BRCA1 and BRCA2 5′ noncoding region variants identified in breast cancer patients alter promoter activity and protein binding

© 2018 The Authors. Human Mutation published by Wiley Periodicals, Inc. The widespread use of next generation sequencing for clinical testing is detecting an escalating number of variants in noncoding regions of the genome. The clinical significance of the majority of these variants is currently unknown, which presents a significant clinical challenge. We have screened over 6,000 early-onset and/or familial breast cancer (BC) cases collected by the ENIGMA consortium for sequence variants in the 5′ noncoding regions of BC susceptibility genes BRCA1 and BRCA2, and identified 141 rare variants with global minor allele frequency \u3c 0.01, 76 of which have not been reported previously. Bioinformatic analysis identified a set of 21 variants most likely to impact transcriptional regulation, and luciferase reporter assays detected altered promoter activity for four of these variants. Electrophoretic mobility shift assays demonstrated that three of these altered the binding of proteins to the respective BRCA1 or BRCA2 promoter regions, including NFYA binding to BRCA1:c.-287C\u3eT and PAX5 binding to BRCA2:c.-296C\u3eT. Clinical classification of variants affecting promoter activity, using existing prediction models, found no evidence to suggest that these variants confer a high risk of disease. Further studies are required to determine if such variation may be associated with a moderate or low risk of BC

Identification of Germline Mutations in Melanoma Patients with Early Onset, Double Primary Tumors, or Family Cancer History by NGS Analysis of 217 Genes

Cutaneous melanoma is the deadliest skin malignity with a rising prevalence worldwide. Patients carrying germline mutations in melanoma-susceptibility genes face an increased risk of melanoma and other cancers. To assess the spectrum of germline variants, we analyzed 264 Czech melanoma patients indicated for testing due to early melanoma (at <25 years) or the presence of multiple primary melanoma/melanoma and other cancer in their personal and/or family history. All patients were analyzed by panel next-generation sequencing targeting 217 genes in four groups: high-to-moderate melanoma risk genes, low melanoma risk genes, cancer syndrome genes, and other genes with an uncertain melanoma risk. Population frequencies were assessed in 1479 population-matched controls. Selected POT1 and CHEK2 variants were characterized by functional assays. Mutations in clinically relevant genes were significantly more frequent in melanoma patients than in controls (31/264; 11.7% vs. 58/1479; 3.9%; p = 2.0 × 10−6). A total of 9 patients (3.4%) carried mutations in high-to-moderate melanoma risk genes (CDKN2A, POT1, ACD) and 22 (8.3%) patients in other cancer syndrome genes (NBN, BRCA1/2, CHEK2, ATM, WRN, RB1). Mutations in high-to-moderate melanoma risk genes (OR = 52.2; 95%CI 6.6–413.1; p = 3.2 × 10−7) and in other cancer syndrome genes (OR = 2.3; 95%CI 1.4–3.8; p = 0.003) were significantly associated with melanoma risk. We found an increased potential to carry these mutations (OR = 2.9; 95%CI 1.2–6.8) in patients with double primary melanoma, melanoma and other primary cancer, but not in patients with early age at onset. The analysis revealed affected genes in Czech melanoma patients and identified individuals who may benefit from genetic testing and future surveillance management of mutation carriers

Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

Polo-like kinases play essential roles in cell cycle control and mitosis. In contrast to other members of this kinase family, PLK3 has been reported to be activated upon cellular stress including DNA damage, hypoxia and osmotic stress. Here we knocked out PLK3 in human non-transformed RPE cells using CRISPR/Cas9-mediated gene editing. Surprisingly, we find that loss of PLK3 does not impair stabilization of HIF1α after hypoxia, phosphorylation of the c-Jun after osmotic stress and dynamics of DNA damage response after exposure to ionizing radiation. Similarly, RNAi-mediated depletion of PLK3 did not impair stress response in human transformed cell lines. Exposure of cells to various forms of stress also did not affect kinase activity of purified EGFP-PLK3. We conclude that PLK3 is largely dispensable for stress response in human cells. Using mass spectrometry, we identify protein phosphatase 6 as a new interacting partner of PLK3. Polo box domain of PLK3 mediates the interaction with the PP6 complex. Finally, we find that PLK3 is phosphorylated at Thr219 in the T-loop and that PP6 constantly dephosphorylates this residue. However, in contrast to PLK1, phosphorylation of Thr219 does not upregulate enzymatic activity of PLK3, suggesting that activation of both kinases is regulated by distinct mechanisms

Le « Pays de Samson ». Eshwa : la grotte dite de Samson ('Araq Isma'in) / Père Raphaël Tonneau

Numérisé par le partenaireAppartient à l’ensemble documentaire : BbLevt0Numérisé par le partenair

Determination of μ-, δ- and κ-opioid receptors in forebrain cortex of rats exposed to morphine for 10 days: Comparison with animals after 20 days of morphine withdrawal

<div><p>Background</p><p>Chronic exposure of mammalian organism to morphine results in adaption to persistent high opioid tone through homeostatic adjustments. Our previous results indicated that in the frontal brain cortex (FBC) of rats exposed to morphine for 10 days, such a compensatory adjustment was detected as large up-regulation of adenylylcyclases I (8-fold) and II (2.5–fold). The other isoforms of AC (III-IX) were unchanged. Importantly, the increase of ACI and ACII was reversible as it disappeared after 20 days of morphine withdrawal. Changes of down-stream signaling molecules such as G proteins and adenylylcyclases should respond to and be preceded by primary changes proceeding at receptor level. Therefore in our present work, we addressed the problem of reversibility of the long-term morphine effects on μ-, δ- and κ-OR protein levels in FBC.</p><p>Methods</p><p>Rats were exposed to increasing doses of morphine (10–40 mg/kg) for 10 days and sacrificed either 24 h (group +M10) or 20 days (group +M10/−M20) after the last dose of morphine in parallel with control animals (groups −M10 and −M10/−M20). Post-nuclear supernatant (PNS) fraction was prepared from forebrain cortex, resolved by 1D-SDS-PAGE under non-dissociated (−DTT) and dissociated (+DTT) conditions, and analyzed for the content of μ-, δ- and κ-OR by immunoblotting with C- and N-terminus oriented antibodies.</p><p>Results</p><p>Significant down-regulation of δ-OR form exhibiting M_w ≈ 60 kDa was detected in PNS prepared from both (+M10) and (+M10/−M20) rats. However, the total immunoblot signals of μ-, δ- and κ-OR, respectively, were unchanged. Plasma membrane marker Na, K-ATPase, actin and GAPDH were unaffected by morphine in both types of PNS. Membrane-domain marker caveolin-1 and cholesterol level increased in (+M10) rats and this increase was reversed back to control level in (+M10/−M20) rats.</p><p>Conclusions</p><p>In FBC, prolonged exposure of rats to morphine results in minor (δ-OR) or no change (μ- and κ-OR) of opioid receptor content. The reversible increases of caveolin-1 and cholesterol levels suggest participation of membrane domains in compensatory responses during opioid withdrawal.</p><p>General significance</p><p>Analysis of reversibility of morphine effect on mammalian brain.</p></div

Na, K-ATPase, caveolin-1 and actin in PNS fractions preparad from experimental groups (±M10) and (±M10/─M20).

<p>Na, K-ATPase <b>(A),</b> [³H]ouabain binding <b>(B),</b> caveolin-1 <b>(C)</b> and actin <b>(D)</b> levels were determined in (+M10), (─M10), (+M10/─M20) and (─M10/─M20) samples of PNS. PNS proteins (20 μg per lane) were resolved under dissociated conditions (+DTT) by standard SDS-PAGE in 10% w/v acrylamide/0.26% w/v bis-acrylamide gels, and immunoblotted. Antibodies H-300 (sc-28800) and I-19 (sc-1616) from Santa Cruz were used for recognition of α subunit of Na, K-ATPase and actin, respectively. [³H]ouabain binding assay was performed at saturating, 18 nM concentration as described in Methods. Caveolin-1 (↓) was identified by Ab C13630-050 from Transduction Laboratories. The human endothelial cell lysate was used as a positive control for caveolin-1 (Transduction Laboratories, in last lane of immunoblots). Results represent the average of three immunoblots of Na, K-ATPase or [³H]ouabain binding assays ± SEM, each performed in quadruplicates (upper columns). Analysis of caveolin-1 and actin was based on signals collected from five and three immunoblots, each performed with four control + four morphine-treated samples of PNS, respectively. 100% on y-axis (upper panels) represents the average intensity of a given immunoblot signal determined in PNS prepared from control, (─M10) rats. The significance of difference between the control and morphine-treated samples was analyzed by Student´s <i>t</i>-test using GraphPad<i>Prizm4</i>. In the lower panels, typical immunoblots are shown (NS, p > 0.05; ***, p < 0.001).</p

Detection of μ-OR, δ-OR and κ-OR in PNS fractions (±M10) treated with <i>N</i>-glycosidase F.

<p>PNS fractions prepared from FBC of rats in groups (+M10) and (─M10) were treated with SDS, Triton X-100 and <i>N</i>-glycosidase F (NGF) as described in Methods. 20 μg of protein was applied per each lane of 10% w/v acrylamide/0.26% w/v bis-acrylamide gels (four + four) and resolved under the dissociated (+DTT) conditions. The OR were recognized by antibodies from Santa Cruz: μ-OR (C-20, sc-7488-R, C-terminus), δ-OR (H-60, sc-9111, N-terminus), and κ-OR (H-70, sc-9112, N-terminus). Results represent the typical immunoblots of NGF-untreated <b>(A)</b> and NGF-treated <b>(C)</b> samples. The significance of difference between NGF-untreated (+M10) versus (─M10) PNS samples <b>(B)</b> and NGF-treated (+M10) versus (─M10) PNS samples <b>(D)</b> was analyzed by Student´s <i>t</i>-test using GraphPad<i>Prizm4</i> (NS, p>0.05). The results represent the average signal of three immunoblots, each performed with four control + four morphine-treated samples of PNS ± SEM. 100% on y-axis (lower panels) represents the average intensity of a given immunoblot signal determined in PNS prepared from control, (─M10) rats.</p

Determination of μ-, δ- and κ-OR in PNS prepared from experimental groups (+M10/─M20) and (─M10/─M20).

<p>PNS fractions prepared from (+M10/─M20) and (─M10/─M20) groups of rats were resolved under dissociated (+DTT) conditions by 1D-SDS-PAGE in 10% w/v acrylamide/0.26% w/v bis-acrylamide gels and the opioid receptors were recognized by antibodies from Santa Cruz: μ-OR (C-20, sc-7488-R, C-terminus), δ-OR (H-60, sc-9111, N-terminus), and κ-OR (H-70, sc-9112, N-terminus). As before, 20 μg of PNS protein was applied per each lane. The results (upper columns) represent the average signal of four immnoblots ± SEM. 100% on y-axis represents the average intensity of a given immunoblot signal determined in PNS prepared from control, (─M10) rats. The significance of difference between the (+M10/─M20) and (─M10/─M20) PNS samples was analyzed by Student´s <i>t</i>-test using GraphPad<i>Prizm4</i>. In the lower panels, typical immunoblots are shown (NS, p>0.05; *, p<0.05).</p

Detection of μ-OR, δ-OR and κ-OR in PNS fractions (±M10/─M20) treated with <i>N</i>-glycosidase F.

<p>PNS fractions prepared from FBC of rats in groups (+M10/─M20) and (— M10/─M20) were treated with SDS, Triton X-100 and <i>N</i>-glycosidase F (NGF) as described in Methods. 20 μg of protein was applied per each lane of 10% w/v acrylamide/0.26% w/v bis-acrylamide gels (four + four) and resolved under dissociated (+DTT) conditions. The OR were recognized by antibodies from Santa Cruz: μ-OR (C-20, sc-7488-R, C-terminus), δ-OR (H-60, sc-9111, N-terminus), and κ-OR (H-70, sc-9112, N-terminus). Results represent the typical immunoblots of NFG-untreated <b>(A)</b> and NGF-treated <b>(C)</b> samples. The significance of difference between NGF-untreated (+M10/─M20) versus (─M10/─M20) PNS samples <b>(B)</b> and NGF-treated (+M10/─M20) versus (─M10/─M20) PNS samples <b>(D)</b> was analyzed by Student´s <i>t</i>-test using GraphPad<i>Prizm4</i> (NS, p>0.05). The results represent the average signal of three immunoblots, each performed with four control + four morphine-treated samples of PNS ± SEM. 100% on y-axis (lower panels) represents the average intensity of a given immunoblot signal determined in PNS prepared from control, (─M10) rats.</p

Density of [³H]naloxon and [³H]diprenorphine binding sites in PNS and PM fractions.

<p><b>A.</b> [³H]naloxon (upper) and [³H]diprenorphine (lower panels) binding was measured in PNS fractions preparared from CTR (─M10), MOR (+M10), 20CTR (─M10/─M20) and 20MOR (+M10/─M10) groups of rats as described in Methods. Results represent the average of three binding assay each performed in pentaplicates. Significance of difference between the control and morphine-treated samples was analyzed by Student´s <i>t</i>-test using GraphPad<i>Prizm4</i>. <b>B.</b> [³H]naloxon (upper) and [³H]diprenorphine (lower panel) binding was measured in PM fractions prepared from the same groups of experimental animals as described in legend to <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186797#pone.0186797.g001" target="_blank">Fig 1A</a></b>. NS, p>0.05.</p