Long-term hydroxytamoxifen treatment of an MCF-7-derived breast cancer cell line irreversibly inhibits the expression of estrogenic genes through chromatin remodeling

Antiestrogen resistance is frequently observed in patients after long-term treatment with tamoxifen, a nonsteroidal antiestrogen widely used for endoc rine therapy of breast cancer. In vitro studies in resistant cells showed t hat the expression of natural estrogen-responsive genes is frequently alter ed. Using MVLN cells, an MCF-7-derived cell model, we previously demonstrat ed that 4-hydroxytamoxifen (OHT) treatment irreversibly inactivated an estr ogen-regulated chimeric luciferase response by a direct effect of the drug and not through a cell selection process (E. Badia et al., Cancer Res., 54: 5860-5866, 1994), In the present study, we present tamoxifen-resistant but still estrogen-dependent clones isolated after long-term treatment of MVLN cells with OHT and show that progesterone receptor (PR) expression was irr eversibly decreased in some of these clones, whereas the PRA:PRB ratio of r esidual PR remained unchanged. The irreversible inactivation of both chimer ic luciferase gene and PR gene expression was associated with the disappear ance of DNase I-hypersensitive sites. In the case of the chimeric gene, at least one of these sites was close to the estrogen responsive element. Geno mic sequencing analysis of a clone with very low PR content did not reveal any methylation on CpG dinucleotides or any mutation in the PR gene promote r region. In all of the resistant clones tested and independently of their PR content, estrogen receptor expression was only lowered by half and remai ned functional, whereas pS2 expression was not modified. We also observed t hat the residual luciferase activity level (1-2%) of the MVLN clones, the l uciferase expression of which had been irreversibly inactivated, was raised cl-fold by trichostatin A treatment. We conclude that long-term OHT treatm ent may modify the chromatin structure and thus could contribute to differe ntially silencing natural target genes.