An improved method for the quantification and recovery of rat uterine nuclear type II [3H]estradiol binding sites immobilized on a glass fiber matrix.

An improved assay for measuring ligand binding to extracted nuclear type II estrogen binding sites which involves preimmobilization on glass fiber filters is described. At least two classes of specific estrogen binding sites have been demonstrated in rat uterus as well as in a variety of other tissues and species and have been designated as type I and type II. Although the endogenous ligand to the type II binding site has recently been identified as methyl p-hydroxyphenyllactate (MeHPLA), tritiated estrogens are generally used for radiolabeling this site due to the susceptibility of MeHPLA to enzymatic hydrolysis in in vitro assays. After extracting the type II site from the nuclear matrix, ligand binding and protein stability appear to be significantly enhanced by first immobilizing the site on an artificial matrix, such as hydroxylapatite, before incubating with radiolabeled ligand. Immobilization of the extracted site on glass fiber filters results in higher specific binding and lower nonspecific binding when compared to hydroxylapatite and a number of other immobilization matrices. The glass fiber ligand exchange procedure for measuring type II binding can also be performed on smaller samples and requires less time than other methods. Type II sites are significantly stabilized when immobilized on glass and exhibit sigmoidal binding curves when incubated with increasing concentrations of [3H]estradiol and [3H]estrone and display inhibition data characteristic of that observed using more traditional assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatographic resolution of the type II estrogen binding site and a tyrosinase-like enzymatic activity from rat uterine nuclei.

Nuclear extracts from estradiol-treated rat uteri which contain type II estrogen binding sites have recently been found to also contain a tyrosinase-like estradiol metabolizing activity. A recent study suggested that both the binding and enzymatic activities are significantly increased in the presence of micromolar concentrations of copper and ascorbate, display a number of common biochemical sensitivities, and share similar ligand/substrate binding affinities. Levels of both activities are significantly increased in uterus in response to hormone (estrogen) stimulation. These and other similarities indicate a possible relationship between the enzymatic and binding activities. A detailed chromatographic examination of these two activities in the present study revealed that while the type II sites and estradiol metabolizing activity exhibited virtually identical chromatographic properties on DEAE-high-performance liquid chromatography they are readily resolved on other chromatographic matrices, including phosphocellulose, DNA-cellulose, and S-Sepharose. These results demonstrate that type II binding sites are distinct from the tyrosinase-like enzyme activity previously described in rat uterine nuclear extracts.

Nuclear type II sites and malignant cell proliferation: inhibition by 2,6-bis-benzylidenecyclohexanones.

Methyl-p-hydroxyphenyllactate (MeHPLA) is a bioflavonoid and/or tyrosine metabolite which may regulate cellular growth and proliferation through interactions with nuclear type II sites. Our current studies suggest that type II sites may function as MeHPLA receptors which are localized on the nuclear matrix, and occupancy of this binding site by MeHPLA directly correlates with the inhibition of normal and malignant cell proliferation. This ligand is inactivated by MeHPLA esterase in mammary tumors, resulting in a deficiency in MeHPLA, high quantities of unoccupied type II sites, and uncontrolled cellular proliferation. For these reasons we synthesized 2,6-bis((3,4-dihydroxyphenyl)methylene)-cyclohexanone (BDHPC) and 2,6-bis((3-methoxy-4-hydroxyphenyl)-methylene)cyclohexanone (BMHPC) for assessment as nuclear type II site and cell growth antagonists. These two esterase stable cyclohexanone derivatives, which bind to nuclear type II sites with high affinity (Kd 1-7 nM), mimic MeHPLA as cell growth-regulating agents. Dose-dependent occupancy of type II sites in MCF-7 human cells by BDHPC and BMHPC directly correlated with the inhibition of cell proliferation, and administration of BDHPC by silastic implant inhibited mouse mammary tumor growth in vivo. These findings demonstrate that esterase-stable type II antagonists such as BDHPC and BMHPC inhibit mammary cancer cell proliferation in vitro and in vivo and support earlier studies demonstrating that MeHPLA and functionally related compounds may regulate malignant cell proliferation at the level of this binding site.