Apoptotic ratios and mitotic abnormalities in 17-β-estradiol-transformed human breast epithelial MCF-10F cells

Treatment of human breast epithelial cells MCF-10F with 17-β-estradiol has been reported to result in E2-transformed cells which have given rise to highly invasive C5 cells that in turn generate tumors in SCID mice. From these tumors, various cell lines, among which C5-A6-T6 and C5-A8-T8, were obtained. Although different phases of the tumorigenesis process in this model have been studied in molecular biology and image analysis assays, no cytological data on apoptotic ratios and mitotic abnormalities have been established to accompany the various steps leading to 17-β-estradiol–treated MCF-10F cells to tumorigenesis. Here we detected that the apoptotic ratio decreases with the transformation and tumorigenesis progress, except for the tumor cell line C5-A8-T8, probably on account of its more intense proliferation rate and a more rapid culture medium consumption. Increased frequency of mitotic abnormalities contributed by tripleand tetrapolar metaphases, and by lagging chromosomes and chromosome bridges observed at the anaphase found by transformation and tumorigenesis progress. However, no difference was found under these terms when the C5-A6-T6 and C5-A8-T8 tumor cell lines were compared to each other. Present findings are in agreement with the nuclear instability and enrichment of dysregulated genes in the apoptotic process promoted by transformation and tumorigenesis in 17-β-estradiol-treated MCF-10F cells.


Cell preparation and staining
The cells were cultivated for 96 hours (MCF-10F cells) and 48 hours (the other cell types) on Permanox ® plastic chamber slides of 4.2 cm 2 and 1.2-2.0 mL working volume (bi-chamber) (Mello et al., 2005) and then fixed in absolute ethanol-acetic acid mixture (3:1, v/v) for 1 minute, rinsed in 70% ethanol for 3-5 minutes, and air dried at room temperature. The preparations were subjected to the Feulgen reaction (4 M HCl at 24 °C, 75 minutes) and counterstained with acid fast green (Mello et al., 2004).

Parameters
Apoptotic ratios and mitotic indices were determined in 2000 cells per preparation of each cell line. Three to four preparations (chambers) of each cell line were examined. Apoptosis was identified based on morphological criteria (Wyllie et al., 1980). As abnormal mitoses, triple-polar and tetra-polar metaphases, metaphases/anaphases showing lagging chromosomes, and chromosomal bridges were considered in total 100 metaphases and 100 anaphases, respectively per cell line. Counts were done in a Zeiss binocular microscope equipped with a 100/1.25 objective.

Statistics
Non-parametric Mann-Whitney and Kruskall-Wallis tests were used to assess the statistical significance of comparisons. The critical level to reject the null hypothesis was considered to be a P value of 5%. Calculations and statistical analyses were performed using the Minitab 12™ software (State College, PA, USA).

Results and Discussion
Cells with an apoptotic morphology (Figure 1a-c) were observed in non-transformed and all transformed MCF-10F cells analyzed here. The apoptotic ratio was found to decrease significantly by the transformation and tumorigenesis progress in the transformed cells, with the exception of the C5-A8-T8 cell line (Table 1). In this case, the apoptotic ratio was 100% higher than that of control MCF-10F cells. On the other hand, C5-A8-T8 cells showed the highest mitotic index values amongst those evaluated here (Table 1). Increase in mitotic index for the case of the C5-A8-T8 cells may have been associated with enhancement in apoptotic ratios, since a higher proliferative rate under in vitro conditions may have lead cells to

Introduction
MCF-10 F, estrogen receptor α-negative immortalized human breast epithelial cells acquire cell transformation properties similarly to those elicited by in vitro treatment with the carcinogen benzo[a]pyrene, when subject to treatment using17-β-estradiol (E2) (Russo et al., 2002a,b;Mello et al., 2007a). This estrogen-elicited event is not abrogated by the estrogen antagonist, ICI 182,780, confirming its independence from the presence of estrogen receptor α (Lareef et al., 2005;Mello et al., 2007a).
The neoplastic progression in E-2-transformed MCF-10F cells is characterized by anchorage-independent growth, colony formation in agar-methocel, an increase in cell proliferation and in invasive capability, loss of heterozygosity in chromosomes 13 and 17, loss of a putative tumor-suppressor gene-containing 9p11-13 locus, and of chromosome 4, deletions in chromosomes 3, 8, 9 (including p15 and p16 genes), and 18 (including one or more tumor-suppressor genes), and gains in chromosomes 1 and 5 (Russo et al., 2002a(Russo et al., ,b, 2006Huang et al., 2007). These events are accompanied by changes in DNA content and nuclear sizes and by supraorganization chromatin remodeling in interphase cells (Mello et al., 2007b).
Although several geometric, densitometric and textural characteristics of the interphase cell chromatin image have been evaluated in MCF-10F cells with the E2-induced neoplastic progression (Mello et al., 2007a(Mello et al., ,b, 2009, no cytological data on apoptotic ratios and mitotic abnormalities were reported for these cells under sequential steps of the transformation/tumorigenesis process. Apoptosis rates are expected to diminish while mitosis abnormalities contributing to nuclear disturbances are expected to become more representative with the E2-induced transformation/ tumorigenesis progress (Huang et al., 2007). Thus, in the present study, apoptotic ratios, mitotic indices, and the frequency of abnormal mitoses were cytologically compared in 17-β-estradiol-transformed E-2, C5, C5-A6-T6, and C5-A8-T8 cells, and non-transformed MCF-10F control cells.
Present findings on mitotic abnormalities are in agreement with increasing nuclear instabilities accompanying transformation and tumorigenesis induced in the MCF-10F cells by 17-β-estradiol (Russo et al., 2006; more rapid culture medium consumption and facilitated cell death. A similar phenomenon has been reported for MCF-10F cells transformed by benozo[a]pyrene and subsequently transfected with the c-Ha-ras oncogene (Barbisan et al., 1999). Although both C5-A6-T6 and C5-A8-T8 cells are tumor-derived cell lines, C5-A8-T8 has been reported as the most rapidly growing tumor amongst various tumors induced in SCID mice by the C5 cells (Russo et al., 2006). In addition, C5-A8-T8 cells show the smallest chromatin entropy probably associated with global DNA methylation in Feulgen-stained interphase   Huang et al., 2007;Mello et al., 2007a). The diminished frequency of apoptotic ratios by cell transformation and tumorigenesis progress, with the exception of the case for C5-A8-T8 cells, is also an expected finding, considering the nature of the cell lines analyzed here and that Gene Ontology analysis has revealed enrichment of dysregulated genes in the apoptotic process in tumorigenic 17-β-estradiol-transformed MCF-10F cells (Huang et al., 2007).