Human cervical cancer (HeLa) cells were purchased from American Type Culture Collection (Rockville, MD), maintained in logarithmic growth, and cultured in DMEM. Cells were cultured in a density between 0.1 and 1.0 × 10⁶/mL. Medium was deemed complete when supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. The cells were cultured in suspension at 37°C and 5% CO₂ in a humidified incubator and carried at 0.1–2.0 × 10⁶ cells/mL, passaging two to three times weekly as needed. Cells were pelleted and resuspended in fresh complete medium in tissue culture plates 24 h before use in experiments to avoid any confounding gene expression that might occur because of handling. Xenoestrogens were dissolved in 1,4-dioxane or DMSO with final concentrations of solvent in control or treated cultures < 0.1%.

Cells were cultured in triplicate at 1.0 × 10⁶/mL for 72 h, treated with different concentrations (0, 20, 40, 60, 80, 90 nM) of 17-β-estradiol, coumestrol, DDT and TCDD. Proliferation was measured by determination of total viable cell mass using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay Kit (Promega, Madison, WI) according to the manufacturer’s instructions. Absorbance at 490 nm was determined on a Bio-Rad (Hercules, CA) plate reader.

Cell cycle analysis was performed as previously described [38,39]. Briefly, 1 × 10⁶ cells /mL were grown in suspension, harvested by centrifugation, washed, and fixed in 1% paraformaldehyde. After washing, the cells were permeabilized in 70% ethanol, washed, and re-suspended in PBS. RNase (Sigma) was added at a final concentration of 5 U/mL. Cells were stained with propidium iodide (PI). Flow microfluorometry was performed and DNA histograms were generated and analyzed using a Becton Dickinson Flowscan (Franklin Lakes, NJ). This method correlates closely with other measures of apoptosis including TUNEL and Annexin V staining while providing additional cell cycle information [41]. This method also allows for enumeration of the percentages of cells in G0/G1 (resting phase), S (DNA synthesis phase), G2M (mitotic phase), and hypodiploid or apoptotic percentages (those cells containing less than the normal amount of DNA) [41].

For Western analysis, 5 × 10⁶ cells were cultured, treated with various concentrations of estrogen, coumestrol, DDT and TCDD compounds. Total protein concentration was determined by the method of Bradford using Bio-Rad Protein Assay reagents (Bio-Rad) in a microtiter assay plate. Total cellular protein (30ug) was electrophoresed on 12.5% SDS-PAGE gel, transferred to a polyvinylidine difluoride membrane (Amersham, Arlington Heights, IL) by electroblotting overnight. Membranes were blocked with 10% electrophoresis grade biotin-depleted non-fat dry milk (BioRad) in 1 X PBS (10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween-20), rinsed in PBS, probed with monoclonal mouse anti-human bcl-2, Cyclin A and D (BD Bioscience San Diego, CA) using 1:1,000 dilution, and washed 3 times in PBS. The secondary antibody was HRP-conjugated goat anti-mouse whole IgG used at 1:1,000 (Transduction Laboratories). All antibodies were diluted in 1% milk in TBS. Membranes were washed three times. Detection of membrane-bound proteins was carried out by enhanced chemiluminesence with an ECL reagent kit (using 0.06 mL/cm² of reagent) and Hybond autoradiography film (both Amersham). Biotinylated standards were used for molecular weight determination and were detected with 1:3,000 streptavidin-horseradish peroxidase (Amersham).

Study results show compound-specific effects on accumulation of viable HeLa cell mass. 17-β-estradiol and TCDD were used as representative controls of endogenous and environmental anti-estrogen compounds. In Figure 1, coumestrol, DDT and TCDD exhibited concentration dependent (20 to 120 nM) suppression on HeLa cell proliferation, indicating the cytotoxic effects of 17-β-estradiol [36–39]. Viable cell mass was assessed by MTT assay (see Methods). Change in total cell number was confirmed in cell cultures by enumeration (not shown). * p < 0.05 was determined using ANOVA with Bonferroni correction for multiple comparisons.

Members of the bcl-2 family are crucial regulators of apoptosis in mammalian cells. The bcl-2 family includes antiapoptotic proteins, such as Bcl-2, and proapoptotic proteins, such as Bax. Since estrogen induced apoptosis is dependent on time and dose, we first tested bcl-2 response to estrogen. HeLa cells demonstrated a dose-dependent decrease (20, 40, 60, 80, 90 nM) in the expression of the antiapoptotic Bcl-2 protein upon estrogen treatment (Figure 2).

Cell accumulation or growth is a homeostatic balance between proliferation and apoptosis [42,43]. Questions have been raised regarding the MTT assay as a measure of viable cell mass, especially for xenoestrogens [44]. Therefore, xenoestrogen effects on cell cycle phase distribution in HeLa cells was also assessed by propidium iodide (PI) staining [38,39]. Representative cell cycle histograms for 17-β-estradiol, coumestrol, DDT and TCDD are shown in Figure 3, 17-β-estradiol and coumestrol had significant cell cycle phase effect on actively growing HeLa cells, causing redistribution from G0/G1 to apoptosis (p < 0.01), whereas TCDD and DDT had minimal effect, when analyzed by PI staining.

Bcl-2, Cyclin A and D cell regulatory proteins modulate cell cycle progression and apoptosis [42,43,46]. Given the observed effects of xenoestrogens on HeLa cell proliferation and cell cycle distribution, examination of bcl-2, Cyclin A and D protein levels was performed. As shown in representative Western blots (Figure 4; n = 3), xenoestrogens DDT, coumestrol, and 17-β-estradiol suppressed bcl-2 protein whereas, anti-estrogen, TCDD did not have a significant effect on bcl-2 expression as compared to the control. DDT, coumestrol and TCDD increased Cyclin A and D protein levels to variable degrees in HeLa cells (Figures 5 and 6 respectively). These preliminary results are consistent with recent reports of potential modulation of bcl-2 by xenoestrogens [47–50], supporting the concept that xenoestrogens may modulate cancer cell biology through associated changes in bcl-2.