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.

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.

Hypofrontality and cognitive impairment in schizophrenia: dynamic single-photon tomography and neuropsychological assessment of schizophrenic brain function.

Regional cerebral blood flow (rCBF) was assessed in 40 chronic male schizophrenic patients (20 medicated, 20 unmedicated) and 31 matched normal controls with Dynamic Single-photon Emission Computed Tomography (D-SPECT). Blind analyses of normalized color-coded tomograms revealed significant bifrontal and bitemporal rCBF deficits in the patient group. Frontal flow deficits were most prominent in paranoid patients (n = 21) and right temporal deficits were most prominent in nonparanoid patients (n = 19). These relative regional declines were observed within the context of significantly elevated hemispheric blood flow in schizophrenics compared with controls. Reduced left frontal rCBF was associated with neuropsychological impairment on the Wisconsin Card Sorting Test and Luria-Nebraska Battery. Increased hemispheric CBF was correlated with the presence of positive schizophrenic symptoms. Medication status was unrelated to rCBF. These findings demonstrate that hypofrontality has important implications for cognitive function in some schizophrenic individuals.

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)

Regional cerebral blood flow in first break and chronic schizophrenic patients and normal controls.

Dynamic 133Xe Single Photon Emission Computed Tomography (SPECT) was used to measure the resting regional cerebral blood flow (rCBF) in 16 neuroleptic free schizophrenic and schizophreniform male patients and 13 age-matched male normal controls. A subgroup consisting of 'first break' patients who had never been exposed to neuroleptic treatment were age-matched to a subgroup of young chronic patients most of whom had been previously exposed to neuroleptics. The age-adjusted rCBF values were compared among first breaks, young chronics, normal controls, and a subgroup of older, more chronic patients. In first break patients, we found significantly lower absolute global cerebral blood flow and significantly lower superior frontal, middle frontal, and middle temporal absolute rCBF compared to normals. We also found significantly lower relative superior frontal rCBF in first breaks vs. normals, and higher relative superior frontal and relative middle frontal rCBF in older chronics vs. the other groups. For relative posterior temporal rCBF there was greater asymmetry (right side > left) in first breaks and young chronics compared to normals and older chronics.

Effects of tricyclic antidepressant treatment on tyramine-O-sulfate excretion in depressed patients.

The urinary excretion of tyramine-O-sulfate following an oral load of tyramine (Tyramine Challenge Test, 'TCT') was measured in a group of fourteen inpatients with unipolar and bipolar major depressive episode. TCT was done both during a pretreatment baseline period and following four weeks of treatment with tricyclic antidepressants. The change in TCT values after treatment correlated with improvement in depression. The previously described ability of TCT to discriminate between endogenous and nonendogenous depressed patients was confirmed at baseline. However, following tricyclic antidepressant treatment, TCT values were not significantly different between endogenous and nonendogenous patients.

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.

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.

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)

Cerebellar blood flow in schizophrenic patients and normal control subjects.

We used 133Xe dynamic single-photon emission computed tomography (DSPECT) to measure the resting cerebellar blood flow in 17 neuroleptic-free schizophrenic and schizophreniform patients and 13 normal control subjects. A subset of these subjects (11 patients and 7 control subjects) additionally underwent activation studies during the Wisconsin Card Sorting (WCS) and Number Matching (NM) tests. Baseline relative cerebellar blood flow was significantly lower in older patients than in age-matched control subjects. For absolute cerebellar flow, there was a significant difference between patients and control subjects in the overall activation response (patients: NM 13.4% increase, WCS 15.7% increase; control subjects: NM 3.1% decrease, WCS 0.0% change). This difference was more pronounced in older subjects. Cerebral blood flow significantly increased during NM (patients: 21.3% increase, control subjects: 6.5% increase) and WCS (patients: 16.5% increase, control subjects: 9.7% increase). The difference in the magnitude of cerebral NM activation between schizophrenic patients and control subjects, although not statistically significant, may call into question the appropriateness of using NM as a control task in schizophrenic patients. Finally, we found no differences between the effects of WCS and NM on cerebellar or cerebral blood flow. Because of the small number of subjects in each group, the results of this study should be interpreted cautiously.

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.

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.

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.

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.