Juglone in Combination with Temozolomide Shows a Promising Epigenetic Therapeutic Effect on the Glioblastoma Cell Line.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor and one of the human malignancies with the highest mortality. Standard approaches for GBM, including gross total resection, radiotherapy, and chemotherapy, cannot destroy all the cancer cells, and despite advances in its treatment, the prognosis for GBM remains poor. The problem is that we still do not understand what triggers GBM. Until now, the most successful chemotherapy with temozolomide for brain gliomas is not effective, and therefore new therapeutic strategies for GBM are needed. We found that juglone (J), which exhibits cytotoxic, anti-proliferative, and anti-invasive effects on various cells, could be a promising agent for GBM therapy. In this paper, we present the effects of juglone alone and in combination with temozolomide on glioblastoma cells. In addition to the analysis of cell viability and the cell cycle, we looked at the epigenetics effects of these compounds on cancer cells. We showed that juglone induces strong oxidative stress, as identified by a high increase in the amount of 8-oxo-dG, and decreases m5C in the DNA of cancer cells. In combination with TMZ, juglone modulates the level of both marker compounds. Our results strongly suggest that a combination of juglone and temozolomide can be applied for better GBM treatment.

Cross-reactivity between histone demethylase inhibitor valproic acid and DNA methylation in glioblastoma cell lines.

Currently, valproic acid (VPA) is known as an inhibitor of histone deacetylase (epigenetic drug) and is used for the clinical treatment of epileptic events in the course of glioblastoma multiforme (GBM). Which improves the clinical outcome of those patients. We analyzed the level of 5-methylcytosine, a DNA epigenetic modulator, and 8-oxodeoxyguanosine, an cellular oxidative damage marker, affected with VPA administration, alone and in combination with temozolomide (TMZ), of glioma (T98G, U118, U138), other cancer (HeLa), and normal (HaCaT) cell lines. We observed the VPA dose-dependent changes in the total DNA methylation in neoplastic cell lines and the lack of such an effect in a normal cell line. VPA at high concentrations (250-500 µM) induced hypermethylation of DNA in a short time frame. However, the exposition of GBM cells to the combination of VPA and TMZ resulted in DNA hypomethylation. At the same time, we observed an increase of genomic 8-oxo-dG, which as a hydroxyl radical reaction product with guanosine residue in DNA suggests a red-ox imbalance in the cancer cells and radical damage of DNA. Our data show that VPA as an HDAC inhibitor does not induce changes only in histone acetylation, but also changes in the state of DNA modification. It shows cross-reactivity between chromatin remodeling due to histone acetylation and DNA methylation. Finally, total DNA cytosine methylation and guanosine oxidation changes in glioma cell lines under VPA treatment suggest a new epigenetic mechanism of that drug action.

Epigenetic on Earth and in Space / Epigenetyka na Ziemi i w kosmosie.

The year 1961 went down in history with exceptional scientific achievements. On May 13, the journal Nature published two articles on the first isolation of messenger ribonucleic acid (mRNA), which is an intermediate product between a gene and a protein. Just two weeks later, on May 27, the first letter of the genetic code, phenylalanine, was discovered. These discoveries made it possible to understand how genetic information is encoded and processed, thus causing the dynamic development of molecular biology. The breakthroughs of 1961 concerned not only nucleic acids. On April 12, the first human, Yuri Gagarin, entered space. Eight years later, in 1969, Neil Armstrong made his first walk on the moon, uttering the famous phrase: It is a small step for man, but a great leap for humanity. The era of conquering and learning about the cosmos has begun, mainly motivated by the natural curiosity of man and the desire to learn about the surrounding reality. The environmental factors in space are very different from terrestrial conditions, which raises questions about their effects on living organisms. In search of answers, a variety of scientific research has been carried out at the International Space Station (ISS) for over twenty years. As space travel is set to become more common in the near future, detailed studies of the effects of long-term space missions on the human body are required. These studies are currently carried out, among others using molecular biology techniques that enable detailed analysis of nucleic acids and proteins, but not only. The breakthrough achievements of 1961 initiated the development both in the field of molecular biology and the science of space, thanks to which today, 60 years after those events, we can combine knowledge and technological achievements from both fields to analyze and understand changes at the molecular level that occur as a result of being in organisms in outer space.

A new molecular mechanism of RNA circularization and the microRNA sponge formation.

A new mechanism of RNA circularization driven by specific binding of miRNAs is described. We identified the 71 CUUCC pentanucleotide motifs distributed regularly throughout the entire molecule of CDR1as RNA that bind to 71 miRNAs through their seed sequence GGAAG. The sequential binding of miR-7 RNAs (71 molecules) brings both ends of CDR1as RNA (1 molecule) together and stimulate phosphodiester bond formation between nucleotides C1 and A1299 at the 5' and 3' end, respectively. The binding of miRNAs to CDR1as RNA results in the unique complex formation, which shows three specific structural domains: (i) two short helixes with an internal loop, (ii) the hinge, and (iii) the triple-helix. The proposed mechanism explains specific RNA circularization and its function as a miRNAs sponge. Furthermore, the existing wet experimental data on the interaction of CDR1as RNA with miR-7 fully supports our observation. Although miR-671 shows the same seed sequence as miR-7, it forms an almost perfect double helix with CDR1as RNA and induces the cleavage of CDR1as, but does not stimulate circularization. To check how common is the proposed mechanism among circular RNAs, we analyzed the most recent circAtlas database counting almost 1.1 million sequences. It turned out that there are a huge number of circRNAs, which showed miRNAs seed binding sequences distributed through the whole circRNA sequences and prove that circularization of linear transcript is miRNA dependent.Communicated by Ramaswamy H. Sarma.

Changes in genomic 5-methylcytosine level mirror the response of orthodox (Acer platanoides L.) and recalcitrant (Acer pseudoplatanus L.) seeds to severe desiccation.

Poor storability of recalcitrant seeds is due to their inability to tolerate low moisture content. Understanding the processes underlying their recalcitrance is a prerequisite to developing a maintenance strategy and prolonging their lifespan. Multiple studies have investigated the differences between orthodox (desiccation-tolerant) and recalcitrant (desiccation-sensitive) seeds. Information on epigenetic regulation, however, is lacking and thus limits our understanding of the processes defining the physiology of seeds. In the present comparative study, changes in the global levels of 5-methylcytosine (m5C) in orthodox and recalcitrant seeds of Acer platanoides L. and Acer pseudoplatanus L. were characterized during progressive stages of severe drying. Concomitant with their differential sensitivity to desiccation stress, we demonstrate variation in the response of embryonic axes and cotyledons to water deficit at the level of DNA methylation. Results indicate that desiccation-induced changes in m5C are both tissue- and seed category-specific and are highly correlated with recalcitrant seed viability. Moreover, we demonstrate that m5C global changes in response to desiccation are not retained in DNA isolated from seedlings, except in seedlings that are derived from strongly desiccated orthodox seeds (moisture content of 3.5%). Finally, the potential utilization of m5C status as a universal seed viability marker is discussed.

Global methylation status of sperm DNA in carriers of chromosome structural aberrations.

Male infertility might be clearly associated with aberrant DNA methylation patterns in human spermatozoa. An association between oxidative stress and the global methylation status of the sperm genome has also been suggested. The aim of the present study was to determine whether the global sperm DNA methylation status was affected in the spermatozoa of carriers of chromosome structural aberrations. The relationships between the 5-methylcytosine (m 5 C) levels in spermatozoa and chromatin integrity status were evaluated. The study patients comprised male carriers of chromosome structural aberrations with reproductive failure (n = 24), and the controls comprised normozoospermic sperm volunteers (n = 23). The global m 5 C level was measured using thin-layer chromatography (TLC) and immunofluorescence (IF) techniques. The sperm chromatin integrity was assessed using aniline blue (AB) staining and TUNEL assay. The mean m 5 C levels were similar between the investigated chromosome structural aberrations carriers (P) and controls (K). However, sperm chromatin integrity tests revealed significantly higher values in chromosomal rearrangement carriers than in controls (P < 0.05). Although the potential relationship between sperm chromatin integrity status and sperm DNA fragmentation and the m 5 C level juxtaposed in both analyzed groups (P vs K) was represented in a clearly opposite manner, the low chromatin integrity might be associated with the high hypomethylation status of the sperm DNA observed in carriers of chromosome structural aberrations.

Inhibition of miR-21 in glioma cells using catalytic nucleic acids.

Despite tremendous efforts worldwide, glioblastoma multiforme (GBM) remains a deadly disease for which no cure is available and prognosis is very bad. Recently, miR-21 has emerged as a key omnipotent player in carcinogenesis, including brain tumors. It is recognized as an indicator of glioma prognosis and a prosperous target for anti-tumor therapy. Here we show that rationally designed hammerhead ribozymes and DNAzymes can target miR-21 and/or its precursors. They decrease miR-21 level, and thus silence this oncomiR functions. We demonstrated that anti-miRNA catalytic nucleic acids show a novel terrific arsenal for specific and effective combat against diseases with elevated cellular miR-21 content, such as brain tumors.

The Sequence and Structure Determine the Function of Mature Human miRNAs.

Micro RNAs (miRNAs) (19-25 nucleotides in length) belong to the group of non-coding RNAs are the most abundant group of posttranscriptional regulators in multicellular organisms. They affect a gene expression by binding of fully or partially complementary sequences to the 3'-UTR of target mRNA. Furthermore, miRNAs present a mechanism by which genes with diverse functions on multiple pathways can be simultaneously regulated at the post-transcriptional level. However, little is known about the specific pathways through which miRNAs with specific sequence or structural motifs regulate the cellular processes. In this paper we showed the broad and deep characteristics of mature miRNAs according to their sequence and structural motifs. We investigated a distinct group of miRNAs characterized by the presence of specific sequence motifs, such as UGUGU, GU-repeats and purine/pyrimidine contents. Using computational function and pathway analysis of their targeted genes, we were able to observe the relevance of sequence and the type of targeted mRNAs. As the consequence of the sequence analysis we finally provide the comprehensive description of pathways, biological processes and proteins associated with the distinct group of characterized miRNAs. Here, we found that the specific group of miRNAs with UGUGU can activate the targets associated to the interferon induction pathway or pathways prominently observed during carcinogenesis. GU-rich miRNAs are prone to regulate mostly processes in neurogenesis, whereas purine/pyrimidine rich miRNAs could be involved rather in transport and/or degradation of RNAs. Additionally, we have also analyzed the simple sequence repeats (SSRs). Their variation within mature miRNAs might be critical for normal miRNA regular activity. Expansion or contraction of SSRs in mature miRNA might directly affect its mRNA interaction or even change the function of that distinct miRNA. Our results prove that due to the specific sequence features, these molecules can also be involved in well-defined cellular processes depending on their sequence contents. The pathway mapping and theoretical gene target identification allowed us to create a biological framework to show the relevance of the specific miRNAs in regulation the distinct type of targets.

tRNA--the golden standard in molecular biology.

Transfer RNAs (tRNAs) represent a major class of RNA molecules. Their primary function is to help decode a messenger RNA (mRNA) sequence in order to synthesize protein and thus ensures the precise translation of genetic information that is imprinted in DNA. The discovery of tRNA in the late 1950's provided critical insight into a genetic machinery when little was known about the central dogma of molecular biology. In 1965, Robert Holley determined the first nucleotide sequence of alanine transfer RNA (tRNA(Ala)) which earned him the 1968 Nobel Prize in Physiology or Medicine. Today, tRNA is one of the best described and characterized biological molecules. Here we review some of the key historical events in tRNA research which led to breakthrough discoveries and new developments in molecular biology.

A New Epigenetic Mechanism of Temozolomide Action in Glioma Cells.

Temozolomide (TMZ) is an oral alkylating chemotherapeutic agent that prolongs the survival of patients with glioblastoma (GBM). Despite that high TMZ potential, progression of disease and recurrence are still observed. Therefore a better understanding of the mechanism of action of this drug is necessary and may allow more durable benefit from its anti-glioma properties. Using nucleotide post-labelling method and separation on thin-layer chromatography we measured of global changes of 5-methylcytosine (m5C) in DNA of glioma cells treated with TMZ. Although m5C is not a product of TMZ methylation reaction of DNA, we analysed the effects of the drug action on different glioma cell lines through global changes at the level of the DNA main epigenetic mark. The first effect of TMZ action we observed is DNA hypermethylation followed by global demethylation. Therefore an increase of DNA methylation and down regulation of some genes expression can be ascribed to activation of DNA methyltransferases (DNMTs). On the other hand hypomethylation is induced by oxidative stress and causes uncontrolled expression of pathologic protein genes. The results of brain tumours treatment with TMZ suggest the new mechanism of modulation epigenetic marker in cancer cells. A high TMZ concentration induced a significant increase of m5C content in DNA in the short time, but a low TMZ concentration at longer time hypomethylation is observed for whole range of TMZ concentrations. Therefore TMZ administration with low doses of the drug and short time should be considered as optimal therapy.

Comprehensive analysis of microRNA expression profile in malignant glioma tissues.

Malignant gliomas represent the most devastating group of brain tumors in adults, among which glioblastoma multiforme (GBM) exhibits the highest malignancy rate. Despite combined modality treatment, GBM recurs and is invariably fatal. A further insight into the molecular background of gliomagenesis is required to improve patient outcomes. The primary aim of this study was to gain broad information on the miRNA expression pattern in malignant gliomas, mainly GBM. We investigated the global miRNA profile of malignant glioma tissues with miRNA microarrays, deep sequencing and meta-analysis. We selected miRNAs that were most frequently deregulated in glioblastoma tissues, as well as in peritumoral areas, in comparison with normal human brain. We identified candidate miRNAs associated with the progression from glioma grade III to glioma grade IV. The meta-analysis of miRNA profiling studies in GBM tissues summarizes the past and recent advances in the investigation of the miRNA signature in GBM versus noncancerous human brain and provides a comprehensive overview. We propose a list of 35 miRNAs whose expression is most frequently deregulated in GBM patients and of 30 miRNA candidates recognized as novel GBM biomarkers.

Mature miRNAs form secondary structure, which suggests their function beyond RISC.

The generally accepted model of the miRNA-guided RNA down-regulation suggests that mature miRNA targets mRNA in a nucleotide sequence-specific manner. However, we have shown that the nucleotide sequence of miRNA is not the only determinant of miRNA specificity. Using specific nucleases, T1, V1 and S1 as well as NMR, UV/Vis and CD spectroscopies, we found that miR-21, miR-93 and miR-296 can adopt hairpin and/or homoduplex structures. The secondary structure of those miRNAs in solution is a function of RNA concentration and ionic conditions. Additionally, we have shown that a formation of miRNA hairpin is facilitated by cellular environment.Looking for functional consequences of this observation, we have perceived that structure of these miRNAs resemble RNA aptamers, short oligonucleotides forming a stable 3D structures with a high affinity and specificity for their targets. We compared structures of anti-tenascin C (anti-Tn-C) aptamers, which inhibit brain tumor glioblastoma multiforme (GBM, WHO IV) and selected miRNA. A strong overexpression of miR-21, miR-93 as well Tn-C in GBM may imply some connections between them. The structural similarity of these miRNA hairpins and anti-Tn-C aptamers indicates that miRNAs may function also beyond RISC and are even more sophisticated regulators, that it was previously expected. We think that the knowledge of the miRNA structure may give a new insight into miRNA-dependent gene regulation mechanism and be a step forward in the understanding their function and involvement in cancerogenesis. This may improve design process of anti-miRNA therapeutics.

Effect of desiccation on the dynamics of genome-wide DNA methylation in orthodox seeds of Acer platanoides L.

5-methylcytosine, an abundant epigenetic mark, plays an important role in the regulation of plant growth and development, but there is little information about stress-induced changes in DNA methylation in seeds. In the present study, changes in a global level of m5C were measured in orthodox seeds of Acer platanoides L. during seed desiccation from a WC of 1.04 to 0.05-0.06 g H2O g g(-1) dry mass (g g(-1)). Changes in the level of DNA methylation were measured using 2D TLC e based method. Quality of desiccated seeds was examined by germination and seedling emergence tests. Global m5C content (R2)increase was observed in embryonic axes isolated from seeds collected at a high WC of 1.04 g g(-1) after their desiccation to significantly lower WC of 0.17 and 0.19 g g(-1). Further desiccation of these seeds to a WC of 0.06 g g(-1), however, resulted in a significant DNA demethylation to R2 » 11.52-12.22%. Similar m5C decrease was observed in seeds which undergo maturation drying on the tree and had four times lower initial WC of 0.27 g g(-1) at the time of harvest, as they were dried to a WC of 0.05 g g(-1). These data confirm that desiccation induces changes in seed m5C levels. Results were validated by seed lots derived from tree different A. platanoides provenances. It is plausible that sine wave-like alterations in m5C amount may represent a specific response of orthodox seeds to drying and play a relevant role in desiccation tolerance in seeds.

The crucial elements of the 'last step' of programmed cell death induced by kinetin in root cortex of V. faba ssp. minor seedlings.

KEY MESSAGE: Kinetin-induced programmed cell death, manifested by condensation, degradation and methylation of DNA and fluctuation of kinase activities and ATP levels, is an autolytic and root cortex cell-specific process. The last step of programmed cell death (PCD) induced by kinetin in the root cortex of V. faba ssp. minor seedlings was explained using morphologic (nuclear chromatin/aggregation) and metabolic (DNA degradation, DNA methylation and kinases activity) analyses. This step involves: (1) decrease in nuclear DNA content, (2) increase in the number of 4',6-diamidino-2-phenylindole (DAPI)-stained chromocenters, and decrease in chromomycin A3 (CMA3)-stained chromocenters, (3) increase in fluorescence intensity of CMA3-stained chromocenters, (4) condensation of DAPI-stained and loosening of CMA3-stained chromatin, (5) fluctuation of the level of DNA methylation, (6) fluctuation of activities of exo-/endonucleolytic Zn(2+) and Ca(2+)/Mg(2+)-dependent nucleases, (7) changes in H1 and core histone kinase activities and (8) decrease in cellular ATP amount. These results confirmed that kinetin-induced PCD was a specific process. Additionally, based on data presented in this paper (DNA condensation and ATP depletion) and previous studies [increase in vacuole, increase in amount of cytosolic calcium ions, ROS production and cytosol acidification "in Byczkowska et al. (Protoplasma 250:121-128, 2013)"], we propose that the process resembles autolytic type of cell death, the most common type of death during development of plants. Lastly, the observations also suggested that regulation of these processes might be under control of epigenetic (methylation/phosphorylation) mechanisms.

The degree of global DNA hypomethylation in peripheral blood correlates with that in matched tumor tissues in several neoplasia.

There are no good blood and serum biomarkers for detection, follow up, or prognosis of brain tumors. However, they are needed for more detailed tumor classification, better prognosis estimation and selection of an efficient therapeutic strategy. The aim of this study was to use the epigenetic changes in DNA of peripheral blood samples as a molecular marker to diagnose brain tumors as well as other diseases. We have applied a very precise thin-layer chromatography (TLC) analysis of the global amount of 5-methylcytosine (m(5)C) in DNA from brain tumors, colon and breast cancer tissues and peripheral blood samples of the same patients. The m(5)C level in tissue DNA from different brain tumor types, expressed as R coefficient, changes within the range of 0.2-1.6 and overlaps with R of that of blood samples. It negatively correlates with the WHO malignancy grade. The global DNA hypomethylation quantitative measure in blood, demonstrates a big potential for development of non-invasive applications for detection of a low and a high grade brain tumors. We have also used this approach to analyze patients with breast and colon cancers. In all these cases the m(5)C amount in DNA cancer tissue match with data of blood. This study is the first to demonstrate the potential role of global m(5)C content in blood DNA for early detection of brain tumors and others diseases. So, genomic DNA hypomethylation is a promising marker for prognosis of various neoplasms as well as other pathologies.

Global changes in DNA methylation in seeds and seedlings of Pyrus communis after seed desiccation and storage.

The effects of storage and deep desiccation on structural changes of DNA in orthodox seeds are poorly characterized. In this study we analyzed the 5-methylcytosine (m(5)C) global content of DNA isolated from seeds of common pear (Pyrus communis L.) that had been subjected to extreme desiccation, and the seedlings derived from these seeds. Germination and seedling emergence tests were applied to determine seed viability after their desiccation. In parallel, analysis of the global content of m(5)C in dried seeds and DNA of seedlings obtained from such seeds was performed with a 2D TLC method. Desiccation of fresh seeds to 5.3% moisture content (mc) resulted in a slight reduction of DNA methylation, whereas severe desiccation down to 2-3% mc increased DNA methylation. Strong desiccation of seeds resulted in the subsequent generation of seedlings of shorter height. A 1-year period of seed storage induced a significant increase in the level of DNA methylation in seeds. It is possible that alterations in the m(5)C content of DNA in strongly desiccated pear seeds reflect a reaction of desiccation-tolerant (orthodox) seeds to severe desiccation. Epigenetic changes were observed not only in severely desiccated seeds but also in 3-month old seedlings obtained from these seeds. With regard to seed storage practices, epigenetic assessment could be used by gene banks for early detection of structural changes in the DNA of stored seeds.

Nucleic acid-based technologies in therapy of malignant gliomas.

Malignant gliomas are the deadliest brain tumors, which are characterized by highly invasive growth, a rampant genetic instability and intense resistance to apoptosis. Such an aggressive behavior of malignant gliomas is reflected in the resistance to chemo- and radiotherapy and weak prognosis in spite of cytoreduction through surgery. Brain tumors preferentially express a number of specific protein and RNA markers, that may be exploited as potential therapeutic targets in design of the new treatment modalities based on nucleic acids. For almost three decades, a possibility to apply DNA and RNA molecules as anticancer therapeutics have been studied. A variety of antisense oligonucleotides, ribozymes, DNAzymes, and aptamers can be designed to trigger the sequence-specific inhibition of particular mRNA of interest. RNA interference (RNAi) is the latest and the most promising technique in the long line of nucleic acid-based therapeutic technologies. Recently, we designed and implemented the experimental therapy of patients suffering from malignant brain tumors based on application of double-stranded RNA (dsRNA) specific for tenascin-C (TN-C) mRNA. That therapeutic agent, called ATN-RNA, induces RNAi pathway to inhibit the synthesis of TN-C, the extracellular matrix protein which is highly overexpressed in brain tumor tissue. In the chapter specific problems of application of nucleic acid-based technologies in glioma tumors treatment will be discussed.

Global DNA methylation changes in blood of patients with essential hypertension.

BACKGROUND: Hypertension is a common disease of the cardiovascular system and one of the main causes of mortality in the world. Its etiopathogenesis and molecular mechanisms are unknown. Epigenetic changes may play a role in its development. Therefore the level of 5-methylcytosine (5mC), a well-known epigenetic marker, was analyzed in DNA from the blood of essential hypertension patients. MATERIAL/METHODS: TLC chromatographic analysis of the DNA nucleotide composition was used to determine 5mC levels in blood DNA samples from 60 patients suffering from essential hypertension (30 with stage 1 and 30 with stage 2 hypertension) and 30 control subjects. RESULTS: The mean levels of 5mC were 1.80 + or - 0.69 in the healthy subjects, 1.14 + or - 0.48 in all the patients with essential hypertension, 1.29 + or - 0.50 in those with stage 1, and 0.99 + or - 0.42 in those with stage 2 of hypertension. Statistically significant differences in 5mC amount in DNA were observed between the control group and the whole patient group, the control group and each subgroup of patients, and the groups of patients with stage 1 and stage 2 of hypertension. The level of 5mC in the DNA of the essential hypertension patients was independent of clinical and biochemical factors. CONCLUSIONS: The level of 5mC in the DNA of patients suffering from essential hypertension is lower than in healthy people and depends of the progression of hypertension.

Promising human brain tumors therapy with interference RNA intervention (iRNAi).

Glioblastoma multiforme (GBM) is the most common type of malignant gliomas, characterized by genetic instability, intratumoral histopathological variability and unpredictable clinical behavior. Disappointing results in the treatment of gliomas with surgery, radiation and chemotherapy have fuelled a search for new treatment modalities. Malignant gliomas express preferentially a number of surface markers that may be exploited as therapeutic targets, such as tenascin-C (TN-C), an extracellular matrix glycoprotein that contributes to tumor cell adhesion, invasion, migration and proliferation. In this paper we describe a novel strategy for human brain tumors therapy based on RNA interference (RNAi) and its application after surgery (intervention with RNAi) to inhibit TN-C synthesis. We present data of 46 patients suffering from brain tumors resected and treated with dsRNA with the sequence homology of tenascin-C mRNA (ATN-RNA). The specific effect of ATN-RNA on TN-C downregulation was proved with antibodies against TN-C in glioblastoma multiforme cultured cells. A significant improvement in overall survival (OS) without loosing the quality of life (QOL) of patients was observed. MRI and CT studies showed tumor growth delay or lack of tumor recurrence. This novel therapy based on RNA interference shows a hopeful therapeutical potential. To our knowledge the intervention with RNAi (iRNAi) method is the first protocol of RNAi application in human brain tumor treatment.

A new and efficient method for inhibition of RNA viruses by DNA interference.

We report here a new method for inhibition of RNA viruses induced by dsDNA. We demonstrated that both long dsDNA molecules and short interfering DNA with a sequence complementary to that of viral RNA inhibited tobacco mosaic virus expression and prevented virus spread. Also, the expression of the HIV-1 gp41 gene in HeLa cells was inhibited by complementary short interfering DNA. We showed that Dicer processed dsDNA, which suggests activation of the cellular machinery involved in silencing of RNA. For the silencing of viral RNA effected with dsDNA, we coined the term DNA interference technology.