Oncology: news from the front edge

How to kill an immortal cell
Dmitry Yuryevich Blokhin,
Doctor of Medical Sciences, Head. Laboratory of Pharmacocytokinetics of the Research Institute for Experimental Diagnosis and Therapy of Tumors of the N. N. Blokhin State Research Center of the Russian Academy of Medical Sciences
"Chemistry and life" No. 3-2009
Published on the website "Elements"This article is about the causes, patterns of development and ways of treating oncological diseases, as well as about the difficulties that oncologists face when developing new means and methods of cancer treatment.

But first it is worth remembering some basic concepts.

A few words about tumor cellsThe human body consists of approximately 100 trillion cells.

Changing this amount is always physiologically justified. For example, with inflammation, the number of white blood cells (leukocytes) increases, which resist the pathogens of infection. With intense physical exertion, the number of muscle cells and muscle mass increase. The process of maintaining an optimal number of cells – cellular homeostasis – is carried out by a complex system of control of cell division (proliferation) and cell death.

Each cell has its own lifespan: red blood cells – about 120 days, white blood cells – from several hours (neutrophils) to several weeks (lymphocytes), and "memory cells", as specialized immune lymphocytes are called, can live for decades. After the expiration of the allotted time, the cell dies. This death is orderly and genetically programmed. The cell death program is activated if the cell is no longer needed by the body (for example, it belongs to embryonic tissue), has aged, has become infected with a virus, has accumulated many mutations or has received other damage that cannot be repaired. In this case, there is a sequential self-assembly of the cell into fragments, which then absorb macrophages or neighboring cells as a nutrient and building substrate. As a rule, the term "apoptosis" is used in the literature to denote programmed cell death.

The cell death program is triggered only after repeated confirmation of the "death signal". The signal can come from the environment surrounding the cell or from its own intracellular "trouble sensors". The cell perceives the external signal with special "death receptors" located on its surface. There are also various internal signals arising from irreversible internal damage to the cell (in most cases, DNA molecules) that prevent its normal division or functioning. But regardless of the source and place of receiving this signal, the same cascade of activation of "suicidal" enzymes is eventually triggered, which complete the execution of the program: effector caspases, DNA-fragmenting factor, etc.

In a healthy and normally functioning organism, a huge number of cells die every second, as many are formed again. But sometimes the process of cellular homeostasis gets out of control and a tumor arises.

A tumor is a pathological growth of tissue consisting of qualitatively altered (atypical) morphology, degree of differentiation and nature of cell growth. Not every increase in tissue volume is a tumor. Edema, for example, is not associated with cell proliferation, but with the accumulation of intercellular fluid, hypertrophied muscles of a bodybuilder are an adaptation of the body to prolonged physical exertion. These changes are transient: after reducing muscle loads, additional tissue undergoes involution (that is, it resolves). The appearance of a tumor is not associated with adaptation, and it is not subject to involution. The tumor, unlike normal tissue, has no pronounced structure, its structure is more or less random. It is formed by cells that do not complete differentiation and bear signs of young, and often embryonic forms.

If the growth of the tumor is limited to the place of its origin, then it is benign. Benign tumors include fibroids, lipomas, epitheliomas, adenomas (the ending -om means "tumor", and the root of the word often comes from the name of the tissue from which the tumor cells originated), papillomas, polyps, pigmented nevi – "hanging moles", warts and many others. Benign tumors, as a rule, do not pose a threat to the patient's life, since they are local in nature. The exception is brain tumors, which, due to the rigidly limited space of the cranium, can mechanically squeeze adjacent areas of the brain and blood vessels, causing paresis, paralysis and even death of the patient.

If the growth of the tumor is not limited to its own tissue and organ, and atypical cells detached from the main node migrate to neighboring and distant organs, causing the appearance of secondary tumor nodes (metastases) there, then such a tumor is malignant.

In addition to the ability to form metastases, that is, to exist outside the usual cellular environment, cancer cells are characterized by uncontrolled division, and they can divide an unlimited number of times without showing signs of aging, and to a large extent lose the ability to programmed cell death. It is the combination of all these signs that distinguishes a cancer cell from a normal one.

Tumor transformation of a cell occurs when it accumulates a certain number of mutations, and not any, but critical for carcinogenesis. So far, scientists do not know exactly how many mutations and in which genes exactly must occur in order for a cell to become a tumor. Obviously, no less than five, and according to the most optimistic forecasts 8-10. It is important that we are not talking about any particular set of mutations: their combinations that determine the tumor transformation can be very different. From a molecular genetic point of view, there are no two completely identical tumors, as well as absolutely identical causes of their occurrence. The uniqueness of each tumor far exceeds the uniqueness of fingerprint patterns.

Scientists have not found a "universal" or "main" mutation necessary and sufficient for the transformation of a normal cell into a cancerous one. However, one gene, changes in which often lead to malignant transformation, is worth mentioning. This gene is called TR53, and its protein product p53 (such an inexpressive designation was made from "protein with a molecular weight of 53 kilodaltons") regulates the activity of more than 150 genes that control the cycle of cell division.

The process of cell division is very complex and fraught with many dangers associated with the emergence and consolidation of somatic mutations, that is, mutations that occur in somatic cells. To avoid such a disaster, there is a system of genetic self-control of cells in the body. At least four control (or verification) points are known at which the correct sequence of events of the replication cycle is analyzed. If something went wrong, then proliferation temporarily stops, and if the damage cannot be repaired, a cell death program is activated that will not allow mutant cells to multiply. The key role in this process is played by the p53 protein, which is often referred to as the "guardian of the genome", and the constantly functioning TR53 gene is referred to as tumor suppressors (inhibiting the development of tumors). But how important it is for tumor suppression is still unclear. On the one hand, the occurrence of inactivating mutations in the TR53 gene or the complete cessation of its expression (gene knockout) causes the destabilization of the genome: the so-called mutatory phenotype of the cell is formed, in which the frequency of occurrence and accumulation of mutations increases sharply. If the mutation of the TR53 gene is inherited from parents, it is present in all cells of the body and is accompanied by the development of the Li-Fraumeni syndrome, in which multiple tumors occur in childhood. Such patients rarely live to adulthood. However, as large-scale genetic studies conducted in laboratories in different countries have shown, only slightly more than half of all human malignant tumors of various localization and stage of development studied carry mutations in the TR53 gene; cells of the second half of the studied array synthesize normal protein p53, which, however, does not prevent them from being malignant!

Hundreds of thousands of mutant cells appear in the human body every day. They are constantly monitored and destroyed by two control systems: the system of cellular genetic self-control, which was discussed above, and the system of nonspecific antitumor immunity.

The antitumor immunity system recognizes mutant cells by the presence on their surface of an extraneous antigen that is not characteristic of this organism or by the absence of one of the absolutely necessary ones. The former include the so-called tumor–associated and viral antigens, and the latter include antigens of the main histocompatibility complex of class I, carrying the information: "I am mine". If these antigens are not present on the cell, it is immediately given a "lethal injection" by the killer cell, which carries out immunological supervision. It forms a channel in the wall of the target cell through which it injects enzymes-granzymes. Granzymes "include" proferments of the caspase class – these are the main performers of the cell death program.

The executive mechanism of the antitumor immunity system is coupled with a mechanism for ensuring genetic self-control. This means that a cell that, as a result of mutation, becomes immune to the action of one control system, will be invulnerable to another. The descendants of such a cell will inherit the acquired trait and begin the formation of a mutant clone – the ability to escape from the system of genetic self-control will allow to avoid death in the future when replicating newly appeared mutations. These cells cannot yet be called tumor-transformed, since they have not yet acquired all the genetic defects necessary for this, but a start has been made: the mutator phenotype has opened up space for further accumulation of mutations.

Since the process of mutagenesis is random, an individual set of mutations will arise in each cell of a mutant clone, and clonal splitting of the population occurs. (The figure shows a diagram of the clonal cleavage of the offspring of a mutant cell. A, B, C and D are random mutations.)

The appearance of new mutations will affect the phenotype of descendants – they will gradually lose their parental traits, but acquire new properties, including those inherent in tumor cells. The most important of them is the ability to an unlimited number of divisions, or reproductive immortality. Without this ability, all other acquired "tumor" properties will not pose a danger: having completed the required number of doubling, the cells will irreversibly lose the ability to divide – the growth of the tumor will stop, followed by its gradual self-destruction. If the cell reaches reproductive immortality, the acquisition of other tumor traits is only a matter of time.

There are cases when a benign tumor that has arisen during its growth for one reason or another becomes malignant – "malignized". So, in place of a benign pigmented nevus, melanoma can form – one of the most malignant skin tumors, as a rule, forming multiple metastases. Malignancy of a benign tumor is not an obligatory process, most of these neoplasms exist in the body for years, grow slowly and mostly cause only cosmetic inconvenience. However, a malignant tumor can develop not only from benign, but also from completely healthy tissue. In these cases, the appearance of a tumor is usually preceded by a "precancer" – a compact cluster of morphologically altered mutant cells. Their descendants can develop into intracranial, "local" cancer, which then spreads and forms infiltrating malignant formations. This is how the progression of the tumor process occurs, the direction of which is the same in all cases – from bad to worse.

Having arisen, the tumor tissue not only grows uncontrollably due to the uncontrolled division of its constituent cells, but also constantly evolves, generating new cell clones, the most malignant of which, that is, better adapted to autonomous existence, displace less malignant ones in the process of competition. It is possible to stop such expansion only by removing the tumor from the body or, at least, limiting its growth.

Treatment of oncological diseasesToday there are three main methods of treating cancer patients: surgical removal of tumor nodes, chemotherapy and radiotherapy, and in the vast majority of cases they have to be combined.

Surgical intervention is effective only when the process is localized and excision of the tumor within healthy tissues does not destroy the functioning of vital organs. In other cases, as well as if there is no primary tumor focus at all, for example, with leukemia, chemotherapy is used, which theoretically should affect tumor cells regardless of their localization.

The idea of "cancer chemotherapy" was first formulated by Paul Ehrlich at the beginning of the XX century. However, the complexity of the problem of selective destruction of tumor cells without harm to normal cells forced Ehrlich to abandon the practical implementation of the idea. And only in the late 40s–early 50s of the last century, doctors discovered chemical compounds that not only stop the division and cause the death of tumor cells in culture, but also inhibit the growth of tumors in the body. The first official cure for cancer was embihin, first used on humans in 1946. Created on the basis of mustard gas, a chemical warfare agent of the First World War, embikhin marked the beginning of a whole family of antitumor drugs of the alkylating type, which are still used today. For more than half a century of its existence, chemotherapy has become an independent field of clinical oncology. However, despite significant advances in this area, complete cure with the help of chemotherapy alone can be achieved only with a limited range of tumor diseases that are highly sensitive to drugs: uterine chorionepithelioma, germinogenic testicular tumors, lymphogranulomatosis, Berkit's lymphoma, acute lymphoblastic leukemia in children. In the chemotherapeutic treatment of patients with Ewing's sarcoma, lymphosarcomas, adenocarcinomas of the breast and ovary, bladder cancer and some other nosological forms, chemotherapy can have a significant clinical effect, but no more than 10% of patients are completely cured. The results of chemotherapy in the treatment of stomach cancer, colon cancer, non-small cell lung cancer look even more modest, and malignant tumors of the esophagus, liver, pancreas and thyroid glands, kidney cancer and cervical cancer show significant resistance to drug treatment. Nevertheless, the use of chemotherapeutic drugs in the complex treatment of these tumors is justified, since it allows after the removal of the tumor to suppress relapses of the disease and the development of metastases, and in the preoperative period helps to reduce the size of the tumor and facilitate its surgical excision.

The reason for such a low clinical effectiveness of medicinal methods of treating tumors is that complete healing can be achieved only by hitting all tumor cells without exception. The requirements for the treatment of infectious and parasitic diseases are not so severe – the immune system "cleans" the survivors of parasites after treatment. And cancer cells are "native" to the body, the immune system does not react to them, and if at least a thousandth of them survive, the tumor will recover to its initial size in just ¹⁰ divisions (210 = 1024). Here lies the main difficulty: it is not possible to destroy all tumor cells with a lightning strike, and in the process of long–term treatment, not only the population is restored, but also its progression, with a change in the properties and spectrum of drug sensitivity - always in the direction "from bad to worse". So chemotherapy is most effective at relatively early stages of tumor development.

The discovery of each new class of chemical compounds with antitumor activity caused a surge of optimism, but each time the results turned out to be much more modest than expected. The first cancer drugs either chemically damaged DNA and protein molecules (alkylating compounds: embihin, melphalan, methyl nitrosourea, cyclophosphamide, etc.), or hindered the process of DNA strand doubling (antimetabolites, the first of which, methotrexate and 5-fluorouracil, created in 1949 and 1956, respectively, are still used in oncology). Later, drugs that affect other intracellular targets appeared: antitumor antibiotics (doxorubicin, bleomycin), substances of plant origin (vinblastine, paclitaxel, etoposide), platinum complex compounds (cisplatin, carboplatin). Despite the fact that these chemical compounds act on a variety of molecular targets in cells, they are united by the ability to selectively suppress the growth and cause the death of tumor cells with relatively little damage to normal tissue cells. In parallel with the search for new antitumor drugs, the molecular mechanisms of action on the cell of drugs already found and used in practice were being studied. With the development of ideas about the mechanisms of antitumor activity of various drugs, it became obvious that the question of the low effectiveness of tumor chemotherapy is inextricably linked with another, no less relevant. According to academician N. N. Trapeznikov, who for many years headed the Oncological Research Center after N. N. Blokhin, if oncologists used to ask why medicines do not work, now the question is posed differently: why do they work? The answer to the last question was found quite recently.

Most of the "first wave" antitumor drugs were selected as a result of an experimental search for chemical compounds that kill mainly tumor cells (they are called substances with potential antitumor activity). To do this, scientists have studied how millions of natural and synthetic substances act on cancer cell cultures. This method is called the random selection method, in scientific terms - randomized screening. Not every one of the selected compounds can subsequently become a medicine. Later, scientists specially synthesized chemical compounds that theoretically should inhibit certain enzymes important for the process of cell division. As a result of these two approaches to the search for drugs, the entire modern arsenal of antitumor drugs was created.

However, the selectivity of chemotherapy drugs is not absolute: in the course of treatment, along with tumor ones, they often affect normal cells, primarily rapidly renewing tissues: bone marrow, epithelium of the gastrointestinal tract and hair follicles of the skin. But if the lesion of the follicles causes only baldness – an annoying, but temporary cosmetic defect, then the mass death of epithelial cells and bone marrow poses a real threat to the lives of patients.

The effectiveness of conservative cancer treatment methods has so far been limited not only by the side toxic effect on normal tissue cells, but also by the drug resistance of tumors. The vast majority of antitumor natural and synthetic chemical compounds act directly on cells, penetrating them and hitting a variety of intracellular molecular targets. Previously, doctors believed that antitumor drugs cause chemical damage to biomacromolecules incompatible with life in the cell – primarily nucleic acids and proteins. However, as our ideas about the mechanisms of programmed cell death developed, it became obvious that almost all antitumor drugs from the "first wave" drugs (embihin, 5-fluorouracil, chlorambucil, methylnitrosourea) to modern (gemzar, fludara, paclitaxel, glivec, rituximab) and even promising (TRAIL, ET-18-OCH3) very effectively activate the cell death program. In other words, cytotoxins do not kill cells, but provoke them to commit suicide. Despite the fact that the functions of genetic self-control are disrupted in the cancer cell, drugs that activate the cell death program mainly affect the tumor cells! This is one of the central paradoxes of tumor chemotherapy: the mutation recognition system, the breakdown of which makes the cell susceptible to mutagenesis and leads to its tumor degeneration, is only part of the "molecular kitchen" that implements the program of cell death. The fact remains that the vast majority of cell lines, that is, permanently maintained in the culture of cancer cells of the same origin, used to search for antitumor drugs, are capable of death as a result of apoptosis.

However, if the action of antitumor drugs is aimed specifically at activating the cell death program, then it should be assumed that a cancer cell in which the genetic program of its own death is damaged or completely lost should be resistant to the action of all known drugs. The proof of this assumption was unexpectedly obtained in our laboratory at the Cancer Center.

A4 cellsAt the very beginning of this century, we studied the activation of the cell death program by monoclonal antibodies to one of the death receptors, Fas.

This receptor appears on the surface of mature lymphocytes, and is also present on some types of malignant lymphoblastic cells. We used monoclonal antibodies to this receptor, which mimic the action of the natural FasL ligand, excite the receptor and activate the cell suicide signal. For experiments, we chose a well-known line of human Jurkat T-lymphoblastic cells isolated many years ago from the blood of a boy with leukemia, on the surface of which there is a Fas receptor. The addition of anti-Fas monoclonal antibodies to the nutrient medium causes the rapid development of apoptosis of these cells. We needed to get a culture in which the cells would be deprived of this receptor or that the receptor turned out to be inactive, that is, cells that are completely resistant to the action of anti-Fas antibodies. To do this, we used a well-known technique of cell selection, growing a culture in the presence of microscopic concentrations of antibodies. As the culture grew, the concentration of antibodies was gradually increased until cells were obtained that grew perfectly in an environment with antibodies. Since the culture obtained as a result of this experiment initially grew in a cup with the number A4, we called it A4, not yet assuming that this purely working name was assigned to a completely unique cell line.

In their appearance and set of surface antigens, A4 cells are similar to the parent Jurkat cells, but do not have a Fas receptor, so anti-Fas antibodies do not stimulate their death. This result was not unexpected. Another thing was puzzling: the resulting clone fully preserved the expression of death receptors of other types inherent in the parent line: ARO-2 for the TRAIL ligand and TNFR-1 for the TNFa cytokine, however, the use of these ligands did not cause any signs of apoptosis in A4 cells, although each of them activated the Jurkat parent cell death program. There could be only one explanation for this phenomenon: the resistance of A4 cells to apoptosis is due not to the absence of a corresponding death receptor, but to disturbances in the cascade of subsequent apoptosis signal transmission reactions.

Since the signaling cascades from "external" (receptors) and "internal" (damage to intracellular targets) signals of apoptosis merge into a common executive mechanism, we tried to launch a program of cellular suicide, acting not on external receptors, but on intracellular triggers. To do this, the cells were treated with cytotoxic drugs of different classes, inducers of oxidative cellular stress (hydrogen peroxide or vitamin K3), X-ray and ultraviolet radiation. In all cases, the proportion of cells with signs of induced apoptosis in the A4 clone population was 2-10 times lower than in Jurkat cells.

It follows from our results that stimuli of a very different nature, activating the Jurkat tumor cell death program, practically do not cause apoptosis of A4 clone cells. Does this fact mean that A4 cells cannot be killed? Of course not. A4 cells can be killed, but with a dose of medication that is incompatible with the patient's life. Cytostatics act on the Jurkat parent line in concentrations one to two orders of magnitude lower. In other words, A4 cells that are not capable of apoptosis exhibit the phenotype of multidrug resistance.

To find out how a cell culture reacts to a particular drug, it is usually treated with drugs in a concentration that causes the death of exactly half of the cells (LD50), and the fate of the second half that survived the toxic attack is monitored. In further studies, we grew cells of both lines in an environment with cytostatics cisplatin, doxorubicin or etoposide (each of these drugs causes apoptosis of Jurkat cells). For A4 cells, LD50 drug concentrations were 30-100 times higher. Counting and morphological analysis of cells that survived the cytotoxic attack showed that Jurkat cells die mainly by the mechanism of apoptosis, and A4 cells – by necrosis, untimely and unnatural death of a cell trapped in impossible conditions for life; their nucleus and cytoplasm swell, and then the nuclear and cell membranes rupture. The fate of the descendants of the cells of both lines that survived under these conditions also turned out to be different: after transplanting into a full nutrient medium, Jurkat cells restored their original appearance and growth rate after three weeks, although there were still quite a lot of dying cells in the culture. Descendants of A4 cells continued to die in huge numbers even after three weeks of cultivation. In their population, both multinucleated cells and cells with micronuclei appeared – the result of an uneven distribution of genetic material in the process of division.

Jurkat cells, which served as the starting material in our study, do not express the p53 protein, so their genome is quite variable and prone to accumulation of additional mutations. It is likely that A4 cells selected from the general population as a result of Fas-mediated selection are a clone that appeared as a result of one or more such mutations, the nature of which has not yet been established. Actually, it is not important and, most likely, represents only one of the many possible options. The result is important: having lost the cell death program, A4 cells were able to survive in the presence of such high concentrations of antitumor drugs that the patient cannot tolerate, therefore, form tumor tissue that is absolutely resistant to drug treatment.

Since the A4 clone was formed spontaneously, it can be assumed that in cancer patients, cells that have lost their cell death program may occur at different stages of tumor progression, regardless of what medications they are treated with. And the entire available arsenal of specific antitumor agents turns out to be powerless in front of such a clone.

This situation is a sad consequence of the methodology used to date for the selection of new antitumor drugs, which uses cell lines that to a greater or lesser extent retain the ability to programmed death. As a result of such screening, the most effective inducers of apoptosis are selected, which do not pose a real danger to tumor cells that have lost their ability to it.

Is there a way out of the impasse? There is no answer to this question today. However, it is worth paying attention to the fact that the part of the A4 culture that survived after treatment with cytotoxins continued to die during several dozen cell divisions that occurred in the absence of drugs. This phenomenon, well-known in radiobiology, is called "reproductive death", which is observed if the genetic damage received by the cell becomes fatal after one or more cycles of DNA doubling. If the death of the cell from external influence occurs before the first division, they speak of interphase death. Why do "unkillable" A4 cells die without external causes? Paradoxically, the cause of their death is the loss of the ability to apoptosis.

"Ordinary" cancer cells retain the ability to apoptosis. Therefore, the part of them that has received dangerous damage self-destructs, but the remaining cells continue to multiply, as in the case of Jurkat, and the tumor lives. And nothing prevents cells with a lost death program from uncontrollably accumulating mutations and other potentially dangerous damage, which may not lead to death in the interphase, but will interfere with the normal process of cell division and destroy the work of genes, which eventually creates a situation incompatible with further life. Therefore, with cells that are not affected by inducers of apoptosis, you can try to fight in a different way: to stimulate the formation of mutations in them, so that the sum of the resulting genetic damage leads to the loss of viability of themselves and especially their descendants. For this purpose, you can try to use supermutagens or chronic exposure to small doses of ionizing radiation. The success of future research largely depends on the correct choice of a model for searching for active compounds.

We believe that the A4 cell line we have obtained, as well as similar ones, may prove to be useful models for the search for fundamentally new antitumor agents, the effect of which will not be limited to the activation of apoptosis. Of course, there is a danger of side effects of such substances on the cells of normal tissues, because mutations will occur in their genomes. But, unlike tumor cells, the mechanism of genetic self-control continues to function in them, which does not allow replicating genetic defects in the next generations. The future will show how promising the use of supermutagens for medicinal purposes will be.

Portal "Eternal youth" http://vechnayamolodost.ru13.07.2009