Dataset Information

Evaluation of active Rac1 levels in cancer cells: A case of misleading conclusions from immunofluorescence analysis.

ABSTRACT: A large number of aggressive cancer cell lines display elevated levels of activated Rac1, a small GTPase widely implicated in cytoskeleton reorganization, cell motility, and metastatic dissemination. A commonly accepted methodological approach for detecting Rac1 activation in cancer cells involves the use of a conformation-sensitive antibody that detects the active (GTP-bound) Rac1 without interacting with the GDP-bound inactive form. This antibody has been extensively used in fixed cell immunofluorescence and immunohistochemistry. Taking advantage of prostate and pancreatic cancer cell models known to have high basal Rac1-GTP levels, here we have established that this antibody does not recognize Rac1 but rather detects the intermediate filament protein vimentin. Indeed, Rac1-null PC3 prostate cancer cells or cancer models with low levels of Rac1 activation still show a high signal with the anti-Rac1-GTP antibody, which is lost upon silencing of vimentin expression. Moreover, this antibody was unable to detect activated Rac1 in membrane ruffles induced by epidermal growth factor stimulation. These results have profound implications for the study of this key GTPase in cancer, particularly because a large number of cancer cell lines with characteristic mesenchymal features show simultaneous up-regulation of vimentin and high basal Rac1-GTP levels when measured biochemically. This misleading correlation can lead to assumptions about the validity of this antibody and inaccurate conclusions that may affect the development of appropriate therapeutic approaches for targeting the Rac1 pathway.

SUBMITTER: Baker MJ

PROVIDER: S-EPMC7535912 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Evaluation of active Rac1 levels in cancer cells: A case of misleading conclusions from immunofluorescence analysis.

Baker Martin J MJ Cooke Mariana M Kreider-Letterman Gabriel G Garcia-Mata Rafael R Janmey Paul A PA Kazanietz Marcelo G MG

The Journal of biological chemistry 20200814 40

A large number of aggressive cancer cell lines display elevated levels of activated Rac1, a small GTPase widely implicated in cytoskeleton reorganization, cell motility, and metastatic dissemination. A commonly accepted methodological approach for detecting Rac1 activation in cancer cells involves the use of a conformation-sensitive antibody that detects the active (GTP-bound) Rac1 without interacting with the GDP-bound inactive form. This antibody has been extensively used in fixed cell immunof ...[more]

PMID: 32817335

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Project description:BackgroundThere is often a huge gap between neurobiological facts and firm conclusions stated by the media. Data misrepresentation in the conclusions and summaries of neuroscience articles might contribute to this gap.Methodology/principal findingsUsing the case of attention deficit hyperactivity disorder (ADHD), we identified three types of misrepresentation. The first relies on prominent inconsistencies between results and claimed conclusions and was observed in two scientific reports dealing with ADHD. Only one out of the 61 media articles echoing both scientific reports adequately described the results and, thus questioned the claimed conclusion. The second type of misrepresentation consists in putting a firm conclusion in the summary while raw data that strongly limit the claim are only given in the results section. To quantify this misrepresentation we analyzed the summaries of all articles asserting that polymorphisms of the gene coding for the D4 dopaminergic receptor are associated with ADHD. Only 25 summaries out of 159 also mentioned that this association confers a small risk. This misrepresentation is also observed in most media articles reporting on ADHD and the D4 gene. The third misrepresentation consists in extrapolating basic and pre-clinical findings to new therapeutic prospects in inappropriate ways. Indeed, analysis of all ADHD-related studies in mice showed that 23% of the conclusions were overstated. The frequency of this overstatement was positively related with the impact factor of the journal.Conclusion/significanceData misrepresentations are frequent in the scientific literature dealing with ADHD and may contribute to the appearance of misleading conclusions in the media. In synergy with citation distortions and publication biases they influence social representations and bias the scientific evidence in favor of the view that ADHD is primarily caused by biological factors. We discuss the social consequences and the causes of data misrepresentations and suggest a few corrective actions.

Project description:QUESTIONS:Molecular phylogenies are being increasingly used to better understand the mechanisms structuring natural communities. The prevalent theory is that environmental factors and biotic interactions promote the phylogenetic clustering and overdispersion of plant communities, respectively. However, both environmental filtering and biotic interactions are very likely to interact in most natural communities, jointly affecting community phylogenetic structure. We asked the following question: How do environmental filters and biotic interactions jointly affect the phylogenetic structure of plant communities across environmental gradients? LOCATION:Eleven Stipa tenacissima L. grasslands located along an environmental gradient from central to south-east Spain, which covers the core of the distribution area of this vegetation type in Europe. METHODS:We jointly evaluated the effects of environmental conditions and plant-plant interactions on the phylogenetic structure -measured with the mean phylogenetic distance index- of the studied communities. As an indicator of environmental conditions, we used a PCA ordination including eight climatic variables. Different metrics were used to measure the following processes: i) competition/facilitation shifts at the entire community level (Species Combination index), and ii) the effect of the microclimatic amelioration provided by the two most important nurses on neighbours composition (similarity indices and comparison of the phylogenetic pattern between canopy patches and bare ground areas). RESULTS:Biotic interactions and, to a less extent, environmental conditions, affected the phylogenetic pattern of the studied communities. While positive plant-plant interactions (both at the community and the scale of individual nurses) increased phylogenetic overdispersion, higher rainfall increased phylogenetic clustering. The opposed effects of environmental conditions and biotic interactions could be the main cause of the overall random phylogenetic structure found in most of these communities. CONCLUSIONS:Our results illustrate, for the first time, how an overall random phylogenetic pattern may not be promoted only by the lack of influence of either environmental filtering or biotic interactions, but rather by their joint and opposing effects. They caution about making inferences about the underlying mechanisms shaping plant communities from the sole use of their phylogenetic pattern. We also provide a comprehensive set of easy-to-measure tools to avoid misleading conclusions when interpreting phylogenetic structure data obtained from observational studies.

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Dataset Information

Evaluation of active Rac1 levels in cancer cells: A case of misleading conclusions from immunofluorescence analysis.

Publications

Evaluation of active Rac1 levels in cancer cells: A case of misleading conclusions from immunofluorescence analysis.

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