High-Level Expression of Wild-Type p53 in Melanoma Cells is Frequently Associated with Inactivity in p53 Reporter Gene Assays

Background: Inactivation of the p53 pathway that controls cell cycle progression, apoptosis and senescence, has been proposed to occur in virtually all human tumors and p53 is the protein most frequently mutated in human cancer. However, the mutational status of p53 in melanoma is still controversial; to clarify this notion we analysed the largest series of melanoma samples reported to date. Methodology/Principal Findings: Immunohistochemical analysis of more than 180 melanoma specimens demonstrated that high levels of p53 are expressed in the vast majority of cases. Subsequent sequencing of the p53 exons 5–8, however, revealed only in one case the presence of a mutation. Nevertheless, by means of two different p53 reporter constructs we demonstrate transcriptional inactivity of wild type p53 in 6 out of 10 melanoma cell lines; the 4 other p53 wild type melanoma cell lines exhibit p53 reporter gene activity, which can be blocked by shRNA knock down of p53. Conclusions/Significance: In melanomas expressing high levels of wild type p53 this tumor suppressor is frequently inactivated at transcriptional level

Towards long-term standardised carbon and greenhouse gas observations for monitoring Europe's terrestrial ecosystems : a review

Research infrastructures play a key role in launching a new generation of integrated long-term, geographically distributed observation programmes designed to monitor climate change, better understand its impacts on global ecosystems, and evaluate possible mitigation and adaptation strategies. The pan-European Integrated Carbon Observation System combines carbon and greenhouse gas (GHG; CO2, CH4, N2O, H2O) observations within the atmosphere, terrestrial ecosystems and oceans. High-precision measurements are obtained using standardised methodologies, are centrally processed and openly available in a traceable and verifiable fashion in combination with detailed metadata. The Integrated Carbon Observation System ecosystem station network aims to sample climate and land-cover variability across Europe. In addition to GHG flux measurements, a large set of complementary data (including management practices, vegetation and soil characteristics) is collected to support the interpretation, spatial upscaling and modelling of observed ecosystem carbon and GHG dynamics. The applied sampling design was developed and formulated in protocols by the scientific community, representing a trade-off between an ideal dataset and practical feasibility. The use of open-access, high-quality and multi-level data products by different user communities is crucial for the Integrated Carbon Observation System in order to achieve its scientific potential and societal value.Peer reviewe

The FLUXNET2015 dataset and the ONEFlux processing pipeline for eddy covariance data

The FLUXNET2015 dataset provides ecosystem-scale data on CO2, water, and energy exchange between the biosphere and the atmosphere, and other meteorological and biological measurements, from 212 sites around the globe (over 1500 site-years, up to and including year 2014). These sites, independently managed and operated, voluntarily contributed their data to create global datasets. Data were quality controlled and processed using uniform methods, to improve consistency and intercomparability across sites. The dataset is already being used in a number of applications, including ecophysiology studies, remote sensing studies, and development of ecosystem and Earth system models. FLUXNET2015 includes derived-data products, such as gap-filled time series, ecosystem respiration and photosynthetic uptake estimates, estimation of uncertainties, and metadata about the measurements, presented for the first time in this paper. In addition, 206 of these sites are for the first time distributed under a Creative Commons (CC-BY 4.0) license. This paper details this enhanced dataset and the processing methods, now made available as open-source codes, making the dataset more accessible, transparent, and reproducible.Peer reviewe

Climate control of terrestrial carbon exchange across biomes and continents

Peer reviewe

Knockdown of TRP2 by shRNA does not affect p53 expression level.

<p>(A) TRP2 and p53 expression in indicated melanoma cell lines was determined by western blot. Tubulin was used as a loading control. (B) The indicated melanoma cell lines were transduced with three different lentiviral TRP2 shRNAs (TRP2_#1, #2, #3) and on day four after shRNA infection the efficiency of the knockdown and p53 expression were analyzed by immunoblotting; a scrambled (scr) shRNA was used as a control.</p

Ectopic re-expression of TRP2 does not rescue the p53 activation induced by TRP2-shRNA_#2.

<p>Indicated melanoma cell lines were stably transduced with a lentiviral p53 reporter construct and a modified TRP2 expression construct coding for TRP2 mRNAs in which the shRNA-binding site is modified by six silent mutations (TRP2in). On day 4 following infection total cell lysates were analyzed for TRP2 and p53 expression by immunoblotting (lower part) with tubulin used as a loading control. In the upper part the corresponding mean GFP fluorescence intensity is depicted, normalized to the relative reporter vector load of the cell lines determined by real time PCR.</p

Effects of TRP2 inhibition by shRNA on transcriptional activity of p53.

<p>Indicated melanoma cell lines were stably transduced with a lentiviral pGreenFire reporter construct encoding for green fluorescence protein (GFP) under the control of a p53 responsive element (4× CGACATGCCCGGGCATGT). The cells were then infected with the different lentiviral supernatants carrying the shRNA expression construct KH1 containing either a scrambled or a sequence targeting TRP2. Mean GFP activity four days post infection is depicted, normalized to the relative vector load of the cell lines determined by Real time PCR.</p

TRP2 and p53 expression in melanoma tissue.

<p>(A) Representative immunohistochemical staining for TRP2 and p53 in consecutive sections of a primary melanoma; scale bars: upper panel 1 mm, lower panel 100 µm. (B) Linear regression analysis of p53 and TRP2 histology scores derived from the analysis of 172 melanomas (139 primary and 33 metastatic melanoma) evaluated by a histopathologist. Staining intensity was scored between 0 and 3 and the extent of positivity between 0 and 4. By multiplying both values a minimum score of 0 and a maximum of 12 was derived; linear regression analysis comparing p53 and TRP2 histology scores revealed borderline-significant (p = 0,352) positive correlation and a coefficient of determination (R²) of 0,0258. (C) Since the number of individual dots in B cannot be visualized the distribution of individual p53 and TRP2 histology scores are displayed in the table.</p