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.

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 miR146a, 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.Breast cancer is the most common malignancy in women. In the United States, the incidence of invasive breast cancer, the most serious form of breast cancer, was estimated as 182,460 new cases and 40,480 deaths in 2008.1

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.

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-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.

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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.

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 b-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 3 10 28 ) being only 30% of the recombination rate at 2 dpg (9.14 3 10 28 ). 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.