The regulation of mitochondrial DNA copy number in glioblastoma cells

As stem cells undergo differentiation, mitochondrial DNA (mtDNA) copy number is strictly regulated in order that specialized cells can generate appropriate levels of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to undertake their specific functions. It is not understood whether tumor-initiating cells regulate their mtDNA in a similar manner or whether mtDNA is essential for tumorigenesis. We show that human neural stem cells (hNSCs) increased their mtDNA content during differentiation in a process that was mediated by a synergistic relationship between the nuclear and mitochondrial genomes and results in increased respiratory capacity. Differentiating multipotent glioblastoma cells failed to match the expansion in mtDNA copy number, patterns of gene expression and increased respiratory capacity observed in hNSCs. Partial depletion of glioblastoma cell mtDNA rescued mtDNA replication events and enhanced cell differentiation. However, prolonged depletion resulted in impaired mtDNA replication, reduced proliferation and induced the expression of early developmental and pro-survival markers including POU class 5 homeobox 1 (OCT4) and sonic hedgehog (SHH). The transfer of glioblastoma cells depleted to varying degrees of their mtDNA content into immunocompromised mice resulted in tumors requiring significantly longer to form compared with non-depleted cells. The number of tumors formed and the time to tumor formation was relative to the degree of mtDNA depletion. The tumors derived from mtDNA depleted glioblastoma cells recovered their mtDNA copy number as part of the tumor formation process. These outcomes demonstrate the importance of mtDNA to the initiation and maintenance of tumorigenesis in glioblastoma multiforme.     

Immortalization of bovine umbilical vein endothelial cells: a model for the study of vascular endothelium

Endothelial cells perform a large array of physiological functions that are influenced by their cellular heterogeneity in the different vascular beds. Vein endothelial cells isolated from the umbilical cords are commonly used to study vascular endothelium. Primary cultures of these cells, however, have low proliferative capacity and a limited life span. We have immortalized bovine umbilical vein endothelial cells (BUVEC) by transfection with an expression vector containing the human papillomavirus type 16 E6E7 oncogenes. Expression of E6E7 extended the life span of BUVEC from 40 to more than 1-20 cell replication cycles with no signs of senescence. Four immortalized clones were isolated and found to maintain endothelial cell properties, such as the uptake of acetylated low density lipoprotein, the expression of the von Willebrand protein, the binding of endothelial cell-specific lectins and proliferative responses to the specific endothelial cell mitogen, vascular endothelial growth factor. Moreover, clone BVE-E6E7-1, like its wild-type counterparts, expressed prolactin mRNA and decreased its proliferation in response to the anti-angiogenic 16-kDa fragment of prolactin. This clone showed little signs of genetic instability as revealed by centrosome and chromosome number analysis. Thus, immortalized E6E7 BUVEC cell lines retain endothelial cell characteristics and could facilitate studies to investigate the action of regulatory factors of vascular endothelium. Moreover, being the first non-human umbilical vein endothelial cell lines, their use should provide insights into the mechanisms governing species-related heterogeneity of endothelial cells.     

The Bactericidal Action of Peroxides; An E.P.R. Spin-Trapping Study

E.P.R. spin trapping has been employed to study radical production during the bactericidal action of three peroxide compounds (peracetic acid, 4-percarboxy-N-isobutyltrimellitimide and magnesium monoperoxyphthalate) upon both Gram negative (Escherichia Coll) and Gram positive (Staphylococcus Aureus) bacteria. Use of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has allowed direct detection of both carbon-centred and hydroxyl radicals, which are produced at varying rates for the different bacteria/peracid systems studied. The inhibition of bactericidal action, by DMPO and two antioxidants, Vitamin C and Trolox C, indicates that radicals are the lethal species and evidence is presented which suggests that radical production is internal to the bacterial cell. Hydroxyl radicals are believed to be the lethal species. The effect of added iron chelators and haem protein inhibitors indicates that iron species and haem proteins in particular are involved. A marked variation is found in observed hydroxyl-radical adduct signals with both the nature and concentration of peracid. A strong inverse correlation is found between the concentration of the observed radical adduct signal and the relative strength of the peroxide as a bactericide; use of a stable nitroxide as a radical scavenger confirms that strong bactericides produce radicals at a much faster rate than weak bactericides. Plots of radical generation versus time are correlated with % bacterial kill, offering further evidence that hydroxyl radicals are the lethal species.     

Functional gene expression profiling in yeast implicates translational dysfunction in mutant huntingtin toxicity

Huntington disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the huntingtin (htt) protein. To uncover candidate therapeutic targets and networks involved in pathogenesis, we integrated gene expression profiling and functional genetic screening to identify genes critical for mutant htt toxicity in yeast. Using mRNA profiling, we have identified genes differentially expressed in wild-type yeast in response to mutant htt toxicity as well as in three toxicity suppressor strains: bna4Δ, mbf1Δ, and ume1Δ. BNA4 encodes the yeast homolog of kynurenine 3-monooxygenase, a promising drug target for HD. Intriguingly, despite playing diverse cellular roles, these three suppressors share common differentially expressed genes involved in stress response, translation elongation, and mitochondrial transport. We then systematically tested the ability of the differentially expressed genes to suppress mutant htt toxicity when overexpressed and have thereby identified 12 novel suppressors, including genes that play a role in stress response, Golgi to endosome transport, and rRNA processing. Integrating the mRNA profiling data and the genetic screening data, we have generated a robust network that shows enrichment in genes involved in rRNA processing and ribosome biogenesis. Strikingly, these observations implicate dysfunction of translation in the pathology of HD. Recent work has shown that regulation of translation is critical for life span extension in Drosophila and that manipulation of this process is protective in Parkinson disease models. In total, these observations suggest that pharmacological manipulation of translation may have therapeutic value in HD.     

Inhibition of p21 Activated Kinase (PAK) Reduces Airway Responsiveness In Vivo and In Vitro in Murine and Human Airways

The p21-activated protein kinases (Paks) have been implicated in the regulation of smooth muscle contractility, but the physiologic effects of Pak activation on airway reactivity in vivo are unknown. A mouse model with a genetic deletion of Pak1 (Pak1(-/-)) was used to determine the role of Pak in the response of the airways in vivo to challenge with inhaled or intravenous acetylcholine (ACh). Pulmonary resistance was measured in anesthetized mechanically ventilated Pak1(-/-) and wild type mice. Pak1(-/-) mice exhibited lower airway reactivity to ACh compared with wild type mice. Tracheal segments dissected from Pak1(-/-) mice and studied in vitro also exhibited reduced responsiveness to ACh compared with tracheas from wild type mice. Morphometric assessment and pulmonary function analysis revealed no differences in the structure of the airways or lung parenchyma, suggesting that that the reduced airway responsiveness did not result from structural abnormalities in the lungs or airways due to Pak1 deletion. Inhalation of the small molecule synthetic Pak1 inhibitor, IPA3, also significantly reduced in vivo airway responsiveness to ACh and 5-hydroxytryptamine (5-Ht) in wild type mice. IPA3 inhibited the contractility of isolated human bronchial tissues to ACh, confirming that this inhibitor is also effective in human airway smooth muscle tissue. The results demonstrate that Pak is a critical component of the contractile activation process in airway smooth muscle, and suggest that Pak inhibition could provide a novel strategy for reducing airway hyperresponsiveness.     

Evolution of <Emphasis Type="Italic">DUX</Emphasis> gene macrosatellites in placental mammals

Macrosatellites are large polymorphic tandem arrays. The human subtelomeric macrosatellite D4Z4 has 11-150 repeats, each containing a copy of the intronless DUX4 gene. DUX4 is linked to facioscapulohumeral muscular dystrophy, but its normal function is unknown. The DUX gene family includes DUX4, the intronless Dux macrosatellites in rat and mouse, as well as several intron-containing members (DUXA, DUXB, Duxbl, and DUXC). Here, we report that the genomic organization (though not the syntenic location) of primate DUX4 is conserved in the Afrotheria. In primates and Afrotheria, DUX4 arose by retrotransposition of an ancestral intron-containing DUXC, which is itself not found in these species. Surprisingly, we discovered a similar macrosatellite organization for DUXC in cow and other Laurasiatheria (dog, alpaca, dolphin, pig, and horse), and in Xenarthra (sloth). Therefore, DUX4 and Dux are not the only DUX gene macrosatellites. Our data suggest a new retrotransposition-displacement model for the evolution of intronless DUX macrosatellites.