Characterisation of Mammalian Cell Lines for use in Proliferative and Estrogenic Response Assays

Author: Daniel Inglis

Inglis, Daniel, 2011 Characterisation of Mammalian Cell Lines for use in Proliferative and Estrogenic Response Assays, Flinders University, School of Medicine

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Abstract

A significant number of endocrine disrupting chemicals (EDCs), which have the ability to act as estrogens or disrupt the estrogen signalling pathway in humans and other species, have been reported in environmental waters. Bioassays using human cell lines, which contain endogenous or inserted estrogen receptors, are useful to rapidly assess this (xeno)estrogen content of water. This thesis presents a comparative assessment of the suitability of MCF7, ZR-75-1, H23, RL95-2, Ovcar and T-47D cell lines for (xeno)estrogen testing in a 96 well plate format. Cell lines were assessed for attachment time, proliferation rate, optimal time for producing a standard curve relating absorbance to cell number and the expression of ERα and ERβ mRNA and protein. Six hours was the optimum time for attachment and gave the smallest 95% confidence interval (CI) for a standard curve for MCF7, Ovcar, RL95-2 and T-47D cells. ZR-75-1 and H23 cells were best after 4 hours of attachment. Incubation for 24 hours gave the highest 95% CI and produced the least accurate standard curve. The reporting of a single doubling time for each cell line may not be accurate as cell lines had a doubling time that was density related. ERα expression was greatest in MCF7, followed by T-47D and ZR-75-1. ERβ was not detected in any cell line. MCF7 cells are characterised as proliferating in response to (xeno)estrogens primarily via the ERα. The E-screen was originally developed using MCF7 cells, however, many modifications have lead to only semi-standardisation of the methodology. T-47D and ZR-75-1 cells have also been shown to proliferate in response to estrogen and were compared to MCF7 cells in an E-screen assay. The responses of cells using a 4hr or 6hr or an overnight attachment period in estrogen-free pre-treatment before exposure to estrogen for 3, 5 or 7 days were compared. The pre-treatment/attachment period did not affect EC50 values, however the proliferation of MCF7 cells almost doubled and the 95% CI was greatly reduced by reducing the attachment/ pre-treatment time to 6 hours. MCF7 cells are used in the E-screen within a given passage range. The changes in sensitivity and response to estrogen of the three cell lines after extended time in culture (>18 passages) were also assessed. Although each cell line maintained similar EC50 values, the proliferative response decreased and variability increased. The regulation of individual genes by estrogen has been widely studied. VEGF, IGFBP-4, pS2, MaoA and EGR1 were selected from microarray and in vivo data based on the magnitude and speed of response to estrogen exposure. The promoter regions, inclusive of their estrogen responsive elements, were identified. pS2 and MaoA promoter regions were successfully amplified and inserted into the pGL4.21(luc2P/Puro) reporter vector. Stable integration into T-47D cells resulted in non-estrogen inducible expression of the luciferase gene. Transient transfection of the vector into MCF7, ZR-75-1 and T-47D cells also resulted in expression of luciferase that could not be induced by estrogen.

Keywords: xenoestrogen,estrogen receptor,E-screen,proliferation
Subject: Medical Biotechnology thesis

Thesis type: Doctor of Philosophy
Completed: 2011
School: School of Medicine
Supervisor: Fiona Young