Morphologic transformation of human breast epithelial cells MCF-10A: dependence on an oxidative microenvironment and estrogen/epidermal growth factor receptors

Abstract

Background: MCF-10A, immortalized but non-transformed human breast epithelial cells, are widely used in research examining carcinogenesis. The studies presented here were initiated with the observation that MCF-10A cells left in continuous culture for prolonged periods without re-feeding were prone to the development of transformed foci. We hypothesized that the depletion of labile culture components led to the onset of processes culminating in the observed cell transformation. The purpose of this study was to define the factors which promoted transformation of this cell line.

Results: Changes in levels of phenol red (PHR), hydrocortisone (HC), and epidermal growth factor (EGF) with or without estrogen treatment indicated that both oxidative stress- and estrogen receptor alpha (ERα)-mediated pathways contribute to cell transformation. Gene array and Western blotting analyses of cells maintained in our laboratory and of those from other sources documented detectable ERα and ERbeta (ERβ) in this ERα-negative cataloged cell line. Results also indicate the possibility of a direct association of EGF receptor (EGFR) and ERα in these cells as well as the formation and high induction of a novel ternary complex that includes ERβ (ERα/ERβ/EGFR) in cells grown under conditions facilitating transformation.

Conclusions: Our studies resulted in the development of a growth protocol where the effects of chronic, physiologically relevant alterations in the microenvironment on cellular transformation were examined. From our results, we were able to propose a model of transformation within the MCF-10A cell line in which oxidative stress, ER and EGFR play essential roles. Overall, our work indicates that the immediate microenvironment of cells exerts powerful growth cues which ultimately determine their transformation potential.

Figure 1

Effects of PHR, HC, EGF, and E2 on Focus Formation by MCF-10A Cells Cultured for 13 Days. MCF-10A cells, plated at 5 × 10⁵ cells/well, were grown in 6-well tissue culture plates under differing culture and treatment (once a week) conditions and photographed at low magnification (phase contrast; 4×) to enable visual indication of the number of transformed foci after13 days in culture. Photographs are representatives of triplicate samples from one experiment.

Figure 2

Effects of PHR, HC, EGF, and E2 on Focus Formation by MCF-10A Cells Grown in Continuous Culture for 5 Weeks. MCF-10A cells, plated at 5 × 10⁵ cells/well, were grown in 6-well plates under differing culture and treatment (once a week) conditions and photographed (phase contrast; 10×) after 5 weeks in continuous culture. Photographs are representatives of triplicate samples from one experiment.

Figure 3

Effect of PHR Presence in -HC/-EGF Medium on the Appearance of MCF-10A Transformed Foci After 3 Weeks of Continuous Culture. MCF-10A cells were seeded in triplicate at a density of 6.25 × 10⁴ cells/well in 6-well tissue culture plates and grown in -HC/-EGF medium either with or without phenol red for 3 weeks in continuous culture. Cells were stained with neutral red (10 mg/ml) and photographed (10× obj.). Photographs are representatives of triplicate wells per culture condition from one experiment.

Figure 4

Effects of 1 nM 2-Fl-E2 Treatment on Focus Formation by MCF-10A Cells Grown in Continuous Culture for 5 Weeks. MCF-10A cells, plated at 5 × 10⁵ cells/well, were grown in 6-well TC plates in -PHR/-HC/-EGF medium and treated with 1 nM 2-Fl-E2 once a week. Two different areas of one well were photographed (phase contrast; 10×) in order to show variations in cell morphology and focus formation within the same treatment group at 5 weeks in continuous culture. Photographs are representatives of triplicate samples from one experiment.

Figure 5

Morphology of MCF-10A Transformed Foci. MCF-10A transformed foci, which appear under different culture/treatment conditions, were photographed to document morphologic characteristics. The photograph for panel (d) was taken from a single T25 flask from one experiment. All other photographs are representatives of triplicate wells of 6-well tissue culture plates from one experiment.

Figure 6

Effect of HC and/or EGF Withdrawal on MCF-10A Cell Growth and Morphology in PHR-Free Medium after 24 h Culture. MCF-10A cells were either maintained in fully supplemented medium (+HC/+EGF), grown in HC/EGF-depleted medium (-HC/-EGF) for 11 passages, or grown in HC-deficient medium (-HC/+EGF) for 13 passages. Cells grown in different media types were photographed under phase-contrast microscopy 24 hours after plating. Photographs are representatives of triplicate samples of each culture condition from one experiment.

Figure 7

Analysis of MCF-10A Cells for ERα, ERβ, and EGFR. Nuclear proteins extracted from MCF-10A cells that were exposed to differing culture conditions were analyzed by Western blotting for the presence of ERα, ERβ, and EGFR. Numbers in parentheses indicate the number of passages in that particular type of medium. Proteins (42 μg/lane) were electrophoresed onto two 12% Tris-HCl gels (lanes 1-8 and 9-16 respectively) and transferred onto nitrocellulose membranes. Membranes were probed with antibodies directed against ERα (a and b), stripped and re-probed with antibodies directed against ERβ (c and d), and stripped a second time and re-probed with antibodies directed against EGFR (e and f). Results shown are those from one experiment.

Figure 8

Proposed Model for the Transformation of Human Breast Epithelial Cells MCF-10A.