Purification and characterization of nuclear type II [(3)H]estradiol binding sites from the rat uterus: covalent labeling with [(3)H]luteolin.

Type II [3H]estradiol binding sites play an important role in normal and malignant cell growth and proliferation and the delineation of the precise function of the type II site in cell growth has been hampered by the inability to purify, sequence and or clone this protein. The present manuscript describes methodology for the solubilization, purification and tentative identification of type II sites from the estrogen-treated rat uterus. This protein(s) chromatographs as a single major peak on DNA-cellulose, Affigel Blue dye affinity resin and during high performance liquid chromatography (HPLC) on hydroxyapatite. The purified fractions from these columns elicited classical [3H]estradiol binding characteristics (sigmoidal saturation curve, hyperbolic Scatchard plot, and Hill coefficient of approximately 4) for type II sites that are typically observed in crude or highly purified nuclear fractions or extracts. The type II binding activity also eluted as a single major component from a ligand affinity resin (GT-18-Sepharose) prepared by coupling 2,6-bis((3-methoxy-4-hydroxyphenyl)methylene)-cyclohexanone to epoxy-activated Sepharose (Pharmacia). The molecular weight of the type II site ([3H]estradiol binding activity) under non-denaturing or denaturing conditions was estimated to be approximately 10-15 kDa by gel filtration HPLC. Similarly, nuclear type II sites covalently labeled with the bioflavonoid, [3H]luteolin, migrated in the 10 kDa range on SDS PAGE. Thus, under these various experimental conditions, nuclear type II sites detected by [3H]estradiol or [3H]luteolin labeling techniques displayed little heterogeneity and appear much smaller than ERalpha or ERbeta or other steroid hormone receptors.

Hormone-induced refractoriness to mammary carcinogenesis in Wistar-Furth rats.

One of the most consistent results in the epidemiology of human breast cancer is the inverse relationship of risk and early full-term parity. The goal of this study was to investigate the molecular mechanisms through which early full-term pregnancy protects the breast from cancer development. We used Wistar-Furth (WF) rats as our experimental system and mimicked pregnancy using estrogen and progesterone (E/P). Sexually mature female rats were treated with steroid hormones for 21 days and after 28 days of gland involution, the rats were administered MNU. Rats that received a high dose of 20 microg E and 20 mg P exhibited an 82% reduction in the incidence of mammary adenocarcinomas as compared to the rats receiving only blank pellets. Decreasing doses of E/P were partially protective suggesting that complete differentiation of the gland was not required for refractoriness. We measured the RNA expression levels of several target genes involved in the regulation of mammary cell proliferation and/or differentiation including estrogen receptor (ER) and progesterone receptor (PR), cyclins D1 and D2, the cell cycle inhibitors p16, p21 and p27, and the tumor suppressor p53. At the time of MNU treatment we found no significant differences in the expression of these genes, with the possible exception of p21, indicating that hormone treatment did not result in constitutive changes in expression levels. The numbers of apoptotic cells were low and comparable in the hormone exposed and age-matched virgin gland (AMV) at the time of carcinogen challenge and remained low for 8 days after MNU treatment. The number of BrdU-labeled cells at the time of carcinogen challenge were also low in both the AMV (1.8%) and hormone exposed (0.8%) animals. In contrast, cell proliferation in the AMV (5.7%) was significantly different from both the parous involuted (1.2%) and the E/P-treated involuted (1.5%) animals 8 days after MNU treatment. We interpret these data to indicate that hormone treatment results in mammary epithelial cells that have persistent alterations in intracellular pathways governing proliferation responses to carcinogens.

Type II [3H]estradiol binding site antagonists: inhibition of normal and malignant prostate cell growth and proliferation.

A number of studies from our laboratory and others have shown that synthetic and naturally occurring bioflavonoids and related compounds have significant antiproliferative activity in the rat uterus and mouse mammary tumor model systems. This cell regulatory activity is attributed to the fact these compounds mimic methyl p-hydroxyphenyllactate (MeHPLA) as ligands for nuclear type II [3H]estradiol binding sites. The rodent prostate is also an estrogen target tissue which contains type II sites (1,2). Therefore, we assessed the effects of 2,6-bis((3-methoxy-4-hydroxyphenyl)-methylene)-cyclohexanone (BMHPC) on normal and malignant prostatic cell growth and proliferation in vitro and in vivo. This cyclovalone is designed to bind to type II sites with high affinity and mimic MeHPLA as a cell growth antagonist. Oral administration of BMHPC (9.5-38.0 mg/kg body weight per day) to intact adult male Balb/c mice for 14 days resulted in a dose dependent reduction (P<0.01) in prostatic weight relative to controls. No significant treatment effects of BMHPC on seminal vesicular, testicular or body weights were observed. BMHPC also competed for [3H]estradiol binding to type II sites in LNCaP and PC-3 human prostatic cancer cell lines and this ligand inhibited the proliferation of these cells in a dose and time dependent fashion. A direct correlation between type II site occupancy by BMHPC and the inhibition of LNCaP or PC-3 cell proliferation was observed which was reversible (not shown) following removal of BMHPC from the medium. Flow cytometry studies revealed that the type II site antagonist significantly reduced (p<0.01-p<0.001) the numbers of LNCaP and PC-3 cells in G0/G1 and caused an accumulation (p<0.001) of these cells in S-phase and G2/M (p<0.01). These data suggest that BMHPC blocks mitosis. This is consistent with the observed cytostatic activity of BMHPC in a variety of model systems. Oral administration of BMHPC to nude mice bearing subcutaneous PC-3 cell xenografts impeded the growth of these solid tumors in vivo without significant signs of toxicity. These findings demonstrate that BMHPC possesses significant anti-proliferative activity in normal and malignant prostatic tissues and cells, and extend our hypothesis that MeHPLA regulation of cellular proliferation via type II binding site interactions is an important pathway involved in cell growth regulation.

Partial purification and characterization of methyl-p-hydroxyphenyllactate esterase in rat uterine cytosol.

Previous studies from this laboratory have shown that methyl-p-hydroxyphenyllactate (MeHPLA) is a bioflavonoid and/or tyrosine metabolite that inhibits both normal and malignant cell proliferation, presumably, by association with nuclear type II [3H]estradiol binding sites. Conversely, the corresponding free acid, p-hydroxyphenyllactate (HPLA) possesses little, if any, cell regulatory activity. Therefore, factors that control the relative concentrations of MeHPLA and HPLA in normal or malignant mammalian tissues may also influence the rate of cellular proliferation. The experiments in this manuscript describe the characterization and purification of MeHPLA esterase from the rat uterus. These studies demonstrate that MeHPLA esterase activity is relatively homogeneous, eluting as a single peak during DEAE ion exchange chromatography and phenyl agarose hydrophobic interaction chromatography. Affi-gel Blue (Cibacron Blue F3GA) affinity chromatography resulted in a significant purification (approx. 300-fold) of the MeHPLA esterase activity, and a combination of these chromatographic steps resulted in a significant purification (> 4300-fold) of this protein. Further analysis of these esterase preparations on nondenaturing agarose gels allowed us to visualize bands of esterase activity. These studies localized one major low-mobility band of esterase activity in rat uterine cytosol preparations, which was increased by estradiol treatment. Furthermore, estrogen induction of MeHPLA esterase in the rat uterus was completely blocked (p < .01) by luteolin, although this bioflavonoid did not affect esterase activity in cytosol preparations from nonestrogenized rats. These results confirm our earlier studies demonstrating that MeHPLA hydrolysis is under estrogen regulation and that bioflavonoids antagonize estrogen action through pathways involving type II site induction and/or the regulation of MeHPLA hydrolysis in normal and malignant cells.

Bioflavonoids, type II [H-3]estradiol binding sites and prostatic cancer cell proliferation.

Previous studies from this laboratory have shown that bioflavonoids including luteolin and quercetin possess antiestrogenic activity in the female reproductive tract and appear to elicit this activity by interaction with type II [H-3]estradiol binding sites in the cell nucleus. Studies by a number of laboratories including our own have shown that type II sites are present in the rodent prostate gland and therefore, we suspected that administration of bioflavonoids such as luteolin may antagonize prostate growth through type LT site binding interactions as well. The studies presented in this report demonstrate that the mouse prostatic tissue does contain nuclear type II sites and that oral administration of luteolin for 14 days results in a significant (p<0.01) reduction in prostatic weight in intact male mice without significant effects on the seminal vesicular, testis or body weights of these animals. These results suggest that luteolin is a prostate specific antagonist under these experimental conditions. In vitro studies with LNCaP and PC-3 human prostate cancer cell lines demonstrated that luteolin treatment resulted in a dose dependent inhibition of prostate cancer cell proliferation which was maximum 4-8 days following treatment. Whole cell binding studies demonstrated that both LNCaP and PC-3 cells contained very high concentrations of type II [H-3]estradiol binding sites (>200,000 sites/cell) relative to levels previously reported for other tissues and cells and luteolin was capable of interacting with these sites in prostate cancer cells. In fact, there was a direct correlation between the type II site occupancy by luteolin and the inhibition of LNCaP or PC-3 prostatic cancer cell proliferation by this bioflavonoid. Flow cytometric analysis revealed that luteolin treatment caused an accumulation of LNCaP cells in G(2)/M (p<0.01) and reduced the fractions of LNCaP cells in S-phase undergoing apoptosis (p<0.01). Similarly, luteolin treatment also arrested PC-3 cells in G(2)/M (p<0.01) and reduced the proportion of cells in G(0)/G(1) (p<0.05). This being the case, it is not surprising that this bioflavonoid also blocked the growth of subcutaneous PC-3 cell xenografts in athymic nude mice. These data demonstrate that naturally occurring type TI site antagonists such as luteolin are capable of inhibiting normal and malignant prostatic cell growth and proliferation in vitro and in viva and may possess prophylactic as well as therapeutic activities against prostatic proliferative diseases including benign prostatic hyperplasia (BPH) and prostate cancer.

Effects of coumestrol on estrogen receptor function and uterine growth in ovariectomized rats.

Isoflavonoids and related compounds such as coumestrol have classically been categorized as phytoestrogens because these environmentally derived substances bind to the estrogen receptor (ER) and increase uterine wet weight in immature rats and mice. Assessment of the binding affinities of isoflavonoids for ER and subsequent effects on uterine growth suggest these compounds are less active estrogens than estradiol and therefore may reduce the risk of developing breast or prostate cancer in humans by preventing estradiol binding to ER. With the renewed interest in the relationships between environmental estrogens and cancer cause and prevention, we assessed the effects of the phytoestrogen coumestrol on uterotropic response in the immature, ovariectomized rat. Our studies demonstrated that in this animal model, coumestrol is an atypical estrogen that does not stimulate uterine cellular hyperplasia. Although acute (subcutaneous injection) or chronic (multiple injection or orally via drinking water) administration of coumestrol significantly increased uterine wet and dry weights, the phytoestrogen failed to increase uterine DNA content. The lack of true estrogenic activity was characterized by the inability of this phytoestrogen to cause cytosolic ER depletion, nuclear ER accumulation, or the stimulation of nuclear type II sites which characteristically precede estrogenic stimulation of cellular DNA synthesis and proliferation. In fact, subcutaneous or oral coumestrol treatment caused an atypical threefold induction of cytosolic ER without corresponding cytosolic depletion and nuclear accumulation of this receptor, and this increased the sensitivity of the uterus to subsequent stimulation by estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Preliminary assessment of luteolin as an affinity ligand for type II estrogen--binding sites in rat uterine nuclear extracts.

Naturally occurring bioflavonoids such as luteolin compete for [3H]estradiol binding to nuclear type II sites and mimic methyl p-hydroxyphenyllactate (MeHPLA) as ligands for this cell regulatory protein. More importantly, luteolin (3',4',5,7-tetrahydroxyflavone) contains catechol hydroxyl groups on the A and B rings that may form quinones capable of binding covalently to proteins; therefore, we evaluated luteolin as a potential affinity ligand for rat uterine nuclear type II sites. The preliminary experiments presented in this manuscript demonstrate that luteolin and a related bioflavonoid, 4,7-dihydroxyflavone (DHF), are competitive inhibitors of [3H]estradiol binding to type II sites in ammonium sulfate (AmSO4) extracts of rat uterine nuclei. This high affinity (Kd 5-10 nM) interaction is specific for type II sites, and neither compound binds to the estrogen receptor (ER). More importantly, the interaction of luteolin with nuclear type II sites was irreversible, whereas DHF readily exchanged with [3H]estradiol for type II sites in these preparations. These findings suggest that this nonexchangable occupancy of type II sites by luteolin is likely to involve covalent attachment. Spectrophotometric analysis of type II site preparations pretreated with luteolin also confirmed the [3H]estradiol exchange assay data, demonstrating that the ligand attachment is irreversible. Because luteolin did not affect [3H]estradiol binding to the ER in uterine cytosol, we suspect that this bioflavonoid may not be simply randomly interacting with a multiplicity of proteins to generate covalent complexes. These preliminary findings suggest that high-affinity binding of luteolin by type II sites is prerequisite to covalent attachment and that this bioflavonoid may be a suitable affinity ligand for the purification of this protein.

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.

Preliminary characterization and partial purification of rat uterine nuclear type II Binding sites.

These studies represent the first biochemical characterization and purification of nuclear type II binding sites from the rat uterus. Uterine nuclei from estradiol-implanted rats were digested with DNA'se and RNA'se, washed with Na deoxycholate-Tween 40 and extracted with 0.4 M ammonium sulfate (AmSO4). Nuclear type II sites in the AmSO4 extract eluted as a single peak during DEAE ion exchange chromatography, HPLC (Waters DEAE 5PW column) and Sephadex G-100 chromatography with a molecular weight of approximately 37K. DEAE and quercetin-sepharose affinity chromatography resulted in significant purification (greater than 800-fold) of nuclear type II sites with a 49% yield. Type II sites were not recognized by rat ER antibodies (Abbot ER-EIA kit) which immunoadsorbed ER from these preparations. These biochemical and immunological studies suggest that the ER and type II sites are likely to be different proteins.

Methyl p-hydroxyphenyllactate and nuclear type II binding sites in malignant cells: metabolic fate and mammary tumor growth.

Previous studies in our laboratory demonstrated that methyl p-hydroxyphenyllactate (MeHPLA) is an important cell growth-regulating agent which binds to nuclear type II binding sites in normal and malignant cells. Furthermore, this compound is deficient in a variety of rat and mouse mammary tumors and human breast cancer preparations, and this deficiency correlates with the loss of regulatory control. The present studies were performed to examine the metabolic fate of [3H]MeHPLA in mouse mammary tumors. Stable analogs of this compound such as 4,4'-dihydroxy benzylidene acetophenone were also assessed for nuclear type II site binding affinity and their ability to inhibit mammary cancer cell growth and proliferation in vitro and in vivo. The results demonstrate that mouse mammary tumors contain esterase activity which hydrolyzes MeHPLA to p-hydroxyphenyllactic acid, and this was the only major metabolite detected in these tumor preparations in vitro or in vivo. 4,4'-Dihydroxy benzylidene acetophenone, an esterase-stable MeHPLA analog, was found to bind with high affinity to nuclear type II sites but not the estrogen receptor, was capable of occupying type II sites in cultured MCF-7 cells, and inhibited the proliferation of these cells in concentrations which directly correlated with type II binding site occupancy. Similarly, 4,4'-dihydroxy benzylidene acetophenone administration by silastic implant or injection resulted in a dose-dependent inhibition of the growth of transplantable mammary tumors in mice, suggesting that this stable analog mimicks MeHPLA as a cell growth-regulating agent. Taken together, these results suggest esterase hydrolysis of MeHPLA in mammary tumors may result in a deficiency in this compound which correlates with a loss of regulatory control.

Estrogen regulation of methyl p-hydroxyphenyllactate hydrolysis: correlation with estrogen stimulation of rat uterine growth.

We have recently demonstrated that methyl p-hydroxyphenyllactate (MeHPLA) is the endogenous ligand for nuclear type II binding sites in the rat uterus and other estrogen target and non-target tissues. MeHPLA binds to nuclear type II binding sites with a very high binding affinity (Kd approximately 4-5 nM), blocks uterine growth in vivo, and inhibits MCF-7 human breast cancer cell growth in vitro. Conversely, the free acid (p-hydroxyphenyllactic acid, HPLA) interacts with type II binding sites with a much lower affinity (Kd approximately 200 nM) and does not inhibit estrogen-induced uterine growth in vivo or MCF-7 cell growth in vitro. On the basis of these observations, we suggested that one way that estrogen may override MeHPLA inhibition of rat uterine growth may be to stimulate esterase hydrolysis of MeHPLA to HPLA. The present studies demonstrate that the rat uterus does contain an esterase (mol. wt approximately 50,000) which cleaves MeHPLA to HPLA, and that this enzyme is under estrogen regulation. This conclusion is supported by the observations that MeHPLA esterase activity is increased 2-3-fold above controls within 2-4 h following a single injection of estradiol, and is maintained at high levels for 16-24 h following hormone administration. This sustained elevation of MeHPLA esterase activity correlates with estradiol stimulation of true uterine growth and DNA synthesis.

Intracellular [3H]dopamine binding sites in normal and malignant cells: relationships to cell proliferation.

Dopamine interaction with target cells undoubtably involves binding to plasma membrane receptors. However, the well documented cell growth inhibitory activity of this catecholamine suggests nuclear regulation. To evaluate this possibility, we determined the intracellular localization and binding of [3H]dopamine in human retinoblastoma (Y-79 cells), normal mouse fibroblasts (LM-cells), and in the rat uterus. Cytosol and purified nuclear preparations devoid of plasma membrane components contained specific, saturable, high affinity (Kd approximately 20 nM) binding sites for [3H]dopamine. The nuclear binding affinity for dopamine, L-dopa, and L-dopa methyl ester correlated with the inhibitory effects of these compounds on cell proliferation, suggesting that intracellular dopamine binding sites may also be involved in cellular response to catecholamines.

Characterization and partial purification of an estrogen type II binding site in chick oviduct cytosol.

An estrogen binding site of moderate affinity (Kd approximately 10 nM) and high capacity (approximately 25-70 pmol/g of tissue) was measured in DES-stimulated chick oviduct cytosol. Saturation analysis by [3H]estradiol exchange demonstrated that this binding site displayed sigmoidal binding characteristics suggesting a cooperative binding mechanism. Competition analysis with a number of compounds demonstrated that the bioflavonoid luteolin was a better competitor for binding to type II sites in chick than either estradiol or DES. Steroid specificity was demonstrated by the inability of 17 alpha-estradiol, progesterone, testosterone, corticosterone, and the triphenylethylene antiestrogen nafoxidine (U-1100A) to compete for [3H]-17 beta-estradiol binding to chick oviduct cytosol preparations. In addition, the binding site appeared to be sensitive to sulfhydryl reducing reagents as evidenced by a 75% reduction in binding activity in the presence of dithiothreitol. Both prelabeling and postlabeling procedures used in conjunction with Sephacryl S-300 chromatography resulted in a single major peak of type II binding activity representing a molecular weight in the 40,000 range. Type II binding activity was recoverable after precipitation with ammonium sulfate, and this material was subjected to a variety of column chromatography procedures in order to achieve further purification of the type II site. Significant purification of the site was achieved with a bioflavonoid-Sepharose (quercetin-Sepharose) affinity matrix. The purified type II sites eluted from quercetin-Sepharose displayed the same sigmoidal binding curves characteristic of native cytosol.

Methyl p-hydroxyphenyllactate. An inhibitor of cell growth and proliferation and an endogenous ligand for nuclear type-II binding sites.

We previously described and partially characterized endogenous ligands for nuclear type II sites in normal and malignant tissues. Chromatography of these ligands on Sephadex LH-20 revealed that two peaks with binding activity (alpha and beta) could be resolved. The beta-peak component was present in all normal tissues that we examined, but not in malignant tissues, and it inhibited the growth of MCF-7 human breast cancer cells in vitro. Conversely, the alpha-peak component was found to be present in both normal and malignant tissues, and did not inhibit MCF-7 cell growth. The present studies describe the purification and identification of the alpha-peak and beta-peak components in bovine serum and an assessment of the effects of these compounds on normal and malignant cell growth. Gas chromatography-mass spectroscopy analysis of the purified beta-peak component demonstrated that the compound was methyl p-hydroxyphenyllactate (MeHPLA). Competition analysis revealed that MeHPLA binds to nuclear type II sites with a high binding affinity, while physiological levels of this compound blocked estradiol stimulation of uterine growth in vivo and inhibited the growth of MCF-7 human breast cancer cells in vitro. The alpha-peak component was found to be the corresponding acid, p-hydroxyphenyllactic acid (HPLA). This compound interacted with nuclear type II sites with a relatively low affinity and did not block uterotropic response to estradiol or inhibit MCF-7 cell growth. These studies demonstrate that HPLA and MeHPLA are ligands for nuclear type II sites and that MeHPLA may be a very important regulator of normal and malignant cell growth.

Bioflavonoid interaction with rat uterine type II binding sites and cell growth inhibition.

Competition analysis with a number of known bioflavonoids demonstrated that these compounds (luteolin, quercetin, pelargonin) compete for [3H]estradiol binding to cytosol and nuclear type II sites in rat uterine preparations. The inhibition of [3H]estradiol binding to type II sites was specific and these bioflavonoids did not interact with the rat uterine estrogen receptor. Since estradiol stimulation of nuclear type II sites in the rat uterus is highly correlated with cellular hypertrophy and hyperplasia, we assessed the effects of these compounds on the growth of MCF-7 human breast cancer cells in culture and on estradiol stimulation of uterine growth in the immature rat. The data demonstrated that addition of quercetin (5-10 micrograms/ml) to MCF-7 cell cultures resulted in a dose-dependent inhibition of cell growth (DNA/flask). This effect was reversible by removal of quercetin from the culture medium, or by the addition of 10 nM estradiol-17 beta to these cell cultures containing this bioflavonoid. Since estradiol-17 beta (10 nM) stimulated nuclear type II sites and proliferation of MCF-7 cells, we believe bioflavonoid inhibition of MCF-7 cell growth may be mediated through an interaction with nuclear type II sites. This hypothesis was confirmed by in vivo studies which demonstrated that injection of luteolin or quercetin blocked estradiol stimulation of nuclear type II sites in the immature rat uterus and this correlated with an inhibition of uterine growth (wet and dry weight). These studies suggest bioflavonoids, through an interaction with type II sites, may be involved in cell growth regulation.

Cytosol type II sites in the rat uterus: interaction with an endogenous ligand.

Previous studies from our laboratory demonstrated that normal, but not malignant tissues, contain a ligand which competes for [3H]estradiol binding to nuclear type II sites in the rat uterus. Since elevated nuclear levels of type II sites are correlated with estrogen stimulation of uterine growth and DNA synthesis, we believe this ligand may regulate cell growth. The present studies show that the ligand for nuclear type II sites also interacts with type II sites in uterine cytosol. This was demonstrated by dilution experiments which show that greater quantities of type II sites are measured in dilute (10 mg/ml) than in concentrated (40 mg/ml) uterine cytosol. Furthermore, stripping of uterine cytosol with 1% dextrancoated charcoal, or pre-binding cytosol type II sites to hydroxylapetite (HAP) prior to binding analysis, removed the ligand from these preparations such that high levels of type II sites were measured. Following charcoal stripping, cytosol type II sites demonstrated good specificity for estrogenic hormones but not progesterone, corticosterone, or the triphenylethylene anti-estrogen, nafoxidine. Since the level of type II sites in the cytosol always preceded and exceeded the level of this site measured in uterine nuclei at all times following estrogen treatment (0-96 h), we believe cytosol type II sites may function as an type II-ligand binding protein (LBP) which regulates the availability of the ligand for interaction with nuclear type II sites. This is consistent with our observation that type II sites are not depleted from uterine cytosol by estrogen treatment and nuclear type II sites are very tightly associated with the nuclear matrix.