MYC inhibition induces metabolic changes leading to accumulation of lipid droplets in tumor cells

The MYC genes are the most frequently activated oncogenes in human tumors and are hence attractive therapeutic targets. MYCN amplification leads to poor clinical outcome in childhood neuroblastoma, yet strategies to modulate the function of MYCN do not exist. Here we show that 10058-F4, a characterized c-MYC/Max inhibitor, also targets the MYCN/Max interaction, leading to cell cycle arrest, apoptosis, and neuronal differentiation in MYCN-amplified neuroblastoma cells and to increased survival of MYCN transgenic mice. We also report the discovery that inhibition of MYC is accompanied by accumulation of intracellular lipid droplets in tumor cells as a direct consequence of mitochondrial dysfunction. This study expands on the current knowledge of how MYC proteins control the metabolic reprogramming of cancer cells, especially highlighting lipid metabolism and the respiratory chain as important pathways involved in neuroblastoma pathogenesis. Together our data support direct MYC inhibition as a promising strategy for the treatment of MYC-driven tumors.     

Modulation of ENaC,CFTR, and iNOS expression in bronchial epithelial cells after stimulation with Staphylococcus epidermidis (94B080) and Staphylococcus aureus (90B083)

Bacteria affect the respiratory epithelium, which is covered by airway surface liquid (ASL) and mucus. Ion concentrations in the ASL are determined by the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na⁺ channel (ENaC). Neonatal sepsis is a major risk factor for subsequent pulmonary disease in preterm newborns. Predominating are coagulase‐negative staphylococci (e.g., Staphylococccus epidermidis and Staphylococccus aureus). The aim of this study was to investigate modulation of CFTR, ENaC, mucins, proinflammatory cytokines, and inducible nitric oxide synthase (iNOS) in respiratory epithelial cells after S. epidermidis 94B080 and S. aureus 90B083 exposure. Bronchial epithelial cells were incubated with S. epidermidis 94B080 and S. aureus 90B083 (neonatal blood isolates) for 1–36 h. Expression of CFTR, ENaC, iNOS, and mucins was analyzed by real‐time PCR and Western blotting. Release of cytokines was analyzed by ELISA, and production of NO by the Griess assay. Expression of CFTR significantly decreased after 36 h incubation with S. epidermidis and more prominently with S. aureus, whereas S. epidermidis caused a significant increase in the expression of β‐ and γ‐ENaC. Expression of iNOS increased, but NO was not detected. Both staphylococci caused a decrease in the intracellular Ca²⁺ concentration. S. aureus induced increased secretion of IL‐6, IL‐8, and transforming nuclear factor (TNF)‐α in a time‐dependent manner as compared with S. epidermidis. In conclusion, expression of ENaC, CFTR, and iNOS is modulated by exposure to S. aureus 90B083 and S. epidermidis 94B080. S. aureus is more potent in causing release of IL‐6, IL‐8, and TNF‐α by bronchial epithelial cells as compared with S. epidermidis. The mRNA expression for the mucus proteins MUC2, MUC5AC, and MUC5B could not be measured, neither in the presence nor in the absence of bacteria.     

Engineering rotor ring stoichiometries in the ATP synthase

ATP synthase membrane rotors consist of a ring of c-subunits whose stoichiometry is constant for a given species but variable across different ones. We investigated the importance of c/c-subunit contacts by site-directed mutagenesis of a conserved stretch of glycines (GxGxGxGxG) in a bacterial c(11) ring. Structural and biochemical studies show a direct, specific influence on the c-subunit stoichiometry, revealing c(<11), c(12), c(13), c(14), and c(>14) rings. Molecular dynamics simulations rationalize this effect in terms of the energetics and geometry of the c-subunit interfaces. Quantitative data from a spectroscopic interaction study demonstrate that the complex assembly is independent of the c-ring size. Real-time ATP synthesis experiments in proteoliposomes show the mutant enzyme, harboring the larger c(12) instead of c(11), is functional at lower ion motive force. The high degree of compliance in the architecture of the ATP synthase rotor offers a rationale for the natural diversity of c-ring stoichiometries, which likely reflect adaptations to specific bioenergetic demands. These results provide the basis for bioengineering ATP synthases with customized ion-to-ATP ratios, by sequence modifications.     

Generation and characterization of monoclonal antibodies to protein kinase 2 (CK2) beta subunit

Protein kinase 2 (CK2) is a ubiquitous and constitutively active serine/threonine protein kinase with various cell functions. It typically forms tetrameric complexes consisting of two catalytic (alpha and/or (alpha') and two regulatory (beta) subunits. The aim of this study was to produce monoclonal antibodies (MAbs) against the CK2beta subunit and to characterize their suitability for Western blotting, immunoprecipitation, and immunohistochemical applications. Bacterially expressed CK2beta-6His-GST recombinant protein has been used as an antigen. Balb/c mice were immunized and given a final boost, and their spleen cells were collected and fused with SP2/0 myeloma cells using PEG 2000. The fused cells were then selected in the HAT-RPMI medium. Anti- CK2beta high-titer antibody-producing hybridoma cell lines were identified by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution in HT-RPMI medium supplemented with 20% fetal bovine serum (FBS). A total of 10 IgG-producing cell lines were selected and further tested for their reactivity with the CK2beta subunit using ELISA, Western blots, immunoprecipitation, and immunostaining of formaldehyde-fixed paraffin-embedded tissue sections. The results obtained clearly indicate that several clones produce antibodies that recognize specifically recombinant and endogenous CK2beta subunit in Western blotting and immunoprecipitation, and are suitable for immunohistochemical analysis. In summary, the produced antibodies will be useful for researchers investigating signaling pathways involving CK2 kinase and their deregulation in human pathologies.     

Thyroid carcinoma in children and adolescents in Ukraine after the Chernobyl nuclear accident: statistical data and clinicomorphologic characteristics.

BACKGROUND: The increase in the number of childhood thyroid carcinoma cases in Ukraine after the Chernobyl nuclear accident in 1986 prompted the development of a registry of thyroid carcinoma cases at the Institute of Endocrinology and Metabolism in Kiev. In the current study, the authors report the statistical data and clinicomorphologic features of the cases included in this registry. METHODS: To study the incidence, and age and gender distribution of thyroid carcinoma in Ukraine, the authors compiled complete clinical information from cases diagnosed and treated at the Institute of Endocrinology and Metabolism and statistical reports submitted to the registry from 27 regions of Ukraine. Morphologic features of the resected tumors were examined and were included in the database. RESULTS: During the 5 years preceding the Chernobyl nuclear accident, a total of 59 cases of thyroid carcinoma were identified in the birth to 18 years age group (25 in children age < or = 14 years and 34 in adolescents ages 15-18 years). Between 1986 and 1997, the total number of thyroid carcinomas in Ukrainian children and adolescents was 577 (358 children and 219 adolescents). Morphologically, the thyroid tumors overwhelmingly were papillary carcinomas, and the majority of these also showed a follicular and/or solid growth pattern. Lymph node metastases and other extrathyroidal spread were common, thus necessitating total thyroidectomy and lymph node dissections in many patients. CONCLUSIONS: Between 1990 and 1997, a significant increase in the incidence of thyroid carcinoma was noted in children and adolescents in Ukraine; the group most affected was comprised of the individuals who were age < or = 5 years in 1986 (the year of the Chernobyl nuclear accident). The largest number of cases occurred in patients living in areas of thyroid radiation doses of > or =0.50 grays. The morphologic features of those thyroid tumors suggest that they are aggressive tumors with a high frequency of lymph node metastases, venous invasion, and extrathyroidal spread.     

Outcomes and quality of life assessment in patients undergoing endoscopic surgery for pituitary adenomas

The endoscopic technique is increasingly being used for the resection of pituitary adenomas. Its efficacy and safety have been generally accepted, but its impact on the quality of life of the patients treated has not been previously addressed. Most of the studies assessing the quality of life after long-term cure of pituitary adenomas suggest a significantly impaired quality of life (QoL) in all subgroups of pituitary tumours. In our study we analyse QoL data following endoscopic surgery, and attempt comparisons with normative and data from standard approaches. The validated health questionnaire SF-36 was sent to 80 patients who had undergone pure endoscopic resection of a pituitary adenoma. Fifty-four patients returned the completed questionnaire. Outcomes were compared with normative data for the Short Form-36 (SF-36) Heath survey for the general Canadian population. We also compared the results amongst different types of adenomas. We attempted a comparison to previous studies on quality of life for patients who underwent conventional trans-sphenoidal surgery. Our study suggests only minimal impairment of quality of life in patients after successful treatment of pituitary adenomas using the endoscopic approach. There were only very few differences in the perceived quality of life within the different subgroups of adenomas. There was a trend to improved scores in the endoscopic group compared with previous studies in patients treated by conventional approaches. Whilst our data suggest minimal impact on the quality of life for patients after endoscopic removal of pituitary adenomas, further studies with larger number of patients and longer follow-up are required to encourage this finding.     

Hepatic Metastatectomy for Low-Grade Leiomyosarcoma: A Case Report

A patient with multiple bilateral liver metastases from a gastrointestinal leiomyosarcoma underwent staged sequential metastatectomy and at 3 years is tumor-free.