17-β-estradiol affects nuclear image properties in MCF-10F human breast epithelial cells with tumorigenesis

Abstract

Treatment of the human breast epithelial cells MCF-10F with 17-ß-estradiol (E2) induces transformation and tumorigenesis. E2-transformed MCF-10F cells are known to exhibit progressive loss of ductulogenesis, and invasive and tumorigenic phenotypes. Although DNA amounts and chromatin supraorganization change in E2-transformed MCF cells, no comparative study has yet been undertaken in the resulting cells selected for aggressive invasiveness (C5) and tumor generation in a heterologous host. The aim of this study was thus to determine whether changes in Feulgen-DNA content and chromatin supraorganization are involved during E2-induced transformation and tumorigenesis of the MCF-10F cells. Image analysis was performed for nontransformed and E2-transformed MCF cells, highly invasive cells (C5), and for cell lines (C5-A6-T6 and C5-A8-T8) derived from tumors generated by injection of C5 cells in SCID mice. A decrease in Feulgen-DNA amounts and nuclear sizes induced by E2 treatment was accented with selection of the highly invasive tumorigenesis potential. However, in the tumor-derived cells a high variability in cellular phenotypes resulted inclusively in near-polyploidy. Significant changes in textural parameters, including nuclear entropy, indicated chromatin structural remodeling with advancing tumorigenesis. An increased variability in the degree of chromatin packing states in the E2-transformed MCF cells is followed by reduction in chromatin condensation and in contrast between condensed and noncondensed chromatin in the highly invasive C5 cells and tumor-derived cell lines. Studies on epigenetic mechanisms involving DNA methylation and/or the histone code would contribute to a better interpretation of the chromatin supraorganization changes reported herein.

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

“…The entropy values of the Feulgen-stained control and 17-ß-estradiol-transformed MCF-10F cells after treatment with Msp I decreased significantly (Table II), which indicates that -CCGG-sequences are involved in the previously reported chromatin packing states of these cells (Table I) (7). After the different cell types were treated with HpaII, the entropy values significantly decreased for MCF-10F, E2, and C5 cells, but they did not change for the C5-A6-T6 cells and even increased for the C5-A8-T8 cells (Table II).…”

“…The entropy studied in Feulgen-stained preparations of the transformed MCF-10F cells has revealed a variable heterogeneity in chromatin packing states, attaining its highest and lowest expression in E2 and C5-A8-T8 cells, respectively (7) ( Table I). Expressive chromatin remodeling with advancing cell transformation and tumorigenesis has thus been suggested for this cell system (7).…”

“…Since it has been considered that higher order chromatin packing states and nuclear organization in animal cells be affected by epigenetic mechanisms (7,11), epigenetic dysregulation might contribute to the chromatin textural repatterning in transformed MCF-10F cells. Indeed, global genome hypomethylation and regional site hypermethylation have been reported in human breast cancer cells (Fernandez et al, Proc AACR Annual Meeting 48: abs.…”

“…Among the various image analysis textural parameters estimated in Feulgen-stained cells, inclusive in transformed MCF-10F cells, entropy has been shown to be relevant for discrimination of changes in chromatin supraorganization (2,(5)(6)(7). Nuclear entropy as studied by video image analysis has been defined as the number of bits necessary to store the densitometric values per nucleus image or the amount of gray value variability contained in a nucleus (5,7,9).…”

“…Nuclear entropy as studied by video image analysis has been defined as the number of bits necessary to store the densitometric values per nucleus image or the amount of gray value variability contained in a nucleus (5,7,9). This parameter measures the complexity of an image (10), such that a high entropy value for Feulgen-stained cells is found when there is a high contrast between condensed and non-condensed chromatin, whereas a low entropy value represents a homogeneously packed chromatin state (fairly constant gray levels) (7,8) (Fig. 1).…”