Translesion DNA Synthesis and Reinitiation of DNA Synthesis in Chemotherapy Resistance.

Many chemotherapy drugs block tumor cell division by damaging DNA. DNA polymerases eta (Pol η), iota (Pol ι), kappa (Pol κ), REV1 of the Y-family and zeta (Pol ζ) of the B-family efficiently incorporate nucleotides opposite a number of DNA lesions during translesion DNA synthesis. Primase-polymerase PrimPol and the Pol α-primase complex reinitiate DNA synthesis downstream of the damaged sites using their DNA primase activity. These enzymes can decrease the efficacy of chemotherapy drugs, contribute to the survival of tumor cells and to the progression of malignant diseases. DNA polymerases are promising targets for increasing the effectiveness of chemotherapy, and mutations and polymorphisms in some DNA polymerases can serve as additional prognostic markers in a number of oncological disorders.

Translesion DNA Synthesis and Carcinogenesis.

Tens of thousands of DNA lesions are formed in mammalian cells each day. DNA translesion synthesis is the main mechanism of cell defense against unrepaired DNA lesions. DNA polymerases iota (Pol ι), eta (Pol η), kappa (Pol κ), and zeta (Pol ζ) have active sites that are less stringent toward the DNA template structure and efficiently incorporate nucleotides opposite DNA lesions. However, these polymerases display low accuracy of DNA synthesis and can introduce mutations in genomic DNA. Impaired functioning of these enzymes can lead to an increased risk of cancer.

BER gene polymorphisms associated with key molecular events in bladder cancer.

AIM: Base excision repair (BER) gene polymorphisms are known to play an independent role in predisposition to developing different cancers as well as to be associated with clinicopathological traits of the disease modifying its clinical outcomes. One of the underlying mechanisms is presumed to include interplay between BER gene polymorphisms and key mutational, epigenetic and chromosomal events in tumor tissues. The present study was aimed at elucidating potential gene-gene interaction and assessing their mutual effects in bladder cancer (BC). MATERIALS AND METHODS: The earlier obtained data on genotyping patients with verified diagnosis of BC for OGG1 rs1052133 (Ser326Cys) and XRCC1 rs25487 (Arg399Gln) polymorphisms were used for this study. The tumor tissue samples from the same patients were analyzed for mutations, epigenetic variations and losses of heterozygosity in some key genes involved in divergent pathogenic pathways of BC. RESULTS: It was shown that the OGG1 (326 codon) heterozygous genotype as well as the minor 326Cys allele can intensify a mutational response of the RAS locus in urothelial carcinomas in the total cohort of patients simultaneously decreasing the mutation rates in the PIK3CA locus in smokers. The XRCC1 (399 codon) heterozygous genotype as well as the minor 399Gln allele reduced the frequency of LOH in the PTEN and TNKS genes, but did not affect the mutational variability in any locus tested. Both polymorphisms influenced the methylation status, carriers of OGG1 326Ser/Cys or Ser/Cys+Cys/Cys genotypes demonstrating increased frequency of methylated RUNX3 and ISL1 genes whereas the similar effect of XRCC1 polymorphism concerning methylation of p16 and TIMP3 genes. When dividing the total cohort into groups based on the extent of tumor spread, the observed associations were characteristic of non-muscle invasive BC. CONCLUSION: The BER gene polymorphisms contributed to modification of key molecular events in urothelial carcinomas. Their mutual effects mainly manifested in non-muscle invasive BC. The underlying mechanisms as well as possible clinical outcomes need to be further explored to propose novel prognostic biomarkers for BC.

Structural Alterations in Human Fibroblast Growth Factor Receptors in Carcinogenesis.

Fibroblast growth factor (FGF) plays an important role in human embryogenesis, angiogenesis, cell proliferation, and differentiation. Carcinogenesis is accompanied by aberrant constitutive activation of FGF receptors (FGFRs) resulting from missense mutation in the FGFR1-4 genes, generation of chimeric oncogenes, FGFR1-4 gene amplification, alternative splicing shift toward formation of mesenchymal FGFR isoforms, and FGFR overexpression. Altogether, these alterations contribute to auto- and paracrine stimulation of cancer cells and neoangiogenesis. Certain missense mutations are found at a high rate in urinary bladder cancer and can be used for non-invasive cancer recurrence diagnostics by analyzing urine cell pellet DNA. Chimeric FGFR1/3 and amplified FGFR1/2 genes can predict cell response to the targeted therapy in various oncological diseases. In recent years, high-throughput sequencing has been used to analyze exomes of virtually all human tumors, which allowed to construct phylogenetic trees of clonal cancer evolution with special emphasis on driver mutations in FGFR1-4 genes. At present, FGFR blockers, such as multi-kinase inhibitors, specific FGFR inhibitors, and FGF ligand traps are being tested in clinical trials. In this review, we discuss current data on the functioning of the FGFR family proteins in both normal and cancer cells, mutations in the FGFR1-4 genes, and mechanisms underlying their oncogenic potential, which might be interesting to a broad range of scientists searching for specific tumor markers and targeted anti-cancer drugs.

FGFR3 and TP53 mutations in a prospective cohort of Belarusian bladder cancer patients.

AIM: The aim of this study was to determine the frequencies of FGFR3 and TP53 mutations in a prospective cohort of 150 bladder cancer patients and to assess the relationship between their mutational status and clinicopathological variables. MATERIALS AND METHODS: The FGFR3 and TP53 mutations were detected by the SNaPshot method and PCR-single-strand conformational polymorphism analysis followed by DNA sequencing. RESULTS: The activating FGFR3 mutations were found in 71 (47.3%) whereas TP53 mutations were observed in 31 (20.7%) urothelial carcinomas. FGFR3-mutant tumors significantly correlated with lower tumor stage and grade, papillary form of bladder cancer and the absence of metastases while TP53-mutant tumors were strongly associated with higher tumor stage and grade as well as the presence of metastasis. We also found significant inverse correlation between FGFR3 mutations and TP53 alterations in urothelial carcinomas (p=0.03). Four possible genotypes were observed in the whole studied cohort, namely FGFR3mut/TP53wt (41.3%), FGFR3wt/TP53wt (38%), FGFR3wt/TP53mut (14.7%), and FGFR3mut/TP53mut (6%). Tumors with FGFR3wt/TP53wt genotype comprised the subgroup, in which all stages and grades were equally distributed. CONCLUSIONS: Our findings confirm the alternative role of FGFR3 and TP53 mutations in the development of bladder cancer. Together these two genetic markers are attributed to 62% of the tumors studied. Tumors with both wild type genes included urothelial carcinomas of all stages and grades and may develop through another genetic pathway. To elucidate complete molecular profile of bladder tumors further additional studies are needed.

[The polymorphism of DNA repair genes XPD, XRCC1, OGG1, and ERCC6, life expectancy, and the inclination to smoke].

The polymorphism of excision repair genes XPD Asp312Asn, XRCC1 Arg399Gln, OGG1 Ser326Cys, and ERCC6 Met1097Val was analyzed by PCR-RFLP in 370 representatives of the Belarusian population of average, old, and elderly ages. Correlation analysis showed that the frequencies of wild-type homozygous combinations significantly increase with age in the group of subjects over 70 years old in the case of the interaction of two genes, XPD 312 and XRCC1399, or three genes, XPD312, XRCC1399, and ERCC6 1097. In a subgroup of the long-lived, this relationship is manifested in case of a pairwise interaction of gene XPD 312 with XRCC1 399 or ERCC6 1097, as well as an interaction of three genes, XPD 312, XRCC1 399, and ERCC6 1097. The data suggest that the optimum activity of repair processes may favor longevity. It is shown that the frequency of the Asp/Asp genotype is reduced, and the frequency of the Asn allele of the XPD 312 gene is increased in the subgroup of smokers as compared with nonsmokers, which apparently indicates an association of this gene polymorphism with an inclination to smoke. The problem requires further study.

Dihydropyridines decrease X-ray-induced DNA base damage in mammalian cells.

Compounds with the structural motif of 1,4-dihydropyridine display a broad spectrum of biological activities, often defined as bioprotective. Among them are L-type calcium channel blockers, however, also derivatives which do not block calcium channels exert various effects at the cellular and organismal levels. We examined the effect of sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate (denoted here as DHP and previously also as AV-153) on X-ray-induced DNA damage and mutation frequency at the HGPRT (hypoxanthine-guanine phosphoribosyl transferase) locus in Chinese hamster ovary CHO-K1 cells. Using formamido-pyrimidine glycosylase (FPG) comet assay, we found that 1-h DHP (10nM) treatment before X-irradiation considerably reduced the initial level of FPG-recognized DNA base damage, which was consistent with decreased 8-oxo-7,8-dihydro-2'-deoxyguanosine content and mutation frequency lowered by about 40%. No effect on single strand break rejoining or on cell survival was observed. Similar base damage-protective effect was observed for two calcium channel blockers: nifedipine (structurally similar to DHP) or verapamil (structurally unrelated). So far, the specificity of the DHP-caused reduction in DNA damage - practically limited to base damage - has no satisfactory explanation.

Transgenerational accumulation of radiation damage in small mammals chronically exposed to Chernobyl fallout.

The purpose of this investigation has been the analysis of the long-term development of biological damage in natural populations of a model mammalian species, the bank vole (Clethrionomys glareolus, Schreber), which were chronically exposed to low doses of ionizing radiation over 22 animal generations within 10 years following the Chernobyl accident. The time course of the biological end-points (chromosome aberrations in bone marrow cells and embryonic lethality) was compared with the time course of the whole-body absorbed dose rate from external and internal exposure in the studied populations inhabiting monitoring sites in Belarus with different ground deposition of radionuclides. The yield of chromosome aberrations and, in lesser degree, embryonic lethality was associated with the radionuclide contamination of the monitoring areas in a dose-dependent manner. As a main feature of the long-term development of biological damage under low dose rate irradiation, permanently elevated levels of chromosome aberrations and an increasing frequency of embryonic lethality have developed over 22 animal generations. This contrasts with the assumption that the biological damage would gradually disappear since in the same period of time the whole-body absorbed dose rate decreased exponentially with a half-value time of about 2.5-3 years. Furthermore, gravid females were captured, and their offspring, born and grown up under contamination-free laboratory conditions, showed the same enhanced level of chromosome aberrations. Therefore the authors suggest that, along with the biological damage attributable to the individual exposure of each animal, the observed cellular and systemic effects reflect the transgenerational transmission and accumulation, via genetic and/or epigenetic pathways, of damage attributable to the chronic low-dose rate exposure of the preceding generations of animals. They also suggest that the level of the accumulated transmissible damage in the investigated populations will decrease in future due to the further recession of the chronic exposure and as a consequence of selection processes.

Long-term development of the radionuclide exposure of murine rodent populations in Belarus after the Chernobyl accident.

As a determinant of the associated health risks, the behavior of radionuclides in natural ecosystems needs to be better understood. Therefore, the activity concentration of various long-lived radionuclides released due to the Chernobyl accident, and the corresponding contributions to the whole-body dose rate, was studied as a function of time in mammalian indicator species inhabiting the natural forest ecosystems of Belarus, the bank vole (Clethrionomys glareolus) and the yellow-necked mouse (Apodemus flavicollus). The activity concentrations of 137Cs, 134Cs, 90Sr, 238Pu, 239,240Pu, 241Pu and 241Am in soil and in animals were measured at five monitoring sites with different ground deposition of radionuclides at different distances from the destroyed reactor. The observed temporal pattern of the radionuclide activity concentration in the studied animal populations reflects the changes in biological availability of these isotopes for biota, mostly due to fuel particle destruction and appearance of dissolved and exchangeable forms of radionuclides. The time course of 134+137Cs activity concentrations in animal populations appeared as a sequence of increase, peak and decrease. Maximal levels of radiocesium occurred 1-2 years after deposition, followed by an exponential decrease. Concentrations of incorporated 90Sr increased up to the tenth year after deposition. The activity concentrations of transuranic elements (238Pu, 239,240Pu, 241Pu and 241Am) were much lower than those of the other radionuclides, in the studied animals. A considerable activity of 241Am in animals from areas with high levels of contamination was firstly detected 5 years after deposition, it increased up to the tenth year and is expected to increase further in the future. Maximal values of the whole-body absorbed dose rates occurred during the year of deposition, followed by a decrease in the subsequent period. Generally, this decrease was monotonic, mainly determined by the decrease of the external gamma-ray dose rate, but there were exceptions due to the delayed maximum of internal exposure. The inter-individual distributions of radionuclide concentrations and lifetime whole-body absorbed doses were asymmetric and close to log-normal, including concentrations and doses considerably higher than the population mean values.

[DNA-repair modulation using derivatives of 1,4-dihydroisonicotinic acid as an example].

The influence of two derivatives of 1,4-dihydroisonicotinic acid on DNA-repair involved in chemical mutagenesis in Drosophila germ cells has been investigated. The compounds tested decreased the level of EMS-induced chromosome breakage and point mutations due to stimulation of maternal repair of DNA primary damage induced in spermatozoa as well as due to activation of DNA-repair in larvae and imago premeiotic stages of Drosophila males. Deficiency of DNA-repair systems leads to decrease in female and male germ-cell sensitivity to antimutagen action.

[Clastogenicity of ethyl methanesulfonate and dimethyl terephthalate in the micronucleus test and ways for its modification]. / Klastogennost' étilmetansul'fonata i dimetiltereftalata v mikroiadernom teste i puti ee modifikatsii.

In the mouse transplacental test, EMS induced micronuclei and disturbed haemopoiesis in female bone marrow and foetal liver. Dimethyl terephthalate at the tested dose was ineffective in pregnant females increasing however the level of these events in foetuses. Hence, both the alkylating agent and the phthalate derivative penetrate placenta and are dangerous for embryos. The 1,4-dihydropyridine derivative (DHP) decreased the EMS-induced micronucleus frequency in pregnant female somatic cells but it was inefficient in fetuses and did not influence the DMtP effects. The typical dependence of its protective action on the physiological status of organism was revealed. This indicates that the antimutagen inhibits the clastogenesis by the induction or stimulation of endogenous components responsible for antioxidant defense and/or neutralization of electrophilic molecules.

[Genetic efficacy of low doses of ionizing radiation in chronically-irradiated small mammals]. / Geneticheskaia éfffektivnost' malykh doz ioniziruiushchei radiatsii pri khornicheskom obluchenii melkikh mlekopitaiushchikh.

Earlier we have established the genetic effects of low dose chronic irradiation in bank vole (somatic and germ cells, embryos), in pond carp (fertilized eggs, embryos, fry) and in laboratory mice (somatic and germ cells) in the range of doses from near-background to 10 cGy. These low dose effects observed in mammals and fish are not expected from extrapolation of high dose experiments. For understanding reasons this discrepancy the comparative analysis of genetic efficiency of low dose chronic irradiation and the higher doses of acute irradiation was carried out with natural populations of bank vole which inhabited the two sites differing in ground of radionuclide deposition. For comparing efficiency the linear regression model of dose-effect curve was used. Dose-effect equations were obtained for animals from two chronically irradiated bank vole populations. The mean population absorbed doses were in the range 0.04-0.68 cGy, the main part of absorbed doses consisted of external radiation of animals exposed to 137Cs gamma-rays. Dose-effect equations for acute irradiation to 137Cs gamma-rays (10-100 cGy) were determined for the same populations. Comparison of genetic efficiency was made by extrapolation, using regression coefficient beta and doubling dose estimation. For chronic exposure the doubling doses calculated from low-dose experiments are 0.1-2 cGy and the doubling doses determined from high-dose experiments are in the range of 5-20 cGy. Our hypothesis that the doubling dose estimate is calculated in higher-dose ionizing radiation experiments should be much higher than the deduced from the low dose line regression equation was verified. The doubling dose estimates for somatic cells of bank vole and those for germ cells of laboratory mice are in close agreement. The radiosensitivity of bank vole chromosomes were shown is practically the same as that for human lymphocytes since doubling dose estimates for acute irradiation close to each other. For low LET radiation a higher genetic efficiency of chronic low doses in comparison with the higher doses of acute gamma-irradiation (137Cs source) was proved by three methods.

Anticlastogenicity of two derivatives of 1,4-dihydroisonicotinic acid in mouse micronucleus test.

Effects of two derivatives of 1,4-dihydroisonicotinic acid (1,4-DHINA) against the monofunctional alkylating agent ethyl methanesulfonate (EMS) were studied in the micronucleus test in (CBA x C57Bl/6(j)) mice. Adult males and pregnant females were treated with an antimutagen (i.p.) and 12h later they were exposed to EMS (i.p.). The frequencies of micronucleated (MN) polychromatic erythrocytes (PCEs) in mouse bone marrow and foetal liver were analysed 6, 12, 18, 24, 30, 36, 48 or 24, 48 and 72 h after the mutagen injection. In adults, the maximum number of MNPCEs was observed 36 or 24h after the EMS administration. In foetuses, which were treated in a maternal organism, such peak was found at 24h. Pre-treatment of mice with the antimutagens 2,6-dimethyl-3,5-diethoxycarbonyl-4-(Na carboxylate)-1,4-dihydropyridine (DHP) and glutapyrone (GP) decreased the yield of MNPCEs in male bone marrow. Having been observed at a peak of MN induction, the anticlastogenic effect of DHP (1/10 LD(50) or 340 mg/kg) reached 30%. DHP at the doses of 0.5-1mM/kg did not affect the EMS-induced frequency of MNPCEs in bone marrow, whereas GP inhibited it at the similar millimolar concentrations. Simultaneously with maternal bone marrow, foetal liver cells were analysed for MNs in the transplacental test. The anticlastogenic effect of DHP (1/10 LD(50)) was found to be more prolonged and higher in females than in males and to average 50%, but this antimutagen was not efficient in foetuses. Both antimutagens did not change the polychromatic/normochromatic erythrocyte (PCE/NCE) ratio as compared with EMS action. Results presented indicate a peak of EMS-induced micronucleated cells in mouse bone marrow 24 or 36 h and in foetal liver 24h after animal treatment. Two 1,4-DHINA derivatives exhibited anticlastogenic activity in adults, but not in foetuses.

[Biological effects in natural populations of small rodents in radiation-polluted territories. Description of sites. Dynamics of concentration of gamma-emitting radionuclides in populations of two species of small mammals]. / Biologicheskie éffekty v prirodnykh populiatsiiakh melkikh gryzunov na radiatsionno-zagriaznennykh territoriiakh. Opisanie statsionarov. Dinamika kontsentratsii gamma-izluchaiushchikh radionuklikov v populiatsiiakh dukh vidov melkikh mlekopitaiushchikh.

It has been revealed that dynamics of gamma-emitting radionuclide concentration in consecutive generations of two species of wild rodents (European bank vole and yellow-necked mouse) is characterised by the phases of an increase, a maximum content (peaks) and a decrease over 10 years after the Chernobyl accident. The peaks of specific activity of gamma-emitting radionuclides in populations in the areas with different densities of radio-contamination falls not on the first year but on the next ones (1987-1989) after the catastrophe, i.e. are observed in the subsequent (3-8) generations of animals. The revealed shift of maximum of radionuclide concentration in comparison with the maximum of their fallout is likely caused by an increase in radionuclide biological availability.

[Biological effects in natural populations of small rodents in radiation-polluted territories. Dynamics of chromosome aberration frequency in a number of generations of European bank vole (Clethrionomys glareolus, Schreber)]. / Biologicheskie éffekty v prirodnykh populiatsiiakh melkikh gryzunov na radiatsionno-zagriaznennykh territoriiakh. Dinamika chastoty aberratsii khromosom v riadu pokolenii evropeiskoi ryzhei lesnoi polevki (Clethrionomys glareolus, Schreber).

The dynamics of chromosome aberration frequency in bone marrow cells of many generations (14) of bank vole living in the radioactive trace of the Chernobyl catastrophe (1986-1992) has been analysed. The study revealed that the chromosome aberration frequency in voles in the areas with radio-contamination density 220 and 1526 kBq/m2 (for 137Cs) significantly exceeds the control level 3-7 times over the whole period under investigation. The dynamics of the frequency of structural chromosome injuries from 1986 to 1991-1992 is characterised by the tendency to increase in all populations inhabiting the areas with various radio-contamination density (8-1526 kBq/m2).

[The dynamic mutability of the somatic and germ cells of animals inhabiting regions of radioactive fallout]. / Dinamika mutabil'nosti somaticheskikh i polovykh kletok zivotnykh, naseliaiushchikh raiony vypadeniia radioaktivnykh osadkov.

Many generations (1-18) of natural populations of small mammals that inhabited in 1986-1991 areas contaminated by radionuclides, had increased levels of the mutations in somatic cells and gametes. The high frequency of chromosome aberrations in somatic cells of young carps from contaminated ponds was detected in 1988-1992. Radiosensitivity of hereditary structures of animal somatic cells and gametes was increased in subsequent generations as compared with generations that lived in 1986-1988.

[Mechanisms of the inhibitive action of 1,4-dihydroisonicotinic acid derivatives in chemical mutagenesis]. / Mekhanizmy ingibiruiushchego deistviia proizvodnykh 1,4-digidroizonikotinovoi kisloty pri khimicheskom mutageneze.

1.4-Dihydroisonicotinic acid derivatives were used as an example to examine the mechanisms of action of exogenous antimutagens in chemical mutagenesis in eukaryotic organisms. The chemical mutagenesis was stimulated by alkylating agents, mutagens of direct action. The behaviour of alkylating mutagenesis was analysed, which gives an insight into the stages at which antimutagens may be involved in this process. Chemical mutagenesis was demonstrated to be suppressed due to lower molecular doses of the mutagen and by affecting the mechanisms of implementation of chemically induced mutations. Great emphasis was laid on the mechanisms of action of mutagens mediated by the intracellular antimutagenic and reparative systems. A special role is played by the analysis of exogenous antimutagens affecting DNA reparation as the most important step in the process of conversion of primary damages to fixed mutations. The paper presents a review of literature on basic problems of chemical mutagenesis modification.

[1,4-dihydroisonicotinic acid (1,4-DHINA) derivatives, inhibitors of chemical mutagenesis]. / Proizvodnye 1,4-digidroizonikotinovoi kisloty (1,4-DGINK)-- ingibitory khimicheskogo mutageneza.

The paper summarizes the results of studies into the modifying effect of some 1,4-dihydroisonicotinic acid (1,4-DHINA) derivatives during chemical mutagenesis in eukaryotic organisms. The compounds under study are effective in reducing the incidence of point and chromosomal mutations induced by ethylmethane sulfonate (EMS) in Drosophila sex cells, displaying the specificity of the influence depending on some factors. The effects of these antimutagens on the formation and repair of chromosomal breaks were studied during mutagenized sperm storage in Drosophila female receptacula semenis. A relation was found between the female sensitivity to antimutagens and the appropriate modifying effect, on the one hand, and the oocytic genotype, on the other. The protective effect of one of 1,4-DHINA derivatives against alkylating agents (EMS and Thiophosphamide) was confirmed by in vivo experiments in mice and in cultured human lymphocytes. The revealed mechanisms of modified chemical mutagenesis both in the Drosophila sex cells and in the mammalian somatic cells are in favour of those of protective action, which are mediated by the antimutagenic cell system, as well as, perhaps, by repair processes.

[Antimutagenesis as a genetic process]. / Antimutagenez kak geneticheskii protsess.

The concept that antimutagenesis is a normal genetic process whose function is to ensure integrity and stability of hereditary structures has been formulated and substantiated. The phenomenon of antimutagenesis is realized by the antimutagenic cell system, forming the first level of DNA protection from the mutagenic influences of endogenous and exogenous agents. Repair systems constitute the second protective level. The common mechanism of the protective action of exogenous antimutagens is that they act through the antimutagenic system components and/or the repair systems. The resistance of organisms to external factors and damaged to their cell systems is determined by a set of components and the intensity of antimutagenic and repair functioning.