New method to detect histone acetylation levels by flow cytometry

Ronzoni, Simona; Faretta, Mario; Ballarini, Marco; Pelicci, PierGiuseppe; Minucci, Saverio

doi:10.1002/cyto.a.20151

Cited by 64 publications

(73 citation statements)

References 25 publications

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“…We also do not know the key target(s) for HDACi action. Proposed surrogate markers, such as measuring the levels of acetylated histones from peripheral blood cells before and after treatment 16 , should serve as indicators of effectiveness, but these need to be validated clinically and do not always seem to correlate strictly with pharmacokinetic profiles 110,111 . Alternative strategies could be attempted to identify novel predictive markers; gene profiling studies from tumour samples obtained from responders and nonresponders might lead to their identification.…”

Section: Maximal Tolerated Dosementioning

confidence: 99%

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Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer

2006

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| Histone deacetylases (HDACs) are considered to be among the most promising targets in drug development for cancer therapy, and first-generation histone deacetylase inhibitors (HDACi) are currently being tested in phase I/II clinical trials. A wide-ranging knowledge of the role of HDACs in tumorigenesis, and of the action of HDACi, has been achieved. However, several basic aspects are not yet fully understood. Investigating these aspects in the context of what we now understand about HDACi action both in vitro and in vivo will further improve the design of optimized clinical protocols.Histone deacetylases (HDACs) have been intensively scrutinized over the past few years for two main reasons. First, they have been linked mechanistically to the pathogenesis of cancer, as well as several other diseases. Second, small-molecule HDAC inhibitors (HDACi) exist that have the capacity to interfere with HDAC activity and can therefore achieve significant biological effects in preclinical models of cancer. These findings justified the introduction of HDACi into clinical trials. These initial clinical trials have just ended and show encouraging results. At this stage it is crucial to evaluate whether our current knowledge on the mechanisms of tumorigenesis that are linked to HDACs, and on the mechanisms of tumour sensitivity to HDACi, is firm enough to improve the design of further clinical trials. Recent structural and chemical data have emerged that will help in the design of novel HDACi with more desirable properties than the existing ones. However, key areas of investigation that might help to further illuminate the design of successful HDACi-based cancer therapy remain poorly explored. Novel findings indicate that our understanding of how HDACi work will probably change significantly, establishing a new paradigm in the field of intelligent drug design with broad implications for the design of targeted therapies in cancer and possibly other diseases.HDACs: enzymes looking for substrate(s) Four HDAC classes have been identified (FIG. 1a). One of them (class 3 or the so-called sirtuins, from the yeast protein Sir2) constitutes a structurally unrelated, NADdependent subfamily, and will not be considered here; neither will the class 3-specific HDACi, which are less characterized than those for the other classes 1 .An extensive phylogenetic analysis of HDACs has been performed 2,3 . HDACs are members of an ancient enzyme family found in animals, plants, fungi and bacteria. It is thought that HDACs evolved in the absence of histone proteins. Indeed, eukaryotic HDACs can deacetylate non-histone as well as histone substrates, and some HDACs reside in the cytoplasm (where histones are synthesized and acetylated for proper assembly, without the intervention of HDACs) and in mitochondria (where histones are absent).Are HDACs, then, truly HDACs 4 ? We postulate that key HDAC substrates might not be histones, but instead belong to the growing list of acetylated non-histone proteins (FIG. 1b and Supplementary information S1 ...

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Section: Maximal Tolerated Dosementioning

confidence: 99%

“…A potential approach would be the use of pan-specific antibodies that are able to recognize acetylated lysines in any protein context for proteomic approaches, similar to those used to study other post-translational modifications 14 . These antibodies are available and should produce a dynamic map of the acetylome and its function 15,16 .…”

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confidence: 99%

Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer

2006

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“…Cells were then analyzed by FC using a Coulter EPICS XL, and at least 20 000 cells from each sample were analyzed. Histone H4 acetylation was verified by FC as described by Ronzoni et al 32 Culture, RNA extraction and gene expression profile In all, 2 Â 10 6 per ml of purified CD19 þ cells were cultured in RPMI-1640 medium (Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (Sigma-Aldrich) for 4 h with or without 1 mM of VPA. Total RNA was then extracted in a single step using TriPure Isolation Reagent (Roche Applied Science, Vilvoorde, Belgium).…”

Section: Patients and Sample Collectionmentioning

confidence: 99%

Antileukemic activity of valproic acid in chronic lymphocytic leukemia B cells defined by microarray analysis

et al. 2009

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“…Deacetylation of fixed samples subsequently analyzed by flow cytometry has been noted previously 10 and HDACi have been HREC 30025A). Peripheral blood from four patients enrolled on a Phase I study to examine the therapeutic effectiveness of LBH589 in pediatric patients with refractory solid tumors (CLBH589AAU03T) was included in the study to ascertain concordance between in vitro and in vivo data.…”

Section: Discussionmentioning

confidence: 97%

“…Flow cytometry was based on the method of Ronzoni et al 2005. Briefly, 8 × 10 5 leukocytes were resuspended in PBS containing 1% FBS, centrifuged and resuspended in 200 μL 1% paraformaldehyde.…”

Section: Methodsmentioning

confidence: 99%