James L. Wittliff

Specific estrogen-receptors in the neoplastic and lactating mammary gland of the rat

Abstract Specific receptors of 3 H-estradiol-17β were demonstrated by a sucrose gradient assay of cytosols from the lactating mammary gland and the R3230AC mammary adenocarcinoma of the rat. These estrogen receptors, each of which sedimented at approximately 8S, exhibited similar dissociation constants (K d ∼1×10 −9 M) and steroid binding specificities. Ovariectomy of the host had no apparent effect on the number of binding sites/mg protein in the R3230AC tumor.

Correlations between certain biochemical properties of breast cancer and response to therapy: A preliminary report

Biochemical analyses were carried out on tissues from 130 women with mammary carcinoma and 104 patients with benign breast lesions. The tissues were assayed for specific estrogen‐binding proteins (EBP) and for several enzymes involved in carbohydrate and lipid metabolism. Normal breast, fibrocystic disease, and fibroadenomas usually exhibited lower enzyme levels than malignant tissues, and rarely contained specific EBP. Intraductal carcinoma appears to contain certain biochemical characteristics unique to this tissue when compared with benign proliferative breast diseases; these differences may be of value in prediction of response to various types of therapy.

Characterization of a distinct glucocorticoid-binding protein in the lactating mammary gland of the rat.

Abstract Specific substances binding [3H]triamcinolane acetonide were detected in the cytosol fraction of the lactating mammary gland of the rat using sucrose gradient centrifugation. These receptors, which were protein in nature, exhibited sedimentation coefficients of 7–8 S and dissociated into lower molecular weight components sedimenting at 4–5 S when separated on sucrose gradients containing 0.4 M KCl. The cytoplasmic form of the binding protein was relatively specific for glucocorticoids although progesterone inhibited binding significantly. The dissociation constant (Kd) of the receptor-ligand complex was in the range of 10−8 M. p-Chloromercuribenzoate diminished the ligand-binding capacity of the receptor suggesting a role for sulfhydryl groups in the binding reaction. Cytosols from mammary tissue obtained from virgin and pregnant rats revealed a paucity of binding sites as compared to those in the lactating gland. Examination of ligand-binding specificity indicates that these glucocorticoid-binding sites are distinct and easily discriminated from those of either the estrogen receptor of the mammary gland or the triamcinolone-binding component in plasma.

Nuclear interaction of Fusarium mycotoxins with estradiol binding sites in the mouse uterus

By using cell-free preparations of uteri obtained from immature BALB/c mice, it was demonstrated that zearalenone and zearalanol, Fusarium mycotoxins, inhibited [3H]estradiol-17 beta binding to specific sites in cytosol. Significant inhibition was noted from zearalenone at 4 x 10(-6) M and from zearalanol at 4 x 10(-7) M. Unlabeled mycotoxins (5 x 10(-6) M) incubated with intact uteri caused translocation of specific estrogen binding sites into nuclei that were exchangeable with [3H]estradiol-17 beta. Zearalanol was more effective in this regard than zearalenone. Ability of the mycotoxins to compete with estradiol-17 beta for the cytosol receptor and to cause translocation of the receptor to the nucleus in general is correlated with their biological activity. These data suggest that the uterotrophic effects of Fusarium mycotoxins are mediated through their association with estrogen receptors in the uterus.

Further studies of biochemical predictive tests in breast cancer.

This report extends our observations on the relationship between the therapy of breast cancer, estrogen binding and tissue enzymes. We have compared the dextran‐coated charcoal method and the sucrose gradient analysis in determining estrogen receptors, and find a sub‐group of receptor positive tumors—identifiable only by the latter technique—which we suggest may lead to an incorrect prediction of response to hormonal therapy. Data are also presented to suggest that enzyme analyses show promise in predicting responsiveness to present modes of combination chemotherapy of breast cancer.

Determination of radioactivity in polyacrylamide gel slices mounted on solid supports.

A simple, economical method for counting acrylamide gel slices on solid filter paper supports in a toluene-based scintillation cocktail is described. Major advantages of the system include no requirement for either dissolution of the gel or elution of the radioactive material prior to emulsion counting and the direct reutilization of scintillation cocktail and vials. Additionally, 32P-labeled RNA samples can be counted with better relative efficiencies and those labeled with 14C or 33P can be determined at equivalent efficiencies. Tritium was detected less readily, with an absolute efficiency of approximately 10%.

Measurements of DNA and RNA in mammary gland homogenates by the ethidium bromide technique

Abstract A rapid method of determining simultaneously DNA and RNA in mammary gland homogenates using the ethidium bromide technique is discussed. The method utilizes a quantitative extraction of DNA and RNA with 2.0 m sodium chloride, SDS, and EDTA at pH 8.0. Assays of mammary gland RNA and DNA using previously published methods were compared with determinations using the ethidium bromide technique. While the fluorescence method gave lower values for RNA when compared to those obtained using the orcinol or absorbancy ratio (OD 260nm/280nm), DNA measurements agreed well with the values determined by the diphenylamine technique. Extinction coefficient data for total mammary gland RNA isolated using a modified phenol extraction procedure are also presented.

Estrogen Specificity of the Induction of Lipovitellin Synthesis and Evidence for a Specific Estrogen-Binding Component

Using male South African clawed toads, Xenopus laevis, administration of the common estrogens as well as estradiol-17α brought about the synthesis of both total soluble protein and the specific yolk protein, lipovitellin, in liver. This elevation in synthesis was both dose-responsive and estrogen specific; neither hydrocortisone, progesterone, nor testosterone promoted synthesis. The Parke-Davis compound, CN-55, 945–27, a known inhibitor of the binding of estrogen to specific receptor sites in mammalian tissues, inhibited the synthesis of lipovitellin indicating a possible requirement for binding protein in the Xenopus system. In cell-free preparations of liver, 3H-estradiol-17β was bound to a macromolecule fraction which was excluded by columns of Sephadex G-100 or G-200 in buffers of low ionic strength and sedimented at 11–12 S on linear sucrose gradients.

Quantitation of radio-ligand binding data using a desk-top calculator in the program mode☆☆☆

Abstract Quantitation of specific estrogen-binding capacity was facilitated using an inexpensive procedure for the rapid processing of data from radioligand binding measurements from sucrose gradient analyses. Ligand-binding data from each fraction of a gradient were punched into a paper tape in ASCII code and, later, read by a tape reader into an Olivetti Programma 101. Using the total instructional capacity (120 steps) a program was written in symbolic language which describes: (a) the calculation of counting efficiency of the instrument for each fraction, (b) the conversion of counts/minute to disintegrations/minute, (c) the computation of the concentration of [ 3 H]estradiol-17β in each fraction and (d) the summation of the quantity of [ 3 H]estradiol-17β bound by specified regions of the gradient. The program can easily be adapted for use with data generated from separation procedures such as centrifugation, electrophoresis or chromatography in which large number of samples are analysed.

Relationship of glycolytic enzyme activities and response of breast cancer patients to chemotherapy. A preliminary report

Activities of glucosephosphate isomerase, lactate dehydrogenase, and NADP‐isocitrate dehydrogenase were significantly elevated in breast cancer specimens from patients who responded favorably to combination cytotoxic chemotherapy regimens compared with those in carcinomas from patients failing to respond to the same chemotherapy. Presence of estrogen receptors and clinical response to hormonal therapy were also evaluated in neoplasms from these patients. The data suggest that measurement of the enzyme profile, along with estrogen receptor levels, may be useful in selecting a mode of therapy for patients with advanced disease.