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 [h-3] estradiol binding-sites in mcf-7 human breast-cancer cells - binding interactions with 2,6-bis-([3,4-dihydroxyphenyl]-methylene)cyclohexanone esters and inhibition of cell-proliferation.

Previous studies from this laboratory demonstrated that 2,6-bis-([3,4-dihydroxyphenyl]methylene)cyclohexanone (BDHPC) and related compounds mimic methyl p-hydroxyphenyllactate (MeHPLA) as endogenous ligands for nuclear type II [H-3]estradiol binding sites. Occupancy of type II sites by these agents results in the inhibition of malignant cell proliferation in vitro and mammary tumor growth in vivo. The present studies were designed to assess the effects of BDHPC esterification on type II site binding interactions in uterine nuclei and in cultured MCF-7 human breast cancer cells in vitro. The results of these experiments demonstrate that in rat uterine nuclear fractions BDHPC acetate (Kd approximately 100 nM) interacts with type II sites with a 100-fold lower affinity than BDHPC (Kd approximately 0.9 nM) and BDHPC benzoate failed to inhibit [H-3]estradiol binding under these experimental conditions. Conversely, BDHPC and BDHPC acetate displayed very similar binding affinities for type II sites in cultured MCF-7 human breast cancer cells and there was a direct correlation between nuclear type II site occupancy and the inhibition of cellular proliferation by these two compounds. BDHPC benzoate failed to interact with type II sites or inhibit MCF-7 cell proliferation. Taken together, these results suggested that BDHPC acetate, but not BDHPC benzoate, was being hydrolyzed by esterases in MCF-7 cells, releasing the free parent compound. This conclusion was supported by the observations that incubation of BDHPC acetate in mammary tumor cytosol preparations resulted in essentially quantitative hydrolysis to BDHPC as determined by thin layer chromatography (TLC) and by high performance liquid chromatography (HPLC) analysis of tumor cytosol extracts. Conversely, BDHPC benzoate was not hydrolyzed by tumor esterases which is consistent with the inability of this compound to bind to type II sites or inhibit MCF-7 human breast cancer cell proliferation. These experiments confirm and extend the hypothesis that esterase hydrolysis of MeHPLA related compounds represents an important biological step involved in the control of the biological activity of type II site agonists which appear to regulate malignant cell proliferation through this binding interaction.

Combination therapy with a nuclear type-ii site agonist and 5-Fluorouracil - inhibition of mammary-tumor growth.

Methyl p-hydroxyphenyllactate (MeHPLA) is an endogenous ligand for nuclear type II sites which apparently regulates cellular growth and proliferation through this binding interaction. Occupancy of type II sites by MeHPLA agonists such as dihydroxybenzylidene acetophenone (DHBA), 2,6-bis ([3,4-dihydroxyphenyl]-methylene) cyclohexanone (BDHPC) and 2,6-bis ([3-methoxy-4-hydroxyphenyl]-methylene)cyclohexanone (BMHPC) is directly correlated with the inhibition of malignant cell proliferation. Most importantly, these compounds inhibit mammary tumor growth in vivo with minimal non-specific cytotoxicity. Therefore, combination therapy with MeHPLA agonists plus standard anti-neoplastic agents such as 5-Fluorouracil (FU) may result in tumor growth inhibition with minimal non-specific cytotoxicity. The results of these studies demonstrated that low doses of BMHPC (2 mug/mL) and FU (0.2 mug/mL) failed to significantly affect MCF-7 human breast cancer cell proliferation. However, combination therapy with these sub-inhibitory doses of BMHPC plus FU resulted in significant inhibition of cell proliferation in vitro, suggesting that BMHPC acts in an additive or synergistic fashion with FU to inhibit MCF-7 cell proliferation. Similarly, when orally administered to mice at a dose level of 50 mug/mL drinking water, neither BMHPC or FU alone substantially inhibited the growth of estrogen-independent transplantable mammary tumors. However, combination therapy with BMHPC plus FU antagonized tumor growth and no significant treatment effects were observed on fluid consumption or the body weights of these animals. These results demonstrate that MeHPLA agonists such as BMHPC are capable of acting additively or synergistically with FU to maintain therapeutic response with reduced non-specific systemic toxicity.

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.