Inhibition of ERβ induces resistance to cisplatin by enhancing Rad51-mediated DNA repair in human medulloblastoma cell lines

Cisplatin is one of the most widely used and effective anticancer drugs against solid tumors including cerebellar tumor of the childhood, Medulloblastoma. However, cancer cells often develop resistance to cisplatin, which limits therapeutic effectiveness of this otherwise effective genotoxic drug. In this study, we demonstrate that human medulloblastoma cell lines develop acute resistance to cisplatin in the presence of estrogen receptor (ER) antagonist, ICI182,780. This unexpected finding involves a switch from the G2/M to G1 checkpoint accompanied by decrease in ATM/Chk2 and increase in ATR/Chk1 phosphorylation. We have previously reported that ERβ, which is highly expressed in medulloblastomas, translocates insulin receptor substrate 1 (IRS-1) to the nucleus, and that nuclear IRS-1 binds to Rad51 and attenuates homologous recombination directed DNA repair (HRR). Here, we demonstrate that in the presence of ICI182,780, cisplatin-treated medulloblastoma cells show recruitment of Rad51 to the sites of damaged DNA and increase in HRR activity. This enhanced DNA repair during the S phase preserved also clonogenic potential of medulloblastoma cells treated with cisplatin. In conclusion, inhibition of ERβ considered as a supplemental anticancer therapy, has been found to interfere with cisplatin-induced cytotoxicity in human medulloblastoma cell lines.     

High-density mapping of the earliness per se-3Am (Eps-3A m ) locus in diploid einkorn wheat and its relation to the syntenic regions in rice and Brachypodium distachyon L.

In temperate grasses, such as wheat (Triticum sp.) and barley (Hordeum vulgare L.), earliness per se (eps) is defined as the intrinsic difference in flowering time of fully vernalized plants grown under long day conditions. The main task of our investigations was to confirm and refine the chromosomal position of the previously mapped Eps-3A ^m locus in einkorn wheat (Shindo et al. in Genome 45:563?C569, 2002; Hori et al. in Breed Sci 57:39?C45, 2007). To this end, 110 recombinant inbred lines (RILs) were phenotypically and genotypically evaluated. The population had been obtained from a cross between an early flowering X-ray mutant KT3-5 (T. monococcum L.) of cultivated spring type and a wild wheat line KT1-1 of T. boeoticum Boiss. (winter type). Heading time evaluations under greenhouse conditions resulted in a 10-day difference (P?=?0.008) between the mapping parents and a range of 49?days for the RIL population. Quantitative trait locus analyses of heading time, anthesis time, spikelet number and spike length showed the best linkage with marker loci close to the telomeric region of the long arm of chromosome 3A. The highest LOD values for heading time, anthesis time, spikelet number and spike length were 5.96, 5.40, 3.33 and 5.10, respectively (LOD threshold 2.0, P?<?0.05). Linkage analysis of 51 molecular markers delimited the new Eps-3A ^m interval to 0.9?cM, including 14 newly developed markers using collinearity with rice (Oryza sativa L.) and Brachypodium distachyon L. The present study marks a further step towards the molecular elucidation and functional analysis of Eps-3A ^m in wheat and other small grain cereals.     

The effect of hyperosmolar stimuli and cyclophosphamide on the culture of normal rat urothelial cells <Emphasis Type="Italic">in vitro</Emphasis>

Highly concentrated urine may induce a harmful effect on the urinary bladder. Therefore, we considered osmolarity of the urine as a basic pathomechanism of mucosal damage. The influence of both cyclophosphamide (CYP) and hyperosmolar stimuli (HS) on the urothelium are not well described. The purpose was to evaluate the effect of CYP and HS on rat urothelial cultured cells (RUCC). 15 Wistar rats were used for RUCC preparation. RUCC were exposed to HS (2080 and 3222 mOsm/l NaCl) for 15 min and CYP (1 mg/ml) for 4 hrs. APC-labelled annexin V was used to quantitatively determine the percentage of apoptotic cells and propidium iodide (PI) as a standard flow cytometric viability probe to distinguish necrotic cells from viable ones. Annexin V-APC (+), annexin V-APC and PI (+), and PI (+) cells were analysed as apoptotic, dead, and necrotic cells, respectively. The results were presented in percentage values. The flow cytometric analysis was done on a FACSCalibur Flow Cytometer using Cell-Quest software. Treatment with 2080 and 3222 mOsm/l HS resulted in 23.7 ± 3.9% and 26.0 ± 1.5% apoptotic cells, respectively, 14.3 ± 1.4% and 19.4 ± 2.7% necrotic cells, respectively and 60.5 ± 1.4% and 48.6 ± 5.3% dead cells, respectively. The effect of CYP on RUCC was similar to the effect of HS. After CYP the apoptotic and necrotic cells were 23.1 ± 0.3% and 17.9 ± 7.4%, respectively. The percentage of dead cells was 57.7 ± 10.8%. CYP and HS induced apoptosis and necrosis in RUCC. 3222 mOsm/l HS had the most harmful effect based on the percentage of necrotic and apoptotic cells.     

Novel insertion in exon 5 of the TCOF1 gene in twin sisters with Treacher Collins syndrome

Treacher Collins syndrome (TCS) is associated with an abnormal differentiation of the first and second pharyngeal arches during fetal development. This causes mostly craniofacial deformities, which require numerous corrective surgeries. TCS is an autosomal dominant disorder and it occurs in the general population at a frequency of 1 in 50,000 live births. The syndrome is caused by mutations in the TCOF1 gene, which encodes the serine/alanine-rich protein named Treacle. Over 120 mutations of the TCOF1 gene responsible for TCS have been described. About 70% of recognized mutations are deletions, which lead to a frame shift, formation of a termination codon, and shortening of the protein product of the gene. Herewith, a new heterozygotic insertion, c.484_668ins185bp, was described in two monozygotic twin sisters suffering from TCS. This mutation was absent in their father, brother, and uncle, indicating a de novo origin. The insertion causes a shift in the reading frame and premature termination of translation at 167 aa. The novel insertion is the longest ever found in the TCOF1 gene and the only one found among monozygotic twin sisters.     

Dental methacrylates may exert genotoxic effects via the oxidative induction of DNA double strand breaks and the inhibition of their repair

Methacrylate monomers used in dentistry have been shown to induce DNA double strand breaks (DSBs), one of the most serious DNA damage. In the present work we show that a model dental adhesive consisting of 45% 2-hydroxyethyl methacrylate (HEMA) and 55% bisphenol A-diglycidyl dimethacrylate (Bis-GMA) at concentrations up to 0.25 mM Bis-GMA induced oxidative DNA in cultured primary human gingival fibroblasts (HGFs) as evaluated by the comet assay and probed with human 8-hydroxyguanine DNA-glycosylase 1. HEMA/Bis-GMA induced DSBs in HGFs as assessed by the neutral comet assay and phosphorylation of the H2AX histone and sodium ascorbate or melatonin (5-methoxy-N-acetyltryptamine) both at 50 μM reduced the DSBs, they also inhibited apoptosis induced by HEMA/Bis-GMA. The adhesive slowed the kinetics of the repair of DNA damage induced by hydrogen peroxide in HGFs, while sodium ascorbate or melatonin improved the efficacy of H(2)O(2)-induced damage in the presence of the methacrylates. The adhesive induced a rise in the G2/M cell population, accompanied by a reduction in the S cell population and an increase in G0/G1 cell population. Sodium ascorbate or melatonin elevated the S population and reduced the G2/M population. In conclusion, HEMA/Bis-GMA induce DSBs through, at least in part, oxidative mechanisms, and these compounds may interfere with DSBs repair. Vitamin C or melatonin may reduce the detrimental effects induced by methacrylates applied in dentistry.     

Automated 3D structure composition for large RNAs

Understanding the numerous functions that RNAs play in living cells depends critically on knowledge of their three-dimensional structure. Due to the difficulties in experimentally assessing structures of large RNAs, there is currently great demand for new high-resolution structure prediction methods. We present the novel method for the fully automated prediction of RNA 3D structures from a user-defined secondary structure. The concept is founded on the machine translation system. The translation engine operates on the RNA FRABASE database tailored to the dictionary relating the RNA secondary structure and tertiary structure elements. The translation algorithm is very fast. Initial 3D structure is composed in a range of seconds on a single processor. The method assures the prediction of large RNA 3D structures of high quality. Our approach needs neither structural templates nor RNA sequence alignment, required for comparative methods. This enables the building of unresolved yet native and artificial RNA structures. The method is implemented in a publicly available, user-friendly server RNAComposer. It works in an interactive mode and a batch mode. The batch mode is designed for large-scale modelling and accepts atomic distance restraints. Presently, the server is set to build RNA structures of up to 500 residues.     

Binding of the plant hormone kinetin in the active site of Mistletoe Lectin I from Viscum album

The crystal structure of the ribosome inhibiting protein Mistletoe Lectin I (ML-I) derived from the European mistletoe, Viscum album, in complex with kinetin has been refined at 2.7? resolution. Suitably large crystals of ML-I were obtained applying the counter diffusion method using the Gel Tube R Crystallization Kit (GT-R) on board the Russian Service Module on the international space station ISS within the GCF mission No. 6, arranged by the Japanese aerospace exploration agency (JAXA). Hexagonal bi-pyramidal crystals were grown during three months under microgravity. Before data collection the crystals were soaked in a saturated solution of kinetin and diffraction data to 2.7? were collected using synchrotron radiation and cryogenic techniques. The atomic model was refined and revealed a single kinetin molecule in the ribosome inactivation site of ML-I. The complex demonstrates the feasibility of mistletoe to bind plant hormones out of the host regulation system as part of a self protection mechanism.