Clinical relevance of oncogenic driver mutations identified in endometrial carcinoma

PurposeEndometrial carcinoma (EC) is a clinically heterogeneous disease characterized by a number of different histological subtypes, and its heterogeneity may be involved in the accumulation of multiple genetic alterations. The aim of this work was to investigate the comprehensive mutational profile of EC tumors, and examine the associations between somatic mutations and clinicopathological features or survival in EC patients.MethodsA total of 100 surgical tumors were obtained from EC patients who had previously undergone surgery. Genomic DNA samples extracted from fresh-frozen tissues were analyzed using the Ion AmpliSeq Cancer Hotspot Panel v2 Kit, covering 50 tumor-related genes.ResultsValidated mutations were detected in 91 of the 100 tumors (91%) and identified in eight of the most frequently mutated genes, namely PTEN (57%), PIK3CA (51%), TP53 (30%), KRAS (23%), CTNNB1 (21%), FBFR2 (13%), FBXW7(10%) and RB1 (9%). PTEN mutations were found to associated with young age (< 60), early-stage, endometrioid histology, non-recurrence and better overall survival (OS). CTNNB1 mutations were associated with young age, endometrioid histology and better OS. On the other hands, TP53 mutations were associated with late-stage, non-endometrioid histology, high-grade, recurrence and worse OS. FBWX7 mutations were associated with late-stage, vascular invasion and lymph node metastasis. FGFR2 mutations correlated with deep (≥ 1/2) myometrial invasion.ConclusionOur comprehensive mutational profile will be useful for understanding and evaluating the molecular characteristics of EC tumors, and may lead to the establishment of novel treatment strategies that improve the survival of patients with EC in the future.     

Copper-dependent DNA damage induced by hydrazobenzene,an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5′-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.     

Molecular Conformation of 2′-Deoxy-2′-methylidene-cytidine: A Potent Antineoplastic Nucleoside

Abstract

2′-Deoxy-2′-methylidenecytidine (DMDC), a potent inhibitor of the growth of tumor cells, was crystallized with two different forms. One is dihydrated (DMDC·2H₂O) and the other is its hydrochloride salt (DMDC·HCLl). Both crystal and molecular structures have been determined by the X-ray diffraction method. In both forms the glycosidic and sugar conformations are anti and C(4′)-exo, respectively, whereas the conformation about the exocyclic bond is trans for DMDC·2H₂O and gauche ⁺ for DMDC·HCl. Proton nuclear magnetic resonance data of DMDC indicate a preference for the anti C(4′)-exo conformation found in the solid state. These molecular conformations were compared with the related pyrimidine nucleosides. When the cytosine bases are brought into coincidence, DMDC displays the exocyclic C(4′)-C(5′) bond located on the very close position to those of pyrimidine nucleosides with typical overall conformations. On the other hand, the hydroxyl O(3′)-H groups are separated by ca. 3 Å in the cases of DMDC and other pyrimidine nucleosides which have the C(2′)-endo sugar conformation. This result may be useful for the implication about the mechanism of the biological activity of DMDC.     

Plasma B-type Natriuretic Peptide as a Predictor of Cardiovascular Events in Subjects with Atrial Fibrillation: A Community-Based Study

Objectives

Atrial fibrillation (AF) is a significant public health issue due to its high prevalence in the general population, and is associated with an increased risk of cardiovascular (CV) events including systemic thrombo-embolism, heart failure, and coronary artery disease. The relationship between plasma B-type natriuretic peptide (BNP) and CV risk in real world AF subjects remains unknown.

Methods

The subject of the study (n = 228; mean age = 69 years) was unselected individuals with AF in a community-based population (n = 15,394; AF prevalence rate = 1.5%). The CV event free rate within each BNP tertile was estimated, and Cox regression analysis was performed to examine the relative risk of the onset of CV events among the tertiles. The prognostic ability of BNP was compared to an established risk score for embolic events (CHADS2 score). In addition, to determine the usefulness of BNP as a predictor in addition to CHADS2 score, we calculated Net Reclassification Improvement (NRI) and Integrated Discrimination Improvement (IDI) indices.

Results

During the follow-up period 58 subjects experienced CV events (52 per 1,000 person-years). The event-free ratio was significantly lower in the highest tertile (p < 0.02). After adjustment for established CV risk factors, the hazard ratio (HR) of the highest tertile was significantly higher than that of the lowest tertile (HR = 2.38; p < 0.02). The predictive abilities of plasma BNP in terms of sensitivity and specificity for general CV events were comparable to those of CHADS2 score. Adding BNP to the CHADS2 score only model improved the NRI (0.319; p < 0.05) and the IDI (0.046; p < 0.05).

Conclusion

Plasma BNP is a valuable biomarker both singly or in combination with an established scoring system for assessing general CV risk including stroke, heart failure and acute coronary syndrome in real-world AF subjects.     

Base composition and nucleosome density in exonic and intronic regions in genes of the filamentous ascomycetes Aspergillus nidulans and Aspergillus oryzae

We sequenced nucleosomal DNA fragments of the filamentous ascomycetes Aspergillus nidulans and Aspergillus oryzae and then mapped those sequences on their genomes. We compared the GC content and nucleosome density in the exonic and intronic regions in the genes of A. nidulans and A. oryzae. Although the GC content and nucleosome density in the exonic regions tended to be higher than those in the intronic regions, the difference in the distribution of the GC content was more notable than that of the nucleosome density. Next, we compared the GC content and nucleosome density in the exonic regions of 9616 orthologous gene pairs. In both Aspergillus species, the GC content did not correlate with the nucleosome density. In addition, the Spearman's rank correlation coefficient (ρ = 0.51) between the GC content of the exonic regions of the 9616 orthologous gene pairs was higher than that (ρ = 0.31) of the nucleosome densities of A. nidulans and A. oryzae. These results strongly suggest that the GC content in the exons of the orthologous gene pairs has been conserved during evolution but the nucleosome density has varied throughout.     

Facilitated Hyperpolarization Signaling in Vascular Smooth Muscle-overexpressing TRIC-A Channels

The TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation-specific channels and likely mediate counterion movements to support efficient Ca²⁺ release from the sarco/endoplasmic reticulum. Vascular smooth muscle cells (VSMCs) contain both TRIC subtypes and two Ca²⁺ release mechanisms; incidental opening of ryanodine receptors (RyRs) generates local Ca²⁺ sparks to induce hyperpolarization and relaxation, whereas agonist-induced activation of inositol trisphosphate receptors produces global Ca²⁺ transients causing contraction. Tric-a knock-out mice develop hypertension due to insufficient RyR-mediated Ca²⁺ sparks in VSMCs. Here we describe transgenic mice overexpressing TRIC-A channels under the control of a smooth muscle cell-specific promoter. The transgenic mice developed congenital hypotension. In Tric-a-overexpressing VSMCs from the transgenic mice, the resting membrane potential decreased because RyR-mediated Ca²⁺ sparks were facilitated and cell surface Ca²⁺-dependent K⁺ channels were hyperactivated. Under such hyperpolarized conditions, L-type Ca²⁺ channels were inactivated, and thus, the resting intracellular Ca²⁺ levels were reduced in Tric-a-overexpressing VSMCs. Moreover, Tric-a overexpression impaired inositol trisphosphate-sensitive stores to diminish agonist-induced Ca²⁺ signaling in VSMCs. These altered features likely reduced vascular tonus leading to the hypotensive phenotype. Our Tric-a-transgenic mice together with Tric-a knock-out mice indicate that TRIC-A channel density in VSMCs is responsible for controlling basal blood pressure at the whole-animal level.     

Function of a Glutamine Synthetase-Like Protein in Bacterial Aniline Oxidation via γ-Glutamylanilide

Acinetobacter sp. strain YAA has five genes (atdA1 to atdA5) involved in aniline oxidation as a part of the aniline degradation gene cluster. From sequence analysis, the five genes were expected to encode a glutamine synthetase (GS)-like protein (AtdA1), a glutamine amidotransferase-like protein (AtdA2), and an aromatic compound dioxygenase (AtdA3, AtdA4, and AtdA5) (M. Takeo, T. Fujii, and Y. Maeda, J. Ferment. Bioeng. 85:17-24, 1998). A recombinant Pseudomonas strain harboring these five genes quantitatively converted aniline into catechol, demonstrating that catechol is the major oxidation product from aniline. To elucidate the function of the GS-like protein AtdA1 in aniline oxidation, we purified it from recombinant Escherichia coli harboring atdA1. The purified AtdA1 protein produced gamma-glutamylanilide (γ-GA) quantitatively from aniline and l-glutamate in the presence of ATP and MgCl₂. This reaction was identical to glutamine synthesis by GS, except for the use of aniline instead of ammonia as the substrate. Recombinant Pseudomonas strains harboring the dioxygenase genes (atdA3 to atdA5) were unable to degrade aniline but converted γ-GA into catechol, indicating that γ-GA is an intermediate to catechol and a direct substrate for the dioxygenase. Unexpectedly, a recombinant Pseudomonas strain harboring only atdA2 hydrolyzed γ-GA into aniline, reversing the γ-GA formation by AtdA1. Deletion of atdA2 from atdA1 to atdA5 caused γ-GA accumulation from aniline in recombinant Pseudomonas cells and inhibited the growth of a recombinant Acinetobacter strain on aniline, suggesting that AtdA2 prevents γ-GA accumulation that is harmful to the host cell.     

Utilization of Amino Acids as a Sole Source of Nitrogen by Obligate Chemolithoautotroph Thiobacillus ferrooxidans

An obligate chemolithoautotroph, Thiobacillus ferrooxidans API 9–3, could utilize amino acids, other than glycine, methionine and phenylalanine, as a sole source of nitrogen. However, both the growth rate and growth yield were lower than those in Fe²⁺-NH4 -salts medium, suggesting that the ammonium ion was a superior nitrogen source for the strain compared to amino acids. Methionine and phenylalanine strongly inhibited the cell growth on Fe²⁺-NH4-salts medium at 10 mm. [¹⁴C]Glycine could not be taken up into the cells, and this meant the strain could not use glycine as a sole source of nitrogen. The uptake of [¹⁴C]leucine into the cells was dependent on the presence of Fe^{2 +}. When the strain was cultured on Fe^{2 +} - leucine (lOmm)-salts medium lacking an inorganic nitrogen source for 5 days at 30°C, 83.5% and 16.5% of the cellular carbon were derived from carbon dioxide and leucine, respectively, indicating that carbon dioxide was a superior carbon source for the bacterium compared to leucine. The ammonium ion did not inhibit the utilization of leucine for cellular carbon. Leucine uptake was markedly inhibited by inhibitors of protein synthesis, such as chloramphenicol (94.3% at 1 mm), streptomycin (57.2% at 5mm) and rifampin (77.2% at 0.1 mm), respectively. Carbon dioxide uptake was also completely inhibited by chloramphenicol at 4mm. These results suggest that the transport of both amino acids and carbon dioxide into the cells was dependent on protein synthesis.