[Radiation preconditioning of mouse retina results in tolerance to MNU-induced degeneration and stimulates retinal recovery].

Emerging body of data indicate protecting effect of low level of stress (preconditioning) on retina. Our previous studies have revealed a non-linear dose-response relationship for cytotoxic effect of both ionizing radiation and N-methyl-N-nitrosourea (MNU) on mouse retina. Moreover, non-cytotoxic dose of MNU increased tolerance of retina to following challenge dose of MNU. This result displays protection of retina through mechanism of recovery. In the present study we used the mouse model for MNU-induced retinal degeneration to evaluate the adaptive response of the retina to proton irradiation and implication of glial Muller cells in this response. In this paper, we have shown that the recovery of the retina after exposure to genotoxic agents is associated with an increased efficiency of DNA damage repair and lowered death of retinal photoreceptors.

[Damage and functional recovery of the mouse retina after exposure to ionizing radiation and methylnitrosourea].

The eye retina consists of terminally differentiated cells that have lost their ability to proliferate. The death of these cells leads tothe loss of sight. The mice retina is characterized by relatively high resistance to radiation, which is provided by its ability to repair damage caused by environmental factors. The aim of our work was to assess the damaging effect of ionizing radiation and methylnitrosourea (MNU) on the DNA structure in the mouse retina, the functional activity of the retina, and its ability to recover in vivo. The results confirm the ability of the mature retina to structural and functional recovery. Adapting influence of low dose chemical agent increases retina resistance to cytotoxic dose of genotoxicants and prevents degeneration of photoreceptor layer of the retina. The results show the possibility of neurohormesis effect in the mice retina after exposure to ionizing radiation and chemicals.

[Mechanisms of radioresistance in terminally differentiated cells of mature retina].

Retinopathy of animals is induced by many agents damaging DNA. This fact shows that DNA lesions may initiate retinal degeneration. The aim of our work was to study the effects of gamma and proton irradiation, and methylnitrosourea (MNU) on mice retina. We evaluated morphological changes, DNA damage and repair in retina, and expression of 5 proteins participating in apoptosis: p53, ATM, FasR, PARP and caspase 3 active. Dose of 14 Gy is equitoxic in terms of induction of DNA single strand breaks by both gamma and proton irradiation. But protons were 2 fold more effective than gamma-rays in induction of DNA double strand breaks. All breaks were repaired within < or =10 h. Irradiation resulted in increased expression of p53 and ATM. But no sings of cell death and retinal degeneration were observed during 7 days after irradiation. Proton irradiation in dose of 25 Gy resulted in increasing over time destructive changes localized mainly in photoreceptor layer of retina. These changes were followed by increased expression of proapoptotic proteins. A single systemic administration of MNU (70 mg/kg) increased intracellular levels of p53, PARP, FasR, caspase 3 active, which was followed by destructive changes in retina with sings of apoptosis of photoreceptors. As in the case of irradiation, the 2-fold dose reduction of MNU abrogated cytotoxic effect of MNU on retina. High level of spontaneous DNA damage such as apurine and apyrimidine sites were observed in mouse retina. The results of our study demonstrate the occurrence of genotoxic threshold in the initiation of retinal cell death in vivo. Topoisomerase 2 of retina is suggested to translate primary DNA damage to cytotoxic effect.

[Cellular markers based on DNA damage and repair (BER, MMR), expression of MLHI, MSH2, FasR, and cell death of lymphocytes as predictive parameters for clinical response to chemotherapy of melanoma].

Melanoma is a highly aggressive neoplastic disease attributed to transformed melanocytes. The efficacy of regimens of cytotoxic chemotherapy for advanced stage patients does not exceed 20%. Search for lymphocyte markers of patients' sensitivity to chemotherapy provides a rational basis for development of cytotoxic chemotherapy. Using blood lymphocytes we evaluated efficacy of BER and MMR, expression of MLH1, MSH2 and FasR, and cell death in melanoma patients relative to clinical response to chemotherapy. We found that LDCI-chemotherapy (lomustine, dacarbazine, cisplatin and interferon gamma), induced AP sites and DNA ss-breaks which repaired trough BER pathway. However, neither initial DNA damage nor the rate of their repair correlated with clinical response. This result prompts us to think that this type of damage is not crucial in cytotoxic effect of LDCI-regimen of chemotherapy. DNA ds-breakes appeared downstream ss-breakes were attributed to repair of 06-methylguanine by MMR mechanism in PHA-stimulated lymphocytes. The number of ds-breakes appeared by 48 correlated with positive clinical response of patients to chemotherapy. The same link was observed between clinical response and the number of dead lymphocytes. However, there was no correlation between clinical response and expression of MLHI + MSH2 and FasR. These results imply possible contribution of crosslink repair through NER pathway to formation of DNA ds-breaks as well as to cytotoxicity of LDCl-therapy. The observed link between high level of secondary ds-breaks and positive response to chemotherapy indicates the potential of these instruments to serve as prognostic end point in clinical trials.

[Effectiveness of DNA repair and expression of MLH1, MSH2 and FASR in lymphocytes of patients with chemotherapy-responsive, disseminated cutaneous melanoma].

Patients with advanced malignant melanoma have poor prognosis as conventional chemotherapy induces complete response in a very small fraction (not more than 20%). One of research strategies aimed at raising its efficiency is the search for markers predicting individual response to chemotherapy. Our study was concerned with evaluation of the potential of DNA damage, repair (BER, MMR), expression of proteins MLH1, MSH2 and FasR as prognosticators of chemotherapy. These parameters were assessed in lymphocytes sampled from the blood of patients with metastatic cutaneous melanoma before and after one cycle of chemotherapy with lomustine, dacarbazine, cisplatin and interferon-gamma (LDCI). Clinical response was evaluated after a full course of therapy. We established that the major DNA damage induced by chemotherapy occurred on the levels of AP sites and single strand (SS) breaks. Despite the individual variations in BER efficacy, complete repair of SS breaks was reported in lymphocytes of all patients 30 days after the first cycle of chemotherapy. As a consequence, this type of damage and relevant BER efficacy did not correlate with clinical response. Conversely, the number of DNA double strand breaks detected in lymphocytes after the first cycle of chemotherapy was in good correlation with positive clinical response (p < 0.001). This parameter does not fully represent MMR function and, if coupled with cytotoxic effect of chemotherapy on lymphocytes, may be used as a predictive marker for clinical response to LDCI chemotherapy regimens for melanoma.

[Genetic markers of melanoma].

Melanoma is among the most aggressive malignancies. Tumors with a thickness of 4 mm can produce metastases, and the mean survival of the patients is 9 months. The review presents modern classification of the melanoma types based on cytological and morphological indices (Clark model). Alterations of genes in melanomas are discussed in detail. These genes include tumor suppressors, proliferative response genes (oncogenes), and transcription factors. Alterations in the Wnt signaling, MAPK cascade, and Fas signaling pathways are considered. Changes in the mismatch repair (MMR) genes are also analyzed. From practical perspective, understanding the genetic alterations provides identification of potential targets for therapeutic exposure and enables prognosis of the tumor response to chemotherapy.

[The regularities of the induction of DNA double strand breaks and DNA repair in human lymphocytes after irradiation by accelerated heavy ions with different energy].

The regularities of the induction of DNA double strand breaks (DSB) in human lymphocytes after irradiation by different doses of accelerated lithium and carbon ions (33 and 480 MeV/nucleon, LET = 20 and 10.6 keV/microm, respectively) and gamma-rays 60Co by using of comet assay were investigated. It was shown that the dependence of DSB formation increases linearly with growing of the dose of lithium and carbon ions and gamma-rays. The biological effectiveness of carbon ions with high energy was similar with gamma-rays, lithium ions possess greater biological effectiveness in comparison with gamma-rays and value of RBE of lithium ions amount 1.6 +/- 0.1. The kinetic of DNA repair from DSB in human lymphocytes after irradiation by lithium and carbon ions and gamma-rays was studied. It is revealed that the reparation proceeds effectively with heavy ion and gamma-ray irradiation by exponential kinetics.

[Retina radioresistance: whole-body exposure of mice to gamma-irradiation induces the retina DNA damage, accumulation of p53 protein followed by DNA repair rather than the apoptosis].

Whole-body irradiation of mice with gamma-rays at 14 Gy causes DNA single and double strand breaks effectively repaired later. p53 is accumulated during the repair period. There is still some amount of DNA breaks 48-72 hours after the irradiation. Despite p53 accumulation and residual DNA lesions in the cells, mice retina demonstrated no morphological destructive changes or apoptosis signs. Retina resistance to apoptotic signals could derive from efficient repair of radiation-induced lesions in transcriptionally active regions of the genome of differentiated cells.

[Methylnitrosourea as challenge mutagen in assessment of the DNA mismatch repair (MMR) activity: association with some types of cancer].

Total repair capability is a widely used phenotypic marker of predisposition to cancer. Evaluation of this parameter implies using a challenge mutagen in an in vitro system to unmask latent genetic instability and repair insufficiency in the target cells. Traditionally, these investigations involve two tests, evaluation of mutagenic susceptibility (chromosomal aberrations) and genotoxic effect (DNA comet assay). The present study was focused on analysis of the effect of methylnitrosourea (MNU) on resting and PHA-stimulated lymphocytes from healthy donors and patients with gynecological cancer. Cytotoxic effect of MNU (apoptotic lymphocyte death) was estimated using two parameters, interaction of the cells with the annexin V-FITC complex, and morphological changes of the nuclei after their staining with the mixture of two DNA tropic dyes. The genotoxic effect of MNU, namely, secondary double-strand DNA breaks, was scored using the neutral comet assay, modified for the calculation of the comets produced exclusively by BrUdr-labeled proliferating lymphocytes. The proportion of these comets was represented as the proliferative cell index. It was shown that resting lymphocytes were resistant to genotoxic and cytotoxic effects of MNU. The response of proliferating cells to the action of MNU was expressed as the development of secondary DNA breaks (P <0.01), along with the increased frequency of apoptosis (P <0.05). The genotoxic effect of MNU on stimulated lymphocytes of gynecological cancer patients was fourfold lower compared to healthy donor lymphocytes. In response to the MNU action, patient lymphocytes did not change their proliferative index, while in healthy donor lymphocytes proliferative index was two times decreased in response to the MNU action. The data obtained pointed to the association between the cytotoxic response of the lymphocytes to the action of MNU and gynecological cancer. Since only proliferating lymphocytes response to the genotoxic effect of MNU, and the effect is revealed a day after the mutagen action, it is suggested that this phenomenon is associated with postreplicative repair, MMR, the substrate of which is O6-methylguanin. The MMR deficiency in patient lymphocytes determines their tolerance to the action of MNU. Genotoxic effect of lymphocytes to the action of MNU can serve as a marker of MMR, as well as of the MMR deficiency-associated gynecological cancer.

[Mismatch repair (MMR) efficiency and MSH2 gene mutation in human colorectal carcinoma cell line COLO320HSR].

Colorectal cancer (CC) is one of two diseases, in which the link between cancer proneness and DNA repair deficiency appears to be proved. A strict relationship between mismatch repair (MMR) gene mutations, microsatellite instability (MSI) has been found in familiar colorectal cancer (Lynch syndrome). Tumorigenesis at familiar cancer is initiated by biallelic mutations in the major MMR genes, namely MSH2 or MLH1. One of these mutations is an inherited germline alteration and the other is a somatic one. The initiating mutation in sporadic colorectal tumors was not still identified although biochemical and genetic signs of MMR deficiency are observed in tumor cells. Two currently used colorectal tumor cell lines HCT116 and COLO320HSR were derived from hereditary and sporadic tumors accordingly. HCT116 cell line exhibits MMR-deficiency due to biallelic deletion in MLHL. As a consequence this shows MSI phenotype and a near-diploid karyotype. COLO320HSR cell line is characterized by MSS phenotype with mostly imbalanced aberrations. This indicates MMR proficiency in these cells. However, both MMR-deficient HCT116 and COLO320HSR cells reveal near-diploid karyotype. Earlier we have shown that the number of secondary DNA double strand breaks, induced by methylnitrosourea (MNU), represent functional activity of cellular MMR. In the present study, using this approach we evaluated sensitivity to MNU and MMR activity in two colorectal tumor cell lines (HCT 116, COLO320HSR) and compared them to that in the HeLa cell line, which have MMR-proficient phenotype. We showed that cell line COLO320HSR exhibits low MMR activity, close to the level of MMR-activity in HCT116 cell line. We found a mutation in MSH2-G520A gene in COLO320HSR. This neutral mutation apparently is not related to polymorphism as we failed to identify the same mutation in any of MSH2 gene sequences of lymphocytes from 30 patients with sporadic colorectal cancer.

[Comparison of geno- and cytotoxicity of methylnitrosourea on MMR-proficient and MMR-deficient human tumor cell lines].

Deficient mismatch repair (MMR) is identified as a mutation of one of four major MMR genes and(or) microsatellite instability. These genomic changes are used as markers of MMR status of the heredity nonpolyposis colorectal cancer (HNPCC) spectrum tumors--familial and sporadic tumors of colon and extracolonic cancers fulfilling Amsterdam clinical criteria II. MMR-deficiency results in mutator phenotype and resistance to geno- and cytotoxicity of alkylating agents. The main cytotoxic damage to DNA in response to chemical methylation is O6-methylguanine (O6-mG). The secondary DNA strand breaks, which are formed during the MMR functioning, are proposed to be required for methylation induced cytotoxicity. We have assumed that the secondary double stand breaks (DSB) upon DNA methylation are able to represent functional efficiency of MMR in cells. The purpose of the paper was to test this assumption on human tumor cells differing in MMR-status and pulse-treated with methylnitrosourea (MNU). We used 3 cell lines: HeLa (MMR-competent endometrial tumor cells), HCT116 (MMR-deficient colorectal carcinoma cells), and Colo320 (sigmoid intestine tumor cells with uncharacterized MMR status). DSBs were evaluated with neutral comet assay. Cytotoxicity/viability was evaluated with MTT-asay and apoptotic index (frequency of morphologically determined apoptotic cells). We show that 1) cytotoxic effect of MNU (250 microM) on HeLa cells was exhibited 3 days after pulse-treatment of cells with MNU; 2) DSBs occurred 48 h after the drug treatment but prior to the onset of apoptosis of HeLa cells; 3) MMR-deficient HCT116 cells were resistant to the drug: no decreased viability, DSBs and apoptosis were observed during 3 days after cell treatment. Both cell lines exhibited high sensitivity to etoposide, classical inductor of unrepairable DSBs and p53. Etoposide has been found to induce DSBs in 6-12 h, which was followed by apoptosis (in 24 h). Colo320 cells exhibited intermediate position between HeLa and HCT116 cell lines in regard to sensitivity to MNU according to MTT-assay and the number of secondary DSBs formed in MNU-treated cells. Nevertheless, in contrast to HeLa cells, these breaks did not induce apoptosis in Colo320 cells. Our data confirm the assumption about case/effect relationship between secondary DNA double strand breaks, induced by monofunctional methylating agent MNU, and functioning of MMR in human tumor cells.

[Binding of SSB-protein from Ehrlich ascites carcinoma cells with DNA and polyribonucleotides]. / Sviazyvanie SSB-belka iz kletok astsitnoi kartsinomy érlikha s DNK i poliribonukleotidami.

Binding of SSB-protein from Ehrlich ascites tumor to ssDNA from M13 phage leads to its compactization. The structure of the complex at the protein/DNA ratios far from the saturation level looks like "beads-on the string". DNA that was fully saturated with protein forms collapsed globular structure. Binding of the protein to the dsDNA from phage lambda increases its flexibility and decreases the coil dimensions; no "beads-on the string" structure are seen. The protein possess slight destabilizing effect on hairpin helices of M13DNA. Competition studies demonstrate that the binding properties of protein with polyribonucleotide lattices and DNA's decrease in ranking as follows: poly(rG) greater than or equal to poly(rI) greater than or equal to ssDNA greater than dsDNA greater than poly(rA) congruent to approximately poly(rU). Thus SSB-protein from Ehrlich ascites tumor differs significantly from its presumed prokaryotic analogs.

[The adaptive reaction of the blood lymphocytes in persons subjected to chronic radiation exposure at low doses]. / Adaptivnaia reaktsiia limfotsitov krovi liudei, podvergshikhsia khronicheskomu vozdeistviiu radiatsii v malykh dozakh.

The changes in the PHA-stimulated human blood lymphocytes of the peoples from Moscow and from polluted after Chernobyl disaster Bryansk region were studied. The decrease in lymphocyte mitotic index in residents from polluted regions was revealed. Moscow population was not distinguished from Bryansk region population by the frequency of cells with micronuclei. There was also no difference in frequency of micronuclei after 1 Gy irradiation of the these populations. The adaptive response (irradiation with a dose of 0.05 Gy followed by 1.0 Gy irradiation 5 hours later) was studied. The decrease in the number of individuals with adaptive response and appearance of the individuals with increased radiosensitivity in the population of polluted regions were observed. There was no such subpopulation among the Moscow residents. So, firstly, low dose chronic irradiation is not an adaptive factor; secondly, the determination of adaptive response can be methodological procedure for the revealing the changes which were induced by low dose chronic irradiation (accompanied by other environment agents).

[DNA repair and apoptosis]. / Reparatsiia DNK i apoptoz.

DNA damage induced by exo- and endogenous agents triggers two opposite mechanisms--DNA repair and programmed cell death. The latter contains a phase of DNA degradation. Both mechanisms compete for DNA as a substrate and for the cell energy supply. The interaction and competition of these processes influence the pattern of cell death (from pure apoptosis to necrosis). Synergetic and competitive relations between DNA-repair and apoptosis are reviewed.

[The DNA-comet method for individual cells. The principle and use of the method]. / Metod DNK-komet individual'nykh kletok. Printsip i primenenie metoda.

The DNA comet assay allows to evaluate the damage in genomes of individual cells. This article summaries the literary evidence and the author's experience in development and application of the technique. Principles of the method are reviewed in addition to protocols for neutral and alkaline conditions; examples of application of the method are given.

[Hyperthermia induced signal for apoptosis and pathways of its transduction in the cell]. / Signal k apoptozu, indutsirovannyi gipertermiei, i puti ego peredachi v kletke.

Hyperthermia (HT) is a physiological agent able to induce apoptosis in normal and tumor cells. The ability of HT to damage different cellular components is underlying the mechanism of apoptogenic activity of HT. The review is aimed to consider the studies representing mainly HT production of genotoxic initial signal and pathways of its transduction to programmed cytotoxic event or apoptosis. In this regard we analyse the membrane effect of HT, HT-induced chromatin and nuclear matrix structure changes, DNA damage, effect of HT on DNA-repair mechanism, and the role of heat shock proteins in apoptosis and cancerogenesis.

[The microelectrophoresis of the DNA in individual intact and gamma-irradiated thymocytes]. / Mikroélektroforez DNK individual'nykh intaktnykh i gamma-obluchennykh timotsitov.

The in vitro gamma-irradiated mouse thymocytes were embedded in low melting agarose at 37 degrees C. After getting at 4 degrees C, the cells were lysed in neutral detergent solution containing proteinase K and ethidium bromide. Microscopic visualization of single lysed and stained cells showed the presence of the central "core" (nuclear matrix) surrounded with "halo" (relaxed nuclear DNA). During electrophoresis (2-5 V/sm, 5 min) this "halo" migrated towards the anode forming a "tail". The use of microdensitometric system provided measuring the size of the tail (L) and quantity of migrated DNA (S) for individual cells as well as obtaining the distribution of these parameters among the cells. The latter may be characteristic of heterogeneity of the cell population. It was shown that L and S increased linearly with the dose irradiation at least between 0.2 and and 5.0 Gy. In irradiated thymocyte (3 Gy) the DNA repair occurred within 10-20 min, but residual DNA damage could be observed even after 60 min of incubation. These damages may initiate the degradation of DNA in irradiated thymocytes that was observed after the repair of DNA.

[Spontaneous death of mononuclear cells, obtained from health donors and patients with systemic lupus erythematosus]. / Spontannaia gibel' mononuklearnykh kletok, poluchennykh ot zdorovykh donorov i bol'nykh sistemnoi krasnoi volchankoi.

Unstimulated human peripheral blood mononuclear cells (MNC) underwent death during incubation in vitro. According to morphological criteria the type of death was identified as apoptosis. There was a good correlation between a fraction of apoptotic cells qualified morphologically, and fraction of "apoptotic" comets. The use of DNA-comets for studying the spontaneous death in vitro of MNC from patients with systemic lupus erythematosus (SLE) showed that 24 h after isolation cells from SLE patients demonstrated a higher level of apoptosis than MNC from normal donors. It is likely that the increased apoptosis of SLE-MNC in vitro may reflect changes occurring in those cells in vivo which are bound with pathogenesis of disease. In this context the comet assay may be promising in diagnostics and monitoring of therapeutic treatments.

[The characteristics of the structural organization and spontaneous DNA damage in the cells of 2 lymphoma L5178Y lines differing in their radiosensitivity]. / Osobennosti strukturnoi organizatsii i spontannaia povrezhdenoost' DNK v kletkakh dvukh linii limfomy L5178Y, razlichaiushchikhsia po radiochuvstvitel'nosti.

Nucleotide particles, resulting from mild lysis procedure, obtained from two strains of murine L5178Y lymphoma cells, differing in radiosensitivity (L-R and LY-S) showed differences in the sedimentation rate and in the halo rewinding in the presence of 40 micrograms/ml Ethidium bromide (EB). Microgel electrophoresis of DNA after an exhaustive lysis of cells (in the presence of sodium laurylsarcosine and proteinase for 20-24 h at room temperature) disclosed not only heterogeneity in DNA migration length among LY-R cells (LR) and LY-S cells (LS), but also differences between two strains with respect to this parameter. L-value for LY-S cells exceeds that for LY-R strain by 20 per cent, and the ratio LS/LR remains constant regardless of the voltage applied. Thus, the cell distribution pattern according to L-value is the intrinsic parameter for both LY-R and LY-S cells, and DNA of LY-S cells is suggested to bear more background strand breaks as compared with the radioresistant LY-R cells. LY-R cells irradiated with 10 Gy repair their DNA completely after 60-90 min of incubation at 37 degrees C but DNA migration pattern for irradiated and repaired LY-S cells is characterized by the predominance of DNA with a higher L-value than that for intact cells. Hence, we suggest that the higher radiosensitivity of LY-S cells might be compatible with a relatively high background DNA breakage in these cells.

[Mechanism of radiation death of human peripheral blood lymphocytes, assessed by the DNA-comet method]. / Mekhanizm radiatsionnoi gibeli limfotsitov perifericheskoi krovi cheloveka, otsenivaemoi metodom DNK-komet.

Radiation-induced death of human peripheral blood lymphocytes (HPBL) was studied using the comet assay. It has been shown that gamma-irradiation induced simultaneously two forms of death, necrosis and apoptosis which may be discriminated by the comet assay. Three groups of comets formed by HPBL are specified: symmetric/slowly asymmetric comets attributed to viable cells (C0/C1-class of comets); comets reminding "tear-drop" (C3/C4-class of comets) which are characterized by decreased total fluorescence of comet and formed by dying/dead (necrotic or apoptotic) cells; intermediate form of comets (C2-class), containing head, tail and attributed to cells committed to apoptosis (containing high molecular weight fragments of DNA). Kinetics of C2-comets permits detection of early stage of apoptotic death of irradiated HPBL.