| The objective of this dissertation was to investigate the effects of estrogen on breast cancer progression by exposing MCF-7 human breast carcinoma cells to either natural or environmental estrogens in vitro. The first aim, the identification of novel estrogen regulated genes, was achieved with the use of differential display. A 248 bp cDNA fragment, C7, hybridized to a 1.1 Kb transcript, exhibited a 4-fold up-regulation by estrogen, and had sequence similarity to thyroid receptor interacting protein 7 and partial homology to HMG-14/-17. Full-length cDNA clones were then obtained by screening a MCF-7 cDNA library. Several cDNA clones, 800–900 bp in length and encoding a putative protein of 99 amino acids, were obtained. Using the longest full-length cDNA clone, 3B5, ubiquitous expression was found in normal human tissues with increased levels of expression in estrogen-responsive tissues. Two genomic clones were isolated to examine the sequence composition upstream of the gene. Predicted cDNA sequence analyses of the 3B5 gene suggested that its product could bind mononucleosome complexes. A glutathione-S-transferase (GST) fusion protein, GST-3B5, was then constructed, and in vitro binding assays were performed. The results indicated that GST-3B5 associated with mononucleosome complexes. The second aim was to study the effects of four synthetic pyrethroid compounds commonly used as pesticides, sumithrin, fenvalerate, d-trans allethrin, and permethrin, on cellular signaling in vitro. The results showed that sumithrin and fenvalerate were estrogenic as measured by induction of pS2 expression and stimulation of MCF-7 cell proliferation whereas d-trans allethrin was anti-estrogenic. Since the estrogen receptor pathway interacts with other signaling pathways, the effects of pyrethroid compounds on erbB-2 and erbB-3 expression and protein kinase C (PKC) activity were examined. Permethrin decreased erbB-2 mRNA levels, and all four pyrethroid compounds decreased erbB-3 expression by approximately 50% as determined by northern analyses. However, the effects of pyrethroid compounds on PKC activity remain inconclusive. In conclusion, our studies suggest that the 3B5 protein may affect transcriptional regulation by altering chromatin structure similar to HMG-14/-17. Moreover, we demonstrated that the ER, erbB-2 and erbB-3 cell signaling pathways in MCF-7 cells were disrupted upon exposure to pyrethroid. |
