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BackgroundThe mitotic spindle is a complex mechanical apparatus required for accurate segregation of sister chromosomes during mitosis. We designed a genetic screen using automated microscopy to discover factors essential for mitotic progression. Using a RNA interference library of 49,164 double-stranded RNAs targeting 23,835 human genes, we performed a loss of function screen to look for small interfering RNAs that arrest cells in metaphase.ResultsHere we report the identification of genes that, when suppressed, result in structural defects in the mitotic spindle leading to bent, twisted, monopolar, or multipolar spindles, and cause cell cycle arrest. We further describe a novel analysis methodology for large-scale RNA interference datasets that relies on supervised clustering of these genes based on Gene Ontology, protein families, tissue expression, and protein-protein interactions.ConclusionThis approach was utilized to classify functionally the identified genes in discrete mitotic processes. We confirmed the identity for a subset of these genes and examined more closely their mechanical role in spindle architecture.

The use of isogenic tumor cells lines expressing either wild-type mTOR cDNA or a mutant that does not bind RAD001 demonstrates that the effects of RAD001 are through inhibition of mTOR function. Findings indicate that molecular rationale for combining DNA damaging agents with RAD001, showing that a general effect on a major anabolic process may dramatically enhance the efficacy of an established drug protocol in the treatment of cancer patients with solid tumors.

A threshold level of the triple helix forming oligonucleotides (TFOs), consisting of three or four 2'-AE residues, are 10-fold more active than those with just 2 2'-AE residues in a hypoxanthine phosphoribosyltransferase (HPRT) activity monitored gene knockout assays. Further, the frequencies of HPRT activity with the active TFOs are 300-400 fold higher than the background activity.

A study was conducted to examine chemically modified nucleic acids as possible antisense compounds and to analyze the structure of DNA duplexes supporting 2'-O-modified RNA analogs. Diffraction data were determined at room temperature using a Rigaku R-AXIS-IIc image plate system. Results indicated that anomeric factors and gauche effects influenced the conformational rigidity of 2'-O-substituents.

In HPLC, temperature programming and isocratic separation were investigated for resolving a mixture of nucleotide polymers. A mixture of short oligonucleotides pd[(A).sub.12-18] was resolved in less than 4 min by utilizing isocratic separation. The method is sensitive to organic modifier concentration; a 0.5% change of organic modifier in the mobile phase leads to more than doubling of the total analysis time. Temperature programming was used to optimize the separation of larger oligonucleotides pd[(A).sub.25-30] and pd[(A).sub.40-60]. When the column temperature was changed by programming during the separation, the analysis time was 75% less than for an isocratic/isothermal run. The low amounts of ion-pairing agent in the mobile phase (between 5 and 10 mM) make these methods suitable for electrospray ionization mass spectrometry.

An interesting new approach to capillary affinity gel electrophoresis (CAGE) has been developed for the selective capture and separation of homopolymer and heteropolymer oligonucleotides. The combination of selectivity of bioaffinity recognition and high-resolution power of capillary gel electrophoresis allows the on-line sequence- and size-specific separation of oligonucleotides. Both rigid gel formulations and viscous replaceable polymer solutions having user-defined, single-stranded oligonucleotides covalently attached as recognition sequences are used. Contrary to most known affinity systems in capillary electrophoresis, which operate in a continuous mode, binding and release are accomplished in two steps, effectively separating the affinity from the separation step. At low temperature, oligonucleotides with complementary sequences in the analyte solution will bind to the immobilized recognition sequence while unrelated oligonucleotides will continue to migrate. This step is a preseparation, removing all nonspecific solutes from the sample. The release of the bound solutes is achieved at elevated temperature, allowing a probe of cross-reactivity for a given biorecognition element. Applications for high-resolution separations of short oligonucleotides and their mismatches are shown, and the potential for on-line preconcentration and separation of dilute analyte solutions, thus effectively enhancing the sensitivity, is demonstrated.

The chemical modification 2′- O-methyl of nucleosides is often used to increase si RNA stability towards nuclease activities. However, the metabolic fate of modified nucleosides remains unclear. Therefore, the aim of this study was to determine the mass balance, pharmacokinetic, and absorption, distribution, metabolism, and excretion ( ADME)-properties of tritium-labeled 2′- O-methyluridine, following a single intravenous dose to male CD-1 mice. The single intravenous administration of [5-3H]-2′- O-methyluridine was well tolerated in mice. Radioactivity was rapidly and widely distributed throughout the body and remained detectable in all tissues investigated throughout the observation period of 48 h. After an initial rapid decline, blood concentrations of total radiolabeled components declined at a much slower rate. [3H]-2′- O-Methyluridine represented a minor component of the radioactivity in plasma (5.89% of [3H]- AUC0-48 h). Three [3H]-2′- O-methyluridine metabolites namely uridine (M1), cytidine (M2), and uracil (M3) were the major circulating components representing 32.8%, 8.11%, and 23.6% of radioactivity area under the curve, respectively. The highest concentrations of total radiolabeled components and exposures were observed in kidney, spleen, pineal body, and lymph nodes. The mass balance, which is the sum of external recovery of radioactivity in excreta and remaining radioactivity in carcass and cage wash, was complete. Renal excretion accounted for about 52.7% of the dose with direct renal excretion of the parent in combination with metabolism to the endogenous compounds cytidine, uracil, cytosine, and cytidine. [ABSTRACT FROM AUTHOR]

Summary: Excess levels of circulating amino acids (AAs) play a causal role in specific human pathologies, including obesity and type 2 diabetes. Moreover, obesity and diabetes are contributing factors in the development of cancer, with recent studies suggesting that this link is mediated in part by AA activation of mammalian target of rapamycin (mTOR) Complex 1. AAs appear to mediate this response through class III phosphatidylinositol 3-kinase (PI3K), or human vacuolar protein sorting 34 (hVps34), rather than through the canonical class I PI3K pathway used by growth factors and hormones. Here we show that AAs induce a rise in intracellular Ca2+ ([Ca2+]i), which triggers mTOR Complex 1 and hVps34 activation. We demonstrate that the rise in [Ca2+]i increases the direct binding of Ca2+/calmodulin (CaM) to an evolutionarily conserved motif in hVps34 that is required for lipid kinase activity and increased mTOR Complex 1 signaling. These findings have important implications regarding the basic signaling mechanisms linking metabolic disorders with cancer progression. [Copyright &y& Elsevier]

Functional genomics is inundating the pharmaceutical industry with large numbers of potential gene targets from several sources such as gene expression profiling experiments (DNA microchips, proteomics) or database mining. Oligonucleotide-based RNA-knock down technologies such as antisense or RNA interference can aid in the filtering and prioritization of target candidates in the drug discovery process. [ABSTRACT FROM AUTHOR]

Abstract: Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27Kip1 was shown to be required for the activation of key cyclin-dependent kinases, thereby triggering the onset of DNA replication and cell cycle progression. Although the SCFSkp2 ubiquitin ligase has been reported to mediate p27Kip1 degradation, the nature of the human ubiquitin-conjugating enzyme involved in this process has not yet been determined at the cellular level. Here, we show that antisense oligonucleotides targeting the human ubiquitin-conjugating enzyme Cdc34 downregulate its expression, inhibit the degradation of p27Kip1, and prevent cellular proliferation. Elevation of p27Kip1 protein level is found to be the sole requirement for the inhibition of cellular proliferation induced upon downregulation of Cdc34. Indeed, reducing the expression of p27Kip1 with a specific antisense oligonucleotide is sufficient to reverse the anti-proliferative phenotype elicited by the Cdc34 antisense. Furthermore, downregulation of Cdc34 is found to specifically increase the abundance of the SCFSkp2 ubiquitin ligase substrate p27Kip1, but has no concomitant effect on the level of IkBα and β-catenin, which are known substrates of a closely related SCF ligase. [Copyright &y& Elsevier]

The chemical modification 2'-O-methyl of nucleosides is often used to increase siRNA stability towards nuclease activities. However, the metabolic fate of modified nucleosides remains unclear. Therefore, the aim of this study was to determine the mass balance, pharmacokinetic, and absorption, distribution, metabolism, and excretion (ADME)-properties of tritium-labeled 2'-O-methyluridine, following a single intravenous dose to male CD-1 mice. The single intravenous administration of [5-(3)H]-2'-O-methyluridine was well tolerated in mice. Radioactivity was rapidly and widely distributed throughout the body and remained detectable in all tissues investigated throughout the observation period of 48 h. After an initial rapid decline, blood concentrations of total radiolabeled components declined at a much slower rate. [(3)H]-2'-O-Methyluridine represented a minor component of the radioactivity in plasma (5.89% of [(3)H]-AUC 0-48 h). Three [(3)H]-2'-O-methyluridine metabolites namely uridine (M1), cytidine (M2), and uracil (M3) were the major circulating components representing 32.8%, 8.11%, and 23.6% of radioactivity area under the curve, respectively. The highest concentrations of total radiolabeled components and exposures were observed in kidney, spleen, pineal body, and lymph nodes. The mass balance, which is the sum of external recovery of radioactivity in excreta and remaining radioactivity in carcass and cage wash, was complete. Renal excretion accounted for about 52.7% of the dose with direct renal excretion of the parent in combination with metabolism to the endogenous compounds cytidine, uracil, cytosine, and cytidine.

The excitation of nociceptive sensory neurons by ATP released in injured tissue is believed to be mediated partly by P2X3 receptors. Although an analysis of P2X3 knock-out mice has revealed some deficits in nociceptive signaling, detailed analysis of the role of these receptors is hampered by the lack of potent specific pharmacological tools. Here we have used antisense oligonucleotides (ASOs) to downregulate P2X3 receptors to examine their role in models of chronic pain in the rat. ASOs and control missense oligonucleotides (180 microg/d) were administered intrathecally to naive rats for up to 7 d via a lumbar indwelling cannula attached to an osmotic minipump. Functional downregulation of the receptors was confirmed by alphabeta-methylene ATP injection into the hindpaw, which evoked significantly less mechanical hyperalgesia as early as 2 d after treatment with ASOs relative to controls. At this time point, P2X3 protein levels were significantly downregulated in lumbar L4 and L5 dorsal root ganglia. After 7 d of ASO treatment, P2X3 protein levels were reduced in the primary afferent terminals in the lumbar dorsal horn of the spinal cord. In models of neuropathic (partial sciatic ligation) and inflammatory (complete Freund's adjuvant) pain, inhibition of the development of mechanical hyperalgesia as well as significant reversal of established hyperalgesia were observed within 2 d of ASO treatment. The time course of the reversal of hyperalgesia is consistent with downregulation of P2X3 receptor protein and function. This study demonstrates the utility of ASO approaches for validating gene targets in in vivo pain models and provides evidence for a role of P2X3 receptors in the pathophysiology of chronic pain.