Profiling cytotoxic microRNAs in pediatric and adult glioblastoma cells by high-content screening, identification, and validation of miR-1300.

MicroRNAs play an important role in the regulation of mRNA translation and have therapeutic potential in cancer and other diseases. To profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen in adult and pediatric GBM cells using a synthetic oligonucleotide library representing all known human microRNAs. Bioinformatics analysis was used to refine this list and the top seven microRNAs were validated in a larger panel of GBM cells using state-of-the-art in vitro assays. The cytotoxic effect of our most relevant candidate was assessed in a preclinical model. Our screen identified ~100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 transfected cells due to cytokinesis failure followed by apoptosis. This was also observed in two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In GBM cells, where miR-1300 is normally not expressed, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target. ECT2 siRNA phenocopied the effects of miR-1300, and ECT2 overexpression led to rescue of miR-1300 induced binucleation. We showed that ectopic expression of miR-1300 led to decreased tumor growth in an orthotopic GBM model. Our screen provides a resource for the neuro-oncology community and identified miR-1300 as a novel regulator of endomitosis with translatable potential for therapeutic application.

The prevalence of PAX2 mutations in patients with isolated colobomas or colobomas associated with urogenital anomalies.

The PAX2 gene is mutated in patients with ocular colobomas, vesicoureteral reflux (VUR), and kidney anomalies (renal-coloboma syndrome, OMIM 120330). The three abnormalities which make up this syndrome also occur in isolation, but the causal genes are not known. PAX2 encodes a transcription factor of the paired box class of DNA binding proteins, important for the development of the urogenital tract, optic nerve and adjacent retina, inner ear, and CNS. In this paper we have investigated the prevalence of PAX2 mutations in patients with ocular colobomas, microphthalmos, or retinal anomalies, either in isolation or with associated urogenital anomalies. Using PCR-SSCP, most or all exons of PAX2 were examined in blood DNA from 99 patients who have either ocular anomalies alone or a combination of ocular and urogenital conditions. PAX2 mutations were not detected in patients with ocular colobomas, either in isolation or with associated abnormalities, except in one patient with typical renal-coloboma syndrome. We conclude that PAX2 mutations are unlikely to be common in patients with ocular colobomas in isolation or in patients with ocular colobomas and associated anomalies, except for patients with typical renal-coloboma syndrome where PAX2 is known to be the aetiological cause.

Cloning and characterization of the human PAX2 promoter.

PAX2, a member of the PAX gene family of developmental transcription factors, is expressed at high levels in the developing eyes, ears, central nervous and urogenital systems, as well as in Wilms' tumor and renal cell carcinoma. Expression of PAX2 in the urogenital system is associated with proliferating cells of the ureteric bud and the differentiating nephrogenic mesenchyme. To date, little is known about the molecular mechanisms controlling the regulation of PAX2 expression. This report describes the cloning and characterization of the human PAX2 gene promoter and localization of the transcription start sites in fetal kidney and Wilms' tumor. We identified two transcription start sites in a Wilms' tumor sample, which were found to be different from that in fetal kidney. The activity of a deletion series of the PAX2 promoter was assessed in NIH-3T3, COS-7, 293, and Madin-Darby canine kidney cells. Although some differences were observed in the activity of each promoter construct, the profile of activity for the promoter fragment series was similar in each experiment, regardless of cell type. The WT1 tumor suppressor protein, which has previously been shown to repress murine Pax2 expression in vitro, was shown to also repress expression from the human PAX2 promoter.

The use of the BiPAP biphasic positive airway pressure system in acute spinal cord injury.

In recent years there has been increasing demand on our Intensive Care Unit (ICU) facilities, mainly due to improved resuscitation techniques in the pre-hospital management of spinal cord injury (SCI). This has resulted in an increasing number of high tetraplegic and paraplegic patients with respiratory problems who have survived the initial injury, but have subsequently required ventilatory support, often for several weeks. In view of the continuing pressure on ICU beds and a consequent need for alternative means of providing ventilatory support within the spinal centre rather than within the ICU setting, there was a requirement to provide a simple means of ventilatory support suitable for use within the ward setting. Ventilatory assistance using BiPAP appeared to fulfil these criteria, enabling patients to be managed at reduced cost. We present our experience using this system in 28 acute SCI patients over a 4 year period.

Differential regulation of the human Wilms tumour suppressor gene (WT1) promoter by two isoforms of PAX2.

PAX2 is a member of the paired box family of genes with an important role in kidney, genital tract and eye development. Another gene essential for kidney and genital tract development is the Wilms tumour gene, WT1. PAX2 and WT1 encode transcription factors expressed during fetal kidney development in patterns that overlap both spatially and temporally. The overlap of PAX2 and WT1 expression in fetal kidney prompted us to determine whether PAX2 regulates the WT1 gene. To investigate this possibility, the WT1 promoter and a series of WT1 promoter deletion fragments were cloned into a luciferase reporter vector, and used in co-transfection experiments with PAX2 expression constructs. PAX2 transactivated the WT1 promoter up to 35-fold in CHO-K1 cells, and from four- to sevenfold in 293 cells. Two regions of the WT1 promoter were required in the same promoter construct for efficient transactivation by PAX2 in CHO-K1 cells, and purified recombinant PAX2 protein was found to bind to two sites in the WT1 promoter, at -205/-230 and +377/+402. Removal of WT1 promoter sequences containing the -205/-230, or +377/+402 binding sites abolished transactivation of the WT1 promoter by PAX2 in CHO-K1 cells, and had a differential effect on transactivation of the WT1 promoter in 293 cells, depending on the PAX2 isoform used. A fragment containing the -205/-230 site alone could be transactivated by PAX2. These findings suggest that PAX2 is a tissue-specific modulator of WT1 expression, and is involved in cell growth control via WT1.

Further delineation of renal-coloboma syndrome in patients with extreme variability of phenotype and identical PAX2 mutations.

Renal-coloboma syndrome is a recently described autosomal dominant syndrome of abnormal optic nerve and renal development. Two families have been reported with renal-coloboma syndrome and mutations of the PAX2 gene. The PAX2 gene, which encodes a DNA-binding protein, is expressed in the developing ear, CNS, eye, and urogenital tract. Ocular and/or renal abnormalities have been consistently noted in the five reports of patients with renal-coloboma syndrome, to date, but PAX2 expression patterns suggest that auditory and CNS abnormalities may be additional features of renal-coloboma syndrome. To determine whether additional clinical features are associated with PAX2 mutations, we have used PCR-SSCP to identify PAX2 gene mutations in patients. We report here four patients with mutations in exon 2, one of whom has severe ocular and renal disease, microcephaly, and retardation, and another who has ocular and renal disease with high-frequency hearing loss. Unexpectedly, extreme variability in clinical presentation was observed between a mother, her son, and an unrelated patient, all of whom had the same PAX2 mutation as previously described in two siblings with renal-coloboma syndrome. These results suggest that a sequence of seven Gs in PAX2 exon 2 may be particularly prone to mutation.

Genomic structure of the human PAX2 gene.

PAX2 is one of nine PAX genes that have been described in vertebrates. Each PAX gene contains a conserved paired box domain that was first identified in Drosophila. PAX2 encodes a transcription factor that has a critical role in the development of the urogenital tract, the eyes, and the CNS. Recently, we reported a mutation of PAX2 in patients with optic nerve coloboma, vesicoureteric reflux, and renal anomalies. To facilitate further analysis of PAX2 mutations in human disease, we have now determined the complete structure of the human PAX2 gene. Five genomic lambda clones containing human PAX2 gene sequences were isolated. Sequencing and restriction mapping of these clones showed that human PAX2 was composed of 12 exons spanning approximately 70 kb. Two alternatively spliced exons and a dinuclotide repeat polymorphism were also determined in PAX2. These data will be useful in characterizing the role of PAX2 in human disease.

Mutation of the PAX2 gene in a family with optic nerve colobomas, renal anomalies and vesicoureteral reflux.

Paired box (PAX) genes play a critical role in human development and disease. The PAX2 gene is expressed in primitive cells of the kidney, ureter, eye, ear and central nervous system. We have conducted a mutational analysis of PAX2 in a family with optic nerve colobomas, renal hypoplasia, mild proteinuria and vesicoureteral reflux. We report a single nucleotide deletion in exon five, causing a frame-shift of the PAX2 coding region in the octapeptide domain. The phenotype resulting from the PAX2 mutation in this family was very similar to abnormalities that have been reported in Krd mutant mice. These data suggest that PAX2 is required for normal kidney and eye development.

Alternative messenger RNA forms and open reading frames within an additional conserved region of the human PAX-2 gene.

PAX-2 is a member of a family of genes containing a highly conserved paired box domain. The paired box domain encodes a DNA binding motif, indicating that PAX proteins may function as transcriptional regulators, participating in a hierarchical network of gene regulation during embryogenesis. In this report, we provide evidence that there is an additional conserved region near the predicted COOH-terminus of PAX-2, PAX-5, and PAX-8. We also describe alternative splicing of a conserved 83-nucleotide segment in the 3' coding sequence of the PAX-2 gene. PAX-2 transcripts that contain the 83-nucleotide insertion encode a putative protein with an alternative COOH-terminus. The presence of an additional conserved region and alternative splicing within the predicted COOH-terminal region of several PAX genes has implications for the evolutionary origins and for the DNA binding site specificity of PAX-2, PAX-5, and PAX-8.

Properties of single potassium channels in vesicles formed from the sarcolemma of frog skeletal muscle.

The patch-clamp method was used to study unitary delayed rectifier K+ channels in large vesicles formed from the membrane of frog skeletal muscle. Channels were activated by depolarizing pulses. Single-channel conductance was about 15 pS in physiological [K+]o and was doubled by raising [K+]o to 120 mM. TEA+ caused an apparent reduction in single-channel current, which we attribute to a rapid block. When depolarizations were repeated at brief intervals, records with and without channel openings were ordered non-randomly, providing evidence for a slow process which was probably inactivation. In multichannel patches the relation between variance and mean current, binomial analysis, and the distribution of times for single and double openings were all consistent with channels behaving independently. Open times were distributed exponentially. Mean open time, tau o, increased with depolarization so that 1/tau o was an exponential function of voltage. First latency histograms peaked at times later than zero and could not be fitted by a scheme having only two closed states. Channel openings occurred in bursts and closed time histograms could be fitted by the sum of three exponentials. Our results imply a scheme with at least three closed states, an open and an inactivated state.

A new preparation for recording single-channel currents from skeletal muscle.

Spherical vesicles of sarcolemma (media diameter, 78 microns) were prepared from frog skeletal muscle by prolonged exposure to enzymes (collagenase, then protease) and to isotonic KCl solutions, which promote swelling of muscle fibres. Patch clamp recording was used to measure unitary K and Na currents from this preparation. Na and K channels had conductances around 15 pS and their kinetic properties appeared unaltered by vesicle formation.

The effects of injection of calcium ions and calcium chelators on calcium channel inactivation in Helix neurones.

1. The effects of intracellular injection of Ca, EGTA and EGTA/Ca buffers on inward currents flowing through the Ca channel in Helix aspersa neurones were studied under voltage clamp. 2. Inward currents were reversibly reduced by Ca injection. The extent of the reduction was dependent on the size and duration of the injection. Recovery from injection was exponential with a time constant around 18 s at 18-20 degrees C. 3. In our salines, which contained tetraethylammonium chloride and 4-aminopyridine, no outward current was activated by Ca injection at the holding potential. A given Ca injection reduced the inward current by the same fraction in 25 mM- and 2 . 5 mM-Sr and also at different test potentials. We conclude that Ca injection does not activate an outward current. 4. Mean resting ionized internal Ca concentration, [Ca]i, measured with a Ca-sensitive electrode was 1.9 X 10(-7) M. Our injections increased this by less than 10(-5) M, as expected if most of the injected Ca is bound. Constant-field or Eyring rate theory considerations suggest that this rise in [Ca]i would not significantly affect the inward current through open Ca channels and we conclude, therefore, that Ca injection causes Ca channel inactivation. 5. The effect of Ca injection was blocked by prior injection of EGTA. Extracellular application of carbonyl cyanide m-chlorophenylhydrazone increased the effect of Ca injection. 6. Ca injection does not inactivate Ca channels by lowering pHi since large H+ injections only caused small irreversible decreases in inward current. 7. EGTA injection increased peak Ca current (ICa) by around 30% and decreased the rate and extent of inactivation. Some inactivation remained, however, even after maximal EGTA injections. 8. Injection of EGTA/Ca buffers with free [Ca] less than 1.8 X 10(-7) M increased peak ICa, while buffers with free [Ca] greater than 8.9 X 10(-7) M decreased ICa. 9. Our results support the suggestion that Ca ions cause Ca inactivation by binding to a site which is accessible from the inside of the cell, and also suggest that there is some steady-state inactivation present at physiological [Ca]i. A simple model is presented which describes the decline of Ca current in terms of Ca binding to a site accessible from the cytoplasm.