECTION OF CELL APOPTOSIS
The discovery of a regulated process of cell death - apoptosis - made it possible to influence its individual stages in a certain way for the purpose of regulation or correction.
The biochemical processes of apoptosis development can be hypothetically divided into several stages:
The action of a factor that causes apoptosis;
Transmission of a signal from a receptor molecule to the cell nucleus;
Activation of apoptosis-specific genes;
Synthesis of apoptosis-specific proteins
Activation of endonucleases
DNA fragmentation (Fig. 2.4).
Currently, it is believed that if a cell dies by apoptosis, then the possibility of therapeutic intervention is implied; if due to necrosis, then such intervention is impossible. Based on knowledge of the regulation of programmed cell death, a wide range of drugs are used to influence this process in various types of cells.
Thus, information about receptor-mediated regulation of cell apoptosis is taken into account when treating hormone-dependent tumors.
Androgen blocking therapy is prescribed for prostate cancer.
Breast cancer often undergoes regression with the use of estrogen receptor antagonists.
Information about the biochemical signal-transmitting pathways for the regulation of apoptosis allows the effective use of antioxidant therapy, drugs that regulate calcium concentrations, activators or inhibitors of various protein kinases, etc. for the purpose of correcting apoptosis in various types of cells.
Awareness of the role of apoptosis in cell death has intensified the search for pharmacological effects that protect cells from apoptosis.
Inhibitors of specific proteases are being actively studied as pharmacological agents. These are usually tri- or tetrapeptides containing aspartic acid (Asp). The use of such proteases for therapeutic purposes is limited by their low ability to penetrate cells. However, despite this, in vivo experiments have successfully used Z-VAD-FMK, a broad-spectrum inhibitor of ICE-like proteases, to reduce the infarct area in stroke models.
In the coming years, we can expect the emergence of new drugs for the treatment and prevention of various diseases, the basis of which will be the principle of regulation of apoptosis processes.
The most effective approaches for correcting apoptosis are those associated with the regulation of apoptosis-specific genes. These approaches underlie gene therapy, one of the promising areas for treating patients with diseases caused by dysfunction of individual genes.
The principles of gene therapy include the following steps:
Identification of the DNA sequence that will be treated;
Determining the type of cells in which treatment will be carried out;
Protection of DNA from hydrolysis by endonucleases;
Transport of DNA into the cell (nucleus).
Gene therapy approaches allow
Strengthen the work of individual genes (transformation of genes that inhibit apoptosis, for example the bcl‑2 gene),
Reduce their expression. To selectively inhibit gene expression, the technique of antisense oligonucleotides (antisenses) is currently used. The use of antisenses reduces the synthesis of certain proteins, which affects the regulation of the apoptosis process.
The mechanism of action of antisenses is being actively studied. In some cases, short (13–17 bases) antisense oligonucleotides, having sequences complementary to the nucleotide sequences of messenger RNA (mRNA) of individual proteins, can effectively block genetic information at a stage preceding transcription (Fig. 2.5). These oligonucleotides bind to DNA and form a triplet helical structure. Such binding may be irreversible or cause selective release of the triplet complex, which ultimately leads to inhibition of gene expression and cell death. In other cases, complementary binding of the antisense to the mRNA occurs, which causes disruption of translation and a decrease in the concentration of the corresponding protein.
Triplet complex
Rice. Regulation of gene expression by antisense oligonucleotides.
It has now been convincingly shown that technology using antisenses is of great importance for the regulation of individual genes in cell culture. Successful suppression of the bcl-2 gene in cell culture experiments raises hopes for the future use of antisenses for the treatment of cancer patients. Many in vitro experiments have shown that antisenses cause inhibition of cell proliferation and differentiation. This result confirms the prospects for the therapeutic use of this technology.