IRREVERSIBLE LOSS OF THE ESTROGEN-RECEPTOR IN T47D BREAST-CANCER CELLS FOLLOWING PROLONGED ESTROGEN DEPRIVATION

The development of antioestrogen resistance is a major clinical obstac le encountered in the treatment of breast cancer. By long-term growth in oestrogen-free medium, we have derived an oestrogen-independent, an ti-oestrogen resistant cell line from the oestrogen receptor (ER)-posi tive, oestrogen-dependent T47D human breast cancer cell line. This cel l line grows maximally in oestrogen-free medium and is resistant to al l tested antioestrogens. This cell line does not express any measurabl e amounts of ER mRNA or protein and, in short-term studies, these cell s show no response to either oestrogens or antioestrogens. However, re turn of these cells to oestrogen-containing medium for more than 8 wee ks resulted in the re-expression of ER mRNA and protein. Subsequent li miting dilution subcloning of the T47D:C4 line revealed two phenotypic ally distinct clones, one which did not express measurable ER after lo ng-term growth in oestrogen-containing medium and one which expressed ER mRNA and protein after a number of weeks in oestrogen-containing me dium. In the absence of oestrogen, both types of cells are ER-negative as determined by Northern and Western blotting and lack of any oestro gen-dependent responses. The clone which re-expresses the ER (T47D:C4: 5W) now responds to E(2) with a 50% increase in growth and a 30-fold i nduction of an ER-reponsive luciferase reporter construct. Long-term g rowth of the stably ER-negative clone (T47D:C4:2W) causes no measurabl e oestrogen-mediated responses, as assessed by ER expression, growth s timulation or luciferase induction. Interestingly, ER mRNA can be dete cted in both cell types by using reverse transcriptase-polymerase chai n reaction (RT-PCR). This suggests that the ER mRNA present in the T47 D:C4:2W clone is either inefficiently translated or is present at such a low level as to be functionally irrelevant. These novel clonal cell lines will prove to be invaluable in the study of the regulation of E R expression and regulatory pathways leading to oestrogen-independent growth.