Investigation of hormone-receptor interactions by means of fluorescence labeling.

Fluorescent-labeled hormones can be used to study hormone-receptor interactions by means of fluorescence polarization, visualization by fluorescence microscopy, or separation methods, e.g., dextran-coated charcoal. Subcellular fragments, single cells, and tissue preparations are amenable to study; in this work rat uterine cytosol was used unless otherwise noted. Estrone labeled with fluorescein at position 17 gives 50% inhibition in the radiometric dextran-coated charcoal assay at 8.3 X 10(-7) M as compared to 3.4 and 3.5 X 10(-8) M for diethylstilbestrol and estradiol, respectively. Scatchard plots from fluorescence polarization are hyperbolic and consistent with two classes of binding sites having association constants 5.6 X 10(10) and 6.4 X 10(7) M-1. Binding by high-affinity sites, which were present at about 3 times the concentraion of "specific" sites (radiometric dextran-coated charcoal assay), was abrogated by estradiol or diethylstilbestrol. Kinetic measurements showed that binding sites that can be blocked by excess estradiol or diethylstilbestrol are those that are both slowly associating and slowly dissociating. Staining of tissues by estrone labeled with fluorescein at position 17 as seen in the fluorescence microscope showed specificity. In normal rat uterus only epithelial cells were stained. In one human infiltrating ductal carcinoma only the malignant ductoid elements stained, while in another there was essentially no staining.

Fluorescence polarization measurement of the hormone-binding site interaction.

Fluorescence polarization methodology has been applied to the binding of fluorescent-labeled prolactin, growth hormone and estradiol to subcellular fractions prepared from rabbit mammary and uterine tissue. Equilibrium measurements treated by Scatchard plots have shown that there are high affinity sites (K approximately 10(9) 1 mol-1), as well as lower affinity sites (K approximately 10(8) 1 mol-1) for both hormones. The binding of the fluorescent labeled hormone to microsomal or cytosol fractions has been shown to be inhibited by the prior addition of native, unlabeled hormone. Kinetic results on the interaction of prolactin with the microsomal fraction are consistent with a bimolecular reaction involving significant structural rearrangements during the reaction (not diffusion controlled). The forward rate constant calculated from data on initial rates was found to be 1.7 X 10(5) 1 mol-1 sec-1. Stopped flow kinetic measurements on the reaction between fluorescent-labeled estradiol and cytosol binding sites show that at low temperatures, the reaction goes in two distinct steps separable in time. The second step may be the reaction found by others (utilizing sedimentation velocity methods) which precedes translocation of the hormone-binding site complex to the nucleus. Fluorescence polarization makes it possible to observe both the formation and dissociation of hormone-binding site complexes over a time scale down to a fraction of a second and at concentrations down to the nanogram per nl range.

Intracellular supravital stain delocalization as an assay for antibody-dependent complement-mediated cell damage.

The ability of hamster SV40 tumor cells to concentrate Neutral Red into localized intracytoplasmic vacuoles and granules has been correlated with the ability of those cells to adhere and to replicate in tissue culture. Cells damaged by complement-fixing anti-tumor antibody and metabolic inhibitors first undergo delocalization of the dye, which appears as a diffuse stain throughout the nucleus and cytoplasm. More severely damaged cells lose the stain entirely, at a stage of progressive cell damage correlated with Trypan Blue uptake. A rapid and sensitive cytotoxic assay procedure has been based upon Neutral Red staining behavior, and the assay has been used to study antibody-dependent, complement-mediated cytotoxic anti-SV40 tumor activity in the sera of normal hamsters.

An improved fluorescence probe cytotoxicity assay.

The phenanthridine dye, ethidium bromide, which is actively excluded by viable cells, undergoes a significant fluorescence enhancement at 5900 A upon binding intracellular double-stranded polyribonucleotides. A rapid and sensitive assay of antibody mediated cytotoxicity to cells grown in vitro has been developed using this phenomenon. In this communication, we describe this fluorescence probe cytotoxicity assay and a sensitive electro-optical system designed to measure the fluorescence enhancement of ethidium bromide as it intercalates with intracellular polyribonucleotides. Basic characteristics of the fluorescence enhancement resulting from the interaction of ethidium bromide and non-viable cells are presented as well as examples of this assay as it has been used to study surface membrane neoantigens of cells tranformed by the oncogenic DNA virus, SV40.

In vitro desensitization of sensitized murine lymphocytes by a serum factor (soluble antigen?).

The possibility that lymphocytes sensitized to cell-associated antigens may be rendered hyporeactive toward cells expressing these antigens by prior incubation with such antigens in soluble form was investigated. Mice were sensitized to the foreign histocompatibility antigens of an allogeneic strain of mice; lymphocytes from such sensitized mice were incubated with serum from normal mice of the sensitizing, sensitized, and F(1) hybrids of the two strains. These lymphocytes were washed and their in vitro activity against preparations of cells of sensitizing strain origin was measured by modifications of a standard assay. Sera from the sensitizing strain, and from F(1) hybrids of the sensitizing and sensitized strains (which would be expected to contain the soluble histocompatibility antigens to which the lymphocytes were sensitized), abrogated the in vitro activity of lymphocytes against target cells of the sensitizing strain, while serum from the sensitized strain did not. Soluble tumor-specific or histocompatibility antigens may be responsible in part for the specific abrogation of sensitized lymphocyte activity by serum from tumorbearing, successfully allografted, allogeneically pregnant, or chimeric animals.

Hormone binding by cells and cell fragments as visualized by fluorescence microscopy.

Fluorescent-labeled hormones offer an alternative approach to radio-labeling in studying the binding of hormones to intact cells or cell fragments. The binding of fluorescent-labeled hormones may be followed quantitatively by measurement of the polarization or the binding may be directly visualized in the fluorescence microscope. The binding of both fluorescein labeled prolactin and estradiol to a variety of whole cells or to microsomal fragments has been observed by fluorescence microscopy. No staining was observed with fresh cells whereas all cell types investigated, after freeze-thawing, stained at physiological levels (10-9M) of either hormone. Microsomal preparations from the mammary tissue of mid-pregnant rabbits likewise stained at low levels of prolactin. Inhibition of staining was not produced even by 10-6 M unlabeled hormone.

Isolation of an SV40 induced sarcoma transformation associated antigen.

A transformation associated antigen was isolated from an SV40 induced hamster sarcoma by sequential silica gel column chromatography and preparative silica gel 60 thin layer chromatography after tissue extraction with chloroform:methanol (2:1, v/v). It migrated with an rf of 0.21 on silica gel 60 thin layer chromatography plates predeveloped and developed in chloroform:methanol:water:glacial acetic acid (10:10:1.5:0.5, v/v) and an rf of 0.27 on cellulose F254 thin layer chromatography plates developed in the same solvent system. Antigenicity was determined using a fluorescence probe cytotoxicity assay to measure inhibition of antibody mediated complement dependent damage to homologous cultured transformed cells. Although compositional analysis of this substance is not complete, it appears to be a polar lipid and would support the concept that transformation associated antigens may be gene plus substrate specific rather than strictly gene specific.

Fluorescein-labeled estradiol: a probe for anti-estradiol antibody.

A fluorescent estradiol derivative binds strongly to antiestradiol antibody. The binding, measured by fluorescence polarization, is inhibited by estradiol and by diethylstilbestrol. Tentatively characterized as N-(estradiol-6-iminooxyacetyl) fluorescein amine, the derivative was prepared from estradiol-6-iminooxyacetic acid, dicyclohexylcarbodiimide, and fluorescein amine, and isolated by TLC. It has a fluorescence emission similar to that of fluorescein and an absorption spectrum consistent with a fluorescein: estradiol molar ratio of 1:1.