A suppressive role of guanine nucleotide-binding protein subunit beta-4 inhibited by DNA methylation in the growth of anti-estrogen resistant breast cancer cells.

BACKGROUND: Breast cancer is the most common malignancy in women worldwide. Although the endocrine therapy that targets estrogen receptor α (ERα) signaling has been well established as an effective adjuvant treatment for patients with ERα-positive breast cancers, long-term exposure may eventually lead to the development of acquired resistance to the anti-estrogen drugs, such as fulvestrant and tamoxifen. A better understanding of the mechanisms underlying antiestrogen resistance and identification of the key molecules involved may help in overcoming antiestrogen resistance in breast cancer. METHODS: The whole-genome gene expression and DNA methylation profilings were performed using fulvestrant-resistant cell line 182R-6 and tamoxifen-resistant cell line TAMR-1 as a model system. In addition, qRT-PCR and Western blot analysis were performed to determine the levels of mRNA and protein molecules. MTT, apoptosis and cell cycle analyses were performed to examine the effect of either guanine nucleotide-binding protein beta-4 (GNB4) overexpression or knockdown on cell proliferation, apoptosis and cell cycle. RESULTS: Among 9 candidate genes, GNB4 was identified and validated by qRT-PCR as a potential target silenced by DNA methylation via DNA methyltransferase 3B (DNMT3B). We generated stable 182R-6 and TAMR-1 cell lines that are constantly expressing GNB4 and determined the effect of the ectopic GNB4 on cell proliferation, cell cycle, and apoptosis of the antiestrogen-resistant cells in response to either fulvestrant or tamoxifen. Ectopic expression of GNB4 in two antiestrogen resistant cell lines significantly promoted cell growth and shortened cell cycle in the presence of either fulvestrant or tamoxifen. The ectopic GNB4 induced apoptosis in 182R-6 cells, whereas it inhibited apoptosis in TAMR-1 cells. Many regulators controlling cell cycle and apoptosis were aberrantly expressed in two resistant cell lines in response to the enforced GNB4 expression, which may contribute to GNB4-mediated biologic and/or pathologic processes. Furthermore, knockdown of GNB4 decreased growth of both antiestrogen resistant and sensitive breast cancer cells. CONCLUSION: GNB4 is important for growth of breast cancer cells and a potential target for treatment.

A dual role of miR-22 modulated by RelA/p65 in resensitizing fulvestrant-resistant breast cancer cells to fulvestrant by targeting FOXP1 and HDAC4 and constitutive acetylation of p53 at Lys382.

Antiestrogen resistance is a major challenge encountered during the treatment of estrogen receptor alpha positive (ERα+) breast cancer. A better understanding of signaling pathways and downstream transcription factors and their targets may identify key molecules that can overcome antiestrogen resistance in breast cancer. An aberrant expression of miR-22 has been demonstrated in breast cancer; however, its contribution to breast cancer resistance to fulvestrant, an antiestrogen drug, remains unknown. In this study, we demonstrated a moderate elevation in miR-22 expression in the 182R-6 fulvestrant-resistant breast cancer line we used as a model system, and this elevation was positively correlated with the expression of the miRNA biogenesis enzymes AGO2 and Dicer. The level of phosphorylated HER2/neu at Tyr877 was also upregulated in these cells, whereas the level of RelA/p65 phosphorylated at Ser536 (p-p65) was downregulated. Knockdown of HER2/neu led to an induction of p-p65 and a reduction in miR-22 levels. Luciferase assays identified two NF-κB binding motifs in the miR-22 promoter that contributed to transcriptional repression of miR-22. Activation of RelA/p65, triggered by LPS, attenuated miR-22 expression, but this expression was restored by sc-514, a selective IKKß inhibitor. Inhibition of miR-22 suppressed cell proliferation, induced apoptosis and caused cell cycle S-phase arrest, whereas enhancing expression of p21Cip1/Waf1 and p27Kip1. Surprisingly, ectopic expression of miR-22 also suppressed cell proliferation, induced apoptosis, caused S-phase arrest, and promoted the expression of p21Cip1/Waf1 and p27Kip1. Ectopic overexpression of miR-22 repressed the expression of FOXP1 and HDAC4, leading to a marked induction of acetylation of HDAC4 target histones. Conversely, inhibition of miR-22 promoted the expression of both FOXP1 and HDAC4, without the expected attenuation of histone acetylation. Instead, p53 acetylation at lysine 382 was unexpectedly upregulated. Taken together, our findings demonstrated, for the first time, that HER2 activation dephosphorylates RelA/p65 at Ser536. This dephosphoryalted p65 may be pivotal in transactivation of miR-22. Both increased and decreased miR-22 expression cause resensitization of fulvestrant-resistant breast cancer cells to fulvestrant. HER2/NF-κB (p65)/miR-22/HDAC4/p21 and HER2/NF-κB (p65)/miR-22/Ac-p53/p21 signaling circuits may therefore confer this dual role on miR-22 through constitutive transactivation of p21.

High and low dose radiation effects on mammary adenocarcinoma cells - an epigenetic connection.

The successful treatment of cancer, including breast cancer, depends largely on radiation therapy and proper diagnostics. The effect of ionizing radiation on cells and tissues depends on the radiation dose and energy level, but there is insufficient evidence concerning how tumor cells respond to the low and high doses of radiation that are often used in medical diagnostic and treatment modalities. The purpose of this study was to investigate radiation-induced gene expression changes in the MCF-7 breast adenocarcinoma cell line. Using microarray technology tools, we were able to screen the differential gene expressions profiles between various radiation doses applied to MCF-7 cells. Here, we report the substantial alteration in the expression level of genes after high-dose treatment. In contrast, no dramatic gene expression alterations were noticed after the application of low and medium doses of radiation. In response to a high radiation dose, MCF-7 cells exhibited down-regulation of biological pathways such as cell cycle, DNA replication, and DNA repair and activation of the p53 pathway. Similar dose-dependent responses were seen on the epigenetic level, which was tested by a microRNA expression analysis. MicroRNA analysis showed dose-dependent radiation-induced microRNA expression alterations that were associated with cell cycle arrest and cell death. An increased rate of apoptosis was determined by an Annexin V assay. The results of this study showed that high doses of radiation affect gene expression genetically and epigenetically, leading to alterations in cell cycle, DNA replication, and apoptosis.

dsRNA expression in the mouse elicits RNAi in oocytes and low adenosine deamination in somatic cells.

Double-stranded RNA (dsRNA) can enter different pathways in mammalian cells, including sequence-specific RNA interference (RNAi), sequence-independent interferon (IFN) response and editing by adenosine deaminases. To study the routing of dsRNA to these pathways in vivo, we used transgenic mice ubiquitously expressing from a strong promoter, an mRNA with a long hairpin in its 3'-UTR. The expressed dsRNA neither caused any developmental defects nor activated the IFN response, which was inducible only at high expression levels in cultured cells. The dsRNA was poorly processed into siRNAs in somatic cells, whereas, robust RNAi effects were found in oocytes, suggesting that somatic cells lack some factor(s) facilitating siRNA biogenesis. Expressed dsRNA did not cause transcriptional silencing in trans. Analysis of RNA editing revealed that a small fraction of long dsRNA is edited. RNA editing neither prevented the cytoplasmic localization nor processing into siRNAs. Thus, a long dsRNA structure is well tolerated in mammalian cells and is mainly causing a robust RNAi response in oocytes.

microRNAome changes in bystander three-dimensional human tissue models suggest priming of apoptotic pathways.

The radiation-induced bystander effect (RIBE) is a phenomenon whereby unexposed cells exhibit molecular symptoms of stress exposure when adjacent or nearby cells are traversed by ionizing radiation (IR). Recent data suggest that RIBE may be epigenetically mediated by microRNAs (miRNAs), which are small regulatory molecules that target messenger RNA transcripts for translational inhibition. Here, we analyzed microRNAome changes in bystander tissues after α-particle microbeam irradiation of three-dimensional artificial human tissues using miRNA microarrays. Our results indicate that IR leads to a deregulation of miRNA expression in bystander tissues. We report that major bystander end points, including apoptosis, cell cycle deregulation and DNA hypomethylation, may be mediated by altered expression of miRNAs. Specifically, c-MYC-mediated upregulation of the miR-17 family was associated with decreased levels of E2F1 and RB1, suggesting a switch to a proliferative state in bystander tissues, while priming these cells for impending death signals. Upregulation of the miR-29 family resulted in decreased levels of its targets DNMT3a and MCL1, consequently affecting DNA methylation and apoptosis. Altered expression of miR-16 led to changes in expression of BCL2, suggesting modulation of apoptosis. Thus, our data clearly show that miRNAs play a profound role in the manifestation of late RIBE end points. In summary, this study creates a roadmap for understanding the role of microRNAome in RIBE and for developing novel RIBE biomarkers.

Alterations of microRNAs and their targets are associated with acquired resistance of MCF-7 breast cancer cells to cisplatin.

Cancer cells that develop resistance to chemotherapeutic agents are a major clinical obstacle in the successful treatment of breast cancer. Acquired cancer chemoresistance is a multifactorial phenomenon, involving various mechanisms and processes. Recent studies suggest that chemoresistance may be linked to drug-induced dysregulation of microRNA function. Furthermore, mounting evidence indicates the existence of similarities between drug-resistant and metastatic cancer cells in terms of resistance to apoptosis and enhanced invasiveness. We studied the role of miRNA alterations in the acquisition of cisplatin-resistant phenotype in MCF-7 human breast adenocarcinoma cells. We identified a total of 103 miRNAs that were overexpressed or underexpressed (46 upregulated and 57 downregulated) in MCF-7 cells resistant to cisplatin. These differentially expressed miRNAs are involved in the control of cell signaling, cell survival, DNA methylation and invasiveness. The most significantly dysregulated miRNAs were miR-146a, miR-10a, miR-221/222, miR-345, miR-200b and miR-200c. Furthermore, we demonstrated that miR-345 and miR-7 target the human multidrug resistance-associated protein 1. These results suggest that dysregulated miRNA expression may underlie the abnormal functioning of critical cellular processes associated with the cisplatin-resistant phenotype.

Hypomethylation and genome instability in the germline of exposed parents and their progeny is associated with altered miRNA expression.

Recent studies suggest that transgenerational genome instability may be epigenetic in nature and mediated via altered DNA methylation and microRNAome. Here, we investigated the nature and mechanisms underlying the disruption of DNA methylation and microRNA expression status in the germline and progeny of exposed parents. We have found that paternal irradiation leads to upregulation of the miR-29 family in the exposed male germline, which causes decreased expression of de novo methyltransferase, DNA methyltransferase 3a, and profound hypomethylation of long interspersed nuclear elements 1 (LINE1) and short interspersed nuclear elements B2 (SINE B2). Epigenetic changes in the male germline further resulted in deleterious effects in the somatic thymus tissue from the progeny of exposed animals, including hypomethylation of LINE1 and SINE B2. Hypomethylation of LINE1 and SINE B2 in the thymus tissue from the progeny was associated with a significant decrease in the levels of lymphoid-specific helicase (LSH) that is crucial for the maintenance of methylation and silencing of repetitive elements. Furthermore, we noted a significant upregulation of miR-468 that targets LSH and leads to its decreased expression in thymus in the progeny of exposed parents. We suggest that miR-468-mediated suppression of LSH leads to aberrant methylation of LINE1 and SINE B2. In summary, altered microRNAome and hypomethylation of retroelements constitute deleterious effects that may significantly influence genome stability of the parental germline and consequently cause genome instability in the progeny.

Involvement of microRNA-451 in resistance of the MCF-7 breast cancer cells to chemotherapeutic drug doxorubicin.

Many chemotherapy regiments are successfully used to treat breast cancer; however, often breast cancer cells develop drug resistance that usually leads to a relapse and worsening of prognosis. We have shown recently that epigenetic changes such as DNA methylation and histone modifications play an important role in breast cancer cell resistance to chemotherapeutic agents. Another mechanism of gene expression control is mediated via the function of small regulatory RNA, particularly microRNA (miRNA); its role in cancer cell drug resistance still remains unexplored. In the present study, we investigated the role of miRNA in the resistance of human MCF-7 breast adenocarcinoma cells to doxorubicin (DOX). Here, we for the first time show that DOX-resistant MCF-7 cells (MCF-7/DOX) exhibit a considerable dysregulation of the miRNAome profile and altered expression of miRNA processing enzymes Dicer and Argonaute 2. The mechanistic link of miRNAome deregulation and the multidrug-resistant phenotype of MCF-7/DOX cells was evidenced by a remarkable correlation between specific miRNA expression and corresponding changes in protein levels of their targets, specifically those ones that have a documented role in cancer drug resistance. Furthermore, we show that microRNA-451 regulates the expression of multidrug resistance 1 gene. More importantly, transfection of the MCF-7/DOX-resistant cells with microRNA-451 resulted in the increased sensitivity of cells to DOX, indicating that correction of altered expression of miRNA may have significant implications for therapeutic strategies aiming to overcome cancer cell resistance.

Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells.

RNA interference and the microRNA (miRNA) pathway can induce sequence-specific mRNA degradation and/or translational repression. The human genome encodes hundreds of miRNAs that can post-transcriptionally repress thousands of genes. Using reporter constructs, we observed that degradation of mRNAs bearing sites imperfectly complementary to the endogenous let-7 miRNA is considerably stronger in human HEK293 than HeLa cells. The degradation did not result from the Ago2-mediated endonucleolytic cleavage but it was Dicer- and Ago2-dependent. We used this feature of HEK293 to address the size of a pool of transcripts regulated by RNA silencing in a single cell type. We generated HEK293 cell lines depleted of Dicer or individual Ago proteins. The cell lines were used for microarray analyses to obtain a comprehensive picture of RNA silencing. The 3'-untranslated region sequences of a few hundred transcripts that were commonly up-regulated upon Ago2 and Dicer knock-downs showed a significant enrichment of putative miRNA-binding sites. The up-regulation upon Ago2 and Dicer knock-downs was moderate and we found no evidence, at the mRNA level, for activation of silenced genes. Taken together, our data suggest that, independent of the effect on translation, miRNAs affect levels of a few hundred mRNAs in HEK293 cells.

Double-strand break repair in plants is developmentally regulated.

In this study, we analyzed double-strand break (DSB) repair in Arabidopsis (Arabidopsis thaliana) at various developmental stages. To analyze DSB repair, we used a homologous recombination (HR) and point mutation reversion assays based on nonfunctional beta-glucuronidase reporter genes. Activation of the reporter gene through HR or point mutation reversion resulted in the appearance of blue sectors after histochemical staining. Scoring of these sectors at 3-d intervals from 2 to 31 d post germination (dpg) revealed that, although there was a 100-fold increase in the number of genomes per plant, the recombination frequency only increased 30-fold. This translates to a recombination rate at 31 dpg (2.77 x 10(-8)) being only 30% of the recombination rate at 2 dpg (9.14 x 10(-8)). Conversely, the mutation frequency increased nearly 180-fold, resulting in a 1.8-fold increase in mutation rate from 2 to 31 dpg. Additional analysis of DSBs over the early developmental stages revealed a substantial increase in the number of strand breaks per unit of DNA. Furthermore, RNA analysis of Ku70 and Rad51, two key enzymes in two different DSB repair pathways, and further protein analysis of Ku70 revealed an increase in Ku70 levels and a decrease of Rad51 levels in the developing plants. These data suggest that DSB repair mechanisms are developmentally regulated in Arabidopsis, whereby the proportion of breaks repaired via HR substantially decreases as the plants mature.

Homologous recombination in plants is organ specific.

In this paper we analysed the genome stability of various Arabidopsis thaliana plant organs using a transgenic recombination system. The system was based on two copies of non-functional GUS (lines #651 and #11) or LUC (line #15D8) reporter genes serving as a recombination substrate. Both reporter assays showed that recombination in flowers or stems were rare events. Most of the recombination sectors were found in leaves and roots, with leaves having over 2-fold greater number of the recombination events per single cell genome as compared to roots. The recombination events per single genome were 9.7-fold more frequent on the lateral half of the leaves than on the medial halves. This correlated with a 2.5-fold higher metabolic activity in the energy source (lateral) versus energy sink (medial) of leaves. Higher metabolic activity was paralleled by a higher anthocyanin production in lateral halves. The level of double strand break (DSB) occurrence was also different among plant organs; the highest level was observed in roots and the lowest in leaves. High level of DSBs strongly positively correlated with the activity of the key repair enzymes, AtKU70 and AtRAD51. The ratio of AtRAD51 to AtKU70 expression was the highest in leaves, supporting the more active involvement of homologous recombination pathway in the repair of DSBs in this organ. Western blot analysis confirmed the real time PCR expression data for AtKU70 gene.

Sex and tissue-specific differences in low-dose radiation-induced oncogenic signaling.

PURPOSE: The possible adverse health effects of low-dose radiation (LDR) exposure constitute a growing concern. Clinically and environmentally relevant exposures occur predominantly under chronic conditions, notwithstanding that most studies of LDR effects have been performed using a single acute exposure. Sex- and tissue-specificity of the LDR-induced changes have not been considered before. We investigated LDR-related expression patterns in muscle, liver and spleen of male and female mice subjected to acute and chronic LDR exposure. Genes involved in oncogenic signaling were of specific interest, as radiation is a well-known carcinogen. MATERIALS AND METHODS: We analyzed the expression pattern of genes coding for growth factors and growth-factor receptors, cytoplasmic serine/threonine protein kinases, G-proteins and nuclear DNA-binding proteins, and other important components of oncogenic signaling. RESULTS: We found sex- and tissue-specific changes in the expression of Ras superfamily members (Nras, Rab2, Rab34, Vav2), protein kinase C (PKC) isoforms (PKCbeta, PKCmu), AP-1 factor components (Jun, JunB and FosB), Wnt signaling pathway members as well as in a variety of other cellular proto-oncogenes and oncogenes. Importantly, Western blot analysis of JunB, PKCmu and Rab2 proteins supported the transcriptomic data. CONCLUSIONS: Substantially different protein levels were observed in all three tissues (muscle, spleen and liver) of acutely and chronically irradiated female and male animals. Based on the obtained data and available literature, we discuss several possible mechanisms that may contribute to radiation-induced carcinogenesis in various tissues of males and females. From our results we could identify the genes that may serve as sex- and tissue-specific biomarkers of the LDR exposure.

Homologous recombination in plants is temperature and day-length dependent.

Homologous recombination (HR) as a strand break repair mechanism was shown to be influenced by various factors. The balance of different vitamins, macro- and microelements, light spectrum, sodium chloride concentration as well as various environmental mutagens were shown to influence the frequency of HR. In this paper we analysed the influence of temperature (4, 22, and 32 degrees C) and day/night duration on the genome stability of plants. We analyzed the HR frequency in transgenic Arabidopsis thaliana plants carrying beta-glucuronidase based homologous recombination substrate. To find the recombination rate (RR), we related the HR frequency to the number of genomes present in plants grown under different conditions. The RR was also standardized to the transcription activity of the transgene. We found RR to be higher in plants grown at suboptimal temperatures (either 4 or 32 degrees C) as compared to plants grown at 22 degrees C. This negatively correlated with the plant metabolic rate and positively correlated with concentration of peroxide produced by plant. In contrast, the RR in plants grown at different day length (8-24 h) was the lowest in plants grown at the longest day (24 h) and highest in the plants grown at the shortest day (8 h). Over 15-fold difference in the RR between plants grown at the shortest and the longest day was observed. Such a difference in recombination rate was primarily due to the higher transgene activity and higher endoreduplication levels in plants grown at longer days. Our data suggests that even "moderate" changes of environmental conditions may have a significant effect on plant genome stability.

Atrazine induces homologous recombination but not point mutation in the transgenic plant-based biomonitoring assay.

Herbicides, such as atrazine, are extensively used in agriculture in order to suppress growth of weeds. From the soil they inevitably find their way to water supplies, leading to human exposure via drinking water. Therefore, it is extremely important to know whether those chemicals pose any hazard to public health. The genotoxicity of atrazine has been a subject of studies in recent years. However, the data that are currently available are inconclusive. There is a need to examine the genotoxicity of low, environmentally relevant concentrations that are currently assumed to be safe. Up to date, studying the genotoxicity of low concentrations of atrazine has constituted a great challenge due to the lack of appropriate, sensitive test systems. In the present work, we used a new sensitive transgenic plant-based system to study the genotoxicity and mutagenicity of atrazine present at minute concentrations in the liquid media. This system gave us an opportunity to monitor the two main types of rearrangements, the frequency of homologous recombination and point mutations, which are indicators of the genotoxicity of atrazine. Atrazine present at low concentrations was found to be a strong inducer of homologous recombination. On the other hand, it did not have a significant influence on the levels of A --> G and T --> G mutations. These results suggest that the transgenic plant-based biomonitoring system is a useful tool for analyzing the genotoxicity of water contaminated by atrazine. In the future this system can be used to study molecular mechanisms of genotoxicity and mutagenicity atrazine and other triazine herbicides.

Dissimilar genome response to acute and chronic low-dose radiation in male and female mice.

The long-term genetic consequences of chronic exposure to low-dose irradiation constitutes a major concern to the general public and research community, especially as chronic radiation has recently been proven to be much more mutagenic and carcinogenic than previously thought. Here we report the results of the first ever comparison of the effects of acute and chronic whole body low-dose radiation exposure on global gene expression. We found a substantial difference between males and females in the expression of genes involved in signaling, growth control, transcription and other pathways upon acute and chronic radiation exposure. Specifically, we found sex differences in the expression of genes coding for G protein-coupled receptors and nuclear receptors. We also found different induction of PKCdelta, PKCbeta and PKCmu, members of PKC signaling pathway as well as in TGF and WNT signaling in males and females. Very pronounced difference, that was confirmed on the level of protein, was observed in the expression of WNT5A that plays an important role in carcinogenesis and muscle regeneration. WNT5A expression was significantly elevated only in chronically exposed females. We also provide the first evidence of the effect of ionizing radiation on the estrogen receptor in females. Repetitive irradiation of muscle tissue has been linked to development of rhabdomyosarcoma (RMS), which, enigmatically, occurs more frequently in males. Our data may be used to study possible mechanisms of RMS development upon chronic radiation exposure. They may provide some clues about the molecular background of the sex differences of RMS occurrence and may in the future lead to the discovery of new biomarkers for RMS predisposition in the irradiated tissue. Overall, differences in male and female responses to acute and chronic low-dose radiation obtained by this study were more drastic than we could have predicted. If confirmed in other experimental systems, these findings could potentially lead to fundamental changes in radiation safety regulations.

Systemic plant signal triggers genome instability.

Previously, we have shown that infection of tobacco plants with a viral pathogen triggers local and systemic induction of homologous recombination (HR). Here, we have tested the hypothesis of whether free radicals are potentially involved in the induction of the systemic effect. We report a significant induction of HR in tobacco plants treated with radical-generating agents, UVC or rose Bengal (RB). Importantly, the recombination increase was observed in local (treated) as well as systemic (non-treated) tissue. The systemic increase in recombination implies the existence of a signal that is transmitted to non-treated tissue. Several sets of grafting experiments proved the generation of said signal by both RB and UVC exposure. A statistically significant increase in HR was observed in tissue that received a systemic signal via a grafted leaf. Similar data were obtained from transgenic plants naphthalene degrading salicylate 1-hydroxylase (NahG) unable to accumulate salicylic acid (SA). Interestingly, pre-treatment of plants with the radical-scavenging compound N-acetyl-l-cysteine (NAC) led to a significantly lower recombination increase upon grafting after treatment with UVC and RB. Moreover, leaves taken for grafting from NAC-pre-treated plants exhibited a lower level of oxidized organic compounds. Our data suggest the involvement of free radical production in either generation or maintenance of the recombination signal. We discuss potential mechanisms for generation of the signal and possible adaptive advantages of enhanced genomic flexibility following exposure to DNA-damaging agents.

Genome stability of vtc1, tt4, and tt5 Arabidopsis thaliana mutants impaired in protection against oxidative stress.

Reactive oxygen species (ROS) are formed upon normal cellular metabolism or influence of environmental factors and, at normal levels, they play an important physiological role. However, at elevated levels, radicals are toxic and extremely dangerous to all cellular components, including DNA. To efficiently protect themselves, plants have developed sophisticated mechanisms for radical screening and scavenging. In this paper, we analyzed the genome stability of several plant mutants impaired in the protection against free radicals. We crossed the well-known uidA recombination reporter line 651 to flavonoid (tt4 and tt5) and Vitamin C (vtc1)-deficient plants. We found that in all lines tested, both spontaneous and induced (UVC and Rose Bengal (RB)) recombination was higher than in the original 651 parental line. The mRNA expression levels of various DNA repair (RAD1, RAD54-like, MSH3) as well as radical scavenging genes (GPx1, CAT, FSD3) exhibited substantial differences in both control and induced conditions. Our data show that plants impaired in certain aspects of the protection against elevated levels of free radicals induce the production of scavenging enzymes earlier than wild-type (wt) plants, and the higher level of radical species results in the increased incidence of spontaneous double-strand breaks resulting in a higher expression of DNA repair genes.

Methylation changes in muscle and liver tissues of male and female mice exposed to acute and chronic low-dose X-ray-irradiation.

The biological and genetic effects of chronic low-dose radiation (LDR) exposure and its relationship to carcinogenesis have received a lot of attention in the recent years. For example, radiation-induced genome instability, which is thought to be a precursor of tumorogenesis, was shown to have a transgenerational nature. This indicates a possible involvement of epigenetic mechanisms in LDR-induced genome instability. Genomic DNA methylation is one of the most important epigenetic mechanisms. Existing data on radiation effects on DNA methylation patterns is limited, and no one has specifically studied the effects of the LDR. We report the first study of the effects of whole-body LDR exposure on global genome methylation in muscle and liver tissues of male and female mice. In parallel, we evaluated changes in promoter methylation and expression of the tumor suppressor gene p16(INKa) and DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT). We observed different patterns of radiation-induced global genome DNA methylation in the liver and muscle of exposed males and females. We also found sex and tissue-specific differences in p16(INKa) promoter methylation upon LDR exposure. In male liver tissue, p16(INKa) promoter methylation was more pronounced than in female tissue. In contrast, no significant radiation-induced changes in p16(INKa) promoter methylation were noted in the muscle tissue of exposed males and females. Radiation also did not significantly affect methylation status of MGMT promoter. We also observed substantial sex differences in acute and chronic radiation-induced expression of p16(INKa) and MGMT genes. Another important outcome of our study was the fact that chronic low-dose radiation exposure proved to be a more potent inducer of epigenetic effects than the acute exposure. This supports previous findings that chronic exposure leads to greater genome destabilization than acute exposure.

Genotoxicity of 2,4-D and dicamba revealed by transgenic Arabidopsis thaliana plants harboring recombination and point mutation markers.

The phenoxy herbicides 2,4-D and dicamba are released daily into the environment in large amount. The mechanisms of genotoxicity and mutagenicity of these herbicides are poorly understood, and the available genotoxicity data is controversial. There is a cogent need for a novel genotoxicity monitoring system that could provide both reliable information at the molecular level, and complement existing systems.We employed the transgenic Arabidopsis thaliana 'point mutation' and 'recombination' plants to monitor the genetic effects of the herbicides 2,4-D and dicamba. We found that both herbicides had a significant effect on the frequency of homologous recombination A-->G mutation. Neither herbicides affected the T-->G mutation frequency. Interestingly, these transgenic biomonitoring plants were able to detect the presence of phenoxy herbicides at concentrations that were lower than the guideline levels for Drinking Water Quality. The results of our studies suggest that our transgenic system may be ideal for the evaluation of the genotoxicity of herbicide-contaminated water. Moreover, the unique ability of the plants to detect both double-strand breaks (homologous recombination) and point mutations provides tremendous potential in the study of molecular mechanisms of genotoxicity and mutagenicity of phenoxy herbicides.

Pathogen-induced systemic plant signal triggers DNA rearrangements.

Plant genome stability is known to be affected by various abiotic environmental conditions, but little is known about the effect of pathogens. For example, exposure of maize plants to barley stripe mosaic virus seems to activate transposable elements and to cause mutations in the non-infected progeny of infected plants. The induction by barley stripe mosaic virus of an inherited effect may mean that the virus has a non-cell-autonomous influence on genome stability. Infection with Peronospora parasitica results in an increase in the frequency of somatic recombination in Arabidopsis thaliana; however, it is unclear whether effects on recombination require the presence of the pathogen or represent a systemic plant response. It is also not clear whether the changes in the frequency of somatic recombination can be inherited. Here we report a threefold increase in homologous recombination frequency in both infected and non-infected tissue of tobacco plants infected with either tobacco mosaic virus or oilseed rape mosaic virus. These results indicate the existence of a systemic recombination signal that also results in an increased frequency of meiotic and/or inherited late somatic recombination.