Spinophilin: from partners to functions.

Spinophilin/neurabin 2 has been isolated independently by two laboratories as a protein interacting with protein phosphatase 1 (PP1) and F-actin. Gene analysis and biochemical approaches have contributed to define a number of distinct modular domains in spinophilin that govern protein-protein interactions such as two F-actin-, three potential Src homology 3 (SH3)-, a receptor- and a PP1-binding domains, a PSD95/DLG/zo-1 (PDZ) and three coiled-coil domains, and a potential leucine/isoleucine zipper (LIZ) motif. More than 30 partner proteins of spinophilin have been discovered, including cytoskeletal and cell adhesion molecules, enzymes, guanine nucleotide exchange factors (GEF) and regulator of G-protein signalling protein, membrane receptors, ion channels and others proteins like the tumour suppressor ARF. The physiological relevance of some of these interactions remains to be demonstrated. However, spinophilin structure suggests that the protein is a multifunctional protein scaffold that regulates both membrane and cytoskeletal functions. Spinophilin plays important functions in the nervous system where it is implicated in spine morphology and density regulation, synaptic plasticity and neuronal migration. Spinophilin regulates also seven-transmembrane receptor signalling and may provide a link between some of these receptors and intracellular mitogenic signalling events dependent on p70(S6) kinase and Rac G protein-GEF. Strikingly a role for spinophilin in cell growth was demonstrated and this effect was enhanced by its interaction with ARF. Here we review the current knowledge of the protein partners of spinophilin and present the available data that are contributing to the appreciation of spinophilin functions.

Genistein modifies the activation kinetics and magnitude of phosphorylated wild-type and G551D-CFTR chloride currents.

We have studied the mechanism by which genistein activates cystic fibrosis transmembrane conductance regulator (CFTR) in CHO cells expressing wild type or G551D-CFTR. In wild-type CHO cells, after exposure to 2.5 microM forskolin, 25 microM genistein induced a further 2-fold and rapid increase of the forskolin-activated CFTR current. In both types of cells, when forskolin was added after genistein preincubation, whole-cell current density was greatly reduced compared to that measured when genistein was added after phosphorylation of CFTR, and all activation kinetic parameters were significantly altered. Genistein had no effect on the adenylate cyclase activity. Our results suggest that the occupancy of a putative genistein binding site is critical for the gating mechanism of CFTR chloride channels, which, depending on the phosphorylation status of the R-domain, drives CFTR either into a refractory state or alternatively to a highly activated state.

Correction of delF508-CFTR activity with benzo(c)quinolizinium compounds through facilitation of its processing in cystic fibrosis airway cells.

A number of genetic diseases, including cystic fibrosis, have been identified as disorders of protein trafficking associated with retention of mutant protein within the endoplasmic reticulum. In the presence of the benzo(c)quinolizinium drugs, MPB-07 and its congener MPB-91, we show the activation of cystic fibrosis transmembrane conductance regulator (CFTR) delF508 channels in IB3-1 human cells, which express endogenous levels of delF508-CFTR. These drugs were without effect on the Ca(2+)-activated Cl- transport, whereas the swelling-activated Cl- transport was found altered in MPB-treated cells. Immunoprecipitation and in vitro phosphorylation shows a 20% increase of the band C form of delF508 after MPB treatment. We then investigated the effect of these drugs on the extent of mislocalisation of delF508-CFTR in native airway cells from cystic fibrosis patients. We first showed that delF508 CFTR was characteristically restricted to an endoplasmic reticulum location in approximately 80% of untreated cells from CF patients homozygous for the delF508-CFTR mutation. By contrast, 60-70% of cells from non-CF patients showed wild-type CFTR in an apical location. MPB-07 treatment caused dramatic relocation of delF508-CFTR to the apical region such that the majority of delF508/delF508 CF cells showed a similar CFTR location to that of wild-type. MPB-07 had no apparent effect on the distribution of wild-type CFTR, the apical membrane protein CD59 or the ER membrane Ca(2+),Mg-ATPase. We also showed a similar pharmacological effect in nasal cells freshly isolated from a delF508/G551D CF patient. The results demonstrate selective redirection of a mutant membrane protein using cell-permeant small molecules of the benzo(c)quinolizinium family and provide a major advance towards development of a targetted drug treatment for cystic fibrosis and other disorders of protein trafficking.

Rapid quality control for testing the radiochemical purity of 99Tc(m)-tetrofosmin.

We describe a new thin-layer chromatography (TLC) method to evaluate the radiochemical purity of 99Tc(m)-tetrofosmin without the drawbacks of toxicity, solvent ratios and time requirement associated with the standard TLC method. The new method uses miniaturized instant TLC plates impregnated with silica gel (ITLCTM/SG, 2.5 x 10 cm) for the stationary phase and 2-butanone for the mobile phase. The standard TLC method was performed with ITLCTM/SG plates (5 x 20 cm) and dichloromethane/acetone (65:35, v/v). Thirty five preparations were analysed by both methods with a storage phosphor imaging system to determine the percentages of hydrolyzed-reduced 99Tc(m) compound (99Tc(m)O2), 99Tc(m)-tetrofosmin and free 99Tc(m)-pertechnetate (99Tc(m)O4(-)). Using the miniaturized TLC method, 99Tc(m)-tetrofosmin had a mean Rf value of 0.55 (standard deviation, 0.05), while 99Tc(m)O4(-) migrated with the solvent front (Rf=1) and 99Tc(m)O2 remained at the origin of the strips (Rf=0). No significant difference was found between miniaturized and standard TLC methods for the radiochemical purity of 99Tc(m)-tetrofosmin using the Wilcoxon matched-pair signed-rank test (P=0.82). Furthermore, the two methods showed a good correlation as measured by the Spearman rank coefficient (r=0.89) and were in perfect agreement, with a kappa index of +1, for a cut-point between positive and negative set at 90%. In conclusion, the results indicate that the miniaturized TLC method is effective for the routine evaluation of the radiochemical purity of 99Tc(m)-tetrofosmin, without some of the drawbacks of the standard method.

Prognostic impact of cathepsin D and c-erbB-2 oncoprotein in a subgroup of node-negative breast cancer patients with low histological grade tumors.

Some node-negative breast cancer patients, with initially good prognosis, relapse from their cancer and are poorly identified. In the present study, based on prospective data of 197 tumors, we measured cathepsin D (cath D, n=197), pS2 protein (n=125), c-erbB-2 oncoprotein (n=100) and epidermal growth factor receptor (EGF-R, n=99) to better define the risk of relapse of node-negative patients in comparison with that defined by the clinical and histological factors. The median follow-up in surviving patients was 75 months. Univariate analysis indicated that patients with histological grade III tumors (the Scarff, Bloom and Richardson classification) had a much poorer prognosis than those with histological grade I or II tumors (P=0.0027 for relapse-free survival and P=0.0156 for overall survival). When the population of node-negative patients was divided by tertiles, high cath D levels showed a significant association with an early relapse (P=0.0316). Using cut-off values, patients with high cath D (> or =25 pmol/mg protein) or c-erbB-2 oncoprotein (> or =4 Human Neu Unit/microg protein) levels, had a significant worse relapse-free survival (P=0.0147 and 0.0417, respectively). No prognostic information was supported by pS2 protein or EGF-R measurements. In multivariate analysis, histological grade, cath D and c-erbB-2 oncoprotein remained independent predictors of recurrence (P=0.005, 0.0361 and 0.0321, respectively). By combining low levels of cath D and c-erbB-2 oncoprotein in histological grade I or II tumors, we identified a subgroup of patients with a 100% relapse-free survival probability at 6 years of follow-up. Moreover, the subgroup of patients with histological grade I or II tumors and high values of both cath D and c-erbB-2 oncoprotein showed a prognosis as poor as the subgroup defined by histological grade III alone, respectively 66% and 70% relapse-free survival at 6 years of follow-up. In conclusion, the combination of conventional prognostic factor (histological grade) and biochemical factors (cath D and c-erbB-2 oncoprotein) enabled us to identify, in this preliminary study, a subgroup of patients having an increased risk of relapse in a group (node-negative patients with low histological grade tumors) considered as good prognosis.

Properties of CFTR activated by the xanthine derivative X-33 in human airway Calu-3 cells.

The pharmacological activation of the cystic fibrosis gene protein cystic fibrosis transmembrane conductance regulator (CFTR) was studied in human airway epithelial Calu-3 cells, which express a high level of CFTR protein as assessed by Western blot and in vitro phosphorylation. Immunolocalization shows that CFTR is located in the apical membrane. We performed iodide efflux, whole cell patch-clamp, and short-circuit recordings to demonstrate that the novel synthesized xanthine derivative 3, 7-dimethyl-1-isobutylxanthine (X-33) is an activator of the CFTR channel in Calu-3 cells. Whole cell current activated by X-33 or IBMX is linear, inhibited by glibenclamide and diphenylamine-2-carboxylate but not by DIDS or TS-TM calix[4]arene. Intracellular cAMP was not affected by X-33. An outwardly rectifying Cl(-) current was recorded in the absence of cAMP and X-33 stimulation, inhibited by DIDS and TS-TM calix[4]arene. With the use of short-circuit recordings, X-33 and IBMX were able to stimulate a large concentration-dependent CFTR transport that was blocked by glibenclamide but not by DIDS. Our results show that manipulating the chemical structure of xanthine derivatives offers an opportunity to identify further specific activators of CFTR in airway cells.

Development of substituted Benzo[c]quinolizinium compounds as novel activators of the cystic fibrosis chloride channel.

Chloride channels play an important role in the physiology and pathophysiology of epithelia, but their pharmacology is still poorly developed. We have chemically synthesized a series of substituted benzo[c]quinolizinium (MPB) compounds. Among them, 6-hydroxy-7-chlorobenzo[c]quinolizinium (MPB-27) and 6-hydroxy-10-chlorobenzo[c]quinolizinium (MPB-07), which we show to be potent and selective activators of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. We examined the effect of MPB compounds on the activity of CFTR channels in a variety of established epithelial and nonepithelial cell systems. Using the iodide efflux technique, we show that MPB compounds activate CFTR chloride channels in Chinese hamster ovary (CHO) cells stably expressing CFTR but not in CHO cells lacking CFTR. Single and whole cell patch clamp recordings from CHO cells confirm that CFTR is the only channel activated by the drugs. Ussing chamber experiments reveal that the apical addition of MPB to human nasal epithelial cells produces a large increase of the short circuit current. This current can be totally inhibited by glibenclamide. Whole cell experiments performed on native respiratory cells isolated from wild type and CF null mice also show that MPB compounds specifically activate CFTR channels. The activation of CFTR by MPB compounds was glibenclamide-sensitive and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive. In the human tracheal gland cell line MM39, MPB drugs activate CFTR channels and stimulate the secretion of the antibacterial secretory leukoproteinase inhibitor. In submandibular acinar cells, MPB compounds slightly stimulate CFTR-mediated submandibular mucin secretion without changing intracellular cAMP and ATP levels. Similarly, in CHO cells MPB compounds have no effect on the intracellular levels of cAMP and ATP or on the activity of various protein phosphatases (PP1, PP2A, PP2C, or alkaline phosphatase). Our results provide evidence that substituted benzo[c]quinolizinium compounds are a novel family of activators of CFTR and of CFTR-mediated protein secretion and therefore represent a new tool to study CFTR-mediated chloride and secretory functions in epithelial tissues.

Expression, localization, and thyrotropin regulation of cathepsin D in human thyroid tissues.

Enzymatic activity and isoform expression of cathepsin D (cath D) were studied in 107 cytosols from various human thyroid tissues including 21 normal tissues, 12 cold benign nodules, 17 toxic adenomas, 22 samples from Graves' disease patients, and 35 thyroid carcinomas. Cath D assay was optimized for human thyroid tissues. We found that mean cath D specific activities, expressed as units per milligrams protein minus thyroglobulin, were higher in carcinomas (P = 0.0001), toxic adenomas (P = 0.0001), and specimens from Graves' disease patients (P = 0.0001) than in normal thyroid tissues. Mean cath D activity in carcinomas was also significantly different from that in cold benign nodules (P < 0.001) and Graves' disease tissues (P < 0.05) but not from that of toxic adenomas. To determine possible mechanisms by which the observed increase in cath D activity might be regulated, we used Western blotting to measure relative amounts of cath D isoforms in the various thyroid tissues. We found that the 31-kDa major processing form of cath D was significantly increased in carcinomas and toxic adenomas compared with normal tissues (P < 0.01), cold benign nodules (P < 0.05), and Graves' disease tissues (P < 0.05). A positive correlation of cath D activity with relative expression of the 31-kDa form (r = 0.67, P = 0.0001) was observed in 104 thyroid cytosols. These data demonstrate a deregulation at the protein level, with resulting increases in cath D activity. Immunogold labeling of cath D showed particle concentration in lysosomes or phagosomes in both normal follicles and papillary carcinoma cells, indicating that cath D localization was not altered by malignant transformation in human thyroid cells. TSH induced cath D synthesis and secretion in extracellular fluid of normal human thyroid cells in primary culture; TSH had little effect on intracellular cath D level. In conclusion, TSH-induced cath D synthesis may explain high cath D levels in Graves' disease tissues and toxic adenomas, because these tissues possess a permanently stimulated cAMP transduction pathway. Furthermore, the overexpression of cath D in thyroid carcinomas in comparison with normal controls adds further arguments for the potential role of cath D in tumor growth and metastasis.

Inhibitions of protein kinases and protein phosphatases have opposite effects on thyrotropin-stimulated cAMP accumulation in human thyroid cells.

We investigated the effects of inhibitions of protein phosphatases and protein kinases on thyrotropin (TSH) stimulation of cAMP accumulation in human thyroid cells. Okadaic acid (OA) and calyculin-A (CL-A), two potent inhibitors of type-1 (PP-1) and type-2A (PP-2A) protein phosphatases, had a biphasic concentration-dependent response on cAMP formation. An inhibitory effect (41.3% and 47.2% inhibition with OA and CL-A) was first observed at 1 microM OA and 10 nM CL-A, followed by a reduction of this effect with OA (24% inhibition) or by a complete reversal of inhibition with CL-A, at 10-fold higher concentrations of both products. Addition of purified PP-1 and PP-2A to crude membranes from cells preincubated with OA, reversed OA-induced adenylyl cyclase inhibition, confirming that these protein phosphatases regulate TSH-mediated cAMP production. Levels of protein incorporation of 32P were higher with 10 microM OA than with 1 microM OA and did not correlate with the biphasic effect of OA on cAMP production. These results support a dual action of protein phosphorylation in the control of adenylyl cyclase activity stimulated by TSH. H-7, an inhibitor of nucleotide- and calcium/phospholipid-dependent protein kinase (PKC), increased by 197% the stimulation of cAMP accumulation by TSH in thyroid cells. Phorbol 12-myristate 13-acetate (PMA) counteracted the effect of H-7 on cAMP levels, which suggests that PKC is involved in the action of H-7. Moreover, KT5926, an inhibitor of calcium/calmodulin-dependent protein kinase II and myosin light chain kinase, increased basal cAMP levels rather than cAMP levels stimulated by TSH. In light of these results, we suggest that phosphorylation/dephosphorylation cycles regulate basal and TSH-stimulated adenylyl cyclase activities in human thyroid.

[Immunoenzymatic assays of c-erbB-2 oncoprotein and epidermal growth factor receptor in breast cancer: correlation with clinical and biological parameters]. / Dosages immunoenzymatiques de l'oncoprotéine c-erbB-2 et du récepteur du facteur de croissance de l'épiderme dans le cancer du sein: corrélation avec les paramètres cliniques et biologiques.

Two new immunoenzymatic assays for c-erbB-2 oncoprotein and epidermal growth factor receptor (EGF-R) (Oncogene Science) in human breast cancer were validated. Correlations between these assays and some clinical and biological parameters were also studied. The repeatability and reproducibility of standard curves for the two methods gave a coefficient of variation (CV) of less than 4% and about 10% respectively. The accuracy of c-erbB-2 oncoprotein and EGF-R assays was examined by using dilution and recovery tests throughout the standard curves. The linear relations between theoretical and measured values, for these tests, had slopes close to 1 and an intercept near 0. The median value for EGF-R, measured on solubilized membranes of 290 primary tumors, was 0.12 fmol/micrograms protein, the mean value was 0.37 (range 0 to 35.7). For c-erbB-2 oncoprotein, the median value, measured using the same population, was 2.75 human neu unit/micrograms protein, the mean value was 7.85 (range 1 to 125). There was an inverse relationship between EGF-R values and those for the estrogen receptor (ER), progesterone receptor and pS2 protein as well as menopausal status. C-erbB-2 oncoprotein concentrations were positively correlated with ER, pS2 protein and cathepsin D. Furthermore, a significant positive correlation was observed between EGF-R levels and c-erbB-2 oncoprotein levels. In conclusion, immunoenzymatic assays of EGF-R and c-erbB-2 oncoprotein are easy to use, sensitive and reliable. The accurate standardisation of immunoenzymatic assays could contribute to the clinical use of EGF-R and c-erbB-2 oncoprotein as prognostic factors in breast cancer.

Cathepsin D in normal and neoplastic thyroid tissues.

BACKGROUND: Cathepsin D is a widely distributed lysosomal acidic endopeptidase. It is an estrogen-regulated protein that is a prognostic factor in breast cancer. The aim of this study was to measure cathepsin D concentrations in thyroid tissues and to correlate these concentrations with clinical and pathologic parameters. METHODS: Cathepsin D and thyroglobulin concentrations were measured in the cytosol of normal thyroid tissues (n = 14), benign nodules (n = 6), and thyroid carcinomas (n = 32) with an immunoradiometric assay. Statistical analysis was based on the Kruskal-Wallis and Wilcoxon tests and on the Spearman rank correlation coefficient. RESULTS: The mean level of cathepsin D, expressed as picomoles per milligram protein minus thyroglobulin, was higher in the 32 carcinomas, 29.1 +/- 15.5, than in the 14 normal thyroid tissues, 8.4 +/- 2.5 (p < 0.001) or in the 6 benign nodules, 11.2 +/- 7.3 (p = 0.003). Cathepsin D concentrations correlated with tumor size; Spearman rank correlation coefficient was rs = 0.44 (p = 0.012). No significant difference was found regarding histologic type. Cathepsin D concentrations were inversely correlated with the thyroglobulin level in the tumor; Spearman rank correlation coefficient was rs = -0.60 (p < 0.001). CONCLUSIONS: Cathepsin D concentration is higher in thyroid carcinoma than in normal thyroid tissue. Increased cathepsin D concentrations correlate with thyroid tumor size but not with histologic type. Further studies should be done to confirm the potential prognostic value of cathepsin D in patients with thyroid carcinomas.

Estrogen receptors and cathepsin D in human thyroid tissue.

BACKGROUND: To investigate the significance of estrogen receptors (ER) in the pathogenesis of thyroid dysplasia, the authors analyzed, by analogy with breast cancers, ER and three estrogen-regulated proteins: progesterone receptor (PR), cathepsin D, and pS2 protein, in cytosols of 42 human thyroid tissues. METHODS: ER and PR were measured by an immunoenzymatic assay and cathepsin D and pS2 by an immunoradiometric assay. Tissue specimens included 7 normal tissues, 6 benign nodules, 8 toxic adenomas, 7 from patients with Graves disease, and 14 carcinomas. RESULTS: ER was present at very low concentrations, with no statistical difference between neoplastic and nonneoplastic tissues. The mean levels of cathepsin D, expressed as pmol/mg protein minus thyroglobulin, were higher in the 14 carcinomas (P = 0.0003), the 7 specimens from patients with Graves disease (P = 0.006), and the 8 toxic adenomas (P = 0.04) than in the 7 normal thyroid tissues. A significant difference also was observed between the carcinomas (P = 0.003) and six benign nodules. Compared to TNM parameters, cathepsin D concentrations correlated with tumor size: higher cathepsin D levels were found in pT4 than in pT2 and pT3 carcinomas. All the tissues tested were negative for PR and pS2 protein. CONCLUSIONS: The results clearly indicate a significant difference between neoplastic and normal thyroid tissue in terms of the amount of cathepsin D, but not that of ER. This suggests that cathepsin D probably is not regulated by estrogen but simply is a marker of protease activity during invasion by thyroid carcinomas.

Stabilization of phospho-intermediates of rat liver plasma membrane alkaline phosphatase by uncompetitive inhibition. Relation with phosphate uptake into hepatocytes.

Rat liver plasma membrane alkaline phosphatase (ALP) phospho-intermediates, which have molecular masses of 151 and 135 kDa bands, were labelled at physiological pH with either (gamma-32P) ATP or 32Pi. This labeling was stabilized by a potent enzyme inhibitor, bromolevamisole (BL), and not by bromodexamisole (BD). BL augmented the rate and extent of autophosphorylation and slowed down the rate of autodephosphorylation of ALP. The phospho-intermediates labeling presented nearly the same kinetic behaviour with either (gamma-32P) ATP or 32Pi. In the presence of BL a marked decrease of the phosphorylation state of many proteins was observed in hepatocytes. BL also produced a decrease of the 32Pi uptake into hepatocytes and a decrease of the specific radioactivity of cellular ATP. BD had nearly the same effect as BL on protein phosphorylation and 32Pi uptake. These results argued against a direct involvement of ALP in Pi transport across hepatocyte plasma membrane.

Analysis and prevention of recurrent goiter.

The current study was done to analyze our experience with recurrent goiter. Prevention must be stressed because reoperations of the thyroid gland present technical difficulties and are associated with an increased risk of hypoparathyroidism and permanent hoarseness. Nodular recurrences occurred in 36 of 1,456 patients (2.5 percent) who underwent thyroidectomy between 1968 and 1983. All patients had the initial operation at Jean Bernard Hospital, Poitiers, France, and had follow-up evaluation from five to 20 years. Multinodular goiter accounted for 70 percent of the recurrences. Sixty percent of the recurrences were in patients with multinodular goiters. Recurrent goiter was usually first detected about eight years after thyroidectomy. Thirty patients with recurrence had reoperations. Two patients had paralysis of the vocal cord and one patient had permanent hypoparathyroidism. Recurrent goiter may occur because of the development of new nodules (true recurrence) or because of the growth of "residual" or persistent macroscopic or microscopic nodules left at the previous thyroid operation. Intraoperative digital palpation of the entire thyroid gland is essential for detecting residual macroscopic thyroid nodules, and all enlarged nodules should be removed. Thyroid-stimulating hormone (TSH) suppressive therapy is recommended by some authorities to prevent "true" recurrences, although its efficacy is debated. Since recurrence is uncommon in the current series, perhaps TSH suppressive therapy should only be used in high-risk patients. In the current experience, only the multinodular character of the nodules in euthyroid patients has a significant correlation with subsequent development of recurrent goiter (p < 0.01), and one must consider patients with multinodular goiter at risk for recurrence. Once TSH treatment is begun, it will logically be continued for life. Total thyroidectomy has been recommended by some endocrine surgeons for treating patients with multinodular goiter. We prefer subtotal thyroidectomy and reserve total thyroidectomy for patients when no normal thyroid tissue can be preserved because only 2.5 percent of the patients in the current study had recurrent goiter. Prevention of residual nodules is probably best assured by systematic palpation during operation of the two thyroid lobes. This considerably lessens the risk of recurrence. Since nodular recurrences occurred in only 2.5 percent of the patients in the current study, although multinodular goiter must be considered at risk for recurrence, we do not recommend systematic total thyroidectomy in multinodular goiter.

Effect of bromolevamisole and other imidazo [2,1-b] thiazole derivatives on adenylate cyclase activity.

We studied the effect of bromolevamisole (BL) and other imidazo [2,1-b] thiazole derivatives--bromodexamisole (BD) and levamisole (LV)--on adenylate cyclase (AC) activity. BL and BD both inhibited forskolin-activated human thyroid AC, while LV had no effect. This inhibition was non-stereospecific and the IC50 values, as measured with 1 mM ATP and 40 microM forskolin, were 0.95 and 0.80 mM for BL and BD, respectively. In contrast, human thyroid alkaline phosphatase (ALP) inhibition was stereospecific, with IC50 values of 0.0012 mM for BL and 0.9 mM for BD. LV was a 10-fold weaker inhibitor of ALP than BL. These results show that ALP inhibition is not correlated with forskolin-activated AC inhibition. Furthermore, in the presence of a competitive inhibitor of GTP (0.1 mM guanosine 5'-O-(2-thiodiphosphate), BL retained its antagonizing effect on forskolin-activated AC which suggests a direct action on the catalytic subunit. The inhibition was of the mixed type, indicating a complex interaction between BL and AC. Glucagon-activated AC activity in rat liver membranes was also inhibited by BL, although to a slightly lesser degree than thyroid stimulating hormone (TSH)-activated AC from human thyroid for a given BL concentration. In cultured human thyroid cells, BL (0.25 mM) induced a potent decrease in cAMP accumulation after 2 hr of stimulation by TSH. Taken together, these results show that BL inhibits AC and that this inhibition is not organ-specific.

Comparative measurement of progesterone receptors in breast cancer by biochemical and immunoenzymatic assays.

The measurement of progesterone receptors (PR) by enzyme immunoassay (Abbott Laboratories, EIA monoclonal) and biochemical assay using a tritiated ligand (promegestone, R5020) was studied and compared by using the statistical method of Passing and Bablok. In order to improve the reliability of the biochemical method, data were analysed using a hyperbolic model which avoids the need to determine non-specific binding experimentally. The comparison of hyperbolic (Y) and Scatchard (X) plots gave a regression curve of Y = 0.93 x + 1.34 fmol/mg of protein. Cytosols from 70 human breast cancers homogenized in the absence of KCl were assayed for PR by both the EIA (Y) and biochemical (X) methods. The linear regression obtained gave Y = 1.21 X + 1.97 fmol/mg of protein. In the presence of 0.4 M KCl-Tris buffer, the corresponding result for 80 human breast cancers was Y = 3.11 X + 1.91 fmol/mg of protein. The slopes of the two regression lines obtained in the presence or absence of KCl were significantly different. Results of the biochemical and EIA methods were similar in the absence of KCl, whereas EIA gave higher values when KCl was used. This discrepancy probably stem from the methodological differences between the two methods: the biochemical assay measures active steroid binding sites while EIA measures antigenic activity. The authors conclude that clinical studies are required before using high-salt extraction buffer in routine PR determination by the EIA method; this will result in improvements in the determination of the hormone dependence and/or the prognosis of human breast cancers.

Enhancement of mitogen-induced lymphocyte proliferation by some inhibitors of alkaline phosphatase and diamine oxidase.

We selected various compounds [bromolevamisole, levamisole, cimetidine, L-homoarginine, 2,3,5,6-tetrahydroimidazo-(2,1-b)thiazole (IT), imidazole, theophylline] previously reported as inhibitors of alkaline phosphatase (ALP) and/or diamine oxidase (DAO) and studied their activity on concanavalin A (ConA)-induced mouse spleen cell lymphocyte proliferation. According to the Ki values, the decreasing order of potency for ALP inhibition was: bromolevamisole, levamisole, theophylline, cimetidine, IT, imidazole and L-homoarginine. The order of potency was different for DAO inhibition. Cimetidine was the most potent inhibitor of DAO, followed by bromolevamisole, levamisole, IT, imidazole and L-homoarginine. Theophylline had no inhibitory effect on DAO. We show that these compounds, except theophylline, enhance ConA-induced lymphocyte proliferation. Similarly, all the compounds except imidazole and theophylline, significantly inhibited ALP at concentrations which enhanced lymphocyte proliferation as measured by (3H)-thymidine uptake. DAO inhibition correlated with DNA synthesis only for IT and cimetidine. These observations suggest that ALP and DAO play a negative role in the proliferation process; however, the degree of enhancement of ConA-induced proliferation did not correlate strictly with the degree of ALP and DAO inhibition.

Comparative inhibition of human alkaline phosphatase and diamine oxidase by bromo-levamisole, cimetidine and various derivatives.

Analogues of bromo-levamisole and guanidine derivatives including cimetidine are examined in vitro in order to investigate their comparative inhibition, towards alkaline phosphatase (ALP) from human liver and diamine-oxidase (DAO) from human placenta. Bromo-levamisole, considered as a potent selective uncompetitive inhibitor of ALP (Ki, 2.8.10(-6) M at pH 10.5) is shown to be a noncompetitive inhibitor of DAO (Ki = 7.10(-4) M). According to the structure-inhibition relationship, the imidazole ring is important for ALP and DAO inhibition. The phenyl ring of bromo-levamisole is required for ALP inhibition but not for DAO inhibition, which is mediated mainly by aminoguanidine or guanidine groups. These results have allowed the selection of cimetidine, an H2-antagonist but also an immunomodulating compound, as inhibitor of these two enzymes. Cimetidine is an uncompetitive inhibitor of ALP (Ki = 3.2.10(-3) M at pH 10.5), and a good inhibitor of DAO (I50 = 3.8.10(-4) M). The Ki of ALP is commonly calculated at pH 10.5, but to study the role of the enzyme at the physiological pH, the inhibition has also been performed at pH 7.4. The Ki values are only slightly affected by this pH variation. So far several compounds, including levamisole, imidazole, theophylline and aminoguanidine are known to possess immunomodulating activities in vivo and/or in vitro and inhibit ALP and/or DAO. Therefore, it seems reasonable to assume that the inhibition of enzymes is involved in the immunomodulating effects of these drugs, when the ranges of active concentrations are similar for these properties.