Spelsberg, T. C.

Abstract A micro version of a nuclear binding assay to assess the biological activity of receptors for steroid hormones was developed for application to small (needle) biopsies of human tumors for the purpose of predicting responses to steroid therapy. This easier assay requires 10-fold less tissue than the original nuclear binding assay described for progesterone receptors in the avian oviduct, endometrium, and endometrial carcinomas (Spelsberg TC, et al., Endocrinology 1987;121:631). We describe the application of this micro assay to normal avian oviduct and cancers of the human breast, and we demonstrate a tissue specificity and saturation of nuclear binding. The micro assay reliably measured as little as 0.5 mg equivalents of tissue per assay tube. Results for breast tumors determined to be estrogen-receptor-positive by the standard dextran-coated charcoal method were also determined with this nuclear binding assay. As described previously for progesterone receptors in endometrial carcinomas, some receptor-positive breast biopsies displayed negligible capacity for nuclear binding. Therefore, with the present assay we have identified nonfunctional receptors in these biopsies, which may be useful for accurate prediction of patients' responses to therapy with hormones.

1. Varied doses of labelled or unlabelled progesterone were injected into immature chicks which had previously been stimulated with oestrogen. The concentrations of nuclear bound [3H]progesterone were correlated with the effects of the hormone on endogenous RNA polymerase I and II activities in isolated oviduct nuclei. 2. The extent of nuclear localization of [3H]progesterone in oviduct (a progesterone target tissue) was shown to be much greater than in lung (non-target tissue). The conccentration of bivalent cations in solvents used in the nuclei isolations has a marked effect on the amount of bound hormone in the nuclei. 3. Evidence for the existence of several classes of binding sites for progesterone in the oviduct nuclei is given. These classes represent about 1000) 10000 and 100000 molecules of the hormone per cell nucleus and are saturated by injecting approx. 10, 100 and 1000 mug of progesterone respectively. 4. When saturation of the first (highest affinity) class of nuclear sites occurs, a marked inhibition in RNA polymerase II (but not RNA polymerase I) activity was observed. When the second class of sites was saturated, alterations in both RNA polymerase I and II activities were observed. Binding to the third class of nuclear binding sites was not accompained by further changes in polymerase activity. It is suggested that the first two classes of nuclear binding sites may represent functional sites for progesterone action in the chick oviduct.

Abstract Background: We have previously demonstrated that endoxifen is the most potent tamoxifen metabolite responsible for eliciting the anti-estrogenic effects of this drug in breast cancer cells expressing ERα (Wu, Hawse et al. Cancer Res 2009;69:1722). These studies support the clinical findings which demonstrate that women with genetically impaired CYP2D6 metabolism have significantly reduced endoxifen levels and a higher risk of breast cancer recurrence. More recently, data has emerged from several laboratories suggesting that expression of ERβ in breast tumors leads to increased benefits from tamoxifen therapy. However, the role of ERβ in mediating endoxifen action has yet to be explored.Methods: Using U2OS osteosarcoma cells, as well as Hs578T and MCF7 breast cancer cells, stably expressing ERβ, we examined the effects of endoxifen on ERβ protein levels by western blotting, ERβ transcriptional activity by real-time RT-PCR, and on ERβ inhibition of breast cancer cell growth through the use of proliferation assays.Results: We have discovered that endoxifen stabilizes ERβ protein, unlike its targeted degradation of ERα, and induces ERα/ERβ heterodimerization in a concentration dependent manner. We have also found that endoxifen is a more potent inhibitor of estrogen induced gene expression in MCF7 cells stably expressing ERβ. Concentrations of endoxifen that mimic patients with impaired CYP2D6 activity (20-40 nM) do not significantly alter E2-induced cell proliferation in MCF7 cells expressing only ERα. However, these concentrations of endoxifen significantly reduce the estrogen induced proliferation rate of MCF7 cells expressing ERβ.Conclusions: Our data demonstrate that endoxifen stabilizes ERβ protein, induces ER heterodimerization and more potently inhibits the actions of estrogen in breast cancer cells expressing ERβ. The presence of ERβ sensitizes cells to the anti-estrogenic effects of endoxifen and growth inhibition occurs at substantially lower endoxifen concentrations (20-40 nM) than in cells that express only ERα (100 nm). Our data suggest that tamoxifen therapy may be beneficial even in patients with reduced CYP2D6 metabolism when ERβ is expressed. These studies also suggest that the increased effectiveness of tamoxifen therapy in patients whose tumors express ERβ may be due to the effects of endoxifen. Finally, these findings underscore the need to further elucidate the role of ERβ in the biology and treatment of breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 405.

Data are presented to explain discrepancies in the literature involving the in vitro binding of steroid receptor complexes to isolated nuclei and chromatin. The type of binding in vitro of the progesterone-receptor complex to nuclei, chromatin, or DNA of hen organs is largely determined by the ionic strength of the medium. Low ionic conditions (0.01 to 0.05 molar potassium chloride) result in a nonspecific, nonsaturable binding, while high ionic conditions (0.15 to 0.20 molar potassium chloride) create a tissue-specific, saturable binding. Pure DNA binds the steroid receptor complex extensively in low salt but very little in the higher salt conditions.

This report describes a rapid regulation of the expression of the c-jun protooncogene by the antiestrogen tamoxifen (Tam). The c-jun protooncogene codes for an important component of the AP-1 transcription factor complex, which regulates the expression of many unlinked genes. Repeated experiments have shown that Tam rapidly increases the steady-state c-jun mRNA levels in the avian oviduct but decreases the levels in the liver. The Tam effects are time- and dose-dependent. These results are supported by other studies that have demonstrated that 17 beta-estradiol decreases steady-state levels of c-jun protooncogene mRNA in oviducts of animals fully withdrawn from estradiol. The effect of Tam in the avian oviduct is in contrast to the reported effects of Tam on the expression of practically all other genes in the avian oviduct and other animal tissues. Transcription analyses using nuclear runoff experiments with oviduct nuclei demonstrate a decrease in the c-jun gene transcription within minutes after Tam treatment with a return to 75% of control values by 4 hr. The fact that Tam transiently decreases the transcription of the c-jun gene but increases the steady-state c-jun mRNA levels suggests that Tam must alter both transcriptional and post-transcriptional events. The results support a role of the c-jun protooncogene as a regulatory gene in the cascade model for steroid action whereby steroids rapidly regulate the regulatory genes, which in turn regulate many other structural genes.