Entropy of Feulgen-stained 17-β-estradiol-transformed human breast epithelial cells as assessed by restriction enzymes and image analysis

Abstract

Abstract. MCF-10F human breast epithelial cells when transformed with 17-ß-estradiol (E2) give rise to highly invasive C5 cells that generate adenocarcinomas in SCID mice. From these tumors, cell lines such as C5-A6-T6 and C5-A8-T8 have been derived. Variable patterns of chromatin supraorganization have been demonstrated for these cells during the transformation/tumorigenesis progress, when assessing chromatin entropy by image analysis in Feulgenstained preparations. Since epigenetic dysregulation might contribute to the chromatin textural repatterning in transformed MCF-10F cells, the association of the variable chromatin packing states with global DNA methylation was investigated in these cells after their treatment with restriction enzymes followed by Feulgen staining and chromatin entropy evaluation by image analysis. The results indicate that although -C m CGG-sequences may affect chromatin supraorganization in some of the analyzed cell types (perhaps due to localized hypermethylation), not all the chromatin condensation patterns in these cells with transformation and/ or tumorigenesis are associated with DNA methylation (e.g. E2 cells). Chromatin supraorganization remodeling in C5-A6-T6 and C5-A8-T8 cells may be attained by different mechanisms, with C5-A6-T6 chromatin packing states perhaps being associated with local DNA hypermethylation or other epigenetic factors, and C5-A8-T8 likely being associated with global DNA hypomethylation, as reported in the literature for other cell types. Thus, we assume that a variable epigenetic modulation affecting the higher-order packing states of chromatin in the estrogentransformed MCF-10F cell model could be evident with the chromatin entropy study by image analysis.

Spatial distribution of AT- and GC-rich DNA within interphase cell nuclei of Triatoma infestans Klug

“…This finding was likely due to the lower sensitivity of the enzymatic assay compared to the immunological test for the detection of minute levels of -C m CGG-sequences in whole T. infestans nuclei. The Msp I/Hpa II assay was useful for the detection of GC-rich DNA and/or -C m CGG-sequences in chromosomes or whole nuclei where these sequences are very representative (Lima-de-Faria et al, 1980;Mello et al, 2000Mello et al, , 2009bMampumbu and Mello, 2006).…”

“…In addition, GC-richness and the occurrence of methylated -CCGG-sequences, which affect extensive global genome sites, can be investigated in chromosomes and interphase chromatin by using the isoschizomeric restriction enzymes Msp I and Hpa II (Lima- de-Faria et al, 1980;Mezzanotte et al, 1983;Bianchi et al, 1986;Gonsálvez et al, 1995;Mello et al, 2000Mello et al, , 2009b. While both enzymes cleave the sequence -CCGG-, Hpa II will not cleave this site if the central cytosine of the internal CG dinucleotide is methylated (Nelson and McCleland, 1991).…”

“…In general, after the restriction enzyme recognizes, cleaves and removes determined DNA sequences from fixed chromosomes or interphase chromatin, specific images are generated (Mezzanotte et al, 1983;Bianchi et al, 1986;Sentis et al, 1989;Gonsálvez et al, 1995;Mello et al, 2000). The richness in -CCGG-sequences excised by Msp I/Hpa II, and the presence of methylated CpG sequences not cut by Hpa II, can be evaluated by quantifying the response to the Feulgen reaction, specific for DNA, under the action of these enzymes, using video or microspectrophotometric image analysis (Mello et al, 2000(Mello et al, , 2009bMampumbu and Mello, 2006).…”

Polyploidy and chromatin remodeling in hepatocytes from insulin‐dependent diabetic and normoglycemic aged mice

“…Values for SDtd, entropy, and energy indicated lower variability in chromatin condensation levels (more homogeneously packed chromatin) (8,11,16,17,39,40) under moderate hyperglycemia in the NOD mice. No apparent changes in chromatin texture were verified in the hepatocytes of NOD mice under severe hyperglycemia in comparison with the normoglycemic control.…”

“…The minimum area to be measured in this apparatus corresponded to four pixels. The software provided the following quantitative information: nuclear area (lm 2 ), mean gray value per nucleus (subsequently converted into optical density, OD 5 absorbance), standard deviation of the total densitometric values per nucleus or absorbance variability per nucleus (SDtd), entropy (number of bits needed to store the densitometric values per nucleus image) (15,16), and energy. The energy parameter was calculated from the densitometric histogram: in contrast to entropy, large values are found in images with large regions of uniform density (15,17).…”