Some properties of the ecdysteroid receptor in the salivary gland of the ixodid tick, Amblyomma hebraeum.

Salivary gland degeneration in ixodid ticks is triggered by an ecdysteroid hormone. We used [3H]ponasterone A (PoA) as a specific ligand to detect the ecdysteroid receptor in the salivary glands of large, partially fed female ticks (Amblyomma hebraeum Koch; Acari: Ixodidae). Binding of [3H]PoA was thermolabile and sensitive to pronase, but not to DNase or RNase, indicating that the ligand binds to a protein. Scatchard analysis of [3H]PoA binding strongly suggested the presence of an ecdysteroid receptor in cytosolic and nuclear extracts of the tissue. The Kd and Bmax for PoA binding in cytosol were 0.72 +/- 0.09 nM and 175 +/- 12 fmol/mg protein, respectively (n = 8). Corresponding figures for nuclear extract were 1.1 +/- 0.5 nM and 282 +/- 35 fmol/mg protein, respectively (n = 3; P > 0.05 compared to cytosol). The relative ability of unlabeled ecdysteroids to compete for [3H]PoA binding was (in descending order): PoA > muristerone A > makisterone A > 20-hydroxyecdysone > mesylinokosterone > ecdysone. The Kd estimated for 20-hydroxyecdysone (probably the natural hormone) correlates very well with its physiological potency in inducing salivary gland degeneration in vivo and in organ culture. None of the vertebrate steroids tested (estradiol, testosterone, progesterone, and corticosterone) was able to displace PoA binding at a concentration 10(5) times higher than PoA. The cytosolic form of the receptor migrated to the 3.2 S region of a 10-40% sucrose density gradient.

Androgen receptor in the harderian glands of the golden hamster: characterization and the effects of androgen deprivation, the pituitary, and gender.

The harderian glands of the golden hamster were found, by a competitive binding assay using [3H]mibolerone as the ligand, to have a high affinity androgen receptor. In intact male hamsters, this receptor was present in both cytosolic and nuclear KCl-extractable fractions. Castration or hypophysectomy led to 3- to 5-fold increases in the concentrations of cytosolic receptor with decreased dissociation constants. Hypophysectomy with maintenance of prolactin levels (by removal of pituitaries and their implantation either in the sella turcica or under the kidney capsule) had no effect on androgen receptor binding, compared to hypophysectomy alone. Female hamsters had androgen receptor levels which were 2 to 4 times higher than those of intact males. Hypophysectomy led to elevated receptor binding in ovariectomized female hamsters and this rise was prevented by maintaining prolactin levels. Binding of [3H]mibolerone in male glands was effectively inhibited by 5 alpha-dihydrotestosterone, whereas the parent molecule, testosterone, required approximately a 10-fold greater molar excess to achieve the same amount of inhibition. Estradiol and progesterone were relatively poor inhibitors of the observed binding of [3H]mibolerone, while dexamethasone was ineffective. Sucrose gradient studies indicated that the harderian androgen receptor migrated to the 8S region, as expected for this receptor in molybdate-containing gradients. These results indicate that the androgen receptor in the hamster harderian gland is a 5 alpha-dihydrotestosterone receptor.

Correlation between c-erbB-2 amplification and risk of recurrent disease in node-negative breast cancer.

Drawing upon the comprehensive population-based Northern Alberta Breast Cancer Registry containing 704 patients with histologically negative axillary lymph nodes who have been followed for 5-16 years, we have undertaken a retrospective case-control study to evaluate the utility of genomic amplification of specific protooncogenes [c-erbB-2 (nee HER-2/neu), c-erbA, c-myc, int-2, and hst-1] as predictive indicators of clinical outcome in node-negative disease. To this end, 115 women with node-negative breast cancer who had recurred at any time up to 16 years posttreatment (cases) were matched pairwise for appropriate clinicopathological variables (size of primary tumor, menopausal state, estrogen receptor status, anniversary year of treatment, and patient age) with a second group of 115 women (controls) selected from a cohort of 502 node-negative patients who had not relapsed during long-term follow-up. Tumor DNA extracted from archival formalin-fixed, paraffin-embedded tissue blocks were analyzed for protooncogene copy number by slot-blot hybridization. Taking a gene copy number of 3 as the cutoff, 27 of the 230 tumor samples examined contained from 3- to 22-fold elevation in c-erbB-2 genomic equivalents. Twenty-one of the 27 tumors amplified for c-erbB-2 were derived from cases and 6 from controls, signifying that 18% of the node-negative patients who had relapsed harbored excessive copies of the protooncogene in their malignant tissue compared to only 5% for the patients who had remained in remission. Accordingly, the occurrence of amplification of c-erbB-2 proved to be a statistically significant predictor of poor prognosis, especially disease-free interval (P = 0.006). Moreover, this genetic alteration appeared to be independent of and to have greater predictive power than most commonly used prognostic factors. Our findings also indicated that as a clinical test, measurement of c-erbB-2 amplification suffers from low sensitivity; however, when greater than 6 gene copies are present, the test has a positive predictive value for recurrence of 70%. Concurrent analysis of tumor DNA blots with probes for the other four protooncogenes examined revealed that their amplification, which others have reported to arise often, especially in node-positive disease, was seldom found even in our high-risk case group (2-3%). In short, our data strongly suggest that amplification of c-erbB-2 may contribute to the pathogenesis of some forms of node-negative breast cancer and thus may serve as a useful genetic marker to identify a subset of high-risk patients.

Immunoassay for estrogen receptor does not detect inactivated receptor.

Accurate quantification of estrogen receptor (ER) is essential for optimal clinical characterization of individual cases of breast cancer. If breast tumors are mishandled, the relatively labile ER protein may lose its steroid-binding capacity (become inactivated) and not be measurable by the routine steroid-binding assay. We tested whether the commercial enzyme immunoassay of Abbott Laboratories could quantify inactivated ER. Samples of powdered breast tumors from humans were exposed to various temperature and homogenization conditions known to inactivate ER, and any remaining ER was quantified by both the immunoassay and the steroid-binding assay. For all inactivation conditions tested, the two assay methods detected the same proportions of remaining ER. We conclude that the inactivation reaction for ER also alters one or both of the antigenic site(s) necessary for the immunoassay. Hence, for breast tumors mishandled to the extent of inactivating ER, the immunoassay offers no advantage over the more conventional steroid-binding assay for quantifying any remaining ER.

The levo enantiomer of o,p'-DDT inhibits the binding of 17 beta-estradiol to the estrogen receptor.

It has been discovered previously that the known estrogenic activity of o,p'-DDT resides with the levo enantiomer. Since it has been presumed that this relatively weak estrogenic activity of o,p'-DDT is mediated by the estrogen receptor, the ability of the resolved enantiomeric forms of o,p'-DDT to inhibit the binding of 17 beta-estradiol to the receptor was investigated. Competitive binding assays including the use of double-reciprocal plots and sucrose gradient analyses revealed that the levo and not the dextro enantiomer could inhibit the estradiol binding to the estrogen receptor. Thus the in vivo estrogenic activity of levo o,p'-DDT correlates with its apparent ability to interact with the estrogen receptor.

Effects of olfactory bulbectomy, melatonin, and/or pinealectomy on three sublines of the Dunning R3327 rat prostatic adenocarcinoma.

Conventional antiandrogen therapy for prostatic cancer generally results in the death of androgen-dependent cells, resulting in shrinkage of the tumor, followed by regrowth of the tumor as androgen-insensitive cells take over. Because of reported antigonadotropic and antineoplastic effects of the pineal hormone melatonin (MEL), we hypothesized that this indole might provide an effective therapy for prostate cancer, as it would be effective against both populations of tumor cells. We used three sublines of the Dunning R3327 rat prostatic adenocarcinoma to determine whether MEL could suppress the growth of these tumors and, if so, by what mechanisms this occurs. In one experiments, we compared the growth of a well-differentiated slow-growing Dunning tumor in rats given MEL combined with the potentiating procedure olfactory bulbectomy (BULBX), with that in rats pinealectomized (PINX) or untreated. Tumor growth in BULBX-MEL rats was significantly suppressed over that in the other two groups, as were the weights of the gonads and accessory sex glands. Tumor morphology, DNA concentration, and androgen receptor concentration and distribution were identical in untreated controls and in BULBX-MEL rats, suggesting that the treatment affected all populations of tumor cells equally. With another strain of well-differentiated slow-growing Dunning tumor, we examined the effects of MEL in rats with and without BULBX. Reproductive parameters were not suppressed in BULBX-MEL rats and, while there was a trend toward slower tumor growth in this group, this was not significant. Intact rats given MEL grew larger tumors than did control rats but, again, differences were not significant. In a third experiment, we examined a fast-growing androgen-insensitive anaplastic Dunning tumor. PINX was without effect on this tumor, but BULBX-MEL resulted in a significant suppression of one of the constants in the logistic equation fitted to the growth curves. This indicates that there were some direct antitumor effects of BULBX-MEL on this tumor strain. We conclude that MEL suppresses growth of some Dunning tumor strains.

Applicability of nuclear androgen receptor quantification to human prostatic adenocarcinoma.

It has been proposed that concentrations of nuclear androgen receptor may be predictive of tumor hormone dependence in cases of advanced human prostatic cancer. We have investigated the ability of this receptor population to reflect patient prognosis during endocrine therapy in 12 cases of stage D disease. KCl-extractable, nuclear matrix-bound and total nuclear androgen receptor concentrations showed a significant positive correlation with duration of patient survival (p less than 0.05) while cytosolic and total cellular androgen receptor concentrations were not significantly correlated with survival. However, use of selected threshold concentrations of receptors revealed that only cytosolic, nuclear KCl-extractable and total cellular receptors could significantly differentiate long-term and short-term survivors. Even given the small number of patients studied, the potential use of this androgen receptor assay as an index of both tumor hormone-dependence and patient prognosis was evident. Therefore, in order to make these androgen receptor assays more applicable, we attempted to simplify the methods for use on readily available tissues. Similar amounts of nuclear androgen binding were observed in crude and purified nuclear pellets, in nuclei treated with DNase and KCl in differing orders or in nuclei from tissue homogenized using glass or Polytron homogenization procedures. More importantly, nuclear androgen receptor concentrations in specimens of prostatic cancer or benign hyperplasia taken by needle biopsy or transurethral resection involving electrocautery did not differ from those of parallel specimens taken by Thompson cold punch. Simplified nuclear androgen receptor assays of needle biopsy or electrocautery specimens are accurate and should prove clinically applicable.

Relationship between concentrations of extractable and matrix-bound nuclear androgen receptor and clinical response to endocrine therapy for prostatic adenocarcinoma.

The relationship between androgen receptor concentrations and clinical response to endocrine therapy for prostatic adenocarcinoma was investigated for 13 stage D patients. Both cytoplasmic and nuclear-androgen receptors were quantified. For nuclear androgen receptor, nuclei were isolated and treated with high ionic strength buffer (0.6 M KCl) to yield a KCl-extractable fraction; the nuclei were then treated with DNase I to yield nuclear matrices. Electron microscopy confirmed the relative nuclear purity and revealed matrix morphology. An hydroxylapatite binding assay and methyltrienolone (R1881) were used to quantify androgen receptor in cytosol, the KCl-extract and matrix preparations. Following 6 months of hormonal therapy, the clinical status of patients was re-evaluated and the patients were grouped according to disease response. The androgen receptor data obtained prior to therapy were compared for the disease response groups. The mean concentrations of cytoplasmic androgen receptor, KCl-extractable nuclear androgen receptor and nuclear matrix-bound androgen receptor, respectively, in those patients with disease progression or death (no. = 6), were 671 +/- 232, 45 +/- 17 and 119 +/- 34 fmol. per gm. of tissue +/- S.E.M., and for those with disease regression or stabilization (no. = 7), 1427 +/- 435, 193 +/- 53 and 611 +/- 92 fmol. per gm. of tissue +/- S.E.M. While cytoplasmic androgen receptor concentrations were not related to clinical status, both extractable and matrix-bound nuclear androgen receptor concentrations were significantly higher in the group which responded to hormonal therapy. These results suggest that nuclear-extractable and nonextractable androgen receptor concentrations are useful indices for the prediction of hormone-dependence of prostatic cancer.

Heparin-mediated inactivation and transformation of mammary cytoplasmic glucocorticoid receptor.

Glucocorticoid receptor of lactating mouse mammary gland cytosol was exposed to heparin when the receptor was either steroid-free or steroid-bound. Heparin caused a dose-dependent and time-dependent loss of steroid binding activity (inactivation) of the steroid-free receptor; this heparin-induced inactivation was inhibited by molybdate. In contrast, steroid-bound receptor maintained its steroid binding capacity in the presence of heparin but the heparin caused transformation of receptor as detected by increased binding to DNA-cellulose and ATP-Sepharose. Heparin also converted steroid-bound receptor from the 7-8S form to the 4S form. Molybdate inhibited both the heparin-induced transformation and associated conversion to the 4S form.

Androgen binding sites on nuclear matrix of normal and hyperplastic human prostate.

To further characterize human prostatic androgen receptor, nuclei were isolated from normal prostate (no. = 3) and benign prostatic hyperplasia specimens (no. = 10). High ionic strength (0.6 M KCl) treatment of nuclei released nuclear extractable androgen receptor and DNase I digestion then yielded nuclear matrices. Androgen receptor was quantified in the nuclear extract and nuclear matrix preparations by Scatchard analysis of specific R1881 binding. Only 1 of the 3 normal tissues had extractable androgen receptor (113 fmol. per gm. of tissue) while the mean concentration of extractable androgen receptor for BPH was 189 fmol. per gm. of tissue. The mean concentrations of matrix-bound androgen receptor were 325 fmol. per gm. of tissue and 548 fmol. per gm. of tissue for normal and hyperplastic prostate, respectively. The androgen binding sites on nuclear matrix may represent the functional intranuclear androgen receptor and a characterization of these sites may provide an understanding of the etiology of BPH.

Estrogen receptor in human benign prostatic hyperplasia.

Estrogens have been proposed as a major etiological factor in the pathogenesis of benign prostatic hyperplasia in man. The presence of estrogen receptor in benign prostatic hyperplasia would support this concept. Using the receptor stabilizer, sodium molybdate, and a hydroxylapatite assay we assayed human benign prostatic hyperplasia for the presence of cytosolic estrogen receptor. For comparison, we assayed estrogen receptor in cytosols of prostatic cancer and normal tissue, and we also measured androgen receptor and progesterone receptor concentrations in the 3 tissue types. Estrogen receptor was present in 8 of 15 benign prostatic hyperplasia specimens at a mean concentration of 9.2 fmol./mg. protein for the estrogen-receptor-positive samples. Sucrose gradient analysis of the estrogen receptor of benign prostatic hyperplasia revealed that it sedimented in the region of 8S, and steroid specificity studies confirmed that the binding to estrogen receptor was estrogen-specific. Estrogen receptor was also found in normal (3 of 3) and malignant (4 of 6) tissues, and all tissues were positive for androgen receptor. The presence of estrogen receptor in human benign prostatic hyperplasia supports the proposal that circulating estrogens may have a role in the pathogenesis of this disorder.

Interaction of chick oviduct progesterone receptor with the 2',3'-dialdehyde derivative of adenosine 5'-triphosphate.

Avian oviduct progesterone receptor was treated with the 2',3'-dialdehyde derivative of ATP (oATP) in an attempt to demonstrate the presence of nucleotide binding sites on the receptor. oATP, when added to cytosol, inhibited binding by transformed receptor to ATP-Sepharose, DNA-cellulose, phosphocellulose, or isolated nuclei in an irreversible manner. oATP did not disrupt the steroid-receptor complex, but it did alter the ionic properties of the receptor. This was demonstrated by an increased affinity of receptor for DEAE-cellulose and for hydroxylapatite. oATP mimicked the effect of ATP on progesterone receptor with regard to two properties: it altered the rate of receptor inactivation that occurs in the absence of progesterone, and it promoted receptor conversion from an 8S complex to lower sedimenting forms (4-6 S). The action of oATP on the receptor could be blocked by the addition of pyridoxal 5'-phosphate, which has been shown previously to interact with the progesterone receptor. A partial interference of oATP action was also observed when ATP was added. These results indicate that oATP interacts with the progesterone receptor and may be used as an affinity-labeling agent for receptor characterization.

Transformation of mammary cytoplasmic glucocorticoid receptor under cell-free conditions.

The transformation of glucocorticoid--receptor complex in the cytosol from lactating mouse mammary tissue was studied by using elevated temperature and KCl as promoters of the transformation reaction. The transformed receptor was identified from the nontransformed receptor by the following criteria: (a) increased binding to DNA--cellulose, (b) increased binding to ATP--Sepharose, (c) higher affinity for the steroid as determined by steroid dissociation kinetics, and (d) different sedimentation profiles on sucrose gradients containing KCl and sodium molybdate. A greater percentage of the nontransformed receptor was converted to the transformed state by an increased KCl concentration as opposed to increased temperature. Pretreatment of cytosol with 10 mM sodium molybdate prevented both the temperature- and salt-mediated transformation of the receptor.

An empirical basis for the competition by dexamethasone to progesterone receptors as estimated with the synthetic progestin R5020.

Normal mammary glands of mice contain both progesterone and glucocorticoid receptors and the levels of both these receptors are modulated as a function of development. Measurement of progesterone receptors using the synthetic progestin, R5020, has led to conflicting data both with regard to the presence of progesterone receptor during certain developmental stages of the mammary gland and the ability of the glucocorticoids to compete for specific R5020 binding sites. In this report we have identified experimental conditions which allow for the separate measurements of the progesterone and glucocorticoid receptors in the same cytosol. If sulfhydryl reducing agents such as dithiothreitol are excluded from the assay buffer, R5020 binds to only a single class of high affinity sites in mammary cytosol of virgin mice and these binding sites exhibit a strict steroid specificity characteristic of progesterone receptors. In contrast, if dithiothreitol is included in the buffers, R5020 binds not only to the high affinity sites but also to certain saturable lower affinity sites; these lower affinity sites for R5020 also bind glucocorticoids such as dexamethasone. These findings should facilitate more accurate quantitation of both progesterone and glucocorticoid receptors in normal and neoplastic tissues and also be applicable to studies on the mechanism(s) of progesterone action.

Distinction between progestin- and glucocorticoid-binding sites in mammary glands. Apparent lack of cytoplasmic progesterone receptors in lactating mammary glands.

The cytosol fraction of the lactating mammary glands of mice does not appear to contain detectable amounts of progesterone receptors. Mixing experiments indicate that the absence of receptors is not due to interference by other factors in the cytosol. However, in the cytosol of mammary glands, there is specific binding or progestins to certain low-affinity sites which have characteristics of specific glucocorticoid-binding sites.

Differing estrogenic activities for the enantiomers of o, p'-DDT in immature female rats.

Female rats (20-21 days) were given single intraperitoneal injections of (+/-)-o,p'-DDT, (--)-o,p'-DDT, or (+)-o,p'-DDT. At 18 h their uteri were excised and the estrogen sensitive parameters, uterine wet weight, and uterine glycogen content were measured. For o,p'-DDT, the levo enantiomer is the more active estrogen in immature female rats. Optical resolutions of other racemic environmental xenobiotics may be important in the evaluation of their biological effects.