Aurélie Trussardi-Régnier

Raman microspectroscopy detects epigenetic modifications in living Jurkat leukemic cells

AIMS Classical biochemical and molecular methods for discerning cells with epigenetic modifications are often biologically perturbing or even destructive. We wondered whether the noninvasive laser tweezer Raman spectroscopy technique allowed the discrimination of single living human cells undergoing epigenetic modifications. MATERIALS & METHODS Human Jurkat leukemic cells were treated with inhibitors of histone deacetylases (trichostatin A and MS-275). Epigenetic changes were monitored through histone electrophoresis, nuclear image cytometry and laser tweezer Raman spectroscopy. RESULTS Treatment of Jurkat cells with histone deacetylase inhibitors increased histone acetylation and induced chromatin organization changes. Characteristic vibrations, issued from laser tweezer Raman spectroscopy analyses, mostly assigned to DNA and proteins allowed discerning histone deacetylase inhibitor-treated cells from control with high confidence. Statistical processing of laser tweezer Raman spectroscopy data led to the definition of specific biomolecular fingerprints of each cell group. CONCLUSION This original study shows that laser tweezer Raman spectroscopy is a label-free rapid tool to identify living cells that underwent epigenetic changes.

The DNA Hypomethylating Agent, 5-aza-2'- Deoxycytidine, Enhances Tumor Cell Invasion Through a Transcription-Dependent Modulation of MMP-1 Expression in Human Fibrosarcoma Cells

In diseases such as cancer, cells need to degrade the extracellular matrix (ECM) and therefore require high protease levels. Thus, aberrant tissue degradation is associated to matrix metalloproteinases (MMPs) overexpression resulting from different mechanisms including epigenetic events. One of the most characterized epigenetic mechanisms is DNA methylation causing changes in chromatin conformation, thereby decreasing the accessibility to the transcriptional machinery and resulting in a robust gene silencing. Modulation of DNA methylation by DNA hypomethylating agents such as 5‐aza‐2′‐deoxycytidine (5‐azadC) is widely used in epigenetic anticancer treatments. Here, we focus on the effects of this drug on the expression level of MMP‐1, ‐2, and ‐9 in human HT1080 fibrosarcoma cells. We demonstrate that 5‐azadC increases MMP expression at both mRNA and protein levels, and promotes invasion potential of HT1080 cells. Using broad‐spectrum and specific MMP inhibitors, we establish that MMP‐1, but not MMP‐2 and ‐9, plays a key role in 5‐azadC‐enhanced cell invasion. We show that 5‐azadC induces MMP‐1 expression through a transcriptional mechanism without affecting MMP‐1 promoter methylation status. Finally, we demonstrate that 5‐azadC treatment increases the nuclear levels of Sp1 and Sp3 transcription factors, and modulates their recruitment to the MMP‐1 promoter, resulting in chromatin remodeling associated to 5‐azadC‐induced MMP‐1 expression. All together, our data indicate that the hypomethylating agent 5‐azadC modulates, mainly via Sp1 recruitment, MMP‐1 expression resulting in an increased invasive potential of HT1080 cells.

Regulation of MMPs during melanoma progression: from genetic to epigenetic.

Melanoma is the most severe skin cancer characterized by a bad prognosis at metastatic stages due to resistance to most classical chemotherapies. Invasion of melanoma cells into the surrounding microenvironment locally and at distance of the primary tumour, is facilitated by expression of proteases that degrade the extracellular matrix. Matrix metalloproteinases (MMP) have been long thought as potential therapeutic targets as they are involved in several steps of tumour progression. However, based on this general concept, broad spectrum MMP inhibitors showed weak anticancer potential. Furthermore, MMPs are also expressed by stroma and infiltrating cells. Although, inflammatory conditions lead to uncontrolled expression of MMPs leading to massive matrix destruction, these enzymes are also essential for immune cells to migrate towards the tumour site, and hence mount an anti-tumoral response. During stromal reaction, MMPs also act as non-matrix deteriorating enzymes, and thus modulating the inflammatory response through limited proteolysis of cytokines and chemokines. MMPs contribution to these processes depends on their activity and their expression. Besides the classic control level of transcription by a variety of growth factors and cytokines, the contribution of epigenetic mechanisms on MMPs expression was demonstrated of great importance to extend our knowledge about the role of these enzymes in a specific context such as melanoma progression. Understanding MMPs regulation by epigenetic drugs in melanoma and infiltrated cells will provide a new platform to develop efficient therapies. The therapeutic implication of epigenetic mechanisms to switch a pro-tumoral inflammatory towards an immune anti-tumoral response will be an exciting challenge in which MMPs expression could play a major role.

Non-optical bimorph-based tapping-mode force sensing method for scanning near-field optical microscopy

A non‐optical bimorph‐based tapping‐mode force sensing method for tip–sample distance control in scanning near‐field optical microscopy is developed. Tapping‐mode force sensing is accomplished by use of a suitable piezoelectric bimorph cantilever, attaching an optical fibre tip to the extremity of the cantilever free end and fixing the guiding portion of the fibre to a stationary part near the tip to decouple it from the cantilever. This method is mainly characterized by the use of a bimorph, which carries out simultaneous excitation and detection of mechanical vibration at its resonance frequency owing to piezoelectric and anti‐piezoelectric effects, resulting in simplicity, compactness, ease of implementation and lack of parasitic optical background. In conjugation with a commercially available SPM controller, tapping‐mode images of various samples, such as gratings, human breast adenocarcinoma cells, red blood cells and a close‐packed layer of 220‐nm polystyrene spheres, have been obtained. Furthermore, topographic and near‐field optical images of a layer of polystyrene spheres have also been taken simultaneously. The results suggest that the tapping‐mode set‐up described here is reliable and sensitive, and shows promise for biological applications.

Variation of the epidermal expression of glucocorticoid receptor‐beta as potential predictive marker of bullous pemphigoid outcome

Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disease in Western countries. Although topical and/or systemic glucocorticoids treatment efficacy is widely recognized, up to 30% of patients with BP may undergo a relapse during the first year of treatment. We investigated the protein expression of the total glucocorticoid receptor and GRβ isoform in the skin biopsy specimens from patients with BP and wondered whether such investigation at baseline provided a tool to predict disease outcome. Total GR and GRβ protein expressions were detected by immunohistochemistry at baseline on 12 patients who later relapse and 11 patients who remained on remission in comparison with 14 control patients. The expression of GRβ in the epidermis of patients with BP who later relapse was significantly higher than that in the epidermis of patients with BP controlled upon corticosteroid treatment, which was also higher than control patients. Thus, our results suggest that increased protein expression of GRβ in skin epithelial cells is predictive of reduced steroid treatment efficacy, and therefore of increased risk of disease relapse in patients with BP.

Interest of new alkylsulfonylhydrazide-type compound in the treatment of alcohol use disorders

RationaleRecent preclinical research suggested that histone deacetylase inhibitors (HDACIs) and specifically class I HDAC selective inhibitors might be useful to treat alcohol use disorders (AUDs).ObjectiveThe objective of this study was to find a new inhibitor of the HDAC-1 isoenzyme and to test its efficacy in an animal model of AUDs.MethodsIn the present study, we prepared new derivatives bearing sulfonylhydrazide-type zinc-binding group (ZBG) and evaluated these compounds in vitro on HDAC-1 isoenzyme. The most promising compound was tested on ethanol operant self-administration and relapse in rats.ResultsWe showed that the alkylsulfonylhydrazide-type compound (ASH) reduced by more than 55% the total amount of ethanol consumed after one intracerebroventricular microinjection, while no effect was observed on motivation of the animals to consume ethanol. In addition, one ASH injection in the central amygdala reduced relapse.ConclusionsOur study demonstrated that a new compound designed to target HDAC-1 is effective in reducing ethanol intake and relapse in rats and further confirm the interest of pursuing research to study the exact mechanism by which such inhibitor may be useful to treat AUDs.