Oestrogen receptor gene structure and function in breast cancer

The mechanisms underlying loss of oestrogen responsiveness in breast cancer are not well-defined. Potential mechanisms include loss of receptor expression, alterations in the oestrogen receptor (ER) gene producing proteins with abnormal function, or changes to receptor-dependent or -independent pathways controlling cell proliferation. Examination by Southern analysis of the ER gene in a series of ER-negative and -positive breast tumour biopsies failed to provide evidence of gross rearrangements and in only only one of thirty seven tumour DNA samples was significant gene amplification observed. No restriction fragment length polymorphisms were detected for the restriction enzymes EcoRI, Pst I or Hind III. Methylation of the ER gene as assessed by Hpa II and Msp I restriction enzyme digests varied between tumours but the degree of methylation was not correlated with levels of expression of the receptor protein. Similar findings applied in a series of ER-negative and -positive breast cancer cell lines and clonal lines of MCF-7 cells, which were developed as an in vitro model for the acquisition of oestrogen and antioestrogen resistance. In this model there was no evidence that changes to ER receptor function and/or structure at the level of the ER gene, mRNA, ligand binding, and ability to induce progesterone receptor might account for the development of hormone resistance. However, the ability of ER to interact with a DNA sequence containing the vitellogenin promoter oestrogen response element, as assessed by gel retardation assay, was impaired in the clone showing the greatest degree of oestrogen and antioestrogen resistance.     

Oestrogen receptor (ESR) polymorphisms and breast cancer susceptibility

The allele frequencies of three restriction fragment length polymorphisms at the oestrogen receptor (ESR) locus were compared between breast cancer patients and controls. Leucocyte or tumour DMA from 238 and 122 patients, respectively, and leucocyte DNA from 672 controls was analysed. Alleles having the XbaI restriction site detected by the M72 probe (covering exon 2 and flanking introns) were significantly more frequent in patients than in controls (P = 0.033). Within the breast cancer population, the presence of the XbaI restriction site was associated with late onset of the disease but this association was only of borderline significance. The allele frequencies of the BstUI polymorphism in exon 1 and the PvuII polymorphism in intron 1 did not differ between cases and controls. However, alleles with the PvuII restriction site were more frequent in patients with progesterone receptor negative primary tumours than in patients with progesterone receptor positive primary tumours (P = 0.027). There was no significant association between any of the ESR polymorphisms and the oestrogen receptor status of the primary tumours. The results indicate that the ESR gene or a gene closely linked to it is involved in the development of at least a subset of breast carcinomas.     

Oestrogen receptors in chick oviduct. Characterization and subcellular distribution.

Macromolecular components with properties of oestrogen receptors have been identified in the 0.5 M KCl nuclear soluble, the nuclear insoluble and the cytosol fractions of laying hen and immature (2--4 weeks, untreated by hormone) chicken oviduct. 7n the 0.5 M KCl extract of laying hen oviduct nuclei, a receptor, of protein nature according to the effects of enzymic treatments, has been identified. It exhibits high affinity for oestradiol with an apparent equilibrium association constant KA = 4 - 109 M-1 at 4 degrees C. The binding of [3H] oestradiol is abolished by 1 muM oestriol, oestrone and diethylstilboestrol, but not by the same concentration of progesterone, testosterone, and cortisol. Sucrose gradient ultracentrifugation studies in the presence of 0.5 M KCl indicate a sedimentation coefficient of 4.3 S, and there is partial aggregation in low-ionic-strength medium. The estimated number of binding sites per nucleus is about 5000, as calculated from DNA content of chick diploid genome. Most of the binding sites were found to be occupied by endogenous oestrogen(s). Oestradiol dissociates from the receptor according to an apparent two-step mechanism. The half-life time for the faster dissociation step is 18 h at 0 degrees C, 25 min at 20 degrees C and 10 min at 30 degrees C, and for the slower one is 180 h, 115 min and 60 min, respectively. In the 0.5 M KCl extract of immature chicken oviduct nuclei, there are approximately 500 receptor sites per nucleus; their affinity for oestradiol is the same as in the case of laying hen soluble nuclear receptor. After repeated extractions of nuclei with 0.5 M KCl medium, a substantial quantity of oestrogen binding sites remains in the residual fraction. Binding characteristics of this insoluble nuclear receptor resemble those of the soluble nuclear receptor: high affinity for oestradiol (KA = 7 - 10(8) M-1 at 37 degrees C) and specificity for oestrogens. The estimated number of binding sites are approximately 2000/cell for laying hen, and approximately 1000/cell for immature chicken. In the high-speed supernatant fraction of laying hen oviduct homogenates, an oestrogen receptor is also present, but its concentration is low (less than or equal to 100 sites/cell) and at the limits of sensitivity of the methods used. In the cytosol of immature chicken oviduct, there are approximately 2500 oestradiol receptor sites per cell.     

Treatment and survival on advanced breast cancer.

The final results of a clinical trial comparing endocrine with cytotoxic drug treatment for advanced breast cancer were analysed. Although cytotoxic treatment gave a significantly higher response rate with a remission duration comparable to that obtained with endocrine treatment, the sequence in which the two treatments were given did not appear to influence survival--except possibly in women with rapidly progressing disease, when cytotoxic treatment is preferred.     

Specific progesterone receptors in human breast cancer.

We have identified a specific progesterone receptor in 11 of 33 human breast cancer cytosols. Since progesterone itself binds to glucocorticoid receptor, to corticosteroid binding globulin (CBG), and to nonspecific components as well as to its own receptor, we have used a synthetic progestin, R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), whose binding specificity is restricted to progesterone receptor. Bound R5020 sediments at 8 S in sucrose gradients; binding is competed by excess unlabeled R5020 or progesterone. The receptor is distinct from glucocorticoid receptor and CBG as determined by competition studies using dexamethasone and hydrocortisone. The dissociation constant for R5020 obtained by Scatchard analysis of dextran-coated charcoal assays is approximately 2 times 10- minus 9 M.     

Oestrogen regulates cathepsin D mRNA levels in oestrogen responsive human breast cancer cells.

A cDNA library was constructed from the mRNA of the ZR-75 oestrogen responsive human breast cancer cell line and screened for oestrogen regulated mRNA sequences. Of the recombinants isolated, 30 contained cDNA that corresponded to a single mRNA species of 2.1 kb that was induced between 10 and 15 fold by oestradiol treatment. The sequence of the entire open reading frame and 3' non-coding region of the mRNA was determined and shown to encode the aspartyl protease cathepsin D. The induction of cathepsin D mRNA is specific for oestrogen and is maximal at 3 X 10(-10) M. Cathepsin D mRNA levels were increased by oestrogen in 3 oestrogen responsive breast cancer cell lines. Cathepsin D mRNA was expressed but not regulated in an oestrogen receptor negative breast tumour cell line, BT 20, and in 2 other malignant cell lines, Hela and A431.     

Delay in diagnosis and long-term survival in breast cancer.

The records of all 1591 women with a histologically confirmed primary breast neoplasm who received their primary treatment at the main referral centre in British Columbia and were diagnosed in the years 1945, 1950, 1955, 1960, 1965, 1970, or 1975 were reviewed. The interval from appearance of the first symptom to diagnosis decreased from 1945 to 1960, but no change was seen from 1960 to 1975. An analysis of survival from the date of first symptom showed that long-term survival was greater in patients with a shorter delay between the appearance of symptoms and diagnosis. The demonstration that shorter delay does improve survival, even when assessed from the appearance of the first symptom, yet delay times have not been falling recently, suggests that educational efforts are inefficient.