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.

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.

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.

[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.

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.

[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.

A comparative study of the antimutagenic effects of antioxidants on chemical mutagenesis in Drosophila melanogaster.

The 1,4-dihydropyridine derivative 2,6-dimethyl-3,5-diethoxycarbonyl-4-(Na carboxylate)-1,4-dihydropyridine (1,4-DHP) was studied for antimutagenic effects in the dominant lethal test and in the sex-linked recessive lethal test of Drosophila melanogaster. The observed effects were compared with those of the radioprotectors cysteine and cysteamine and with those of the phenolic antioxidant butylated hydroxytoluene (BHT). In a wide range of concentrations, including low ones, 1,4-DHP reduces the frequency of EMS-induced genetic damage (point mutations and chromosome breakage). A reduction of the mutation rate induced by EMS in adults could be observed independently of the developmental stages (larvae or imago) pretreated with 1,4-DHP. The protective effect of this new antimutagen against the alkylating agent depended on both the 1,4-DHP dose and the level of the EMS-induced mutation rate. The effect of 1,4-DHP was more pronounced than that of the studied radioprotectors. It is concluded that dihydropyridine-type compounds are able to protect eukaryote germs cells from genetic damage produced by direct-acting mutagens such as EMS.

Mutagenic effects of dimethyl terephthalate on mouse somatic cells in vivo.

The mutagenic activity of dimethyl terephthalate (DMtP) was evaluated in the micronucleus test in mice. A clear clastogenic effect was obtained at all concentrations studied (0.2-1.0 mmole/kg body weight). The maximum number of micronuclei occurred 24 h after a single intraperitoneal (i.p.) injection. The time-course for the DMtP-induced micronuclei was in agreement with the available data on the rapid excretion of phthalates from the mammalian body. The dose-effect response was best described by a linear equation with a logarithmic component. The emergence of the latter term was related to the toxic effects of DMtP at higher concentrations on bone marrow erythropoietic function. A comparison of the effects induced by DMtP and by methyl nitrosourea indicated that DMtP cannot be considered a strong mutagenic compound. We have compared the sensitivity of the mouse micronucleus test and that of Drosophila dominant-lethal test by contrasting the effects obtained at similar exposure doses. This comparison leads to the conclusion that the micronucleus test is capable of responding to far lower phthalate concentrations than the Drosophila dominant-lethal mutation test. Our results testify to the ability of dimethyl terephthalate to cause genotoxic damages in vivo in both somatic and germinal cells of higher organisms. Thus, the chemical in question may be of potential genetic hazard to man.

[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.

[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).

[Sensitivity of individual Drosophila to the mutagenic action of ethyl methanesulfonate]. / Izuchenie chuvstvitel'nosti otdel'nykh osobei Drozofily k mutagennomi deistviiu étilmetansul'fonata.

The genetic effect of some factors is generally evaluated as an average response of all individuals, without taking into account their potential differences. The presence of individual sensitivity in separate Drosophila organisms to the mutagenic influence of ethyl methanesulfonate was shown when analysing recessive sex-linked lethal mutations in germ cells of males. Different sensitivity of separate individuals to mutagens reflects the existence of cryptic genetic variability in Drosophila strains on a large scale. It is advisable to take into account individual sensitivity of organisms to mutagenic factors, when conducting mutation research and studying genetic consequences of biosphere pollution.

[Mutagenic action of the dimethyl ester of terephthalic acid on murine somatic cells in vivo]. / Mutagennoe deistvie dimetilovogo éfira tereftalevoi kisloty na somaticheskie kletki myshi in vivo.

Mutagenic activity of dimethyl terephthalate (DMtP) was evaluated using the bone marrow micronucleus test in mice. Clear clastogenic effect with the highest response in 24 h after a single i.p. injection was obtained at all concentrations used (0.2-1.0 mM/kg). The time-course for the micronuclei induced by DMtP was in agreement with the literature data on fast excretion of phthalates from mammal body. The dose-response curve for DMtP-induced micronuclei was linear in form with the logarithmic component. The emergence of the latter was related to the elevation of the chemical's concentration to the level at which DMtP starts to exert toxic influence on bone marrow erythropoietic function. The comparison of the effect induced by DMtP with that of methyl nitrosourea indicated that DMtP could not be considered as a strong mutagenic compound. Susceptibility of the micronucleus test was compared with that of Drosophila dominant lethal test in terms of the concentrations at which equal clastogenic effect was seen. This comparison made it possible to conclude that the micronucleus test in mice was able to respond to much lower phthalate concentrations, as compared with the test in Drosophila. The results provided the evidence of capacity of dimethyl terephthalate to cause alterations of genetical structures in both somatic and germinal cells of two highly organized species in vivo.

[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.

[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.

[Cytogenetic effects of 1,4-dihydropyridine in various test systems]. / Tsitogeneticheskii éffekt 1,4-digidropiridina v razlichnykh test-sistemakh.

Cytogenetic effect of 1,4-dihydropyridine was studied in different test-systems. The preparation is shown to decrease the level of complete sex-chromosome losses in Drosophila and chromosome aberration frequency in Allium fistulosum seedlings. The preparation does not affect spontaneous mutability of bone marrow cells in mice, high doses of the preparation have no mutagenic potential. Thus, 1,4-dihydropyridine shows antimutagenic activity reducing the chromosome mutation level in sex and somatic cells of eucaryotic organisms. Absence of the effect on mice chromosomes may testify to the specificity of 1,4-dihydropyridine action.

[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.

[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.

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.