Ageing of Technical Air and Technical Air with 7.5% C5-Fluoroketone by Free-Burning Arcs

This paper reports on the effect of ageing by free-burning arcs in 7.5% C5-fluoroketone (C5-FK) with 92.5% technical air in comparison to that in technical air (80% N2, 20% O2) at 1.3bar absolute pressure. The gases are aged by applying a series of arcs dissipating an accumulated energy of around 315kJ. It is found that the arc voltages in technical air and technical air with C5-FK are in the same range and do not vary significantly as a function of ageing or current amplitude (∼40-900 A). Contact erosion in both mediums is found to be similar if the discharge procedure is same. However, erosion increases significantly if ageing is performed in a short contact gap that needs more arcing operations to achieve similar level of arcing energy accumulation. Furthermore, gas decomposition by-products are analysed using gas chromatography coupled with mass-spectrometry

Use of the in situ proximity ligation assay (PLA) for studies of CKS2 interactions in HeLa cells

Cyclin-dependent kinases regulatory subunit 2 (CKS2) is overexpressed and associated with aggressiveness of several cancers. The reason for this is not clarified. CKS2 is known to bind the cell cycle regulatory proteins cyclin-dependent kinase 1 and 2 (CDK2 and CDK2) and mitochondrial single-stranded DNA-binding protein (SSBP1). SSBP1 participates in the biogenesis of mitochondria. These interactions have not previously been explored by in situ proximity ligation assay (PLA). The purpose of this study was to investigate cellular localization of CKS2 in HeLa cells. Further, co-localization and interactions of CKS2 with CDK1, CDK2 and SSBP1 were explored by immunofluorescence cytochemistry and PLA. Several fixation methods were tested for optimization of the PLA. Immunofluorescence cytochemistry showed that CKS2 was distributed in large foci in the nucleus and in small foci in the cytoplasm. In addition, CKS2 and CDK1 were localized at the centrosomes both in interphase and mitosis. The nuclear CKS2 foci were mainly restricted to weakly Hoechst stained areas, and they were co-localized with CDK1 and CDK2. Co-localization between CKS2, CDK1 and CDK2 was also apparent in the cytoplasm in interphase and mitosis. A small part of the cytoplasmic CKS2 and CDK1 foci co-localized with a SSBP1. PLA functioned well for both formalin and methanol fixated cells. However, formalin fixated cells required treatment with an extra reagent, e.g. a detergent buffer or methanol for production of PLA signals in the nucleus. Methanol fixation alone produced reproducible nuclear signals and was used for the examination of CKS2-CDK interactions, whereas combined formalin and methanol fixation were used for studying CKS2-SSBP1 and CDK1-SSBP1 interactions. PLA confirmed that CKS2 interacted with CDK1 and CDK2 in the nucleus and with CDK1, CDK2 and SSBP1 in the cytoplasm. The distribution of the CDK1-SSBP1 interactions in the cytoplasm resembled that of the CKS2-SSBP1 interactions. The results indicated that the large CKS2 foci in the nucleus were localized in euchromatin, which is transcriptionally competent DNA, and thus support the hypothesis that CKS2 has a role in the transcription of DNA. CKS2 probably interacted with CDK1 and CDK2 in these areas. Some of the cytoplasmic CKS2 and CDK1 foci interacted with SSBP1, probably within the mitochondria. In addition, the co-localization of CKS2 and CDK1 at the centrosomes lends support to the hypothesis of CKS-dependent activation and inactivation of the CDK1-CCNB complex prior to and in the mitosis

Reference MicroRNAs for RT-qPCR Assays in Cervical Cancer Patients and Their Application to Studies of HPV16 and Hypoxia Biomarkers

MicroRNA (miRNA) expressions in tumor biopsies have shown potential as biomarkers in cervical cancer, but suitable reference RNAs for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays in patient cohorts with different clinicopathological characteristics are not available. We aimed to identify the optimal reference miRNAs and apply these to investigate the potential of miR-9-5p as human papilloma virus (HPV) 16 biomarker and miR-210-3p as hypoxia biomarker in cervical cancer. Candidate reference miRNAs were preselected in sequencing data of 90 patients and ranked in a stability analysis by RefFinder. A selection of the most stable miRNAs was evaluated by geNorm and NormFinder analyses of RT-qPCR data of 29 patients. U6 small nuclear RNA (RNU6) was also included in the evaluation. MiR-9-5p and miR-210-3p expression was assessed by RT-qPCR in 45 and 65 patients, respectively. Nine candidates were preselected in the sequencing data after excluding those associated with clinical markers, HPV type, hypoxia status, suboptimal expression levels, and low stability. In RT-qPCR assays, the combination of miR-151-5p, miR-152-3p, and miR-423-3p was identified as the most stable normalization factor across clinical markers, HPV type, and hypoxia status. RNU6 showed poor stability. By applying the optimal reference miRNAs, higher miR-9-5p expression in HPV16- than HPV18-positive tumors and higher miR-210-3p expression in more hypoxic than less hypoxic tumors were found in accordance with the sequencing data. MiR-210-3p was associated with poor outcome by both sequencing and RT-qPCR assays. In conclusion, miR-151-5p, miR-152-3p, and miR-423-3p are suitable reference miRNAs in cervical cancer. MiR-9-5p and miR-210-3p are promising HPV16 and hypoxia biomarkers, respectively

Mitochondrial Function of CKS2 Oncoprotein Links Oxidative Phosphorylation with Cell Division in Chemoradioresistant Cervical Cancer

CDK regulatory subunit 2 (CKS2) has a nuclear function that promotes cell division and is a candidate biomarker of chemoradioresistance in cervical cancer. The underlying mechanisms are, however, not completely understood. We investigated whether CKS2 also has a mitochondrial function that augments tumor aggressiveness. Based on global gene expression data of two cervical cancer cohorts of 150 and 135 patients, we identified a set of genes correlated with CKS2 expression. Gene set enrichment analysis showed enrichment of mitochondrial cellular compartments, and the hallmarks oxidative phosphorylation (OXPHOS) and targets of the MYC oncogene in the gene set. By in situ proximity ligation assay, we showed that CKS2 formed complex with the positively correlated MYC target, mitochondrial single-stranded DNA binding protein SSBP1, in the mitochondrion of cervix tumor samples and HeLa and SiHa cervical cancer cell lines, indicating a role in mitochondrial DNA (mtDNA) replication and thereby OXPHOS. CDK1 was found to be part of the complex. Flow cytometry analyses of HeLa cells showed cell cycle regulation of the CKS2-SSBP1 complex consistent with mtDNA replication activity. Moreover, repression of mtDNA replication and OXPHOS by acute hypoxia decreased CKS2-SSBP1 complex abundance and expression of MYC targets. By immunohistochemistry, cytoplasmic CKS2 expression was found to add to the prognostic impact of nuclear CKS2 expression in patients, suggesting that the mitochondrial function promotes tumor aggressiveness. Our study uncovers a novel link between regulation of cell division by nuclear pathways and OXPHOS in the mitochondrion that involves CKS2 and promotes chemoradioresistance of cervical cancer

Hypoxia-independent gene expression signature associated with radiosensitisation of prostate cancer cell lines by histone deacetylase inhibition

Background: Histone deacetylase inhibitors (HDACis) like vorinostat are promising radiosensitisers in prostate cancer, but their effect under hypoxia is not known. We investigated gene expression associated with radiosensitisation of normoxic and hypoxic prostate cancer cells by vorinostat. Methods: Cells were exposed to vorinostat under normoxia or hypoxia and subjected to gene expression profiling before irradiation and clonogenic survival analysis. Results: Pretreatment with vorinostat led to radiosensitisation of the intrinsically radioresistant DU 145 cells, but not the radiosensitive PC-3 and 22Rv1 cells, and was independent of hypoxia status. Knockdown experiments showed that the sensitisation was not caused by repression of hypoxia-inducible factor HIF1 or tumour protein TP53. Global deregulation of DNA repair and chromatin organisation genes was associated with radiosensitisation under both normoxia and hypoxia. A radiosensitisation signature with expression changes of 56 genes was generated and valid for both conditions. For eight signature genes, baseline expression also correlated with sensitisation, showing potential as pretreatment biomarker. The hypoxia independence of the signature was confirmed in a clinical data set. Conclusions: Pretreatment with HDACi may overcome radioresistance of hypoxic prostate tumours by similar mechanisms as under normoxia. We propose a gene signature to predict radiosensitising effects independent of hypoxia status