Breast cancer is a hormonally responsive cancer and hormones, including estrogen, are required for breast cancer growth. The evolution of breast cancer into an estrogen-independent growth phenotype is thought to be an important step in the progression of breast cancer to hormone-independence and endocrine therapy resistance. Understanding the factors that contribute to the development of an estrogen-nonresponsive growth phenotype is of major importance in terms of breast cancer therapeutics. Resistance to endocrine therapies may be due to a number of factors, including loss of estrogen receptor-alpha (ER-) expression, but most tumours that have developed resistance to endocrine therapy remain ER- positive. The mechanisms responsible for the development of estrogen-independence in the presence of continued expression of ER-_ are poorly understood. In order to address this, a breast cancer cell model of apparent estrogen-independence was developed. An estrogen-nonresponsive cell fine, T5-PRF, was developed from T5 (ER-) positive and estrogen-responsive) human breast cancer cells by chronically depleting the cells of estrogen in long-term culture. The T5-PRF cells are insensitive to the growth-stimulatory effects of estrogen while still retaining expression of the (ER-) The tissue matrix consists of linkages and interactions of the nuclear matrix (NM), cytoskeleton and extracellular matrix. This system is a dynamic structural and functional component of the cell that maintains and coordinates cell function and gene expression. The (ER-) is localized to the NM and studies suggest that alterations in NM proteins may influence gene expression. Three proteins (identified as cytokeratins 8, 18 and 19) present in the NM-intermediate filament (NM-IF) fraction were found to be regulated by estrogen in T5 human breast cancer cells. However, T5-PRF, estrogen-nonresponsive cells, overexpressed these three proteins compared to T5 cells, and these proteins were no longer regulated by estrogen in T5-PRF cells. Treating T5 cells with antiestrogens resulted in a significant reduction in these proteins, while no effect was seen in T5-PRF cells, supporting the conclusion that these three NM-IF proteins are regulated by estrogen in T5 human breast cancer cells and may play a role in estrogen action in human breast Comm cells. T5-PRF cells were also found to have significantly increased ligand-independent (ER-) activity. In addition, an ER-_ mRNA variant with an inframe deletion of exons 3 and 4 was detected in T5-PRF, but not T5, human breast cancer cells. Recombinant expression of this (ER-) variant in T5 human breast cancer cells increased estrogen-dependent and -independent reporter gene expression, suggesting that the presence of this (ER-) mRNA variant may contribute to anestrogen-independent growth phenotype. Furthermore, T5-PRF human breast cancer cells contained elevated mitogen-activated protein kinase (MAPK) activity. The MAPK signal transduction pathway can be activated by estrogen in human breast cancer cells and may be involved in the regulation of ER-_ transcriptional activity through both ligand-dependent and independent pathways, suggesting that increased activity of MAPK may contribute to the ligand-independent activity of the ER-_ in T5-PRF cells. In conclusion, T5-PRF human breast cancer cells contain several changes compared to parental T5 cells, which may all contribute to an estrogen-nonresponsive growth phenotype and affect the transcriptional activity/regulation of the (ER-_)