ABSTRACT
OBJECTIVE: Chromatin texture patterns of tumour cell nuclei can serve as cancer biomarkers, either to define diagnostic classifications or to obtain relevant prognostic information, in a large number of human tumours. Epigenetic mechanisms, mainly DNA methylation and histone post-translational modification, have been shown to influence chromatin packing states, and therefore nuclear texture. The aim of this study was to analyse effects of these two mechanisms on chromatin texture, and also on correlation with gelatinase expression, in human fibrosarcoma tumour cells. MATERIALS AND METHODS: We investigated effects of DNA hypomethylating agent 5-aza-2'-deoxycytidine (5-azadC) and histone deacetylase inhibitor trichostatin A (TSA) on nuclear textural characteristics of human HT1080 fibrosarcoma cells, evaluated by image cytometry, and expression of gelatinases MMP-2 and MMP-9, two metalloproteinases implicated in cancer progression and metastasis. RESULTS: 5-azadC induced significant variation in chromatin higher order organization, particularly chromatin decondensation, associated with reduction in global DNA methylation, concomitantly with increase in MMP-9, and to a lesser extent, MMP-2 expression. TSA alone did not have any effect on HT1080 cells, but exhibited differential activity when added to cells treated with 5-azadC. When treated with both drugs, nuclei had higher texture abnormalities. In this setting, reduction in MMP-9 expression was observed, whereas MMP-2 expression remained unaffected. CONCLUSIONS: These data show that hypomethylating drug 5-azadC and histone deacetylase inhibitor TSA were able to induce modulation of higher order chromatin organization and gelatinase expression in human HT1080 fibrosarcoma cells.
Subject(s)
Cell Nucleus/genetics , Epigenesis, Genetic , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Biomarkers, Tumor/metabolism , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/enzymology , Chromatin/genetics , Chromatin/metabolism , Chromatin Assembly and Disassembly/drug effects , DNA Methylation , Decitabine , Disease Progression , Fibrosarcoma/enzymology , Fibrosarcoma/pathology , Histone Deacetylase Inhibitors/pharmacology , Histones/genetics , Histones/metabolism , Humans , Hydroxamic Acids/pharmacology , Image Cytometry , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase Inhibitors/pharmacology , Neoplasm Metastasis/genetics , Neoplasm Metastasis/pathology , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolismABSTRACT
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.
