[Selenium and cancer: from prevention to treatment]. / Selén a rakovina: od prevencie k liecbe.

Selenium (Se) is an essential dietary component for all animals, including human beings, that is regarded as a protective agent against cancer. Although the mode of its anticancer action is not yet fully understood, several mechanisms, such as antioxidant protection through selenoenzymes, stimulation of DNA repair, and apoptosis in tumor prestages have all been proposed. Despite the unsupported results of the last "SELECT" trial, the cancer-preventing activity of Se has been demonstrated in a majority of epidemiological studies. Moreover, recent studies suggest that Se has a potential to be used not only in cancer prevention but also in cancer treatment, where in combination with other anticancer drugs or radiation it may increase the efficacy of cancer therapy. In combating cancer cells, Se acts as a prooxidant rather than an antioxidant, inducing apoptosis through the generation of oxidative stress. Thus, inorganic Se compounds, having high redox potency, represent a promising option in cancer therapy.

Further characterization of the role of Pso2 in the repair of DNA interstrand cross-link-associated double-strand breaks in Saccharomyces cerevisiae.

DNA interstrand cross-links (ICL) are thought to be one of the most lethal forms of DNA damage. Therefore, they present a colossal challenge for the DNA damage response and repair pathways. In Saccharomyces cerevisiae, ICL repair utilizes factors from all of the three major repair groups: nucleotide excision repair (RAD3 epistasis group), post-replication repair (RAD6 epistasis group) and recombinational repair (RAD52 epistasis group). Moreover, there are additional factors significantly influencing the repair of ICL in this organism. These have been designated PSO1-10 based on the psoralen sensitive phenotype of the corresponding mutants. Phenotype of the pso2 mutant suggests that Pso2 is not involved in incision step of ICL repair, but it rather functions in some downstream event such as processing of DNA ends created during generation of ICL-associated double-strand breaks (DSB). In order to address the question whether function of Pso2 in the repair of ICL-associated DSB could be mediated through protein-protein interactions, we have conducted a comprehensive two-hybrid screen examining a possibility of interaction of Pso2 with Yku70, Yku80, Nej1, Lif1, Dnl4, Rad50, Mre11, Xrs2, Rad51, Rad52, Rad54, Rad55, Rad57, Rad59 and Rdh54. Here we show that Pso2 associates with none of the above DSB repair proteins, suggesting that this protein very likely does not act in the repair of ICL-associated DSB via crosstalk with DSB repair machinery. Instead, its function in this process seems to be rather individual.

Disruption of the RAD51 gene sensitizes S. cerevisiae cells to the toxic and mutagenic effects of hydrogen peroxide.

The RAD51 gene was disrupted in three different parental wild-type strains to yield three rad51 null strains with different genetic background. The rad51 mutation sensitizes yeast cells to the toxic and mutagenic effects of H2O2, suggesting that Rad51-mediated repair, similarly to that of RecA-mediated, is relevant to the repair of oxidative damage in S. cerevisiae. Moreover, pulsed-field gel electrophoresis analysis demonstrated that increased sensitivity of the rad51 mutant to H2O2 is accompanied by its decreased ability to repair double-strand breaks induced by this agent. Our results show that ScRad51 protects yeast cells from H2O2-induced DNA double-strand breakage.

Effect of expression of the Escherichia coli nth gene in Saccharomyces cerevisiae on the toxicity of ionizing radiation and hydrogen peroxide.

PURPOSE: To examine the contribution of endonuclease III (Nth)-repairable lesions to the cytotoxicity of ionizing radiation (IR) and hydrogen peroxide (H2O2) in the yeast Saccharomyces cerevisiae. MATERIALS AND METHODS: A selectable expression vector containing the E. coli nth gene was transformed into two different wild-type strains (7799-4B and YNN-27) as well as one rad52 mutant strain (C5-6). Nth expression was verified by Western analysis. Colony-forming assay was used to determine the sensitivity to IR and H2O2 in both stationary and exponentially growing cells. RESULTS: The pADHnth-transformed wild-type (77994B) strain was considerably more resistant than vector-only transformants to the toxic effects of IR, in both stationary and exponential growth phases, although this was not the case in another wild-type strain (YNN-27). In contrast, there were no significant effects of nth expression on the sensitivity of the wild-type cells to H2O2. Moreover, nth expression caused no effects on the H2O2 sensitivity in the rad52 mutant cells, but it led to a slight increase in sensitivity in these cells following IR, particularly at the highest dose levels used. CONCLUSIONS: Whilst other damage-processing systems may play a role, DNA lesions that are substrates for Nth can also make a contribution to the toxic effects of IR in certain wild-type yeast. Hence, DNA double-strand breaks should not be considered the sole lethal lesions following IR exposure.

Increased DNA double strand breakage is responsible for sensitivity of the pso3-1 mutant of Saccharomyces cerevisiae to hydrogen peroxide.

Escherichia coli endonuclease III (endo III) is the key repair enzyme essential for removal of oxidized pyrimidines and abasic sites. Although two homologues of endo III, Ntgl and Ntg2, were found in Saccharomyces cerevisiae, they do not significantly contribute to repair of oxidative DNA damage in vivo. This suggests that an additional activity(ies) or a regulatory pathway(s) involved in cellular response to oxidative DNA damage may exist in yeast. The pso3-1 mutant of S. cerevisiae was previously shown to be specifically sensitive to toxic effects of hydrogen peroxide (H2O2) and paraquat. Here, we show that increased DNA double strand breakage is very likely the basis of sensitivity of the pso3-1 mutant cells to H2O2. Our results, thus, indicate an involvement of the Pso3 protein in protection of yeast cells from oxidative stress presumably through its ability to prevent DNA double strand breakage. Furthermore, complementation of the repair defects of the pso3-1 mutant cells by E. coli endo III has been examined. It has been found that expression of the nth gene in the pso3-1 mutant cells recovers survival, decreases mutability and protects yeast genomic DNA from breakage following H2O2 treatment. This might suggest some degree of functional similarity between Pso3 and Nth.

Repair of oxidative DNA damage--an important factor reducing cancer risk. Minireview.

Oxygen free radicals formed during normal aerobic cellular metabolism generate a variety of DNA lesions including modified bases, abasic sites and single strand breaks with blocked 3' termini. If left unrepaired, these damages may contribute to a number of degenerative processes, including cancer and aging. In most organisms, the repair of oxidative DNA lesions is supposed to be handled by the base excision repair (BER) pathway. BER is a multistep process that involves the sequential activity of several proteins, many of them were isolated and functionally characterized using the simple prokaryotic and lower eukaryotic model systems, Escherichia coli and Saccharomyces cerevisiae, respectively. As the amino acid sequence of DNA repair proteins is often well conserved from bacteria to man, our understanding of BER in higher eukaryotes drives extensively from the microbial models, namely from the yeast S. cerevisiae. Thus, results obtained on a simple yeast model are a source of new information, which can be used as a paradigm for all eukaryotic cells.

Oxidative stress in microorganisms--I. Microbial vs. higher cells--damage and defenses in relation to cell aging and death.

Oxidative stress in microbial cells shares many similarities with other cell types but it has its specific features which may differ in prokaryotic and eukaryotic cells. We survey here the properties and actions of primary sources of oxidative stress, the role of transition metals in oxidative stress and cell protective machinery of microbial cells, and compare them with analogous features of other cell types. Other features to be compared are the action of Reactive Oxygen Species (ROS) on cell constituents, secondary lipid- or protein-based radicals and other stress products. Repair of oxidative injury by microorganisms and proteolytic removal of irreparable cell constituents are briefly described. Oxidative damage of aerobically growing microbial cells by endogenously formed ROS mostly does not induce changes similar to the aging of multiplying mammalian cells. Rapid growth of bacteria and yeast prevents accumulation of impaired macromolecules which are repaired, diluted or eliminated. During growth some simple fungi, such as yeast or Podospora spp., exhibit aging whose primary cause seems to be fragmentation of the nucleolus or impairment of mitochondrial DNA integrity. Yeast cell aging seems to be accelerated by endogenous oxidative stress. Unlike most growing microbial cells, stationary-phase cells gradually lose their viability because of a continuous oxidative stress, in spite of an increased synthesis of antioxidant enzymes. Unlike in most microorganisms, in plant and animal cells a severe oxidative stress induces a specific programmed death pathway--apoptosis. The scant data on the microbial death mechanisms induced by oxidative stress indicate that in bacteria cell death can result from activation of autolytic enzymes (similarly to the programmed mother-cell death at the end of bacillary sporulation). Yeast and other simple eukaryotes contain components of a proapoptotic pathway which are silent under normal conditions but can be activated by oxidative stress or by manifestation of mammalian death genes, such as bak or bax. Other aspects, such as regulation of oxidative-stress response, role of defense enzymes and their control, acquisition of stress tolerance, stress signaling and its role in stress response, as well as cross-talk between different stress factors, will be the subject of a subsequent review.

MNNG-induced [corrected] RecBCD dependent DNA degradation in recA13 mutant cells is not the basis of their hypersensitivity to this agent.

We have examined the hypersensitivity of Escherichia coli recA13 mutant cells to killing by N-methyl-N'-nitro-N-nitro-soguanidine (MNNG) and have shown out that despite MNNG-induced adaptation they remained vastly more sensitive to the cytotoxic effect of this agent than wild type cells. Because this might have been a consequence of a different extent of induction of the adaptive response in the recA13 background, we have measured O6-alkylguanine-DNA alkyltransferase (ATase) activity in extracts of adapted and non-adapted recA13 mutant and wild type cells. Adaptation increased ATase levels by 28- and 34-fold in wild type and recA13 mutant cells, respectively. Thus, the adaptive response was no less inducible in recA13 mutant cells than in wild type cells. This indicates that the extreme sensitivity of recA13 cells to MNNG is not caused by an inability to repair the principal toxic lesions induced in DNA. Low doses of MNNG caused substantial degradation of cellular DNA in recA13 mutant cells but not in the wild type cells. This DNA degradation is shown to be the RecBCD-enzyme dependent. Since recA13 recB21 double mutants were even more sensitive to MNNG than recA single mutants, DNA degradation appears not to be the cause of the MNNG-hypersensitivity in recA13 cells.

Role of PSO genes in the repair of photoinduced interstrand cross-links and photooxidative damage in the DNA of the yeast Saccharomyces cerevisiae.

Recent progress in elucidating the molecular structure of the PSSO genes PSO2 to PSO7 is presented. Their role in DNA repair and mutagenesis is discussed in the light of the putative proteins encoded in the respective ORFs and with the knowledge of recent progress in biological and biochemical experimentation. The role of the RecA protein in some steps of DNA repair in Saccharomyces cerevisiae is presented and discussed.

Searching for a functional analogy between yeast Pso4 and bacterial RecA proteins in induced mitotic recombination.

The pso4-1 mutant of S. cerevisiae is phenotypically similar to the recA mutant of E. coli; it is sensitive to DNA cross-linking agents and defective in both recombination and mutagenesis. In this paper we have measured the effect of the recA gene expression on the frequency of mitotic crossing-over and mitotic gene conversion in response to DNA damage induced by photoactivated 8-methoxypsoralen (8-MOP + UVA), ultraviolet radiation (UV) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The diploid pso4-1 mutant and the repair wild type strain were transformed with the multicopy plasmid carrying the recA gene placed under the control of the ADH1 promoter. The results showed that RecA is not able to restore block in induced mitotic recombination in pso4-1 cells after DNA damaging agents used. Thus RecA protein is not able to substitute Pso4 protein in homologous mitotic recombination indicating that they have probably different functions in this process.

Further characterization of the yeast pso4-1 mutant: interaction with rad51 and rad52 mutants after photoinduced psoralen lesions.

The pso4-1 mutant was characterized as deficient in some types of recombination, including gene conversion, crossing over, and intrachromosomal recombination. The mode of interaction between pso4-1 and rad51 and between pso4-1 and rad52 mutants indicated that the PSO4 gene belongs to the RAD52 epistasis group for strand-break repair. Moreover, the presence of the pso4-1 mutation decreased 8-MOP-photoinduced mutagenesis of the rad51 and rad52 mutants. Complementation tests using heterozygous diploid strains showed that the pso4 protein might interact with the rad52 protein during repair of 8-mop photolesions. The pso4-1 mutant, even though defective in inter- and intra-chromosomal recombination, conserves the ability for plasmid integration of circular and linear plasmid DNA. On the other hand, similar to the rad51 mutant, pso4-1 was able to incise but did not restore high-molecular-weight DNA during the repair of cross links induced by 8-MOP plus UVA. These results, together with those of previous reports, indicate that the PSO4 gene belongs to the RAD52 DNA repair group and its product participates in the DNA rejoining step of the repair of cross-link lesions, which are crucial for induced mutagenesis and recombinogenesis.

Biological consequences of E.coli RecA protein expression in the repair defective pso4-1 and rad51::URA3 mutants of S. cerevisiae after treatment with N-methyl-N'-nitro-N-nitrosoguanidine.

RecA protein of E.coli is a multifunctional protein participating in genetic recombination, recombinational repair and mutagenesis. We used E.coli recA gene as a probe for complementation of repair defects after treatment of N-methyl-N'-nitro-N-nitrosoguanidine in the pso4-1 and rad51::URA3 mutants of S. cerevisiae. We tried to find the role of the RecA protein in S. cerevisiae mutants defective in different repair pathways. The RecA protein had no effect on survival of haploid and diploid pso4-1 mutants, but it had a significant effect on MNNG induced mutagenesis, which was increased to the wild type level. No effect of the RecA protein on survival was observed in rad51::URA3 mutant after MNNG treatment. However, in this case the RecA protein decreased the induced mutagenesis. We can suppose that the RecA protein, with its multifunctional enzymatic activity, can bind to special intermediates and initiate function of different repair pathways depending on repair defects of the mutants studied.

Expression of Escherichia coli recA and ada genes in Saccharomyces cerevisiae using a vector with geneticin resistance.

Construction of E. coli-yeast shuttle plasmids containing the neo selection gene is described. The protein-coding regions of the E. coli ada or recA genes under the control of the ADH1 promoter and terminator were ligated into the SphI unique site of pNF2 to produce pMSada and pMSrecA, respectively. The plasmids were used for transformation of the haploid and diploid pso4-1 strains of S. cerevisiae and their corresponding wild types. Transformants were obtained by selection for geneticin (G418) resistance. Crude protein samples were extracted from the individual transformants. Both the RecA and Ada proteins were present in all strains containing the recA and ada genes on plasmids, respectively. Thus the geneticin selection system was successfully used for the preparation of model yeast strains.

Expression of the E.coli ada gene in S.cerevisiae provides cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine in rad6 but not in rad52 mutants.

The Escherichia coli ada gene protein coding region under the control of the yeast alcohol dehydrogenase promoter in the extrachromosomally replicating yeast expression vectors pADHO6C and pVT103LO6C was introduced into the wild-type yeast strains, YNN-27 and FF-18733, and the repair deficient mutants LN-1 (rad1-1), VV-5 (rad6-1), C5-6 (rad52-1) and FF-18742 (rad52::URA3). This resulted in the expression of 3950, 1900, 1870, 1620, 1320 and 1420 fmol ada-encoded ATase/mg protein respectively: transformation with the parent vectors resulted in ATase activities of 3-17 fmol/mg protein. The wild-types, rad1-1 and rad6-1 yeast expressing the bacterial ATase showed increased resistance to the toxic and mutagenic effects of N-methyl-N'-nitro-N- nitrosoguanidine (MNNG). Expression of ATase in the rad52-1 and rad52::URA3 mutants neither complemented their sensitivity, nor reduced the mutagenic effects of this agent. These results suggest that whilst a portion of the toxic and mutagenic lesions induced by MNNG can be repaired in yeast by the E.coli Ada protein in a RAD1- and RAD6-independent manner, the RAD52 gene product may be essential for the complete functioning of the Ada ATase. This is the first suggestion of a possible cofactor requirement for ATase.

The Escherichia coli recA gene increases UV-induced mitotic gene conversion in Saccharomyces cerevisiae.

The effect of the Escherichia coli RecA protein on mitotic recombination in the diploid D7 strain of Saccharomyces cerevisiae damaged by UV radiation was investigated. The D7 strain was transformed by two modified versions of the pNF2 plasmid: one, containing the ADH-1 promoter, and the other containing the recA gene tandemly arranged behind the ADH-1 promoter region. Immunological analysis proved the presence of the 38-kDa RecA protein in D7/pNF2ADHrecA transformants. We observed a positive effect of recA gene expression on mitotic gene conversion, mainly at higher doses of UV radiation. The results indicate that a RecA-like activity could participate in steps preceding mitotic conversion events in yeast.

The E. coli recA gene can restore the defect in mutagenesis of the pso4-1 mutant of S. cerevisiae.

The E. coli recA gene was introduced into the pso4-1 mutant of S. cerevisiae and transformants were treated with 8-MOP+UVA and 254-nm UV light. The results showed that the recA gene increased the resistance to the toxic effect of 8-MOP+UVA and restored the frequency of reversion of the pso4-1 mutants after both treatments. The presence of the recA gene stimulated expression of the small subunit of the ribonucleotide reductase (Rnr2) in the pso4-1 mutants. Thus the E. coli recA gene is functional in yeast. Moreover, it was shown that the pso4-1 mutant is epistatic to pso1-1 and rad6-1, which belong to a mutagenic repair pathway. We propose here that the PSO4 gene has some role in the control of mutagenic repair in yeast.

A recA-ada hybrid gene inducible by DNA damage.

A damage-inducible expression vector was constructed in which the original recA structural gene was replaced by the protein-coding region of the ada gene. The O6-alkylguanine-DNA alkyltransferase encoded by the ada gene can be measured by a rapid and highly sensitive assay. The introduction of this construct into an appropriate host cell provides an effective bacterial assay for genotoxins.

UV-induced mutability in repair-deficient rad6-1 strains of Saccharomyces cerevisiae is caused by a suppressor gene.

The RAD6 gene is a multifunctional gene required for DNA repair, induced mutagenesis and sporulation. The survival and revertibility of two loci in four rad6-1 mutant strains of different origin after UV irradiation were followed. As expected, all the rad6-1 strains tested were more sensitive to UV radiation in comparison with RAD6 strains. The reversion frequency per survivor in trp1-289 and arg4-17 alleles was significantly higher in all four rad6-1 mutant strains than in wild-type strains after equal doses of UV radiation. On the basis of genetic analysis we suggest that the phenomenon of increased frequency of induced mutagenesis is caused by a suppressor gene.

The Escherichia coli recA gene increases resistance of the yeast Saccharomyces cerevisiae to ionizing and ultraviolet radiation.

The Escherichia coli recA protein coding region was ligated into an extrachromosomally replicating yeast expression vector downstream of the yeast alcohol dehydrogenase promoter region to produce plasmid pADHrecA. Transformation of the wild-type yeast strains YNN-27 and 7799-4B, as well as the recombination-deficient rad52-1 C5-6 mutant, with this shuttle plasmid resulted in the expression of the bacterial 38 kDa RecA protein in exponential phase cells. The wild-type YNN27 and 7799-4B transformants expressing the bacterial recA gene showed increased resistance to the toxic effects of both ionizing and ultraviolet radiation. RecA moderately stimulated the UV-induced mutagenic response of 7799-4B cells. Transformation of the rad52-1 mutant with plasmid pADHrecA did not result in the complementation of sensitivity to ionizing radiation. Thus, the RecA protein endows the yeast cells with additional activities, which were shown to be error-prone and dependent on the RAD52 gene.