Programmed cell death. Alexander Shtil
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Abstract of the dissertationin medicine on the topic Drug resistance of tumor cells mediated by P-glycoprotein: mechanisms of urgent formation and approaches to overcoming
As a manuscript
Shtil Alexander Albertovich
DRUG RESISTANCE OF TUMOR CELLS MEDIATED BY P-GLYCOPROTEIN: EMERGENCY MECHANISMS AND APPROACHES TO OVERCOME
Moscow 2003
The work was carried out at the State Institution Russian Oncological Research Center named after N.N. Blokhin Russian Academy of Medical Sciences, Moscow
Official opponents:
Doctor of Medical Sciences, Professor A.M. Garin,
Doctor of Biological Sciences, Professor, Honored Scientist of the Russian Federation A.N. Salrin,
Doctor of Biological Sciences, Professor N.S. Sergeeva,
Leading institution: Russian Medical Academy of Postgraduate Education of the Ministry of Health of the Russian Federation.
The defense of the dissertation will take place on December 25, 2003 at a meeting of the specialized Academic Council D.001.017.01 at
RONC named after. N.N.Blokhin Russian Academy of Medical Sciences at the address: 115478, Moscow, Kashirskoe highway, 24.
The dissertation can be found in the library of the Russian Cancer Research Center named after. N.N. Blokhin RAMS.
Scientific secretary of the specialized Academic Council
Doctor of Medical Sciences
Yu.V. Shishkin
General characteristics of the work The relevance of the topic
I. Multidrug resistance of tumor cells: biological mechanisms and significance in oncology.
Despite significant advances in pharmacology, including the development of technologies for creating drugs with expected properties, the success of chemotherapy for tumors is limited by the most important feature of living systems - the ability to respond to changes in the external environment. One of the manifestations of such elasticity is the development of resistance of tumor cells to drugs [used in chemotherapy. The wide prevalence and long-term, persistent nature of cell adaptation to external influences suggest that overcoming drug resistance may be associated not only with the search for more effective drugs: there is probably no drug to which cells would not be able to develop resistance. Only elucidation of the biological mechanisms of resistance to various types of stress will again serve to develop strategies for overcoming drug resistance, a necessary condition for increasing the effectiveness of treatment of cancer patients.
Multidrug resistance (MDR) of tumors - the preservation of viability by tumor cells in response to the influence of various drugs - is one of the main reasons for the progression of the disease: the tumor is insensitive to chemotherapy, regardless of the combination of different drugs. The MDR phenomenon has a long-term and stable character: resistance mechanisms are inherited over generations of cells. Thus, MDR is one of the key factors in tumor progression.
There are two main types of cell resistance to toxins. Primary g.e. resistance observed before exposure to chemotherapy) is due to the expression of defense mechanisms during tumor progression. Yes, activation
anti-apoptotic mechanisms conferring resistance to immune effectors may be related to drug resistance. Secondary (acquired) resistance occurs in cells exposed to stress. Before these influences, the drive mechanisms in such cells are poorly expressed or absent; surviving after treatment with one toxin, cells acquire resistance to many substances - MDR (Riordan, Ling, 1985). Further selection consolidates the acquired phenotype over generations of cells.
The most important mechanism of MDR is the reduced accumulation of toxins in the cell, due to the excretion of substances into the intercellular environment. This transport is carried out by the integral protein of the plasma membrane P-glycoprotein (Pgp) due to the energy of ATP hydrolysis (Juliano, Ling, 1984). Numerous data indicate that increases in MDRI and Pgp mRNA are often factors in the resistance of multiple tumor types to treatment (Linn et al., 1995; Stavrovskaya et al., 1998).
II. Development of MDR in tumor cells: biological mechanisms as targets for prevention
It is reasonable to assume that the increase in the amount of MDRI mRNA is due to amplification of this gene. This MDR mechanism has been identified in cultured cell lines selected for survival in the presence of toxins (Roninson, 1991). However, when analyzing human tumors, MDRI gene amplification was not detected either in primary tumors or in neoplasms after treatment. The probable cause of clinical MDR is overexpression of MDRI and Pgp with unchanged gene structure (preservation of copy number and nucleotide sequence), i.e. epigenetic asset! phenotype. In cultures of human tumor cells, an increase in the level of MDRI mRNA and the amount of Pgp was noted after a single treatment of the chemotherapy drug
different in chemical structure and mechanisms of action (Chaillon and Erwin, 1993). Evidence was obtained of the accumulation of MOI 1 mRNA in metastases of cancer in the lung tissue already 20-50 minutes after the start of intraoperative lung infusion with doxorubicin (Abolhoda et al., 1999). These results suggest the possibility of epigenetic activation of MDR in experimental and clinical situations: an increase in MDR1 and Pgp mRNA in tumors can occur without amplification of the MDR1 gene.
This type of biological regulation - urgent activation of the phenotype - involves the induction of transcription of the gene (genes) encoding the corresponding phenotype, and/or post-transitional control (stabilization of NK, regulation of protein synthesis and functioning). In relation to MDR, this type of regulation means the possibility of induction of the MYR gene\ (cell fusions and the relatively rapid development of resistance in [ear cells in response to stress. The inducibility of the MYR 1 gene suggests the development of signaling pathways from the cell periphery to the nucleus. Such pathways could be stress-implementing signaling mechanisms: protein kinase C KS), phospholipases and intracellular Ca2+, mitogen-activated neuropsinases, nuclear factor kappa B (NkB).Signal transmission to the gulatory region of the KYR\ gene and the transcript ensures activation of gene compression.
The study of MDR regulation also has a fundamental practical aspect. Inhibition of these mechanisms through pharmacological and/or therapeutic interventions would prevent the development of MDR during myotherapy.
III. Overcoming the formed MDR of tumor cells.
If blocking the MYR gene activating signals can prevent the formation of MDR in primary sensitive cells, then such
the approach is not applicable to overcome already formed resistance. The traditional method of combating secondary MDR is the use of Pgp modulators in combination with cytostatics (Lehne, 2000). However, the use of Pgp inhibitors is limited by side effects (heart rhythm disturbances, immunological imbalance). Equally important, the effectiveness of modulator+cytostatic combinations may be reduced due to blocking at least some cell death mechanisms during MDR selection.
Overcoming formed MDR is achievable if two conditions are met: 1) the concentration of the drug must be sufficient to activate the effekgor mechanisms of cell death, 2) the functions of these mechanisms must be preserved in cells with MDR. The first condition is met if the drug overcomes the Pgp barrier. However, it is required to prove that achieving a critical intracellular concentration of the agent is sufficient to activate the death of a cell that is resistant to many influences. Survival mechanisms operating in resistant cells should serve as targets for the elimination of the latter.
To implement the second condition, approaches aimed at the lysis of resistant cells as a mechanism for inducing their death seem promising. Vaccination of mice with syngeneic myeloma cells transfected with cDNAs of certain cytokines leads to the development of a cytotoxic T-lymphocyte (CTL)-mediated immune response and rejection of the inoculated tumor in immunized animals (Dranoff et al., 1993; Levitsky et al., 1996). CTLs lyse cells using granzyme B and perforin. Since granzyme B activates caspase 3, one of the distal effectors of apoptosis, and perforin causes primary damage to the plasma membrane (necrosis), one can hope that CTLs will be effective if proximal death mechanisms are blocked; triggering of distal links of apoptosis in combination with necrosis
leads to the death of cells resistant to antitumor drugs that induce programmed cell death.
Formulation of the problem
MDR is a clinically unfavorable phenomenon, the overcoming of which requires knowledge of the mechanisms of its development and the ways in which cell death occurs; it is necessary to study both aspects of the problem. First, it is necessary to study the mechanisms of MDR formation in MDR1/Pgp-negative human cells; the study of these mechanisms will serve to prevent the development of resistance in primarily sensitive cells. Secondly, analysis of the death processes operating in MDR cells will create the basis for overcoming resistance in situations where secondary MDR has formed.
The purpose of the study is to establish the mechanisms of urgent formation of lymph nodes in human tumor cells and to develop approaches to overcome this hell of resistance.
1. To optimize models for the development of MDR in human tumor cell cultures in response to the effects of chemotherapy drugs and experimental agonists and antagonists of signaling mechanisms.
2. Determine the main mechanism for the urgent development of MDR when cells are treated with antitumor agents: amplification of the MDR gene, selection of Pgp-positive cells, or de novo induction of MDR.
3. Explore the pathways of intracellular signal transmission that regulate the activation of MDR - protein kinase C, phospholipase C, intracellular Ca2+, mitogen-activated protein kinases, NFkB).
4. To identify the role of transcriptional activation and post-transcriptional regulation (mRNA stability) of MDR gene expression in the acute development of MDR in response to the effects of chemotherapy.
5. Develop ways to prevent the development of MDR in tumor cells by combining chemotherapy drugs with blockers of CR-activating signaling pathways and gene transcription inhibitors
6. To study the kinetics of activation of initiating and effector casps, changes in the transmembrane potential of mitochondria, proteolytic cleavage of poly(ADP)ribose polymerase, internucleosomal DNA fragmentation and integrity of the plasma membrane in parent cells and variants with MDR when treated with a drug not transported by P-glycoprotein.
7. Use vaccination with tumor cells expressing
cytokines to generate an immune response against MDR cells. »
Provisions submitted for defense.
1. The formation of Pgp-mediated MDR - an urgent cell response to many influences - is mediated by epigenetic activation of the MDR 1 gene. This activation is due to numerous mechanisms of intracellular signal transmission, induction of the gene promoter and stabilization of mRNA and can be prevented by inhibitors of these signals.
2. Overcoming Pgp-mediated MDR may be associated with a targeted effect on the plasma membrane of resistant cells. Pgp does not protect cells from disruption of the integrity of the plasma membrane - necrosis.
Scientific knowledge
1. For the first time, the idea of the formation of MDR as an urgent cell response to an exogenous stimulus has been substantiated;
2. For the first time, the mechanism of development of a specific drug resistance phenotype, P£p-mediated MDR, has been studied in detail: epigenetic activation of the MYR 1 gene encoding this protein.
3. For the first time, a model of urgent activation of the MHS gene has been developed,
accompanied by the acquisition of a stable phenotype of Pgp-mediated MDR in cultured human tumor cells; 1. Signal transduction pathways, mechanisms of transcription activation and post-transcriptional regulation of the MOK 1 gene in cells exposed to antitumor drugs have been identified. 5. For the first time, classes of pharmacological substances - blockers of intracellular signal transmission - have been characterized to prevent the formation of RCR-mediated MDR in tumor cells. 5. For the first time, death mechanisms operating in cells with PgP-mediated MDR were studied, and an approach to overcoming resistance was developed, which involved primary damage to the integrity of the plasma membrane.
Practical value.
1. Development of methods to prevent the urgent development of Pgp-mediated MDR in cultured tumor cells when exposed to chemotherapeutic drugs.
2. Preclinical trials of modified genetically engineered vaccines to overcome MDR.
Approbation of work.
The dissertation was discussed on June 30, 2003 at a joint conference of the departments of tumor cell genetics, cytogenetics with the group of molecular genetics, viral and cellular oncogenes, molecular endocrinology, antitumor immunity, biochemical pharmacology, medical research, experimental diagnostics and biotherapy of tumors; Departments of Schmunology, Hematology, Chemotherapy, Clinical Pharmacology, Advanced Treatment Methods of the Russian Cancer Research Center named after. N.N.Blokhsha RAMS.
The main materials of the dissertation were presented at the following conferences: 2nd international symposium "Cylostatic Drug Resistance", (K Germany, 1991); Gordon Conference "Advances in Chemotherapy" (New York, London, USA, 1994); "Molecular Toxicology" (Copper Mountain, USA, 1995); "Inducible Genomic Responses" (Stevenson, USA, 1996); "Nucleic Acids - Integrating Molecular Diagnostics and Therapy" (San Diego, USA, 1996); annual conferences of the American Association of Cancer Research (1994-2001): 6th and 7th congresses "Advances in Oncology", (Hersonissos, Greece, 2001,2002); "The structure and functions of the cell nucleus" (St. Petersburg, 2002), as well as at seminars at Oncotech, Inc. (Irvine, USA, 1996), Salk Institute (LaJolla, USA, 1997), Lee Moffitt Cancer Center (Tampa, USA, 1997), The Jackson Laborati (Bar Harbor, USA, 1997), Sloan-Kettgering Cancer Center, New Yo] USA, 1999), the universities of Copenhagen (2002), Innsbruck (2002) and Gronings! (2003), Moscow State University. M.V. Lomonosov (2002), Research Institute of Experimental Pathology, Oncology and Radiobiology named after. R.E. Kavetsky (Kyiv, 2002).
Publications.
Structure and scope of the dissertation.
The dissertation is presented on 181 pages of typescript and consists of the introduction of the chapters “Literature Review”, “Materials and Methods of Research”, “Research Results” (two parts), discussion and conclusions. The work contains 44 figures and 6 tables. Bibliographic material includes links to 270 literature sources.
Materials and methods of research.
Laboratory animals and cell lines. Balb/c mice were used. For experiments on MDR activation, we used human cell lines H9 (T-cell leukemia), K562 (promyelocytic leukemia), SW<
(colon cancer), as well as sublinigo K562Í/S9, in which Pgp is expressed without selecting cells for resistance to toxins (Mechetner et al., 1997). For experiments on creating antitumor immunity, myeloma lines MPC11, J558 and S194 were used. To obtain sublines with MDR, stepwise selection of MPC11 cells for resistance to doxorubicin was carried out. The independent MPC1 subles lDoxlO-1 and MPCllDoxlO-2 proliferated in the presence of 100 nM doxorubic acid.
MDR study: MDRI gene RNA, Pgp quantity and function.
To activate MDR, we used liggosin-1P-arabinofuranoside (cytosar, Ara C), doxorubicin, vincristine, nocodazole, blsomycin, sphingomyelinase, Ca2+ ionophore A23187, thapsigargin, 2-deoxyglucose, trichostatin A and phorbol ester 12-0-tetradecanoylmyristate. 13-acetate (TPA). To inhibit the activation of MDR, actinomycin D, α-amanitin, ecteinascidin 743 (ET743), chelerythrine, bis-indolylmaleimide I, calphostin C, BARTA/AM, TMB-8, pyrrolidindptiocarbamate (PDTC), tosyl-L-phenylchloromethyl ketone (TPCMK), sodium salicylate, salicylic acid, PD98095. Inhibitors were added to the cells over 30 min. before adding activators. The level of MDR\ mRNA in cells was studied by reverse transcription polymerase chain reaction (PCR) (Noonan et al., 1990; Shtil et al., 2000) using primers: MDRV. straight: 5"-ССС ATS ATT GCA ATA GCA GG-3"; reverse: 5"-GTT CAA ACT TCT GCT SCT GA-3". Product length 167 bp. p2-microglobulin: direct: 5"-ASS CCC ACT GAA AAA GAT GA-3"; reverse: 5"-ATC TTC AAA CCT CCA TGA TG-3". Product length 120 bp.
The amount of Pgp and its transport function were determined by flow cytometry with the monopoly antibody UIC2 (Mechetner et al., 1997). Fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG antibodies were used as secondary antibodies. For teaching Pgp-
dependent transport, fluorescent substrates Pgp-rhodamine 123 (in experiments on MDR induction) (Neyfakh, 1988) and calcein acetoxymethyl ester (in experiments with myeloma cells) (Holló et al., 1996; Shtü et al., 1999) were used.
Cell death assay. Cell survival in the presence of toxins was studied by the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2P-tetrazolium (MTT test) (Mossman, 1983 ; Sidorova et al., 2002). V-FITC annexations were used to determine the number of apoptotic cells; necrotic cells were detected with propidium iodide (PI). The transmembrane electrical potential of mitochondria was determined by the fluorescence of the JC-1 probe (Zamzair et al., 1997). To determine genomic DNA fragmentation, cells were lysed in a buffer containing sodium citrate, NP-40, RNase A, and PI; the suspension was analyzed on a flow cytometer (Shtil et al., 1999; Fragmented DNA was detected in the sub-Gl region. To study the formation of free forms of oxygen in cells, dichlorofluorescein diacetate ester (DCFDA) was used, which penetrates the cytoplasm and fluoresces after oxidation by intracellular metabolites.
PKC activity in the cytosol and particle fractions was determined by radioactive method by phosphorylation of myelin basic protein after cell lysis and separation of fractions by centrifugation (Shtil et al. 2000).
The activity of mitogen-activated protein kinases ERK1/2 and JNK1 was determined by phosphorylation of specific substrates (myelin basic protein and c-Jun) after cell lysis and immunoprecipitation of the kinases (et al., 1996).
Reporter plasmids, expression vectors, transfection.
K562 and B\Y620 cells were trifected with a plasmid carrying a region of the troximal promoter of the MOL 1 gene -1202/+118 nt. relative to the transcription start site, cloned into the pOB2b vector. The reporter protein was Pge/1y luciferase. To test the transactivation naivety of NaκB, cells were trifected with a promoter-reporter construct containing binding sites for OTkB (5xNκB-lgocyferase). To suppress luciferase activity, the cells were simultaneously injected with a sacmid carrying the Rental luciferase under the control of the SIV40 promoter. In some experiments, the activity of luciferase Pne]\y was related to the concentration of total SEL in the transfected cells. For transfection, we used Spofeknsh or Lipofectamine, as well as the “gene gun” (for myeloma cells) Nabillevich et al., 1996). Vectors expressing the p50 and p65 IκB subunits under the control of the BU40 promoter were used for cotransfection with the parasite - 1202/+118-luciferase The activity of luciferases in cell 1 cells was studied by the chemo-pominescence method.
Immunization of mice. MPC11 cells and sublines with MDR were irradiated (40 ~r), trifected with a plasmid carrying granulocyte-macrophage-salinity-stimulating factor (GM-CSF) cDNA and injected subcutaneously into mice (1.5 x 105 years per animal). Control mice were injected with the same number of irradiated cells transfected with the vector without insert. After 7 days, fresh cells were irradiated and trifected with a plasmid carrying interleukin-12 (IL-12) DNA or irradiated cells transfected with a chasmid without insert (control). After another 7 days (a total of 14 days after the first vaccination), the animals were inoculated with fresh cells subcutaneously (10th per week) (Tisher et al., 1998).
CTL activity in mixed culture with myeloma cells. The spleens were removed 11 days after injection of IL-2-transfected myeloma cells into mice. Splenocytes were cultured for 5 days at 37°C, 5%
COg with fresh irradiated cells of the line used for immunization. Fresh myeloma cells were then loaded with 51Cr (CTL target cells). CTL activity was assessed by the release of mSG into the medium after incubation with targets. To inhibit perforin processing, splenocytes were incubated with concanamycin A and then with targets (Kataok et al., 1996).
Research results
Urgent activation of Pgp-mediated MDR.
MDRI mRNA accumulates in cells in response to the effects of antitumor drugs (Chaudhary and Roninson, 1993). To clarify whether this effect is associated with the selection of Pgp-positive cells or with the induction of MDR, experiments were carried out on MES/Pgp-negative H9 cells. The cells were treated with Ara C, a non-Pgp transported drug used in patients with breast cancer and hematological malignancies. Expression of MDR\ in untreated cells is not detected after 25 cycles of PCR, whereas in Ara C-treated cells an increase in MDRI mRNA is observed after 3-6 hours. impact (Fig. 1, A). An increase in MDRI mRNA can be observed even faster - after just 1 hour of exposure, if the cytosar concentration is increased to 75 µM. The increase in MDRI mRNA persists in cells that survive a single exposure to Ara C for at least 6 weeks.
We next examined whether the amount of Pgp increases in parallel with the accumulation of MDRI mRNA. In Fig. Figure 1 B shows that Ara C-treated cells express Pgp. It is important that exposure to Ara C causes a shift to the right of the entire population, which indicates that almost any cell in culture is capable of accumulating Pgp. Ara C-treated cells express functionally active Pgp: in these cells, elimination
rhodamine 123 is more potent than in intact cells; the effect is removed by verapamil, a blocker of Pgp-dependent transport.
O 1 3 6 10 16 24 HOURS.
fluorescence (Ig) -
Fig.1. Increase in MDRX and Pgp mRNA after a single exposure to Ara C. A: H9 cells treated with 10 μM Ara C. MDR1 and p2-microglobulin (B2M) mRNA were determined by PCR after reverse transcription. B: cells treated with 10 µM Ara C for 24 hours (no<яя панель). Контроль - необработанные клетки (верхняя панель). Таким образом, накопление иРНК MDR\ наблюдается в течение первых
asy exposure to an agent not transported by Pgp. It means that
The most likely mechanism for this effect is the induction of a phenotype
not selection of “pre-existing” resistant cells.
In Fig. Figure 2 shows the dependence of cell survival on the concentration of vincristine, a chemotherapy drug transported by Pgp. Cells that survived
Rice. 2. A single exposure to cptozar leads to the formation of a stable Pgp-transported drug.
H9 cells were treated with 10 µM Ara C for 24 hours, resuspended in fresh medium and incubated for 12 days. After restoration of logarithmic cell growth, their sensitivity to vincristine was studied in comparison with cells not treated with cytosar (control). Results of 4 experiments (MTT test)
Thus, a single exposure of Pgp-negative cells to a non-Pgp-transported chemotherapy drug leads to rapid - within one cell cycle - accumulation of MDRI gene mRNA, functionally competent Pgp and, most importantly, the development of resistance to the Pgp-transported agent. Primary sensitive cells acquire Pgp-mediated MDR. The mechanism of this phenomenon is not the selection of Pgp-positive cells, but the activation of the de novo phenotype. How does Pgp-mediated formation occur? MDR: due to activation of MDRX gene transcription or stabilization of mRNA, do both mechanisms function?
In the experiments presented in Fig. 3, H9 cells were treated with Ara C in the presence! transcription inhibitors - actinomycin D, a-amanitin and ecteinascidin 743 (ET743). All inhibitors tested prevented the Ara C-induced increase in MDRI mRNA levels.
Rice. 3. Transcription inhibitors prevent the accumulation of MDR1 RNA. H9 cells were treated with 10 μM Ara C for 24 hours. without or in the presence of actinomycin D, α-amanitin or ET743. The results of 3 experiments are summarized.
To study the half-life (stability) of mRNA, cells were treated with Ara C for 10 hours. and transferred to fresh medium or medium with actinomycin D and incubated for another 36 hours. In untreated cells, MDRI mRNA turned out to be short-lived: its half-life was ~30 min. Treatment with Ara C extended the half-life of mRNA to 6 hours. Thus, the accumulation of MDRI mRNA, and, consequently, the development of Pgp-mediated MDR in response to cytotoxic stress, is caused not only by the activation of MDR1 transcription, but also by the stabilization of the mRNA of this gene.
Mechanisms of intracellular signal transmission in MDR activation Figure 4 shows that a single treatment of H9 cells with TFA, a PKC agonist, led to the induction of the MDRI gene. Specific PKC inhibitors - chelerythrine, calphostin C and bis-indolylmaleimide I - prevented MDR activation by phorbol ester and chemotherapy. Similar
The data were obtained on K562 cells treated with Ara C, doxorubicin, or TFA in the presence of the indicated PKC inhibitors.
Rice. 4. PKC inhibitors prevent MDRI induction. H9 cells were treated with 10 μM Ara C for 16 h. without or in the presence of PKC inhibitors. The results of 3 experiments are summarized.
In summary, PKC is important for the regulation of MDRI (and therefore the MJI phenotype; this gene can be induced by a PKC agonist, and PKC inhibitors prevent activation of MDRI by anticancer drugs.
The physiological agonist of PKC is diacylglycerol (DAT), which is formed during the hydrolysis of phosphatidylinositol-4,5-diphosphate (PIg) by phosphatidylinositol-specific phospholipase C and/or during the breakdown of phosphatidylcholine under the action of phospholipase C, specific for this phospholipid (Berridge, Irvine, 1984). Activation of MDRI induced by Ara C or doxorubicin can be blocked by neomycin sulfate and U73122, inhibitors of phosphatidylinositol-specific phospholipase C (Fig. 5). Activation of MDRI by phorbol ester is insensitive to neomycin sulfate and U73122 because TFA is a direct PKC agonist, and this kinase functions distal to phospholipase C. Inhibition of phosphatidylcholine-specific
Yusfolipase C (drug B609) did not lead to changes in the expression of MYR 1 Fig. 5). These results indicate the importance of PI2 hydrolysis of the yusphatidylinosengol-specific phospholipazone C in the activation of MYA 1.
Rice. 5. Phospholipase C inhibitors in the induction of MDR\.
H9 cells were treated with 10 μM Ara C for 16 h. without or in the presence of neomycin sulfate, U73122 or D609. The results of 3 experiments are summarized.
Phospholipase C breaks down PI2 into DAT and FIs. The first product activates PKC, the second increases the level of intracellular Ca2+ due to its mobilization from the ectoplasmic reticulum. The role of intracellular Ca2+ in the induction of MDRI is proven by the ability of the Ca2+-specific ionophore A23187 and thapsigargin, a Ca2+-ATPase inhibitor, to activate this gene; the specific Ca2+ chelator BARTA/AM prevented the induction of MDR\ by both chemotherapy and TFA (Fig. 6).
1 2 3 4 5 in 7 8 E 10 11 12 14 14 15 1617 18 19 Rns. b. The role of intracellular Ca2+ in the induction of A) R1.
H9 cells were treated with MOI inducers for 16 hours. alone or in the presence of the calcium chelator BARTA/AM. Tracks: 1-untreated cells, 2,3-A23187; 4.5-
thapsigargin; 6,7-AgaC; 8,9-doxorubicin; 10,11-bleomycin;12,13-2-deoxy-glucose;14,15-nocodazole;16,17-sphingomyelinase;18,19-TFA. Even tracks: inductor odd (except 1): inductor -5 µM VARTA/AM.
To clarify the participation of intra- and extracellular Ca2+ in the activation of MDR, two series of experiments were carried out. First, gene activation was studied under conditions of cell incubation in a medium without Ca2+. Removal of extracellular Ca2+ did not impair the activation of MDRI by chemotherapy and TPA. Secondly, the agent TMB-8, which prevents the entry of Ca2+ into cells and does not change the concentration of intracellular Ca2+, did not affect the induction of MDRI. Thus, intracellular but not extracellular calcium is required for MDRX activation. The above experiments prove the fundamental role of the phospholipase C->DAG-*PKS and PI3->Ca2+ signaling pathways in the activation of the MDRX gene by various substances, including chemotherapy.
However, PKC is not a universal mechanism for activating MDR. PKC activity varies in cells treated with individual MDRX inducers. T< активирует ПКС, Ara С не оказывает существенного влияния на активность этой киназы, а церамид - вторичный мессенджер, накапливающийся в обработанных химиопрепаратами клетках (Bose et al., 1995), ингибирует ее (табл. 1).
" Table 1. Effects of MDRI gene inducers on PKC activity.
Treatment PKC activity, pmol/mg protein/min.
cytosol particle fraction
Control 119±13 59+9
TFA, YuONM 47+10* 153+14*
Ara C, 25 µM 143+16 65+10
Ceramide, 1 µM 138+15 27+8*
Ceramide, YumkM 83+15* 15+7*
*R<0,05 в сравнении с контролем (необработанные клетки). Данные 6 опытов.
PKC inhibitors chelerygrine and bis-indole-maleimide I prevented the activation of MDR\Ara C, doceorubicin and TFA, but not ceramide (Fig. 7). Therefore, activation of PKC is not a prerequisite for the induction of the MDRX gene. Some agents (eg, ceramvd) activate ACL-independent signaling mechanisms or act distal to the ACL.
Rice. 7. Ceramnd activates MDR\ regardless of the ACL intervention.
H9 cells were treated with 25 μM C2-ceramide for 24 hours. alone or in the presence of PKC inhibitors. Control of the effectiveness of inhibitors is their blocking of MDR activation when exposed to 10 µM cytosar.
Such mechangams can be mitogen-activated protein kinases. Doxorubicin and Ara C at concentrations that activate MDR\ increased the activity of JNK1, but not ERK1/2, while TFA activated ERK1/2, but JNK1 activity did not change (Fig. 8). Consistent with these data, the ERKI/2 inhibitor PD98059 abolished MDRI activation by TFA, but not by chemotherapy. Thus, mitogen-activated protein kinases serve as a level of divergence of MDR1-activating signals generated by different substances.
Activation of MAP kinases by doxorubicin and TPA
Rice. 8. Differential activation of MAP kinases by MDR1 inducers. H9 cells were treated with doxorubicin and TFA. ERK1/2 and JNK1 activities were determined after immunoprecipitation and phosphorylation of substrates. The effects of inducers are expressed as the level of activation of each kinase compared to untreated cells, in which activity is set to 1. Data from 3 experiments are summed.
The transcription factor NFkB is a mechanism for the rapid response of cells to external stimuli (Karin, 1995). In a resting cell, this protein is located in the cytoplasm in complex with the inhibitory subunit; in response to exposure, NFkB dissociates from the complex, is transported into the nucleus and activates the gene regulatory regions of which have NFkB binding sites. The NFkB inhibitors PDTC, TPCMC, and salicylates prevented MDRX activation by cytosar, doxorubicin, and TFA (Fig. 9). It is especially important that salicylap, a drug widely used in the clinic, turns out to be an effective blocker of urgent MDRI activation. Moreover, sodium salicylate canceled the long-term (up to 7 days) activation of MDRX by cytosar (Fig. 10), confirming; the importance of NFkB as a mechanism of MDR activation.
Rice. 9. Prevention of MDR1 induction by NFkB inhibitors. H9 cells were treated with 10 μM Ara C, 5 μM doxorubicin, or 10 nM TFA for 16 h. alone or in the presence of inhibitors of NFkB activation. The results of 4 experiments are summarized.
4, wash
L ha S - + +----
salicylate - - + \ s 5 7
days without AGAS
Rice. 10. Long-term effect of sodium salcinlate as an inhibitor of MDRI induction.
H9 cells were treated with 10 μM Ara C for 48 hours. alone or in the presence of sodium salicylate, resuspended in fresh medium and incubated for another 1-7 days.
The importance of establishing the role of NκB is also in the fact that this mechanism “unites” cytoplasmic and nuclear events in the activation of the MYR 1 gene. This connection is confirmed by experiments on activation of the gene promoter; AYUSH exogenous No.kV. Cotransfection of OTkV subunits p50 and p65 resulted in activation of the region -1202/+118 nt. promoter AYUSH (Fig. 11). However, in the indicated region, the canonical sequence 5"-COOIM^YUSS-3" (R-any purine base, aL-any pyrimidine base) for interaction with NaκB or homologous sequences was not found. The following assumptions are possible: 1) NokV interacts with an as yet unidentified sequence in the region -1202/+118 i.i.; 2) NkB activates an intermediate gene (genes), the product of which binds to the region -1202/+118 nt. and induces the MDR1 promoter.
Rice. 11. NFkB - activator of the MDR promoter.
K562 cells were transfected with the indicated constructs and a plasmid carrying Renilla luciferase rei under the SV40 promoter. Firefly luciferase activity reflecting induction of the -1202/+118 nt region of the MDRX promoter (denoted as MDF normalized to Renilla luciferase activity.
Regulation of the MDR1 gene promoter.
Pathways for transmitting L1-activating signals must have a common “convergence point” - the regulatory region of the gene and mRNA. To study the MDRI promoter, we used drugs that modulate the physicochemical state of chromatin - histone deacetylase inhibitors trichostatin A and sodium butyrate, representatives of classes of chemotherapy drugs that directly regulate gene transcription. It is important to compare the mechanism of MDR activation by these agents with Ara C and doxorubicin, drugs for which chromatin is not a direct target. Trichostatin A and sodium butyrate activate the endogenous MDRX gene in H9, K562 and SW620 cells. The increase in mRNA (Fig. 12, left panel) was accompanied by activation of luciferase transcribed from the -1202M18 nt site. MDRI promoter (Fig. 12, right panel).
Rice. 12. Histone deacetylase inhibitors induce the endogenous MDRX gene and promoter (transient transfection).
Slash, H9, K562 and SW620 cells were treated with the indicated drugs for 24 hours. The level of MDRX mRNA was studied in GTCR. Right: K562 cells were transfected with the -1202/+P8 n.o.-luciferase construct. In 24 hours. cells were stimulated with the indicated drugs for 16 hours. The results of 3 experiments are presented.
It has been established that trichostatin A and butyrate activate MDRI due to the interaction of the transcription factor NF-Y with the inverted HAAT box (Jin and Scolto, 1998), which confirms the chain of events “inducer-chromatin modification -> activation of the MDRI promoter.” However, under conditions of belt transfection, the chromatin on the plasmid remains “imperfect.”
To study the role of chromatin in MDRI induction, SW620 cells were transfected with the -1202/+118 nt Firefly luciferase construct and a plasmid carrying the neo gene; then cells were selected for survival in the presence of neomycin. In selectants, as well as during temporary transfection, trichostatin A and sodium butyrate caused the accumulation of endogenous MDRI mRNA and induced transcription from the region -1202/+118 i.i. promoter of this gene. Activation of MDRI by histone deacetylase inhibitors was abolished by ET743, a transcription blocker. This shows a direct connection between an increase in the amount of MDRI mRNA and activation of the promoter of this gene through chromatin acetylation.
These data were obtained when cells were exposed to agents that directly changed the physicochemical state of chromatin. Is the mechanism always identified, since not all chemotherapy drugs (and other exogenous stimuli) are direct regulators of chromatin? We noted that the half-life: MDRI mRNA increases in cells treated with Ara C and TFA. Figure 1 shows that TFA and trichostatin A are powerful inducers of MDRI promoter activity (transcription), while neither Ara C, nor doxorubicin, nor ceramide activated the promoter or caused only a weak effect.
Rice. 13. Effects of MDR activators on MDRI promoter induction. K562 cells were transfected with the -1202/+118 nt Firefly luciferase construct, divided into equal parts and stimulated for 16 hours. the indicated drugs.
Luciferase activity in untreated cells, normalized to the total protein content in cell lysates, was set to 1. The results were reproduced in 3 experiments.
These results suggest that an increase in the level of A/III1 mRNA occurs both as a result of transcription activation (TFA, trichostatin A) and without direct induction of the promoter (cytosar, doxorubicin, ceramide). In the latter case, one should recognize either the mechanisms of mRNA stabilization, or the activation of an intermediate gene, the product of which induces the MIEX promoter or stabilizes the transcript (see experiments with exogenous NaκB).
Table 2. Inhibitors of signaling mechanisms that prevent urgent activation of the MF1 gene tsntozprom.
Drug Intracellular target Inhibitory concentration
chelerythrine PKS 10 µM
calphostin S PKS 1 µM
bis-indolyl-
maleimide I PKS 5 µM
VARTA/AM Ca2+ 5 µM
PDTK No.kV 5 µM
GFKhMK No.kV 25 µM
sodium salicylate No.kV 20 mM
yutirin №kV 10 mM
Ziesters higher plasmatic
fatty acid membrane? Yumkg/ml
1KTSHGOMISHSH B transcript elongation? 500-1000 ng/ml
x-amanitin RNA polymerase II 9 μg/ml
zhgeinascischsh 743 not installed 100 nM
Table 2 shows pharmacological drugs representing groups of signal transduction inhibitors that prevent MDR activation by cygosar or doxorubicin in H9 and K562 leukemia cells.
Overcoming Pgp-mediated MDR: plasma membrane target
cytotoxic action.
Thus, Pgp-mediated MDR can form quite quickly during one cell division, and persist in generations of surviving cells. Many stimuli are inducers of MDR, including the clinical use of antitumor drugs of various chemical groups. Pgp is the hallmark of mechanisms that prevent the activation of programmed death (apopto) (Johnstone et al., 1999). Therefore, a tumor that is primarily resistant to apoptogenic stimuli and rapidly acquires MDR in response to treatment poses a particular challenge for therapy. What are the strategies to overcome multifactorial resistance?
The death of such cells under the influence of immune effectors has been studied. The prerequisite for this approach was data on the rejection of vaccinated myeloma by splenocytes that accumulate during vaccination of mice; the same tumor, modified to express cytokines (Turner et al., 1996). Since the properties of the MPC1 IDoxlO-l and MPC1 1Dox10-2 sublines turned out to be similar, MPCllDoxlO-1 cells are analyzed below.
The MDR phenotype in the selectants was characterized by overexpression of the mdrVo gene, decreased calcein transport, and resistance to doxorubicin and vincristine, Pgp substrates. To create immunity to myelol inoculation, MPC11 and MPCI IDoxlO-l cells were transfected with cDNA of the GM-KS IL-12 cytokines. The number of cells inoculated was five times the minimum 100% tumor-killing dose.
Table 3. Tumor rejection rates in control and
immunized animals.
Mice Grafting
MPC11 MPCllDoxlO-1
Intact 0/15* 0/10
Immunization with MPC11 cells:
irradiated cells 1/15 0/10
■M-CSF/IL-12-transfected cells 15/15** 9/10**
Immunization with MPC1 Xox10-1 cells:
irradiated cells 0/15 0/10
"M-CSF/IL-12-transfected cells 9/10** 10/10**
Tumor rejection rate: in the numerator - the number of mice that did not develop a tumor; in 1 replacement - the total number of vaccinated mice, **P<0,001 по критерию х2 в сравнении с еиммунизированными (интактными и вакцинированными только облученными клетками) ивотными.
Table 3 shows that MPC11 and MPC1 IDoxIO cells are tumor-producing: in 100% of non-immunized animals, tumors appeared 8-10 days after inoculation of the corresponding cells. However, in mice immunized with cytokine-expressing cells, tumors did not appear (vaccination with MPC11) or were rare (vaccination with a resistant subline). Antitumor immunity was long-lasting: tumors did not appear within 5 weeks after inoculation of fresh cells. The incidence of graft rejection was similar between groups immunized with MPC 11 cells and the resistant subline. These results indicate the possibility of death of malignant resistant cells when exposed to the factor(s) detected in in vivo experiments.
Such a factor are CTLs generated in the spleen in response to immunization with cytolytic expressing cells. In Fig. Figure 14 shows that splenocytes from mice immunized with GM-CSF- and IL-12-expressing MPC 11 cells lysed MPC 11 and MPCllDoxlO-1 target cells with almost identical efficiency (Figure 14).
-ér-MPCU/MPCJlDoilO
MRS11/MRSI
effector.target
Rice. 14. CTL lysis of parental and Pgp-positive cells.
Numerator: cells for immunization, denominator: cells to be grafted.
Consequently, antitumor immunity in animals vaccinated with a cytokine-expressing cell vaccine is associated with the sensitivity of MDR cells to the logical action of CTLs.
The killing activity of CTLs is due to the secretion by these cells of the CD95/Fas ligand and/or the granzyme B-perforin tandem (Vetke, 1994). To resolve the question of which of these mechanisms functions in the system under study, cell survival was studied in the presence of the Fas ligand. MPC11 and MPC1 ShoxY-1 cells turned out to be resistant even to relatively high concentrations of the Fas ligand. Thus, granzyme B/perforin should be considered a candidate mechanism for the cytotoxic action of immune UTJs. Indeed, preincubation of immuno-CTL with concanamycin A, an inhibitor of perforin processing, reduced the lysis of target cells MPCllDoxl 0-1 (Fig. 15).
To find ways to overcome MDR, it is important that sublines with MJB retained sensitivity to cytotoxic effects due to disruption of the integrity of the plasma membrane by perforin. Penetration of granzyme B into the cell through pores in the plasma membrane formed by perforin will lead to the activation of effegrine caspases. Dachshund
killer tandem - the simultaneous action of an agent that forms pores in the outer membrane and protease - makes it possible to cause the death of cells that are resistant to a number of external influences. Therefore, the plasma membrane is an important target for therapy aimed at eliminating resistant cells.
T; with k) o o o
n (o n ii) o o
effector: target
Rice. 15. Granznm B/psrforin - mechanism of CTL-mediated death. Concanamycin A (open bars) reduces CTL-mediated lysis of MPC1 XoxI-1 cells. Dark columns indicate lysis of CTL targets without preincubation with concanamycin A. One of 3 representative experiments is shown.
Zerschied and free oxygen radicals in overcoming MDR. If the plasma membrane is the desired target, then how to cause its damage? It is especially desirable that such an effect would be exerted by an actor common to many anticancer drugs. One of these “intermediaries” is ceramvd. In order for ceramide to be effective for threodolization of MDR, it must satisfy at least two requirements: ;) not be transported out of the cell by Pgp; 2) trigger death pathways that function in resistant cells. Is it enough to “bypass” the membrane transporter, the leading mechanism of MDR, to cause the death of cells that are resistant to a number of influences?
Ceramide is not transported by P-gpicoprotein.
Because ceramide accumulates in response to a variety of chemotherapy drugs, we examined whether this metabolite met these conditions. It turned out that the kinetics of ceramide accumulation in parental K562 cells and their Pgp-positive subline K5621/89 are almost the same (Fig. 16). At the same time, K562^B9 cells accumulated significantly less rhodamine 123 than K562 cells (control of Pgp-mediated transport). Consequently, ceramide is not a transport substrate for Pgp.
8000 6000 4000 2000 0
0 0.05 0.2 1 5 C5-BOO!RU-ceramide
Rice. 16. Pgp does not transport short-chain ceramide. K562 and K5621/59 cells were incubated for 30 min. with C5-ceramide conjugated with fluorochrome VSY1RU (top panel) or rhodamine (III)123 (bottom panel). Cell luminescence was studied using flow cytometry. Data from 3 experiments.
There were no differences in the cytotoxicity of synthetic (C2) and natural (Ci) ceramides for K562 and K5621/89 cells, as well as for pairs
parental cells and selectants: MCP-7 and MCP-7Ac1g, KV-3-1 and KV-8-5-11. Thus, ceramide causes the death of Pgp-negative and Pgp-positive cells with equal efficiency.
does not prevent disruption of the integrity of the plasma membrane.
To identify the mechanisms of death of K5621/59 cells under the influence of ceramide, the following parameters were studied: activation of caspases 9 and 3, cleavage of poly-ADP-ribose polymerase (PAKR), internucleosomal DNA degradation, intramembrane translocation of phosphatidylserine (by annexin V binding), transmembrane electrical mitochondrial potential and integrity of the plasma membrane (by incorporation of PI into cells). In Fig. 17 shows that by 24 hours. exposure to ceramide, the percentage of dying cells reached 37+4%. Particularly important was the appearance of “double positive” (annexin-PI4) cells, meaning that in K562LB9 cells, ceramide quite early causes disruption of the permeability of the plasma membrane (necrosis or late apoptosis). At the same time, activation of caspase 3, characteristic of classical apoptosis, was not detected; PAS proteolysis turned out to be a late event (48 hours of exposure).
The effect of ceramide on K5621/B9 cells was practically not accompanied by a change in the transmembrane potential of mitochondria: this indicator in dying cells did not differ from the corresponding value for untreated cells. Internucleosomal DNA fragmentation was also not a leading sign of death: only about 11% of events occurred in the sub-01 region (hypodiploid cells), while the percentage of dying cells (sum of annexin+/PI, annexin7PI+ and double positive) at the same duration exposure was 64 + 4%. Thus, the most important sign of death of K562LB9 cells under the influence of ceramide is an early violation of the integrity of the plasma membrane - necrosis.
time (hours) with ceramide
■ K562,Annexin+ □ K562,Annexin+PI+ BK562i/S9,nH+
IK562.PI+ V K562i/S9,AHHeKCHH+ ■ K562|789,Annexin+PI+
Rice. 17. Indicators of death of K562 and K562L/S9 cells under the influence of ceramide. Cells were treated with 25 μM C2-ceramide for the indicated periods of time. The percentage of annexation of V-positive, propidium iodide (PI+)-positive and “double positive” (annexin + PI+) cells was determined by flow cytometry.
In K562 cells, p53 does not function, and the chimeric protein kinase Br/Ab1 is expressed, i.e. These cells contain molecular determinants of resistance to apoptosis. Therefore, the K562i/S9 subline can be considered as a model of pleiotropic resistance, where Pgp is one of the mechanisms. All the more important is the data on the ability of ceramide to cause death! of such cells by the mechanism of necrosis.
Metabolites that can cause necrosis may be free oxygen radicals. To establish their role in ceramide-induced death of K562/iS9 cells, the cytotoxicity of ceramide in the presence of N acetylcysteine (NAC), an oxygen radical chelator, was studied and the rate of formation of oxygen radicals in cells treated with ceramide was studied. b Fig. 18 shows that NAC abolished the cytotoxicity of ceramide. Thus, free oxygen plays a decisive role in the death of resistant cells when exposed to ceramide. Figure 19 shows the dependence
Luorescence of cells loaded with OCPOA depending on the time of action of ceramide and
"control agent - hydrogen peroxide, O2 donor." H2O2 caused rapid
same after 20 minutes. - increase in cell fluorescence. Ceramide caused
opposite effect: after 15 minutes. the impact was observed to be sharp - by 1.5
Row - reduction of glow. Only by 24 hours. processing intensity
cell fluorescence returned to the level of this indicator in
treated cells and increased by 48-72 hours. impact when the amount
There were already a lot of growing cells (Fig. 17). Therefore, in the processed
ramide cells initially undergoes reduction, and oxidation
occurs later, probably as a result of depletion of resources for the restoration of utricellular substrates.
Fig. 18. NAC blocks K562i/S9 cell death. Cells were treated with Cr-ceramide without or with 5 mM NAC. NAC alone had no effect on cell viability.
Rice. 19. Oxidation in K562|/89 cells under the influence of ceramide. Cells were treated with 25 μM C2-ceramide for the indicated time intervals. Intracellular oxidation was studied by changes in the fluorescence of cells loaded with E>SGOA. H2Og was used as an “oxygen explosion” control. The results of 3 experiments are summarized.
The discussion of the results
Analysis of the mechanisms of activation of the MOI1 gene allows us to state the following: 1) The AUM gene and the MDR phenotype can be induced by short-term exposure of cells to many substances. Overexpression of MBJ\ and accumulation of Pgp are detected in cells that survive after termination! xenobiotic effects. Thus, epigenetic activation of the MYR\ gene provides a stable MDR phenotype.
2) The urgent formation of MDR is regulated by general cell signaling mechanisms. Indeed, activation of phospholipases, mobilization of intracellular Ca2+, induction of NaκB and cascades of stress-activated protein kinases are mechanisms that ensure the cell response to stress. In addition, transcription factors and functionally active sites in
The ipoMOTope of the MDR1 gene, important for the activation of MDR, is not unique to this gene: ■JF-Y, CCAAT box and chromatin-modulating signals are important for the regulation of transcription of most eukaryotic genes. This, apparently, explains the high inducibility of the MDRX gene: this gene is activated by foot stimuli in cells of various histogenesis. In turn, the regulation of MDRX by general stress-implementing mechanisms emphasizes the iviological importance of MDR activation along with other responses of the cell to exogenous influences.
3) The multiplicity and interchangeability of the activation mechanisms of MDR shs.20) are determined by the multi-level nature of the regulation of this phenotype. And each of these levels gives the cell the opportunity to “choose” whether or not to activate MDR. First, the choice is dictated by the nature of the stimulus (different ents act through different mechanisms). Secondly, the specific mechanism depends on the presence of certain signaling molecules and their interaction in networks of this type. Thirdly, the choice occurs at the level of the actual anscription mechanisms (the state of chromatin in the DRI promoter region). From these considerations, the reasons for the high inducibility on MDRX (the wide interchangeability and overlap of signaling and transcriptional mechanisms ensures induction by many stimuli and in different types of cells), and cases of lack of induction are not induced by every stimulus and not in every experimental system).
The scheme for urgent activation of the MDRX gene (using the example of cytosar and TFA) is shown in Fig. 20.
The concept of MDRX induction as a delayed event, involving first the activation of proteins necessary for the induction of toro MDRX, further complicates the picture of epigenetic activation of MDR.<ой каскадный механизм может быть главным или дополнительным,
Regulation of MY1 by chemotherapy and TPA
neomycin, 1173122
activation of transcription, stabilization of mRNA
Rice. 20. Mechanisms of urgent activation of the MY 1 gene.
intensifying or maintaining the rate of transcription. The possibility of a cell choosing different ways of inducing MDR - direct (without activation of intermediate genes) and/or mediated by the induction of other genes - makes it even more difficult to prevent the development of MDR.
Activation of transcription of the gene L)/? 1 is not the only mechanism for the development of MDR. Another important component of epigenetic regulation may be the stabilization of MOK 1 nRNA. It is possible that, in relation to the balance between transcription induction and stabilization of MOK 1 mRNA, the mechanisms of MDR activation under the influence of various inducers and in different cell types are also different. This assumption is consistent with the concept of multi-level regulation of the expression of the MyACh gene (Fig. 19).
4) Targeted prevention of MDR is possible by establishing the mechanisms of transmission of M)L1-activating signals. The need to prevent the development of MDR in the clinic is confirmed by the connection between the development of MDR and the cytotoxicity of drugs. Indeed, the level of mRNA/εγ1 in cells treated with toxins increases with increasing concentrations of the latter. Consequently, the use of high-dose chemotherapy regimens (justified to increase the effectiveness of treatment) can lead to the development of MDR in surviving cells. The concentrations of drugs required for complete resorption of the tumor may be higher than acceptable for the patient, which determines the limit for dose escalation in chemotherapy. The intracellular signaling antagonists identified above may serve as prototypes for future drug MDR blockers used in combination with traditional anticancer drugs. Thus, preventing the urgent development of MDR expands the possibilities of overcoming this clinically unfavorable phenomenon.
What are the approaches to overcoming established MDR? One of the “vulnerable” targets in resistant cells may be the plasma membrane. Violation of its integrity causes the death of resistant cells: Pgp does not prevent death from influences that induce necrosis. A therapeutically important cellular structure has been identified that should be targeted. Obviously, simply membranolytic agents are unsuitable due to uncontrolled toxicity to tissues, including non-tumor ones. It is necessary to find agents that overcome the Pgp barrier and cause damage to the plasma membrane, i.e. create an intracellular concentration of the drug sufficient to activate necrosis. A substance that satisfies this condition may be ceramide, a sphingolipid that accumulates in the cell in response to many stimuli, including antitumor drugs. Ceramide is not transported by Pgp, which ensures that the concentration of this metabolite in resistant cells is sufficient to induce death.
The toxicity of ceramide to resistant cells means that signaling pathways distal to ceramide accumulation in these cells are not blocked by Pgp or other mechanisms associated with MDR selection. Therefore, overcoming resistance must involve finding ways to accumulate ceramide (and possibly other toxic metabolites) in resistant cells. Cytotoxicity of ceramide requires caveolae or their analogues - submembrane formations rich in phospholipids and proteins and accumulate during selection for MDR (La\ et al., 1998). Importantly, selection for MDR may be accompanied by an increase in cell molecular targets for anticancer therapy.
Free forms of oxygen appear to be an essential mechanism of ceramide-induced death. This raises the question of the importance of mitochondria in the survival of MDR cells. Mitochondrial pathway
transmits additional signals to activate effector caspases. ■The litochondrial “loop” is a powerful factor in the potentiation of such signals; “the fall in the electrical potential of mitochondria and especially the release of cytochrome C into the plasma ensure the irreversibility of the death process. Since it is not possible to predict whether selection for resistance will be associated with inactivation of one or another death pathway, it is important that only a “reliable” cascade remains functional in MDR cells.
Our results indicate sensitivity to oxygen adicals in cell lines with Pgp-mediated MDR. It can be assumed that, although primary resistance and selection for drug resistance are determined by many mechanisms, it is still possible to overcome multifactorial resistance by increasing intracellular oxidation. Free oxygen immediately enters into numerous oxidation reactions, leading to damage to structures important for the life of the cell - DNA and membranes. In the above experiments, necrosis turned out to be the leading method of whitening when exposed to oxygen radicals. It does not mean that necrosis is the only way of death induced by free forms of oxygen. In a specific situation, the predominance of this or another type of death should be established; The standard distinction between apoptosis and necrosis does not characterize the variety of mechanisms of death. This evaluation is useful for determining whether the damage to the membrane is primary or whether this process is delayed. However, it seems important to assess the permeability of the plasma membrane in any situation: violation of the integrity of the membrane causes irreversibility. Therefore, the necrotic component of death is important for the generation of MDR cells, since Pgp can protect cells from agents that induce apoptosis, but not from substances that induce apoptosis. causing necrosis.
The last circumstance is not surprising. For the functioning of this protein of the plasma membrane, it requires a certain physico-chemical state due to its integrity. With necrosis, severe damage to the cell will occur, mainly due to disruption of the transport of water and ions. These damages will affect almost any cellular structures, in contrast to apoptotic cascades, which are expressed in the sequential cleavage of substrates - an energy-dependent process that involves the formation of multicomponent protein-lipid complexes in the cell and the strict specificity of the interaction of caspases with substrates. Blocking one or more links of such a cascade (for example, as a result of impaired lipid transport in cells with MNA MS, “correctly” localized signaling complexes are not formed) will interrupt the transmission of signals to underlying mechanisms, resulting in the cell escaping death and, ultimately, the formation of resistance Also for this reason, to combat MDR, it is desirable that the death mechanisms be multiple and include activation of not only caspases, but: other families of proteases (lysosomal, proteasomal, nuclear), as well as signal potentiation (mitochondrial pathway) and disruption of the permeability of the plasma membrane . The more death mechanisms can be activated in resistant cells, the more reliable the final result is overcoming MDR.
1. The MYR\ gene and the P-glycoprotein-mediated MDR phenotype can be induced by a single short-term exposure to MN cells< веществ, в том числе противоопухолевых препаратов. МЛУ закрепляется в
tions of cells that survived after exposure. Epigenetic activation on MOT produces a stable MDR phenotype.
2) The urgent formation of MDR is ensured by activation of the expression of the ER1 gene: induction of its transcription and stabilization of mRNA.
3) The activation of MDR involves the general mechasomes of the cell's response to stress: the sphatidylinositol pathway, protein kinase C, mobilization of intracellular 2+, activation of NaκB, mitogen-activated protein kinases and regulation of the physical state of chromatin. Inhibition of these mechanisms prevents the development of MDR in cells that survive treatment with these tumor drugs.
4) Overcoming the PgP barrier and inducing death mechanisms that function in cells with MDR are necessary and sufficient conditions for the destruction of such cells.
5) Pgp individually and the MDR phenotype as a whole do not ensure the survival of pgp under influences that cause a primary violation of the integrity of the omatic membrane. Thus, the plasma membrane is an apeutic target, and the induction of necrosis is one of the strategies to overcome arterial resistance.
6) The death of cells with MDR, caused by a violation of the integrity of the zymatic membrane (perforation-mediated lysis), is caused by otoxic T-lymphocytes generated in response to immunization with chole cells modified to express cytokines.
7) Intracellular metabolites that can damage the zymatic membrane and cause the death of cells with MDR are free oxygen molecules. Their generation under the influence of antitumor garates is an effective mechanism for overcoming MDR.
Main publications on the topic of the dissertation:
1. Shtil A., Shushanov A., Stavrovskaya A., Moynova E. Frequency of metastasis in Syrian hamster tumor cells selected for low levels of “typical” multidrug resistance. Experimental and Toxicological Pathology, 1994, 46, 257-262.
2. Erokhina M., Shtil A., Shushanov S., Sidorova T., Stavrovskaya A. Partial restoration of the actin cytoskeleton in transformed Syrian hamster fibroblasts selected for low levels of "typical" multidrug resistanceJ/FEBS Letters, 1994,341, 295-298.
3. Stromskaya T., Filippova N., Rybalkina E., Egudina S., Shtil A., Elisseenkova A., Stavrovskaya "A. Alterations in melanin synthesis in human melanoma cells select in vitro for multidrug lesislaaccJ/Experimental and Toxicological Pathology, 199 47, 157-166.
4. Yu R., Shtil A., Tan T.-H., Roninson I., Kong T. Adriamycin activates c-Jun N-terminal kinase in human leukemia cells: a relevance to apoptosis JlCancer Letter: 1996,107, 73 -81.
5. Komarov P., Shtil A., Buckingham L., Balasubramanian M., Piraner O., Emanuel M, Roninson I., Coon J. Inhibition of cytarabine-induced MDR1 (P-glycoprotein gene activation in human tumor cells by fatty acid-PEG-fatty acid diesters, novel inhibitors of P-glycoprotein function. I I International Journal of Cancer, 1996,68 245-250.
6. Walter R., Shtil A., Roninson I., Holian O. 60 Hz electric fields inhibit protein kinase C activity and multidrug resistance gene (MDR1) up-regulation JIRadiatio Research, 1997,147, 369-375.
7. Walter R., Shtil A., Roninson I., Reyes H., Holian 0. Effects of 60 Hz electric fon protein kinase С activity and multidrug resistance gene (MDR1) expression. // Current Surgery, 1997, 54,366- 370.
Stromskaya T., Rybalkina E., Filippova N., Shtil A., Stavrovskaya A. Regulation of MDR1 gene expression and P-glycoprotein function in cultured melanoma and hepatoma cells .//British Journal of Cancer, 1998, 77, 1716-1725 .
Komarov P., Shtil A., Holian 0., Tee L., Buckingham L., Mechetner E., Roninson I., Coon J. Activation of the LRP (lung resistance-related protein) gene by short-term exposure of human leukemia cells to phorbol ester and cytarabine.f/Oncology Research, 1998,10, 185-192.
I. Shtil A, Mandlekar S, Yu R., Walter R., Hagen K., Roninson I., Tan T.-H., Kong T. Differential regulation of mitogen-activated protein kinases by microtubule-binding agents in human breast cancer cells J/Oncogene, 1999, 18, 377-384.
Damiano J., Cress A., Hazlehurst L., Shtil A., and Dalton W. Cell adhesion mediated drug resistance (CAM-DR): role of integrins and resistance to apoptosis in human myeloma cell linesMBlood, 1999,93, 1658- 1667.
Shtil A., Turner J., Durfee J., Dalton W., and Yu H. Cytokine-based tumor cell vaccine is equally effective against parental and isogenic multidrug-resistant myeloma cells: the role of cytotoxic T-lymphocytes. I/Blood, 1999,93, 1831-1837.
Shtil A., Ktitorova O., Kakpakova E., and Holian O. Differential effects of the MDR1 (multidrug resistance) gene-activating agents on protein kinase C: evidence for redundancy of mechanisms of acquired MDR in leukemia cells .//Leukemia and Lymphoma, 2000, 40, 191-195.
Shtil A., Grinchuk T., Tee L., Mechetner E., Ignatova T. Overexpression of the MDR1 gene is associated with a decreased mitochondrial transmembrane potential in K562 human leukemia cells selected for P-glycoprotein-mediated multidrug resistance.//International Journal of Oncology, 2000,17,387-392. Shtil A., Turner J., Dalton W., Yu H. Alternative pathways of cell death to: circumvent pleiotropic resistance in myeloma cells: role of cytotoxic T-lymphocytes. // Leukemia and Lymphoma, 2000, 38, 59-70.
16. Shtil A.A. Signal transduction pathways and transcriptional mechanisms as targets for prevention of the emergence of multidrug resistance in human cancer cells.HCurre, Drug Targets, 2001,2, 57-77.
17. Shtil A. A. Epigenetic activation of multidrug resistance of tumor cells: signal transmission, transcriptional activation and possibilities of prevention. // "Advances of modern biology", 2001, 121, 563-575.
18. Sidorova T.A., Nigmatov A., Kakpakova E.S., Stavrovskaya A.A., Gerassimova G.K., Shtil A.A., and Serebtyakov E.P. Effects of isoprenoid analogues oiSDB-ethylenediamine on multidrug resistant tumor cells alone and in combination with chemotherapeutic dmgs.l/Journal of Medicinal Chemistry, 2002,21,5330-5339.
19. Shtil A. A. Emergence of multidrug resistance in leukemia cells during chemotherapy: mechanisms and prevention.1 ¡Journal of Hematotherapy and Stei Cell Research, 2002, 11, 231-241.
20. Shtil A. A. Multifactorial drug resistance: P-glycoprotein on the apex of the Vyraxmi.llJournal of Hematotherapy and Stem Cell Research, 2002,11,437-43
21. Shtil A. A. P-glycoprotein as a therapeutic target: good news.l/Leukemia, 2002, 2169-2170.
22. Shtil A.A. Development of multidrug resistance as an urgent cell response to exogenous influences. // "Biological membranes (2003,20,236-243.
23. Lehne G., Shtil A. A. Targeting P-glycoprotein for execution of cellular death mechanisms in cancer., H "Targeted Cancer Therapies, An Odyssey", 2003, 203-i
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Shtil, Alexander Albertovich. Drug resistance of tumor cells mediated by P-glycoprotein: mechanisms of urgent formation and approaches to overcoming: abstract of thesis. ... Doctor of Medical Sciences: 14.00.14.- Moscow, 2003.- 46 p.: ill.
Introduction to the work
Relevance of the topic
I. Multidrug resistance of tumor cells:
biological mechanisms and significance in oncology.
Despite significant advances in pharmacology, including the development of technology for creating drugs with expected properties, the success of chemotherapy for tumors is limited by the most important feature of living systems - the ability to respond to changes in the external environment. One of the manifestations of such dasticity is the development of resistance of tumor cells to drugs [used in chemotherapy. The wide distribution and long-term, persistent nature of cell adaptation to external influences suggest that overcoming drug resistance may be associated not only with the search for more effective drugs: there is probably no drug to which cells would not be able to develop resistance. Only elucidation of the nological mechanisms of resistance to various types of stress will serve as a basis for the development of strategies to overcome drug resistance, a necessary condition for increasing the effectiveness of treatment of cancer patients.
Multidrug resistance (MDR) of neoplasms - the preservation of viability by tumor cells in response to the effects of various drugs - is one of the main reasons for progression! Diseases: the tumor is insensitive to chemotherapy, regardless of the combination of drugs used. The MDR phenomenon has a long-term and stable character: resistance mechanisms are inherited over generations of cells. Thus, MDR is one of the key factors in tumor progression.
There are two main types of cell resistance to toxins. Primary g.e. Resistance observed before exposure to chemotherapy drugs is due to the expression of defense mechanisms during tumor progression. Yes, activation
anti-apoptotic mechanisms conferring resistance to immune effectors may be related to drug resistance. Secondary (acquired) resistance occurs in cells exposed to stress. Before these effects, protective mechanisms in such cells are poorly expressed or absent; surviving after treatment with one toxin, cells acquire resistance to many substances - MDR (Riordan, Ling, 1985). Further selection consolidates the acquired phenotype over generations of cells.
The most important mechanism of MDR is the reduced accumulation of toxins in the cell, due to the excretion of substances into the intercellular environment. Such transport is carried out by the integral protein of the plasma membrane P-glycoprotein (Pgp) due to the energy of ATP hydrolysis (Juliano, Ling, 1984)."! human Pgp is encoded by the gene MDRI(multidrug resistance 1), localized on chromosome 7 (Chen et al., 1986). Numerous data indicate that an increase in mRNA MDRI and Pgp often serves as a factor in the resistance of multiple tumor types to treatment (Linn et al., 1995; Stavrovskaya et al., 1998).
II. Development of MDR in tumor cells: biological mechanisms as targets For prevention
It is reasonable to assume that an increase in the amount of mRNA MDRI caused by amplification of this gene. This MDR mechanism has been identified in cultured cell lines selected for survival in the presence of toxins (Roninson, 1991). However, when analyzing human tumors, gene amplification MDR1 was not detected either in primary tumors or in neoplasms after treatment. A likely cause of clinical MDR is overexpression MDRI and Pgp with unchanged gene structure (preservation of copy number and nucleotide sequence), i.e. epigenetic activity of the phenotype. In cultures of human tumor cells, an increase in the level of mRNA MDRI and the amount of Pgp noted during a single treatment of chemotherapy
distinct in chemical structure and mechanisms of action (Chaudhary, Aninson, 1993). Evidence of mRNA accumulation has been obtained MDR\ in metastases of cancer to the lung tissue already 20-50 minutes after the start of intraoperative lung infusion with doxorubicin (Abolhoda et al., 1999). These results (confirm the possibility of epigenetic activation of MDR in experiment and in other situations: increased mRNA MDR\ and Pgp in tumors can occur without gene amplification MDRI.
This type of biological regulation - urgent activation of the phenotype - involves the induction of transcription of the gene (genes) encoding the corresponding phenotype, and/or post-transcriptional control (stabilization of NK, regulation of protein synthesis and functioning). In relation to MDR, this type of regulation means the possibility of gene induction MDR\ cellular stimuli and the relatively rapid development of resistance in r/cholesterol cells in response to stress. Gene inducibility MDRI suggests the development of signaling pathways from the cell periphery to the nucleus. Such pathways could include stress-implementing signaling mechanisms: protein kinase C ІКС), phospholipases and intracellular Ca 2+, mitogen-activated urinary kinases, nuclear factor kappa B (NFkB). Signaling to the gulatory region of a gene MDRI and the transcript ensures activation of gene compression.
The study of MDR regulation also has a fundamental practical aspect. Inhibition of these mechanisms using pharmacological and/or genetic influences would prevent the development of MDR in the process [myoteragash.
III. Overcoming the formed MDR of tumor cells.
If blocking an activating gene MDR\ signals can prevent the formation of MDR in primary sensory cells, then such
the approach is not applicable to overcome already formed resistance. The traditional method of combating secondary MDR is the use of Pgp modulators in combination with cytostatics (Lehne, 2000). However, the use of Pgp inhibitors is limited by side effects (heart rhythm disturbances, immunological imbalance). It is equally important that the effectiveness of modulator + hormone-static combinations may be reduced due to blocking of at least some cell death mechanisms during selection for MDR.
Overcoming formed MDR is achievable if two conditions are met: 1) the concentration of the drug must be sufficient to activate the effector mechanisms of cell death, 2) the functions of these mechanisms must be preserved in cells with MDR. The first condition is met if the drug overcomes the Pgp barrier. However, it is required to prove that achieving a critical intracellular concentration of the agent is sufficient to activate the death of a cell that is resistant to many influences. Survival mechanisms operating in resistant cells should serve as targets for the elimination of the latter.
To implement the second condition, approaches aimed at the lysis of resistant cells as a mechanism for inducing their death seem promising. Vaccination of mice with syngeneic myeloma cells transfected with cDNAs of certain cytokines leads to the development of a cytotoxic T-lymphocyte (CTL)-mediated immune response and rejection of the grafted tumor in immunized animals (Dranoff et al., 1993; Levitsky et al., 1996). CTLs lyse cells using granzyme B and perforin. Since granzyme B activates caspase 3, one of the distal effectors of apoptosis, and perforin causes primary damage to the plasma membrane (necrosis), one can hope that SLs will be effective if proximal death mechanisms are blocked; triggering of distal links of apoptosis in combination with necrosis
leads to the death of cells resistant to antitumor drugs - promoters of programmed cell death.
Formulation of the problem
MDR is a clinically unfavorable phenomenon, the overcoming of which requires knowledge of the mechanisms of its development and the ways in which cell death occurs; it is necessary to study both aspects of the problem. First, it is necessary to study the mechanisms of development of MDR in MD/? l/Pgp-negative human cells, the study of these mechanisms will serve to prevent the development of resistance in primarily sensitive cells. Secondly, analysis of the death processes operating in cells with MDR will create the basis for overcoming resistance in situations where secondary MDR has formed.
The purpose of the study is to establish the mechanisms of urgent formation of lymph nodes in human tumor cells and to develop approaches to overcome this resistance.
To optimize models for the development of MDR in human tumor cell cultures in response to the effects of chemotherapy and experimental agonists and antagonists of signaling mechanisms.
Determine the main ones! mechanism of urgent development of MDR when cells are treated with antitumor agents: gene amplification MDRI, selection of Pgp-positive cells or de novo MDR suppression.
Investigate the pathways of intracellular signal transmission that regulate the activation of MDR - protein kinase C, phospholipase C, cellular Ca +, mitogen-activated protein kinases, NFkB).
To identify the role of transgressional activation and posttransgressional regulation (mRNA stability) of gene expression MDRI in the immediate development of MDR in response to exposure to chemotherapy.
To develop ways to prevent the development of MDR in tumor cells when combining chemotherapy drugs with blockers MDR\- activating signaling pathways and gene transcription inhibitors
To study the kinetics of activation of secreting and effector casps, changes in the transmembrane potential of mitochondria, proteolytic cleavage of poly(ADP)ribose polymerase, internucleosomal DNA fragmentation and integrity of the plasma membrane in parental cells and variants with MDR when treated with a drug not transported by P-glycoprotein.
Use vaccination with cytokine-expressing tumor cells to generate an immune response against MDR cells.
Provisions submitted for defense.
The formation of Pgp-mediated MDR - an urgent cell response to many influences - is mediated by epigenetic activation of the gene MDRI. This activation is due to multiple mechanisms of intracellular signaling, gene promoter induction and mRNA stabilization and can be prevented by inhibitors of these signals.
Overcoming Pgp-mediated MDR may be associated with targeting the plasma membrane of resistant cells. Pgp does not protect cells from disruption of the integrity of the plasma membrane - necrosis.
Scientific background For the first time, the idea of the formation of MDR as an urgent response of a cell to an exogenous stimulus has been substantiated;
For the first time, the mechanism of development of a specific drug resistance phenotype - Pgp-mediated MDR - has been studied in detail: epigenetic activation of the gene encoding this protein MDRI.
3. A model of urgent gene activation was developed for the first time MDRI,
accompanied by the acquisition of a stable phenotype of Pgp-mediated MDR in cultured human tumor cells; \. Signal transduction pathways, mechanisms of transcription activation and post-transcriptional gene regulation have been identified MDRX in cells exposed to antitumor drugs. 5. For the first time, classes of pharmacological substances - blockers of intracellular signal transmission - have been characterized to prevent the formation of Pgp-mediated MDR in tumor cells. 5. For the first time, the death mechanisms operating in cells with Pgp-mediated MDR were studied, and an approach to overcoming resistance was developed, which involved primary damage to the integrity of the plasma membrane.
Practical value.
Development of methods to prevent the urgent development of Pgp-mediated MDR in cultured tumor cells when exposed to chemotherapeutic drugs.
Preclinical trials of modified genetically engineered vaccines to overcome MDR.
Approbation of work.
The dissertation was discussed on June 30, 2003 at a joint conference of laboratories of tumor cell genetics, cytogenetics with a group of molecular genetics, viral and cellular oncogenes, molecular endocrinology, anti-tumor immunity, biochemical pharmacology, medical research, experimental diagnostics and biotherapy of tumors; Department of Schmunology, Hematology, Chemotherapy, Clinical Pharmacology, Advanced Treatment Methods of the Russian Cancer Research Center named after. N.N. Blokhin RAMS.
The main materials of the dissertation were presented at the following conferences: 2nd international symposium "Cytostatic Drug Resistance", (U Germany, 1991); Gordon Conference "Advances in Chemotherapy" (New York, London, USA, 1994); "Molecular Toxicology" (Copler Mountain, USA, 1995); "Inducible Genomic Responses" (Stevenson, USA, 1996); "Nucleic Acids - Integrating Molecular Diagnostics and Therapy" (San Diego, USA, 1996); annual conference of the American Association of Cancer Research (1994-2001): 6th And 7th congresses "Advances in Oncology", (Hersonissos, Greece, 2001,2002); "The structure and functions of the cell nucleus" (St. Petersburg, 2002), as well as at seminars at Oncotech, Inc. (Irvine, USA, 1996), Salk Institute (LaJolla, USA, 1997), Lee Moffitt Cancer Center (Tampa, USA, 1997), The Jackson Laboratory (Bar Harbor, USA, 1997), Sloan-Kettering Cancer Center, New Yo] USA, 1999), universities of Copenhagen (2002), Innsbruck (2002) and Groningey (2003), Moscow State University. M.V. Lomonosov (2002), Research Institute of Experimental Pathology, Oncology and Radiobiology named after. R.E. Kavetsky (Kyiv, 2002).
Publications.
Structure and scope of the dissertation.
Studies of programmed cell death - apoptosis (from the Greek. αποπτωσις
- leaf fall) - in the last decade have entailed several important scientific discoveries: a holistic system for the implementation of this phenomenon was discovered - genes regulating it, as well as surface cell receptors and their ligands that mediate cell death were identified.
In numerous experiments, scientists have proven: programmed death is a mandatory and integral property of any cell of any multicellular system. (Every day, about 5% of the body's cells undergo apoptosis, and new ones take their place. It takes a cell from 15 minutes to 2 hours to disappear without a trace).
In 2002, molecular biologist Sidney Brenner ( Sydney Brenner, Robert Horwitz ( H. Robert Horvitz) and John Sulston ( John Edward Sulston) for discoveries in the field of “genetic regulation of organism development and programmed cell death” was awarded the Nobel Prize in Physiology or Medicine.
Experts believe that further research into apoptosis could contribute to the development of drugs against dangerous diseases such as cancer, stroke, heart attack, Alzheimer's disease, Parkinson's disease and some others.
ALEXANDER ALBERTOVICH STIHL– Doctor of Medical Sciences, Head of the Laboratory of Mechanisms of Tumor Cell Death, Research Institute of Carcinogenesis, Russian Cancer Research Center named after. N.N. Blokhin Russian Academy of Medical Sciences.
Specialist in the field of cellular and molecular oncology.
Scientific interests: mechanisms of programmed cell death (bacteria and eukaryotes, in particular tumor cells).
Questions_Elena Vetrova
March, April
Moscow, 2009
Alexander Albertovich, apoptosis is one of the key biological processes that occurs in the body throughout its life. It plays a critical role in the embryonic development of the body (morphogenesis), and in maintaining dynamic balance in organs and tissues (homeostasis), and in the process of the initiation and development of tumors(carcinogenesis).
In what other cases does the cell suicide program turn on in the body? And how does this program “know” what is good for the body and what is bad?
In Fig. apoptosis. The process starts when the cell's functionality is exhausted. Genes that ensure cell division are blocked, and genes that provide the synthesis of lytic (from the Greek. lytikos - rid , dissolve ) enzymes - stimulated. These enzymes enter the nucleus and lyse (destroy) chromatinchromosomes- a complex of DNA, RNA and proteins. Chromosomes disintegrate and synthesis in the cell stops.
Some external signs of apoptosis in a cell:
pyknosis (nucleus shrinkage);
chromatolysis (reduction of nuclear staining);
karyorrhexis (disintegration of the nucleus into parts);
destruction of the cytoplasm, etc.
The remains are phagocytosed (engulfed) by macrophages.
Programmed death is activated during physiological processes - in the simplest organisms, for example, bacteria, to maintain the size of the cell population that is optimal for given environmental conditions (nutrients, temperature). This effect is observed in biofilms, self-regulating bacterial communities.
In the embryonic development of tissues and organs of higher organisms and in the adult organism, programmed death serves to replace cell populations - aging and losing functions - with young ones.
The program does not know what is good and what is bad for the body, it’s just how life works - death is a side of life...
That is, a side effect of evolution is thatwhat is August Weissmann talking about?German zoologist and evolutionist, wrote at the end of the 19th century...
...Therefore, the death of non-tumor cells during chemotherapy is bad: apoptosis of normal cells is undesirable here, but the program does not distribute between “bad and good”, but allows those who can survive to survive, destroying the more vulnerable, more sensitive... Justice in human understanding it doesn’t work here, and you have to pay for this hopelessness and “unprincipledness.”
Scientists learned about the existence of apoptosis not so long ago, about half a century ago. How did this discovery affect the development of science?
In Fig. activation of the genetic program of apoptosis in a cell under the influence of external factors (signals)
(Fig. enlarges when you click on it with the cursor)
This discovery made it possible in a short time - over about 15 years - to form ideas about the most important biological processes - the transmission of intracellular signals, the regulation of proteolysis - the process of breakdown of proteins and peptides in the body, the regulation of the shape of the nucleus, organelles and membranes.
Study of the role of chromatin proteins in gene expression has intensified.
The ideas about the mechanism of action of cytostatic agents, mainly antitumor drugs and ionizing radiation, on tumor and normal cells are substantiated.
The discovery of the mechanisms of cell death today has focused many scientific directions - from biology and chemistry to the therapy of human, animal and plant diseases.
Yes, this direction is considered one of the most promising in molecular biology. It is optimistically associated with the development of fundamentally new drugs for the treatment of still incurable degenerative diseases, cancer, and even the possibility of defeating old age.
According to some reports, more than four hundred laboratories in the world today are somehow involved in this topic.
The promise of the study of apoptosis for molecular biology is due to the fact that this phenomenon (and, more broadly, cell death) covers a combination of many scientific ideas and directions. A cell, whether simple or complex, is always a complex system, and death is an integral property of living things. Therefore, the study of death is generally promising for understanding the essence of life.
And, indeed, in practical terms, studying the mechanisms of death will make it possible to specify the effect of many drugs.
Relatively recently, the phenomenon was discovered bystander effect (« bystander effect"): when cells dying for any reason send a certain signal to the healthy cells adjacent to them, and they self-destruct. What motivates them? Are there any hypotheses about this?
This is an example of biological regulation in a complex system: in this way, cells kill each other by sending signals to their neighbors. The signal (usually a water-soluble protein) is not harmful for its own cells, but is destructive for cells of a different origin located in the same population - the game is based on this difference in sensitivity.
What is the role of oxygen and hydrogen peroxide in the process of apoptosis?
Very important - oxygen radicals are very active chemically. They trigger reactions that damage proteins, nucleic acids and lipids.
Rice. increases when you click on it with the cursor.
The therapeutic effect of ionizing radiation and anthracycline antibiotics is based on this property. Excitation of special chemical compounds - photosensitizers - by light causes an oxygen explosion and severe cell damage. If such a photosensitizer accumulates in a tumor and the latter is illuminated, the death of tumor cells occurs. In oncology, such therapy is called photodynamic and is widely used, however, for tumors that are accessible to light and not very large in size.
The principle of apoptosis is observed both at the cellular and subcellular levels, in tissues and organs. Academician of the Russian Academy of Sciences Vladimir Skulachev proposed calling this phenomenon phenoptosis. Why in nature do some plants and animals (bamboo after flowering, salmon after spawning) turn on a self-destruction program in the prime of their lives? This is unlikely to be due to irreversible gene mutations.
Probably not related, but this is the mechanism of nature - the language in which they say that the faded bamboo will die... The mechanism is epigenetic - there are no mutations, but there are rapid changes in the physico-chemical properties of chromatin, and an important gene (genes) from working becomes silent . Such a language does not require any destruction - it is enough to attach or detach a chemical group - methyl, phosphate, acetyl - from one protein to another.
Enough hint...
There is a hypothesis that cancer is also a programmed death of a genetically unstable organism containing irreparable damage (and therefore dangerous for evolution). Increasingly, oncologists can be heard saying that the appearance of a completely transformed cell is only a matter of time. If so, then cancer is not a disease, like old age, but a certain result of the development of a living system. But scientists assume, and futurologists predict, a victory over cancer in the future. And indeed, in the end, man has long ceased to expect mercy from evolution. Is it possible to defeat cancer?
Perhaps it cannot be defeated - cancer is one of the mechanisms for removing a non-viable individual, along with other typical pathological processes - inflammation, allergies. Organic life is built on the turnover of organisms - death is inevitable, and mechanisms for its implementation are necessary. Cancer is one such mechanism. The task of scientists is to delay death, not avoid it.
If there is a strong connection between aging and the development of cancer, and apparently no one doubts this, perhaps it is necessary to expand the front of the fight against it to the scale of the fight against aging?
The fight against aging is necessary both as part of the fight against tumors and in other aspects - this is beyond doubt.
The body's anti-cancer defense is based on apoptosis. Why does apoptosis stop working after a series of mutations? Why, at some point, does the body stop resisting and begin to help the tumor (for example, the immune system)?
Because apoptosis, like any biological process, is disrupted by mutations, and also because apoptosis can be easily disrupted without mutations.
What is the role of the group of cytochrome P450 enzymes in the process of carcinogenesis?
3.Sir John Sulston- British biologist, member of the Royal Society of Great Britain. His laboratory compiled a complete description of the order of division of embryonic cells of the nematode Caenorhabditis elegans and traced the fate of absolutely all of its 959 cells during development.
Without a doubt. The existence of such proteins confirms the essential significance of apoptosis in biology - the mechanisms of this phenomenon are not random, they are encoded in the DNA of the cell and come into action when there is an appropriate signal. Without them there is no death.
Recently, American researchers from the Albert Einstein Institute of Medicine discovered that a small fragment of the intracellular protein p115 activates apoptosis.
Before apoptosis, p115 breaks down into two fragments, the smaller of which consists of 205 amino acids. According to scientists, it plays an important role in the process of cell death, since its expression leads to the release of cytochrome C from mitochondria into the cytoplasm of cells, which leads to their death. Scientists hope that this discovery could lead to the development of new drugs to combat cancer and other pathologies characterized by excessive cell proliferation.
In your opinion, which of the latest discoveries in the field of apoptosis are key for medicine and biology?
In Fig. expression of a small fragment of the p115 protein (shown in green) leads to the release of cytochrome C (red) from mitochondria into the cytoplasm of cells, which leads to cell death.
http://www.cbio.ru/article.php?storyid=3319
New approaches to the treatment of proliferative diseases are based on a similar phenomenon - to create conditions under which the cell’s own proteins would work as killers. Thus, proteolytic cleavage of the protein poly(ADP-ribose) polymerase, which occurs during the induction of apoptosis, forms its fragment that binds to DNA and prevents the repair of its damage. The normal protein poly(ADP-ribose) polymerase, necessary for healing DNA damage, becomes destructive to the cell.
The latest discoveries that are now receiving important practical applications for the regulation of cell death include evidence of the possibility of suppressing a specific gene in a cell and the whole organism through the introduction of so-called short hairpin antisense RNA.
Silent gene technology ( silencing genes), when the binding of short double-stranded RNA molecules to the regulatory sequence of the target gene makes it possible to stop the synthesis process in this gene.
Promising vector (viral) gene therapy.
These tools, the product of years of collaboration between chemists and biologists, make it possible to achieve long-term gene shutdown. If the products of such genes are important for cell viability, we induce death with minimal side effects. The latter is especially important in an oncology clinic - often chemoradiotherapy is tolerated by patients more severely than the disease itself...Standard medications are still poorly understood which cells to destroy and in what ways. Gene therapy may be more effective, and the side effects of treatment may be reduced.
Undoubtedly, fruitful, although encountering a number of difficulties, is the concept of target-directed - targeted - (from English in target - goal, target) tumor therapy.
In Fig.
d effect of the drug Herceptin - (manufactured by Roshe / Switzerland). Created on the basis of antibodies that can block HER2 receptor proteins.
Inhibits tumor development.
Stabilizes the patient's condition.
Shortens the period of chemotherapy.
(Fig. enlarges when you click on it with the cursor)
Individual drugs created to inactivate a specific target (protein) in a tumor cell are highly effective and well tolerated over long courses of treatment (examples - Gleevec, Iressa).
Your laboratory is engaged in research into strategies for targeting the pathways of initiation and implementation of tumor cell death. How far have you come in solving these problems?
Specialists from our laboratory have established the mechanisms of tumor cell death under the influence of new drugs created in Russia. In particular, a mechanism has been identified in which it is possible to bypass the drug resistance of tumor cells: new chemical compounds overcome the barrier that otherwise prevents drugs from entering the cell.
In addition to researching the role of chemical compounds in the reprogramming of cells into cancer cells and the death of cancer cells, you are engaged in elucidating the molecular mechanisms of cell death. What remains unclear about these mechanisms? What questions do scientists still have to answer?
It is important to figure out how to avoid inducing apoptosis in non-tumor cells. Killing a cell is not a problem, the problem is how to protect a healthy one...
Is your laboratory working on studying the mechanisms of the killer function of cancer cells? Is it possible, in principle, to block the body-destructive activity of a cancer cell and its effect on normal tissues?
Possible and necessary. Means have appeared to inactivate tumor necrosis factor, a toxin secreted by a cancer cell and causing the destruction of normal cells, a kind of bystander effect. These products are undergoing initial clinical trials.
You worked in the USA for eight years. What distinguishes Russian and Western approaches to fighting cancer? It is known that in the United States significant funds are allocated to combat it and to prevent cancer. And the most important result of these measures is that mortality from cancer is gradually decreasing.
In Russia, the situation with cancer incidence remains difficult. What measures can reverse the unfavorable situation?
The Western scientific style is characterized by a desire for an in-depth study of mechanisms, and not just phenomena, completeness of representation, thanks to broad interdisciplinary connections, and high competition for the creation of important scientific information.
Cancer mortality continues to be a global problem, but for some locations the situation has improved. For example, there have been fewer cases of stomach cancer - dietary conditions have changed, long-term storage of food has become safe with the development of the refrigeration industry, anti-tobacco propaganda has been organized - these public measures are indeed bringing positive results.
On innovation in healthcare(primarily in the field of cancer treatment) planned $10 billion– twice as much as provided for in the current financial year.
And the situation in Russia can be improved by uniting the joint efforts of many social strata and, of course, with funding adequate to such complex and important tasks.
