Regulation of DNA repair mechanism in human glioma xenograft cells both in vitro and in vivo in nude mice

Background

Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells.

Methodology/Principal Findings

Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls.

Conclusion/Significance

Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point.     

Complete genome sequence of the thermophilic sulfur-reducer Desulfurobacterium thermolithotrophum type strain (BSA(T)) from a deep-sea hydrothermal vent

Desulfurobacterium thermolithotrophum L'Haridon et al. 1998 is the type species of the genus Desulfurobacterium which belongs to the family Desulfurobacteriaceae. The species is of interest because it represents the first thermophilic bacterium that can act as a primary producer in the temperature range of 45-75 °C (optimum 70°C) and is incapable of growing under microaerophilic conditions. Strain BSA(T) preferentially synthesizes high-melting-point fatty acids (C(18) and C(20)) which is hypothesized to be a strategy to ensure the functionality of the membrane at high growth temperatures. This is the second completed genome sequence of a member of the family Desulfurobacteriaceae and the first sequence from the genus Desulfurobacterium. The 1,541,968 bp long genome harbors 1,543 protein-coding and 51 RNA genes and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Odoribacter splanchnicus type strain (1651/6)

Odoribacter splanchnicus (Werner et al. 1975) Hardham et al. 2008 is the type species of the genus Odoribacter, which belongs to the family Porphyromonadaceae in the order 'Bacteroidales'. The species is of interest because members of the Odoribacter form an isolated cluster within the Porphyromonadaceae. This is the first completed genome sequence of a member of the genus Odoribacter and the fourth sequence from the family Porphyromonadaceae. The 4,392,288 bp long genome with its 3,672 protein-coding and 74 RNA genes and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Genome sequence of the moderately thermophilic halophile Flexistipes sinusarabici strain (MAS10)

Flexistipes sinusarabici Fiala et al. 2000 is the type species of the genus Flexistipes in the family Deferribacteraceae. The species is of interest because of its isolated phylogenetic location in a genomically under-characterized region of the tree of life, and because of its origin from a multiply extreme environment; the Atlantis Deep brines of the Red Sea, where it had to struggle with high temperatures, high salinity, and a high concentrations of heavy metals. This is the fourth completed genome sequence to be published of a type strain of the family Deferribacteraceae. The 2,526,590 bp long genome with its 2,346 protein-coding and 53 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Genome sequence of the filamentous, gliding Thiothrix nivea neotype strain (JP2(T))

Thiothrix nivea (Rabenhorst 1865) Winogradsky 1888 (Approved Lists 1980) emend. Larkin and Shinabarger 1983 is the type species of the genus Thiothrix in the family Thiotrichaceae. The species is of interest not only because of its isolated location in the yet to be genomically characterized region of the tree of life, but also because of its life-style with gliding gonidia, the multilayer sheath, rosettes, and the embedded sulfur granules. Strain JP2(T) is the neotype strain of the species which was first observed by Rabenhorst in 1865 and later reclassified by Winogradsky in 1888 into the then novel genus Thiothrix. This is the first completed (improved-high-quality-draft) genome sequence to be published of a member of the family Thiotrichaceae. The genome in its current assembly consists of 15 contigs in four scaffolds with a total of 4,691,711 bp bearing 4,542 protein-coding and 52 RNA genes and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Metabolite profiling can assist variability analysis in Trichoderma species

Variability in 41 isolates of Trichoderma belonging to 21 species was observed in the phenolic acid profile of their culture filtrates. The phenolic acid profiles were observed to be very stable in the culture filtrate of Trichoderma species. The similarity in phenolic acid profile was recorded and based on it the species were grouped into three distinct groups, viz. highly similar, moderately similar and least similar. Of the 21 species, seven species showed highly similar trend, whereas two and four species showed moderate and least similarity in their phenolic acid profiles, respectively. Looking into the stability of phenolic acid profile in the culture filtrate of the Trichoderma species the present tool may help in diversity analysis in Trichoderma species originating from different geographical areas.     

Characterisation,genetic diversity and antagonistic potential of 2,4-diacetylphloroglucinol producing Pseudomonas fluorescens isolates in groundnut-based cropping systems of Andhra Pradesh,India

This study is focused on isolation and characterisation of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing Pseudomonas fluorescens isolates from different soils of groundnut-based cropping systems in Andhra Pradesh. In our studies, 21 isolates of P. fluorescens were isolated and confirmed through various biochemical tests, of which five were tested positive for 2,4-DAPGproduction with specific primers. Biocontrol potential of these isolates on groundnut stem rot pathogen (Sclerotium rolfsii) was determined through in vitro dual culture assays. The eight isolates were found effective against S. rolfsii (up to 75% inhibition) in dual culture method. All the five 2,4-DAPG-producing Plant Growth-Promoting Rhizobacteria isolates were highly antagonistic to S. rolfsii. Genetic diversity of these P. fluorescens isolates was determined by random amplification of polymorphic DNA analysis. Overall, our results suggest that the prevalence of 2,4-DAPG-producing fluorescent Pseudomonads in different crop rhizospheres of groundnut-based cropping systems.