Amplicon-dependent CCNE1 expression is critical for clonogenic survival after cisplatin treatment and is correlated with 20q11 gain in ovarian cancer

Genomic amplification of 19q12 occurs in several cancer types including ovarian cancer where it is associated with primary treatment failure. We systematically attenuated expression of genes within the minimally defined 19q12 region in ovarian cell lines using short-interfering RNAs (siRNA) to identify driver oncogene(s) within the amplicon. Knockdown of CCNE1 resulted in G1/S phase arrest, reduced cell viability and apoptosis only in amplification-carrying cells. Although CCNE1 knockdown increased cisplatin resistance in short-term assays, clonogenic survival was inhibited after treatment. Gain of 20q11 was highly correlated with 19q12 amplification and spanned a 2.5 Mb region including TPX2, a centromeric protein required for mitotic spindle function. Expression of TPX2 was highly correlated with gene amplification and with CCNE1 expression in primary tumors. siRNA inhibition of TPX2 reduced cell viability but this effect was not amplicon-dependent. These findings demonstrate that CCNE1 is a key driver in the 19q12 amplicon required for survival and clonogenicity in cells with locus amplification. Co-amplification at 19q12 and 20q11 implies the presence of a cooperative mutational network. These observations have implications for the application of targeted therapies in CCNE1 dependent ovarian cancers.     

Three-dimensional ultrastructural analysis of peroxisomes in HepG2 cells

Peroxisomes in the human hepatoblastoma cell line, HepG2, exhibit distinct alterations of shape, size, and distribution, dependent on culture conditions (cell density, duration in culture, and presence of specific growth factors). Although many cells with elongated tubular peroxisomes are present in thinly seeded cultures, spherical particles forming large focal clusters are found in confluent cultures. The authors have analyzed the ultrastructure and the spatial relationship of peroxisomes of HepG2 cells at different stages of differentiation, using three-dimensional (3D)-reconstruction of ultrathin serial sections, and electronic image processing. Cells were prepared for immunofluorescence using different antibodies against peroxisomal matrix and membrane proteins, as well as for electron microscopy after the alkaline 3,3′-diaminobenzidine staining for catalase. The results indicate that the tubular peroxisomes, which can reach a length of several microns, are consistently isolated, and never form an interconnected peroxisomal reticulum. At the time of disappearance of tubular peroxisomes, rows of spherical peroxisomes, arranged like beads on a string, are observed, suggesting fission of tubular ones. In differentiated confluent cultures, clusters of several peroxisomes are seen, which, by immunofluorescence, appear as large aggregates, but after 3D reconstruction consist of single spherical and angular peroxisomes without interconnections. The majority of such mature spherical peroxisomes (but not the tubular ones) exhibit tail-like, small tubular and vesicular attachments to their surface, suggesting a close functional interaction with neighboring organelles, particularly the endoplasmic reticulum, which is often observed in close vicinity of such peroxisomes.     

Immune checkpoint blockade opens a new way to cancer immunotherapy

Among the main promising systems to triggering therapeutic antitumor immunity is the blockade of immune checkpoints. Immune checkpoint pathways regulate the control and eradication of infections, malignancies, and resistance against a host of autoantigens. Initiation point of the immune response is T cells, which have a critical role in this pathway. As several immune checkpoints are initiated by ligand–receptor interactions, they can be freely blocked by antibodies or modulated by recombinant forms of ligands or receptors. Antibodies against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) were the first immunotherapeutics that achieved the US Food and Drug Administration approval. Preliminary clinical results with the blockers of additional immune checkpoint proteins, such as programmed cell death protein 1 (PD-1) indicate extensive and different chances to boost antitumor immunity with the objective of conferring permanent clinical effects. This study provides an overview of the immune checkpoint pathways, including CTLA-4, PD-1, lymphocyte activation gene 3, T-cell immunoglobulin and mucin domain 3, B7-H3, and diacylglycerol kinase α and implications of their inhibition in the cancer therapy.     

The Inhibition Effect of Lactobacilli Against Growth and Biofilm Formation of <Emphasis Type="BoldItalic">Pseudomonas aeruginosa</Emphasis>

The emergence of antibiotic-resistant and food-spoilage microorganisms has renewed efforts to identify safe and natural alternative agents of antibiotics such as probiotics. The aim of this study was the isolation of lactobacilli as potential probiotics from local dairy products with broad antibacterial and anti-biofilm activities against antibiotic-resistant strains of Pseudomonas aeruginosa and determination of their inhibition mechanism. Antibiotic susceptibility and classification of acquired resistance profiles of 80 P. aeruginosa strains were determined based on Centers for Disease Control and Prevention (CDC) new definition as multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) followed by antibacterial assessment of lactobacilli against them by different methods. Among the 80 P. aeruginosa strains, 1 (1.3%), 50 (62.5%), and 78 (97.5%) were PDR, XDR, and MDR, respectively, and effective antibiotics against them were fosfomycin and polymyxins. Among 57 isolated lactobacillus strains, two strains which were identified as Lactobacillus fermentum using biochemical and 16S rDNA methods showed broad inhibition/killing and anti-biofilm effects against all P. aeruginosa strains. They formed strong biofilms and had bile salts and low pH tolerance. Although investigation of inhibition mechanism of these strains showed no bacteriocin production, results obtained by high-performance liquid chromatography (HPLC) analysis indicated that their inhibitory effect was the result of production of three main organic acids including lactic acid, acetic acid, and formic acid. Considering the broad activity of these two L. fermentum strains, they can potentially be used in bio-control of drug-resistant strains of P. aeruginosa.     

Effects of fixation on the preservation of peroxisomal structures for immunofluorescence studies using HepG2 cells as a model system

Summary The immunofluorescence technique has become an important tool for the investigation of peroxisomes in cell culture. We have used this method for the study of peroxisomes in the human hepatoblastoma cell line HepG2. A marked heterogeneity of peroxisomal forms was detected. Besides spherical (about 100 nm) and rod-shaped structures (about 300 nm) many elongated, undulating tubular forms (up to 5 m) were found. Further observations indicate that the appearance of the peroxisomal forms in immunofluorescence is dependent on the fixation procedure used. Whereas the fixation with methanol-acetone (–20°C) or ethanol results in a punctate pattern with spherical particles, the use of formaldehyde/Triton X-100 fixation shows well-preserved tubules and rods. These observations may be of special importance for studies on the biogenesis of peroxisomes.     

Does acute exposure to mobile phones affect human attention?

Recent studies have indicated that acute exposure to low level radiofrequency (RF) electromagnetic fields generated by mobile phones affects human cognition. However, the relatively small samples used, in addition to methodological problems, make the outcomes of these studies difficult to interpret. In our study we tested a large sample of volunteers (168) using a series of cognitive tasks apparently sensitive to RF exposure (a simple reaction task, a vigilance task, and a subtraction task). Participants performed those tasks twice, in two different sessions. In one session they were exposed to RFs, with half of subjects exposed to GSM signals and the other half exposed to CW signals, while in the other session they were exposed to sham signals. No significant effects of RF exposure on performance for either GSM or CW were found, independent of whether the phone was positioned on the left or on the right side.