p53 is functionally inhibited in clear cell renal cell carcinoma (ccRCC): a mechanistic and correlative investigation into genetic and molecular characteristics.

PURPOSE: Although p53 is rarely mutated in ccRCC, its overexpression has been linked to poor prognosis. The current study sought to elucidate the unique role of p53 in ccRCC with genomic, proteomic, and functional analyses. MATERIALS AND METHODS: Data from the Cancer Genome Atlas (TCGA) were evaluated for genomic and proteomic characteristics of p53; a tissue micro array (TMA) study was carried out to evaluate the association of p53 and phosphorylated p53 (pp53) with clinical outcome. Mechanistic in vitro experiments were performed to confirm a pro-apoptotic loss of p53 in ccRCC and p53 isoforms as well as posttranslational modifications of p53 where assessed to provide possible reasons for a functional inhibition of p53 in ccRCC. RESULTS: A low somatic mutation rate of p53 could be confirmed. Although mRNA levels were correlated with poor prognosis and clinicopathological features, there was no monotonous association of mRNA levels with survival outcome. Higher p53 protein levels could be confirmed as poor prognostic features. In vitro, irradiation of ccRCC cell lines markedly induced levels of p53 and of activated (phosphorylated) p53. However, irradiated ccRCC cells demonstrated similar proliferation, migration, and p53 transcriptional activity like non-irradiated controls indicating a functional inhibition of p53. p53 isoforms and could not be correlated with clinical outcome of ccRCC patients. CONCLUSIONS: p53 is rarely mutated but the wildtype p53 is functionally inhibited in ccRCC. To investigate mechanisms that underlie functional inhibition of p53 may provide attractive therapeutic targets in ccRCC.

High throughput image cytometry micronucleus assay to investigate the presence or absence of mutagenic effects of cold physical plasma.

Promising cold physical plasma sources have been developed in the field of plasma medicine. An important prerequisite to their clinical use is lack of genotoxic effects in cells. During optimization of one or even different plasma sources for a specific application, large numbers of samples need to be analyzed. There are soft and easy-to-assess markers for genotoxic stress such as phosphorylation of histone H2AX (γH2AX) but only few tests are accredited by the OECD with regard to mutagenicity detection. The micronucleus (MN) assay is among them but often requires manual counting of many thousands of cells per sample under the microscope. A high-throughput MN assay is presented using image flow cytometry and image analysis software. A human lymphocyte cell line was treated with plasma generated with ten different feed gas conditions corresponding to distinct reactive species patterns that were investigated for their genotoxic potential. Several millions of cells were automatically analyzed by a MN quantification strategy outlined in detail in this work. Our data demonstrates the absence of newly formed MN in any feed gas condition using the atmospheric pressure plasma jet kINPen. As positive control, ionizing radiation gave a significant 5-fold increase in micronucleus frequency. Thus, this assay is suitable to assess the genotoxic potential in large sample sets of cells exposed chemical or physical agents including plasmas in an efficient, reliable, and semiautomated manner. Environ. Mol. Mutagen. 59:268-277, 2018. © 2018 Wiley Periodicals, Inc.

Histone deacetylase inhibitors, but not vincristine, cooperate with radiotherapy to induce cell death in medulloblastoma.

BACKGROUND: Though ionising radiation (IR) is an efficient means of postoperative treatment for children with medulloblastoma, the disease is incurable in about a third of them. Thus, multimodality regimens have been introduced, typically combining IR with vincristine. MATERIALS AND METHODS: The combination of IR and vincristine was compared to the combination of IR and histone deacetylase inhibitors (HDIs) for their anticancer activity against medulloblastoma cells in vitro. Cytotoxic activities were assessed by measuring propidium iodide uptake and by cell cycle analysis. RESULTS: HDIs augmented the cytotoxic effect of IR, while the combination of vincristine and IR was significantly less cytotoxic than vincristine alone. Cell cycle analyses revealed that vincristine did not interfere with IR-induced G2/M arrest, whereas HDIs abolished the latter. CONCLUSION: These in vitro findings indicate a favourable interaction of IR and HDIs, but an unfavourable one of IR and vincristine, in medulloblastoma, and provide a rationale for comparing the combination of IR with either vincristine or HDIs in vivo.

A new compact disc format of high density array synthesis applied to peptide nucleic acids and in situ MALDI analysis.

A fully automated synthesizer was constructed and designed to perform high speed miniaturized syntheses of compound libraries using the SPOT technique. Utilizing magnetically controlled drop-on-demand ink jet nozzles, an r/phi array format of 2500 spots can be simultaneously dispensed from up to 24 separate reagent valves onto a rotating disc as the solid phase in less than three minutes. In addition, a complete wash station is on board allowing for fully programmable combinatorial syntheses without manual attention. A new carbon black/polypropylene composite solid phase disc was developed and tested for its functionalisation/loading, spot detection, durability and MALDI-TOF target capabilities. The carbon black/polypropylene composite was then successfully employed jointly as the solid phase in the syntheses of short peptide and PNA oligomers and as the target probe holder for MALDI-TOF measurement without transfer of the material. Several protocols for PNA syntheses were also investigated and an optimised PNA methodology for the carbon black/polypropylene composite is reported.