Methyl Methanesulphonate Research Articles

Differential roles of proteins involved in migration of Holliday junctions on recombination and tolerance to DNA damaging agents in Rhizobium etli

The recombination genes involved in Holliday junction migration ( ruvB, recG, radA) and heteroduplex editing ( mutS) were studied in the α-proteobacterium Rhizobium etli. The genes were interrupted with a loxPSp interposon and R. etli mutants, either single or in combination, were constructed by marker exchange. Our results show that these systems play a differential role in sensitivity to DNA damaging agents and recombination in R. etli. RuvB appears to be the main system for tolerance toward agents instigating single- or double-strand breaks (such as UV light, methyl methanesulphonate and nalidixic acid) while the RecG and RadA systems play minor roles in tolerance to these agents. Using five different recombination assays, we have found that a ruvB null mutant showed a notable reduction in recombination proficiency, while a radA mutant was only weakly affected. A null mutation in recG had the opposite effect, enhancing recombination in most of our assays. This effect was more clearly seen in an assay that measured recombination between divergent sequences (i.e. homeologous), but is unaffected by inactivation of mutS. These data indicate that RecG in R. etli limits intra- and intergenomic plasticity.

Antimutagenic effect of sage tea in the wing spot test of Drosophila melanogaster

The present study assayed the antimutagenic potential of Salvia officinalis (sage) in the form of tea infusion, by the somatic mutation and recombination test (SMART) on Drosophila melanogaster. The use of herbal infusions is much common in the human diet, so the aim of the present study was to estimate the antimutagenic effects of the S. officinalis tea rather than essential oils. Methyl methanesulphonate (MMS) was used as the mutagen and positive control. Several types of treatment were performed: short acute treatment with sage infusion or MMS, longer (chronic) treatment with sage solution or MMS, and two combined treatments, i.e. short treatment with sage followed by a longer treatment with MMS and vice versa. Sage infusion used in our experiments showed a clear antimutagenic effect, reducing the frequency of mutations induced by MMS. The inhibition effect of sage tea is obtained and confirmed when pre- or post-treatments with mutagen were used. The results indicate that although sage in this regime decreases the number of mutation events, it is not efficient enough in case of the 2 h sage pre-treatment. Antioxidant activity, suppression of metabolic activation, could be mechanisms through which sage or some of its components act as desmutagen.

No-observed effect levels are associated with up-regulation of MGMT following MMS exposure

The alkylating agents methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS) have non-linear dose–response curves, with a no-observed effect level (NOEL) and a lowest observed effect level (LOEL) for both gross chromosomal damage and mutagenicity. However, the biological mechanism responsible for the NOEL has yet to be identified. A strong candidate is DNA repair as it may be able to efficiently remove alkyl adducts at low doses resulting in a NOEL, but at higher doses fails to fully remove all lesions due to saturation of enzymatic activity resulting in a LOEL and subsequent linear increases in mutagenicity. We therefore assessed the transcriptional status of N-methylpurine-DNA glycoslase (MPG) and O6-methylguanine DNA methyltransferase (MGMT), which represent the first line of defence following exposure to alkylating agents through the respective enzymatic removal of N7-alkylG and O6-alkylG. The relative MPG and MGMT gene expression profiles were assessed by real-time RT-PCR following exposure to 0–2μg/ml MMS for 1–24h. MPG expression remained fairly steady, but in contrast significant up-regulation of MGMT was observed when cells were treated with 0.5 and 1.0μg/ml MMS for 4h (2.5- and 6.5-fold increases respectively). These doses lie within the NOEL for MMS mutagenicity (LOEL is 1.25μg/ml), thus this boost in MGMT expression at low doses may be responsible for efficiently repairing O6methylG lesions and creating the non-linear response for mutations. However, as the LOEL for MMS clastogenicity is 0.85μg/ml, O6-alkylG is unlikely to be responsible for the clastogenicity observed at these concentrations. Consequently, at low doses N7-methylG is possibly the predominant cause of MMS clastogenicity, while O6-methylG is more likely to be responsible for MMS mutagenicity, with MGMT up-regulation playing a key role in removal of O6-alkylG lesions before they are fixed as permanent point mutations, resulting in non-linear dose–responses for direct acting genotoxins.

Localization of Smc5/6 to centromeres and telomeres requires heterochromatin and SUMO, respectively

The Smc5/6 holocomplex executes key functions in genome maintenance that include ensuring the faithful segregation of chromosomes at mitosis and facilitating critical DNA repair pathways. Smc5/6 is essential for viability and therefore, dissecting its chromosome segregation and DNA repair roles has been challenging. We have identified distinct epigenetic and post-translational modifications that delineate roles for fission yeast Smc5/6 in centromere function, versus replication fork-associated DNA repair. We monitored Smc5/6 subnuclear and genomic localization in response to different replicative stresses, using fluorescence microscopy and chromatin immunoprecipitation (ChIP)-on-chip methods. Following hydroxyurea treatment, and during an unperturbed S phase, Smc5/6 is transiently enriched at the heterochromatic outer repeats of centromeres in an H3-K9 methylation-dependent manner. In contrast, methyl methanesulphonate treatment induces the accumulation of Smc5/6 at subtelomeres, in an Nse2 SUMO ligase-dependent, but H3-K9 methylation-independent manner. Finally, we determine that Smc5/6 loads at all genomic tDNAs, a phenomenon that requires intact consensus TFIIIC-binding sites in the tDNAs.

Automated image analysis of cytokinesis-blocked micronuclei: an adapted protocol and a validated scoring procedure for biomonitoring

Micronuclei (MN) frequencies in peripheral blood lymphocytes have been used worldwide as a biomarker of chromosomal damage for genotoxicity testing and biomonitoring studies. Automation of MN analysis would provide faster and more reliable results with minimizing subjective MN identification. We developed an automated facility for the scoring of the in vitro MN cytokinesis-block assay for biomonitoring on Giemsa-stained slides, fulfilling the following criteria: applicable to the cytokinesis-block micronucleus methodology, discriminating between mono-, bi- and polynucleated cells, MN scoring according to HUMN scoring criteria, false-negative MN rate <10% and false-positive (FP) MN rate <1%. We first adapted the slide preparation protocol to obtain an optimal cell density and dispersion, which is important for image analysis. We developed specific algorithms starting from the cell as a detection unit. The whole detection and scoring process was separated into two distinct steps: in the first step, the cells and nuclei are detected; then, in the second step, the MN are searched for in the detected cells. Since the rate of FP MN obtained by the automatic analysis was in the range of 0.5-1.5%, an interactive visual validation step was introduced, which is not time consuming and allows quality control. Validation of the automated scoring procedure was undertaken by comparing the results of visual and automated scoring of micronucleated mono- and binucleated cells in human lymphocytes induced by two clastogens (ionizing radiation and methyl methane-sulphonate), two aneugens (nocodazole and carbendazim) and one apoptogen (staurosporine). Although the absolute MN frequencies obtained with automated scoring were lower as compared to those detected by visual scoring, a clear dose response for MNBN frequencies was observed with the automated scoring system, indicating that it is able to produce biologically relevant and reliable results. These observations, together with its ability to detect cells, nuclei and MN in accordance with the HUMN scoring criteria, confirm the usability of the automated MN analysis system for biomonitoring.

Influence of DNA repair gene polymorphisms on the initial repair of MMS‐induced DNA damage in human lymphocytes as measured by the alkaline comet assay

We have applied the alkaline comet assay to study the functional impact of gene polymorphisms in base excision repair (APEX1 Asp148Glu, XRCC1 Arg194Trp, XRCC1 Arg399Gln) and homologous recombination repair (XRCC3 Thr241Met, NBS1 Glu185Gln), two pathways that play crucial roles in the repair of DNA damage induced by methylmethane sulphonate (MMS). We also examined the effect of polymorphisms in mismatch repair (MLH1 -93 A/G) and nucleotide excision repair (XPD Lys751Gln) as putative negative controls based on the limited roles of these pathways in MMS-induced repair. Phytohemagglutinin-stimulated peripheral lymphocytes from 52 healthy individuals were treated with MMS and allowed to repair for 0, 15, 40, or 120 min after a 6-min washing step. DNA damage was measured as a pseudo-percentage score (comparable to % tail DNA) converted from a total visual score calculated from the distribution of cells with different degrees of damage (normal, mild, moderate and severe). The repair was faster at the beginning of the observation period than towards the end, and was not complete after 2 hr. Presence of the APEX1 148Asp, XRCC3 241Met or NBS1 185Gln alleles were significantly associated with a high pseudo-percentage score (above median) at early time points, with the APEX1 effect being most prolonged (up to 40 min after washing, odds ratio 5.6, 95% confidence interval 2.0-15.5). No significant effects were seen with the XRCC1 Arg194Trp, XRCC1 Arg399Gln, MLH1 -93A/G and XPD Lys751Gln polymorphisms. Our results provide evidence for the functional nature of the variant alleles studied in the APEX1, XRCC3, and NBS1 genes.

Planarian neoblast micronucleus assay for evaluating genotoxicity

Planarian neoblasts are somatic stem cells that have the potential to be used in genotoxicity assays due to their proliferative nature, sensitivity to genotoxic agents, and experimental accessibility. Two freshwater planarian species, Girardia tigrina and Girardia schubarti, were used to develop a neoblast-based micronucleus (MN) assay to assess genotoxicity. Intact or regenerating planarians were exposed to γ-rays, methyl methanesulphonate (MMS), or cyclophosphamide (CP), and neoblast MN frequency was measured. Exposure to the clastogens had no detectable effect on the MN frequency of intact planarian neoblasts. However, for regenerating individuals, active neoblast proliferation was induced by decapitation, and γ-ray doses as low as 0.5 Gy, and MMS and CP concentrations as low as 0.8 μM and 100 mM, respectively, induced a significant increase in MN frequency. Exposure to higher doses of γ-rays consistently resulted in detectable increases in MN frequency. For MMS and CP, concentrations of up to 1.6 μM and 200 mM, respectively, resulted in significant increases in MN frequency, but exposures to higher concentrations led to a decrease to non-significant levels, possibly due to cytotoxic effects of MMS and CP. After completion of regeneration, the MN frequencies returned to those of non-exposed controls, indicating that the neoblast MN assay for regenerating G. tigrina or G. schubarti reflects chromosomal damage caused by acute exposure to clastogenic agents. Upon standardization, this assay may represent an interesting alternative that allows damage caused to freshwater organisms by potentially genotoxic environmental pollutants to be monitored.

Evaluation of the Litron In Vitro MicroFlow ® Kit for the flow cytometric enumeration of micronuclei (MN) in mammalian cells

We have evaluated the performance of the prototype In Vitro MicroFlow ® Kit (Litron Laboratories), which offers a flow cytometric method for scoring micronuclei (MN). This method uses sequential staining to differentiate MN from chromatin fragments derived from apoptotic or necrotic cells. Data were generated using the genotoxins methylmethane sulphonate (MMS), dimethylbenzanthracene (DMBA) and vinblastine, and the non-genotoxins dexamethasone and staurosporine, which are known to induce apoptosis in vitro. The results obtained with these agents were compared with conventional microscopy. For short-duration exposures (3–4 h) both manual and flow methodologies demonstrated good concordance, with concentration-related increases in the percentage of MN for MMS, DMBA and vinblastine. Statistically significant increases were observed at ≥20 and 40 μg/mL, for manual and flow analysis, respectively, for MMS; at 0.5 and 0.75 μg/mL for DMBA; and at 0.035 and 0.04 μg/mL, respectively, for vinblastine. Dexamethasone showed clear negative responses by manual and flow cytometric analysis, with comparable results for both methodologies (all <1.7-fold compared with concurrent vehicle controls). Data for staurosporine, however, were less consistent showing significantly higher flow cytometric MN frequencies compared with those seen after manual analysis. Continuous (24 h) treatments were also conducted with MMS, vinblastine, dexamethasone and staurosporine. There was good concordance between the methodologies for MMS, staurosporine and vinblastine. However, dexamethasone generated discordant results, i.e. microscopic analysis was clearly negative at all doses tested, whereas flow cytometry produced significant increases in MN frequency (up to 8.1-fold at 100 μg/mL compared with the concurrent vehicle control). The inconsistencies observed between flow cytometry and standard microscopy, and the differences in assay sensitivity, particularly for apoptosis-inducing compounds, suggest that the prototype In Vitro MicroFlow ® Kit requires further refinement. Studies to investigate new parameters to address these issues are now under way and will be reported separately.

Allelism of Saccharomyces cerevisiae gene PSO10, involved in error-prone repair of psoralen-induced DNA damage, with SUMO ligase-encoding MMS21

In order to extend the understanding of the genetical and biochemical basis of photo-activated psoralen-induced DNA repair in the yeast Saccharomyces cerevisiae we have identified and cloned 10 pso mutants. Here, we describe the phenotypic characterization and molecular cloning of the pso10-1 mutant which is highly sensitive to photoactivated psoralens, UV(254) (nm) radiation and the alkylating agent methylmethane sulphonate. The pso10-1 mutant allele also confers a block in the mutagenic response to photoactivated psoralens and UV(254) (nm) radiation, and homoallelic diploids do not sporulate. Molecular cloning using a yeast genomic library, sequence analysis and genetic complementation experiments proved pso10-1 to be a mutant allele of gene MMS21 that encodes a SUMO ligase involved in the sumoylation of several DNA repair proteins. The ORF of pso10-1 contains a single nucleotide C-->T transition at position 758, which leads to a change in amino acid sequence from serine to phenylalanine [S253F]. Pso10-1p defines a leaky mutant phenotype of the essential MMS21 gene, and as member of the Smc5-Smc6 complex, still has some essential functions that allow survival of the mutant. DNA repair via translesion synthesis is severely impaired as the pso10-1 mutant allele confers severely blocked induced forward and reverse mutagenesis and shows epistatic interaction with a rev3Delta mutant allele. By identifying the allelism of PSO10 and MMS21 we demonstrate the need of a fully functional Smc5-Smc6 complex for a WT-like adequate repair of photoactivated psoralen-induced DNA damage in yeast.

A yeast-based genetic screening to identify human proteins that increase homologous recombination

To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR.

Crystal Structure and DNA Repair Activities of the AP Endonuclease from Leishmania major

Apurinic/apyrimidinic endonucleases initiate the repair of abasic sites produced either spontaneously, from attack of bases by reactive oxygen species or as intermediates during base excision repair. The catalytic properties and crystal structure of Leishmania major apurinic/apyrimidinic endonuclease are described and compared with those of human APE1 and bacterial exonuclease III. The purified enzyme is shown to possess apurinic/apyrimidinic endonuclease activity of the same order as eukaryotic and prokaryotic counterparts and an equally robust 3′-phosphodiesterase activity. Consistent with this, expression of the L. major endonuclease confers resistance to both methyl methane sulphonate and H 2O 2 in Escherichia coli repair-deficient mutants while expression of the human homologue only reverts methyl methane sulphonate sensitivity. Structural analyses and modelling of the enzyme–DNA complex demonstrates a high degree of conservation to previously characterized homologues, although subtle differences in the active site geometry might account for the high 3′-phosphodiesterase activity. Our results confirm that the L. major's enzyme is a key element in mediating repair of apurinic/apyrimidinic sites and 3′-blocked termini and therefore must play an important role in the survival of kinetoplastid parasites after exposure to the highly oxidative environment within the host macrophage.

Nicotinamidase activity is important for germination

It has been suggested that nicotinamide must be degraded during germination; however, the enzyme responsible and its physiological role have not been previously studied. We have identified an Arabidopsis gene, NIC2, that is expressed at relatively high levels in mature seed, and encodes a nicotinamidase enzyme with homology to yeast and bacterial nicotinamidases. Seed of a knockout mutant, nic2-1, had reduced nicotinamidase activity, retarded germination and impaired germination potential. nic2-1 germination was restored by after-ripening or moist chilling, but remained hypersensitive to application of nicotinamide or ABA. Nicotinamide is a known inhibitor of NAD-degrading poly(ADP-ribose) polymerases (PARP enzymes) that are implicated in DNA repair. We found reduced poly(ADP)ribosylation levels in nic2-1 seed, which were restored by moist chilling. Furthermore, nic2-1 seed had elevated levels of NAD, and germination was hypersensitive to methyl methanesulphonate (MMS), suggesting that PARP activity and DNA repair responses were impaired. We suggest that nicotinamide is normally metabolized by NIC2 during moist chilling or after-ripening, which relieves inhibition of PARP activity and allows DNA repair to occur prior to germination.

Global protein expression profiling of budding yeast in response to DNA damage

Exposure to DNA-damaging agents can activate cell cycle checkpoint and DNA repair processes to ensure genetic integrity. Such exposures also can affect the transcription of many genes required for these processes. In the budding yeast Saccharomyces cerevisiae, changes of global gene expression as a result of a DNA-damaging agent were previously identified by using DNA chip technology. DNA microarray analysis is a powerful tool for identifying genes whose expressions are changed in response to environmental changes. Transcriptional levels, however, do not necessarily reflect cellular protein levels. Green fluorescent protein (GFP) has been widely used as a reporter of gene expression and subcellular protein localization. We have used 4156 yeast strains expressing full-length, chromosome-tagged GFP fusion proteins to monitor changes of protein levels in response to the DNA-damaging agent, methyl methanesulphonate (MMS). Through flow cytometry, we identified 157 proteins whose levels were increased at least three-fold following treatment with MMS. Of 157 responsible genes, transcriptions of 57 were previously not known to be induced by MMS. Immunoblot experiments with tandem affinity-tagged yeast strains under the same experimental conditions confirmed these newly found proteins as inducible. These results suggest, therefore, that the 57 protein expressions are regulated by different mechanisms, such as post-translational modifications, and not by transcriptional regulation.

Cyclosporin a Potentiates the Cytotoxic Effects of Methyl Methanesulphonate in HL-60 and K562 Cells

Methyl methanesulphonate (MMS) is a DNA damaging agent, which induces oxidative stress, ATP depletion, and consequently, cell death, in HL-60 and K562 cells. The cell death induced by MMS predominantly exhibited the morphological and biochemical hallmarks of necrosis. A minor population of dying cells exhibited apoptotic hallmarks, especially in K562 cell cultures. Cyclosporin A (CsA) was used to modulate the MMS-induced cell death. Our results indicated that CsA did not prevent cells from dying, but changed the mode of death from necrotic to apoptotic. Surprisingly, CsA enhanced oxidative stress and increased the overall number of dead cells. Based on these results, we conclude that the modulatory effect of CsA on MMS-induced cell death might arise from an interference by CsA with mitochondrial metabolism, rather than from inhibition of the MMS efflux mediated by P-glycoprotein.

Role of Artemis in DSB repair and guarding chromosomal stability following exposure to ionizing radiation at different stages of cell cycle

We analyzed the phenotype of cells derived from SCID patients with different mutations in the Artemis gene. Using clonogenic survival assay an increased sensitivity was found to X-rays (2–3-fold) and bleomycin (2-fold), as well as to etoposide, camptothecin and methylmethane sulphonate (up to 1.5-fold). In contrast, we did not find increased sensitivity to cross-linking agents mitomycin C and cis-platinum. The kinetics of DSB repair assessed by pulsed-field gel electrophoresis and γH2AX foci formation after ionizing irradiation, indicate that 15–20% of DSB are not repaired in Artemis-deficient cells. In order to get a better understanding of the repair defect in Artemis-deficient cells, we studied chromosomal damage at different stages of the cell cycle. In contrast to AT cells, Artemis-deficient cells appear to have a normal G 1/S-block that resulted in a similar frequency of dicentrics and translocations, however, frequency of acentrics fragments was found to be 2–4-fold higher compared to normal fibroblasts. Irradiation in G 2 resulted in a higher frequency of chromatid-type aberrations (1.5–3-fold) than in normal cells, indicating that a fraction of DSB requires Artemis for proper repair. Our data are consistent with a function of Artemis protein in processing of a subset of complex DSB, without G 1 cell cycle checkpoint defects. This type of DSB can be induced in high proportion and persist through S-phase and in part might be responsible for the formation of chromatid-type exchanges in G 1-irradiated Artemis-deficient cells. Among different human radiosensitive fibroblasts studied for endogenous (in untreated samples) as well as X-ray-induced DNA damage, the ranking order on the basis of higher incidence of spontaneously occurring chromosomal alterations and induced ones was: ligase 4 ≥ AT > Artemis. This observation implicates that in human fibroblasts following exposure to ionizing radiation a lower risk might be created when cells are devoid of endogenous damage.

The protein kinase TOUSLED is required for maintenance of transcriptional gene silencing in Arabidopsis

TOUSLED-like kinases (TLKs) are highly conserved in plants and animals, but direct evidence linking TLKs and transcriptional gene silencing is lacking. We isolated two new alleles of TOUSLED (TSL). Mutations of TSL in ros1 reactivate the transcriptionally silent 35S-NPTII transgene and the transcriptionally silent endogenous loci TSI (TRANSCRIPTIONAL SILENCING INFORMATION). Chromatin immunoprecipitation (ChIP) analysis shows that histone H3Lys9 dimethylation is decreased in the reactivated transgene and endogenous TSI loci in the tsl ros1 mutant. However, there is no change in DNA methylation in the affected loci. Western blot and ChIP assay suggest that TSL might not be responsible for histone H3Ser10 phosphorylation. The tsl seedlings were more sensitive to DNA damage reagent methyl methanesulphonate and UV-B light. Our results provide direct evidence for a crucial role of the TOUSLED protein kinase in the maintenance of transcriptional gene silencing in some genomic regions in a DNA-methylation-independent manner in Arabidopsis.

Ecological–genetic feedback in DNA repair in wild barley, Hordeum spontaneum

Regulation of genetic variation in natural populations is a problem of primary importance to evolutionary biology. In the reported study, the repair efficiency of double strand DNA breaks was compared in six wild barley accessions from Israeli natural populations of H. spontaneum: three from mesic populations (one from Maalot and two from Mount Meron, Upper Galilee) and three from xeric populations (one from Wadi Quilt in the Judean Desert and two from Sede Boqer, in the northern Negev Desert). Pulsed field gel electrophoresis was used to score double-strand breaks of DNA (DSBs) caused by methyl methanesulphonate (MMS) treatment. All six accessions were also tested for heat tolerance: four of these, three xeric and one mesic (from Maalot population), were scored as heat tolerant whereas both accessions from Mount Meron population displayed heat sensitivity. MMS caused a significant increase in the level of DSBs relative to the control in all accessions. The major questions were whether and how the efficiency of DNA repair after mutagenic treatment is affected by the environmental conditions and accession's adaptation to these conditions. Differences were found among the accessions in the repair pattern. Plants of two out of the four heat tolerant accessions did not manage to repair DNA neither at 25 degrees Celsius nor at 37 degrees Celsius. The remaining two heat tolerant accessions significantly repaired the breaks at 37 degrees Celsius, but not at 25 degrees Celsius. By contrast, plants of the two heat susceptible accessions significantly lowered the level of DSBs at 25 degrees Celsius but not at 37 degrees Celsius. Therefore, the accessions that proved capable to repair the induced damages in DNA at one of the two temperatures displayed a pattern that may imply the existence of a negative feedback mechanism in regulation of genetic variation. Such a dependence of DNA integrity on environment and genotype may serve an important factor for maintaining relatively high level of mutability without increasing the genetic load.

A stress regulatory network for co‐ordinated activation of proteasome expression mediated by yeast heat shock transcription factor

Heat shock transcription factor (HSF) mediates the transcriptional response of eukaryotic cells to heat, infection and inflammation, pharmacological agents, and other stresses. Although genes encoding heat shock proteins (HSPs) are the best characterized targets of HSF, recent genome-wide localization of Saccharomyces cerevisiae HSF revealed novel HSF targets involved in a wide range of cellular functions. One such target, the RPN4 gene, encodes a transcription factor that directly activates expression of a number of genes encoding proteasome subunits. Here we demonstrate that HSF co-ordinates a feed-forward gene regulatory circuit for RPN4 activation. We show that HSF activates expression of PDR3, encoding a multidrug resistance (MDR) transcription factor that also directly activates RPN4 gene expression. We demonstrate that the HSF binding site (HSE) in the RPN4 promoter is primarily responsible for heat- or methyl methanesulphonate induction of RPN4, with a minor contribution of Pdr3 binding sites (PDREs), while a Yap1 binding site (YRE) is responsible for RPN4 induction in response to oxidative stress. Furthermore, heat-induced expression of Rpn4 protein leads to expression of Rpn4 targets at later stages of heat stress, providing a temporal controlling mechanism for proteasome synthesis upon stress conditions that could result in irreversibly damaged proteins. In addition, the overlapping transcriptional regulatory networks involving HSF, Yap1 and Pdr3 suggest a close linkage between stress responses and pleiotropic drug resistance.

Lack of in vitro Genotoxicity of Povidone-Iodine in Solution, in Ointment or in a Liposomal Formulation (Repithel®)

The in vitro genotoxicity of povidone-iodine in Repithel^®, Betaisodona^® ointment and Betaisodona^® solution was investigated using CHO-K1 cells. The comet assay and chromosome aberration test were used to characterise the genotoxic potency of povidone-iodine within 4 h of contact with test cells. Methyl methanesulphonate without S9 activation and benzo[a]pyrene with S9 activation were used as positive controls. No higher concentrations than 6% Repithel, 5% Betaisodona ointment and 2% Betaisodona solution in culture medium with 10% fetal bovine serum were tested within 4 h of contact with CHO-K1 cells for the comet assay, because higher concentrations will provide >60% cytotoxicity. All tested concentrations of povidone-iodine formulations (4–6% Repithel, 3–5% Betaisodona ointment and 1–2% Betaisodona solution) are not genotoxic in the comet assay. 10% Repithel, 5% Betaisodona ointment and 2.5% Betaisodona solution in culture medium with 10% fetal bovine serum were final concentrations in the chromosome aberration test, which provide <60% cytotoxicity within 4 h of contact with CHO-K1 cells. All tested concentrations of povidone- iodine formulations (5–10% Repithel, 2.5–5% Betaisodona ointment and 1.25–2.5% Betaisodona solution) are not genotoxic in the chromosome aberration test. Using the comet assay and chromosome aberration test to characterise the genotoxic potency of povidone-iodine within 4 h of contact with CHO-K1 cells, there was no chromosome damage detected.

The role of natural biopolymers in genotoxicity of mutagens/carcinogens elimination

Nowadays naturally occurring compounds with the potential antimutagenic and anticarcinogenic effects are of great importance for their prospective use in cancer chemoprevention and treatment. The new water soluble derivative of microbial polysaccharide beta-D-glucan-carboxymethyl glucan (CMG) belongs to such a category of natural substances. CMG isolated from the cell wall of baker's yeast Saccharomyces cerevisiae is included into the class of biopolymers known as biological response modifiers (BRMs) with a broad range of activities, above all ones interfering with cancer therapy. It was demonstrated on four experimental model systems that biological and consequential medicinal importance of CMG is based on the combined application with another active compound. In the Saccharomyces cerevisiae antimutagenicity assay CMG significantly reduced ofloxacin-induced mutagenicity in the yeast strain D7. CMG exerted bioprotective (anti-toxic and antimutagenic) effect after its simultaneos application with methyl methanesulphonate on the repair-deficient strain uvs10 of the unicellular green alga Chlamydomonas reinhardtii. In the Vicia sativa simultaneous phytotoxicity and anticlastogenicity assay CMG exerted statistically significant anticlastogenic efect against maleic hydrazide-induced clastogenicity in Vicia sativa L. Only in the Salmonella/microsome assay CMG did not exert statistically significant antigenotoxic effect, despite of the fact that it reduced 9-aminoacridine-induced mutagenicity in S. typhimurium TA97, but his(+) revertants decreasing was statistically significant only at the highest CMG concentration used. The data presented unambiguously documented that even biopolysaccharides (e.g., derivatives of beta-glucan) belonging to the most abundant class of natural biopolymers may contribute to cancer prevention and therapy.