Estradiol and tamoxifen interaction at receptor sites at 37 C.

Mature rat uterine cytosol was pretreated with dextran-coated charcoal at 0 C for 2 h. This renders the subsequently formed estradiol-receptor complex thermostable at 37 C and also uncovers antiestrogen binding sites, possibly by removing an endogenous ligand. A sharp distinction is found between tamoxifen and estradiol as receptor ligands in pretreated cytosols in that tamoxifen will inhibit estradiol binding, on incubation at 37 C, only if dithiothreitol (DTT) had been included in the pretreatment solution. The presence of tamoxifen as the sole ligand in cytosol pretreated in the presence of DTT does not protect the estradiol receptor from thermal inactivation and following 37 C incubation tamoxifen is found bound exclusively to antiestrogen binding sites. Incubating the cytosol at 37 C with an equimolar mixture of estradiol and tamoxifen results in a very large increase in receptor-bound estradiol. This effect is attributed to the presence of tamoxifen complexed with antiestrogen sites. Tamoxifen in such equimolar ligand mixture binds to antiestrogen sites and is excluded from receptor sites by the estradiol, whose affinity for these sites is much greater than that of tamoxifen.

Measurement of the estradiol receptor in human breast tissue by the immobilized antibody method.

A simple immobilized antibody procedure for estradiol receptor detection has been applied to samples of human breast tissue cytosols previously assayed by the charcoal-dextran method. Good agreement between the two procedures was realized confirming the validity of the immobilized antibody assay.

The versatile gastrocnemius myocutaneous flap.

The medial and lateral gastrocnemius myocutaneous flaps are described. Their usefulness, as direct flaps without a delay, in reconstruction of the lower extremity is described. In our practice, this flap has supplanted the cross-leg flap for most reconstructions in the lower extremity.

Stabilization of estradiol-receptor complexes by elimination of cytosolic factors.

Treating mature rat uterine cytosol with dextran coated charcoal (DCC) for 2 hrs at 0-4C in the absence of ligand causes the subsequently formed receptor-estradiol complex to be stable at 37C. Receptor binding is increased by the DCC treatment for uteri excised at metestrus or diestrus but remains nearly unchanged for uteri obtained at proestrus or estrus. Results suggest that the DCC removes or inactivates factor(s) present in the cytosol which render the receptor complex thermolabile.

Tamoxifen treatment failures in hormonally responsive breast cancers. Correlation with steroid receptors.

Clinical correlation of treatment response with estrogen (ERP) and progesterone (PgRP) receptors in 81 patients revealed a remission rate of 76% if both receptors were present in the tumor. The response rate was 89% in those patients with positive receptors in whom an endocrine ablative procedure was performed and was 63% in those patients treated with tamoxifen, suggesting that the latter modality may not be effective in the treatment of some hormonally responsive tumors. An assay to determine receptor sensitivity to tamoxifen was developed and correlated with clinical response to tamoxifen treatment. Of the 56 human breast tumors analyzed, 29 tumors were ERP- and PgRP-positive and ten of these were tamoxifen-insensitive. Sixteen tumors were ERP- and PgRP-negative and tamoxifen-insensitive. Of the remaining 11 tumors, four were tamoxifen-sensitive. Nineteen of the 56 patients whose tumors were analyzed were treated for advance breast cancers with tamoxifen. Twelve patients who were ERP- and PgRP-positive and tamoxifen-sensitive responded to this modality of treatment. Seven treatment failures were noted in this group all of whom were ERP-positive and tamoxifen-insensitive. Endocrine ablation failed either prior or subsequently in three of these patients all of whom were PgRP-negative. Two patients who were ERP- and PgRP-positive and tamoxifen-insensitive subsequently responded to an alternative antihormonal treatment.

The formation at 37 C of a nondissociable receptor-estradiol complex.

The receptor-estradiol complex formed in rat uterine cytosol when heated at 37 C converts from a dissociable to a nondissociable form. The conversion is best observed in cytosols pretreated with charcoal at 0 C which renders the subsequently formed receptor-estradiol complexes thermostable at 37 C. In the presence of dithiothreitol the heated complex remains dissociable. Tamoxifen does not form nondissociable complexes with the estradiol receptor. It is proposed that the nondissociable form of the receptor complex is a required phase in the mechanism of estradiol action.

Copper and endogenous mediators of estradiol action.

Divalent copper increases by severalfold specific estradiol binding in rat uterine cytosol at 37 degrees C. Two endogenous substances have now been isolated from the cytosol one of which sharply inhibits the copper effect while the other sharply promotes it. The inhibitor is thermostable, it is adsorbed by dextran coated charcoal and elutes from Sephadex columns with water. The promoter is thermolabile at 60 degrees C, it is not readily adsorbed by the charcoal and elutes from Sephadex columns with KCl. The two substances are thought to be mediators of estradiol action.

Copper and the estradiol receptor.

Copper is an essential element in living organisms and it appears to be involved in estrogen action. This study bears on the manner in which the metal may be linked to the mechanism of this action. Divalent copper was found to induce at 37 degrees C a several fold increase in estradiol binding to the receptors in rat uterine cytosols. An endogenous substance present in the uterine cytosol and separated from it by fractionation on a hydroxylapatite column was found to function as a potent inhibitor of the copper effect. This substance has been found so far also in human breast tissue and in some human breast tumors.

Distinction between malignant and normal breast tissue based on endogenous mediators of estradiol binding.

A component of human breast tumors, isolated by chromatographing the cytosol on hydroxylapatite, increased specific estradiol binding by the cytosolic proteins (23/24) while a similarly isolated component of normal sections of the cancerous breasts inhibited sharply the binding (17/17). Using the same procedures, a component isolated from rat uteri excised at proestrus acted like the one from human breast tumors and a component isolated from uteri at the other phases of the cycle acted like the one from normal breast tissue. The described hormone binding differences are revealed in incubations at 37 degrees C with divalent copper present in the incubation mixture.

Estimation of 5 alpha-dihydrotestosterone receptors in human breast tumors by the immobilized antibody method.

A simple method for estimation of 5 alpha-dihydrotestosterone (DHT) receptors in the cytosol of human breast tumors is described. It is based on the competition of immobilized antibody with the receptors for the labeled hormone. Out of eleven patients with primary breast tumors, four had DHT-receptors, three were negative, and four patients were border line cases.

Breast cancer cells cultured in a system of new design presecrete an extracellular matrix and proliferate within it without cell-cell adhesion.

Breast cancer cells, MCF-7 line, when cultured in microporous, hydrophobic polymer tubes, presecrete ribbon shaped, gauze-like extracellular matrices within which they then reside and proliferate without observable cell-cell adhesion and can do so in the complete absence of serum supplement in the culture media. This cell functioning is attributed to the direct access of oxygen to the cell attachment site.

The relationship between family history, exposure to exogenous hormones, and estrogen receptor protein in breast cancer.

Eight-hundred and thirteen patients were prospectively studied to examine the influence of family history and the prior use of exogenous hormones as covariables in the subsequent expression of estrogen receptor protein (ERP) in the primary tumor of patients with breast cancer. Cases were divided by menstrual status; there were 385 pre- and perimenopausal and 428 postmenopausal patients. The influence of prior exposure to estrogen replacement therapy (ERT) in postmenopausal patients or oral contraceptives (OC) in pre- and perimenopausal patients on tumor ERP was analyzed controlling for family history: none, first degree (1 degree, mother or sister), second degree (2 degrees, grandmother or aunt), or both 1 degree and 2 degrees relatives. The results showed no influence of the prior use of ERT in postmenopausal women on subsequent tumor ERP. Among pre- and perimenopausal women, those with a family history of breast cancer in only a 1 degree relative, showed a borderline significant association between prior OC usage and subsequent tumor ERP. The use of OC was consistently associated with ERP negative tumors (9/9) whereas of 29 patients who had no prior OC exposure 17 had ERP negative tumors (P = 0.04, Fisher's Exact Test). Analysis of the prior exposure to OC, verified with the primary care physician or pharmacist, showed that these patients first used OC at the mean age of 32.2 years, had used OC for a mean duration of 41.9 months and stopped OC use a mean of 79.5 months before being diagnosed as having breast cancer. These results suggest that in a subset of patients with breast cancer, and a first degree relative only who had breast cancer, prior exposure to OC may influence the subsequent ERP status of the tumor. This is not due to exogenous estrogen saturation of receptors as there was a long latent period between exposure and diagnosis. Alternative hypotheses as to the mechanism of selection of subsequent tumor ERP may be either inhibition of ERP positive preneoplastic or tumor cell clones early in the evolution of the tumor or early selection of a tumor capable of endogenous estrogen synthesis with receptor saturation.