Immunological probes for oestradiol receptors in human breast tumours

Monoclonal antibodies have been prepared against a soluble oestradiol receptor (REC) preparation partially purified from human myometrium by oestradiol affinity chromatography. The antibodies were detected by their ability to immunoprecipitate receptor bound [125I] oestradiol. One of the antibodies (D5) has been studied in detail. It will only precipitate REC after activation by salt, heat, low pH or KCNS and will not react with nuclear RE. It will not react with androgen, progesterone or glucocorticoid receptors nor with sex hormone binding globulin; it will only combine with REC from human sources. D5 recognizes a cytoplasmic 29 kdalton protein (p29) that can be separated from both type I and II soluble oestradiol binding proteins. p29 can react with activated REC and is qualitatively and quantitatively related to REC. IRMA and histochemical methods have been developed for quantitating p29 and relating its amount to receptors in human breast tumours. With both methods, highly significant (P less than 0.001) correlations with REC but not RP have been obtained. Both methods indicate that many REC-RP+ tumours contain p29. The histochemical method detects marked cellular heterogeneity in some tumours. The function of p29 is not known. It is an REC-related antigen that may be a previously undetected component of the oestradiol receptor machinery.     

Influence of anti-oestrogens on the specific binding in vitro of (3H)oestradiol by cytosol of rat mammary tumours and human breast carcinomata.

The anti-oestrogenic potential of two nitrogen-mustard-containing compounds, I.C.I. 79792 and I.C.I. 85966, was studied. I.C.I. 85966 usually did not decrease specific binding of [3H]oestradiol by breast-tumour cytoplasmic proteins. I.C.I. 79792 decreased specific [3H]oestradiol binding, but not to the same extent as similar concentrations of I.C.I. 46474, diethylstilboestrol or dibutyldihydrostilboestrol.     

Adrenal androgen concentrations in breast tumours and in normal breast tissue. The relationship to oestradiol metabolism

Concentrations of ADIOL, DHA and DHAS were measured in human breast tumours and normal tissue from the same breast and related to 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) activity in these tissues. ADIOL and DHA were significantly higher in tumour tissue compared to normal tissue from the same breast (paired t-test: P less than 0.05 and P less than 0.01 respectively) whereas the difference between concentrations of DHAS in normal tissue and tumour tissue was not significant. There was a positive correlation between ADIOL and DHA in both tissues (P less than 0.001) but for DHAS the relationship was only significant in normal tissue (ADIOL:DHAS, P less than 0.001; DHA:DHAS, P less than 0.002). An increase in 17 beta-HSD activity was associated with an increase in DHAS concentrations in both normal and tumour tissue (P less than 0.01 and P less than 0.001 respectively) and with an increase in DHA concentrations in normal tissue (P less than 0.05). These results might be explained by an impairment in the balance between sulphatase and sulphotransferase activity in breast tumours.     

Tissue culture of human breast tumours.

Several media have been used in an attempt to assess the most appropriate combination of ingredients to effect good cell growth from human breast tumours. Good epithelial cell growth has been obtained in 60 out of a group of 160 tumours successfully cultured in four types of basic media. The other tumours were fibroblastic in nature or contained a mixture of epithelial and fibroblastic cells.     

Enzyme activities in human breast tumours.

The activities of six enzymes associated with carbohydrate metabolism were measured both in carcinomas and in normal breast tissues. The following differences were observed. 1. The carcinoma showed higher enzyme activities than the normal mammary tissue. 2. The ratios of glutamate dehydrogenase, hydroxybutyrate dehydrogenase, glutathione reductase and catalase to lactate dehydrogenase were lower in carcinomas than in normal tissues. Similarly, the ratios of glutamate dehydrogenase, hydroxybutyrate dehydrogenase, glutathione reductase and catalase to glucose-6-phosphate dehydrogenase were also significantly lower in carcinomas. 3. There were no significant differences in enzyme activities between I and II stage of the disease and the metastatic tissues, however, there were significant differences between I and III stage. The significance of these findings is discussed in terms of the alterations in the balance between the metabolic pathways.     

Inhibitory effect of thiols on the degradation of albumin by human spleen cathepsin D

The degradation of native albumin by human spleen cathepsin D was inhibited by GSH, cysteine and cysteamine. The thiols existing physiologically also inhibited reduced-carboxymethylated albumin, indicating that these thiols react preferentially with the enzyme itself rather than the substrate. The inhibitions of native albumin proteolysis were dose-dependent. These effects of thiols which have not been observed in other animal cathepsin D, suggest an essential function for cathepsin D in the human spleen.     

Intermediate Mr cytosolic components potentiate hepatic 5''-deiodinase activation by thiols.

The role in the activation of microsomal 5'-deiodinase (5'-DI) of rat hepatic cytosolic components of Mr approx. 13,000 (Fraction B) was studied in the presence of various concentrations of thiol compounds such as dithiothreitol (DTT), dihydrolipoamide (DHLA), GSH, and 2-mercaptoethanol (2-ME). Although Fraction B (which was prepared by gel filtration to exclude GSH and GSSG) had no intrinsic 5'-DI activity, could not stimulate microsomal 5'-DI activity in the absence of added thiol and did not contain GSH as a mixed disulphide, it could produce a 3-fold increase in the maximal deiodinase activity achievable with DTT as well as other thiols, with the order being the same as the activation potency of these thiols in the absence of Fraction B (i.e. DHLA greater than DTT greater than 2-ME greater than GSH). These observations suggest that: a component of cytosolic Fraction B, designated 'deiodination factor B' (DFB), operates as an efficient intermediary to enhance activation of microsomal 5'-DI by thiols through a mechanism independent of GSH; thiols may participate in a non-specific thiol-disulphide exchange with inactive (oxidized) DFB to convert it into an active form that contains one or more thiol groups and is more effective than GSH or other thiols in facilitating the re-activation of inactive (oxidized) microsomal 5'-DI thiol (ESI) to its active state (ESH).     

The lectin binding sites on the plasma membrane components of human lymphoblastoid cell lines.

The plasma membrane components of five human B-cell lines and three human T-cell lines were separated by dodecyl sulfate polyacrylamide gel electrophoresis, incubated with the radioactive labeled lectins from lentil, castor bean, wheat germ, Phaseolus bean, peanut, gorse and the Roman snail and the molecular weights of the binding sites determined. The lentil, castor bean and wheat germ lectin bound to multiple components from molecular weights (Mr) 20 000 to 200 000 within the plasma membranes, whereas peanut lectin bound preferentially to glycoproteins of Mr 150 000 and 83 000 in B-cells, and 150 000 and 130 000 in T-cells. The gorse lectin bound to a 220 000 component in B-cells which was not labeled in T-cells.