A mutation in the POU-homeodomain of Pit-1 responsible for combined pituitary hormone deficiency.

Pit-1 is a pituitary-specific transcription factor responsible for pituitary development and hormone expression in mammals. Mutations in the gene encoding Pit-1 have been found in two dwarf mouse strains displaying hypoplasia of growth hormone, prolactin, and thyroid-stimulating, hormone-secreting cell types in the anterior pituitary. A point mutation in this gene was identified on one allele in a patient with combined pituitary hormone deficiency. Mutant Pit-1 binds DNA normally but acts as a dominant inhibitor of Pit-1 action in the pituitary.

Thyrotropin-induced hyperthyroidism caused by selective pituitary resistance to thyroid hormone. A new syndrome of "inappropriate secretion of TSH".

An 18-yr-old woman with clinical and laboratory features of hyperthyroidism had persistently elevated serum levels of immunoreative thyrotropin (TSH). During 11 yr of follow-up there had been no evidence of a pituitary tumor. After thyrotropin-releasing hormone (TRH), there was a marked increase in TSH and secondarily in triiodothyronine (T3), the latter observation confirming the biologic activity of the TSH. Exogenous T3 raised serum T3 and several measurements of peripheral thyroid hormone effect, while decreasing serum TSH, thyroxine (T4), and thyroidal radioiodine uptake. After T3, the TRH-stimulated TSH response was decreased but was still inappropriate for the elevated serum T3 levels. Dexamethasone reduced serum TSH but did not inhibit TRH stimulation of TSH. Propylthiouracil reduced serum T4 and T3 and raised TSH. This patient represents a new syndrome of TSH-induced hyperthyroidism, differing from previous reports in the absence of an obvious pituitary tumor and in the responsiveness of the TSH to TRH stimulation and thyroid hormone suppression. This syndrome appears to be caused by a selective, partial resistance of the pituitary to the action of thyroid hormone. This case is also compared with previous reports in the literature of patients with elevated serum levels of immunoreactive TSH in the presence of elevated total and free thyroid hormones. A classification of these cases, termed "inappropriate secretion of TSH," is proposed.

Ectopic production of the isolated beta subunit of human chorionic gonadotropin.

A material similar to the beta subunit of human chorionic gonadotropin (hCG-beta) was detected in serum (300 ng/ml) and tumor extract from a 75-yr-old man with pancreatic adenosquamous carcinoma. This material was indistinguishable from hCG-beta in three different types of radioimmunoassay that displayed widely varying reactions with glycoprotein trophic hormones and their subunits. In gel chromatography there appeared to be heterogeneity of the serum beta-like immunoactivity, including one component that coeluted with standard hCG-beta tracer and another immunologically indistinguishable component that displayed a slightly lower elution volume. Neither complete human chorionic gonadotropin (hCG) nor its alpha subunit was detected in radioimmunoassays of serum, before or after fractionation, or in tumor extract. The absence of complete hCG was confirmed in a gonadotropin bioassay sensitive to 15 ng of hCG, which showed no bioactivity in serum or tumor extract containing 450 and 90 ng of hCG-beta, respectively. This case probably represents the first demonstration of isolated polypeptide subunit production of ectopic origin and suggests that hCG-beta, as well as other subunits, may prove useful as cancer markers.

Metabolic clearance and production rates of human thyrotropin.

Metabolic clearance (MCR) and production rates (PR) of human thyrotropin (hTSH) were determined by the constant infusion to equilibrium method 57 times in 55 patients. 16 control patients had a mean hTSH MCR of 50.7 ml/min. The mean hTSH MCR was significantly (P < 0.02) higher in 19 euthyroid men (51.6 ml/min) than in 12 euthyroid women (43.0 ml/min), but this apparent sex difference disappeared when the MCR were corrected for surface area, 25.8 (men) versus 25.2 ml/min per m(2) (women). Hypothyroid patients had significantly (P < 0.005) lower hTSH MCR (30.9 ml/min), and hyperthyroid patients had significantly (P < 0.05) higher hTSH MCR (60.9 ml/min) than controls. The hTSH MCR in patients with "decreased thyroid reserve" (40.9 ml/min), hyperfunctioning thyroid nodule (53.8 ml/min), and "empty sella syndrome" (46.6 ml/min) were not significantly different from controls. The mean hTSH PR in controls (104.3 mU/day) was significantly (P < 0.005) different from that in patients with "decreased thyroid reserve" (956 mU/day), hypothyroidism (4,440 mU/day), hyperthyroidism (< 43.9 mU/day) and a hyperfunctioning thyroid nodule (< 38.7 mU/day). In primary hypothyroidism intravenous triiodothyronine therapy (50 mug/day) for 10 days decreased the hTSH PR (from 4,244 to 2,461 mU/day) before changes in the hTSH MCR (from 33.1 to 33.7 mU/day) were observed. These studies have demonstrated that changes in the serum concentration of hTSH are mainly due to altered pituitary hTSH secretion with only a minor contribution from the change in hTSH MCR.

Differences between purified ectopic and normal alpha subnits of human glycoprotein hormones.

"Ectopic" proteins, not distinguished immunologically from the common alpha subunit of the human glycoprotein hormones, were purified approximately 10,000-fold from a gastric carcinoid tumor (A.L.-alpha) and from tissue culture medium of bronchogenic carcinoma cell lines (ChaGo-alpha). The purified A.L.-alpha was homogeneous by sodium dodecyl sulfate (SDS) gel electrophoresis while the purified ChaGo-alpha showed multiple components, some of which represented aggregated species. In SDS gel electrophoresis, the apparent molecular weights of A.L.-alpha (15,000) and dithioerythritol-reduced ChaGo-alpha (13,000) were significantly lower than those of the alpha subunits of human chorionic gonadotropin (hCG-alpha), luteinizing hormone, follicle-stimulating hormone, or thyroid-stimulating hormone (22,000-23,000). Binding experiments with [35S]-SDS suggested that these apparent differences in molecular weight resulted, at least in part, from diminished binding of the SDS by the normal compared to the ectopic alpha subunits. In gel chromatography, the apparent molecular weights of A.L.-alpha (27,000) and ChaGo-alpha (30,000) were slightly higher than those of normal alpha subunits (23,000-24,000). Both A.L.-alpha and ChaGo-alpha were not distinguished from hCG-alpha in ion-exchange chromatography. The composition of A.L.-alpha was similar to that of hCG-alpha in 13 amino acids but showed decreased phenylalanine and increased valine; glucosamine was identified in both A.L.-alpha and hCG-alpha. Under conditions in which hCG-alpha combined with the hCG beta subunit (hCG-beta) to produce 95% of the expected gonadotropin-binding activity in a rat testis radioreceptor-assay, A.L.-alpha incubation with hCG-beta resulted in only 2% of the expected activity, and ChaGo-alpha incubation with hCG-beta produced no detectable activity. These characteristics of ectopic alpha subunits may reflect abnormalities of neoplastic protein synthesis or carbohydrate attachment which result in polypeptides with chemical and immunologic similarity to normal subunits but with differences in physical and combining properties; alternatively, the ectopic subunits may represent as yet unrecognized alpha precursor forms.

Partial target organ resistance to thyroid hormone.

An 8-year old boy with a small goiter, normal basal metabolic rate (BMR), and elevated serum thyroid hormone levels (thyroxine [T(4)] 19.5 mug per 100 ml, free T(4) 4 ng per 100 ml, triiodothyronine [T(3)] 505 ng per 100 ml) was studied. He had measurable serum thyroid-stimulating hormone (TSH) levels (average 5.5 muU per ml), and the thyroxine-binding proteins, hearing, and epiphyseal structures were normal. There was no parental consanguinity nor were there thyroid abnormalities either in the parents or six siblings.Methimazole, 50 mg daily, depressed thyroxine synthesis (T(4) 10.5, free T(4) 2.5) and caused a rise in TSH to 11 muU per ml. After discontinuation of treatment, TSH declined to 4.2 muU per ml and chemical hyperthyroidism returned (T(4) 21.0 mug per 100 ml, free T(4) 4.2, and total T(3) 475 ng per 100 ml, radioactive iodine [RAI] uptake 68%), but studies of BMR and insensible water loss showed the patient to be clinically euthyroid. Thyrotropin-releasing hormone (TRH), 200 mug i.v., caused a brisk rise in TSH to 28 muU per ml, with T(4) rising to 28 mug per 100 ml, free T(4) to 5.6, and T(3) to 730 ng per 100 ml, thus indicating that the pituitary-thyroid system was intact and that the patient's TSH was biologically active. The unusual sensitivity of the pituitary cells to TRH in spite of the markedly elevated serum thyroid hormone levels also suggested that the pituitary was insensitive to suppression by T(3) or T(4). Serum dilution studies gave immunochemical evidence that this patient's TSH was normal. Neither propranolol, 60 mg, chlorpromazine, 30 mg, nor prednisone, 15 mg daily, influenced thyroid indices. Steroid treatment, however, suppressed the pituitary response to TRH, T(3) in doses increased over a period of 12 days to as much as 150 mug daily caused a rise in serum T(3) to above 800 ng per 100 ml, a decline of T(4) to euthyroid levels (T(4) 9.5 mug per 100 ml, free T(4) 1.6 ng per 100 ml), suppression of the RAI uptake from 68% to 35%, and marked blunting of the responses to TRH, but the BMR and insensible water loss remained normal. The data suggest that the patient's disorder is due to partial resistance to thyroid hormone.

Differential expression of mutant and normal beta T3 receptor alleles in kindreds with generalized resistance to thyroid hormone.

Thyroid hormone resistance (THR) is primarily an autosomal dominant inherited disease characterized by resistance of pituitary and peripheral tissues to the action of thyroid hormone. We investigated whether the heterogeneous phenotypic features that occur not only among kindreds but also within the same kindred might be due to the expression of differing ratios of mutant and normal receptors in tissues. Using an allele-specific primer extension method, we determined the relative expression of normal and mutant mRNAs from the fibroblasts of affected and unaffected members of two kindreds with TRH: A-H and N-N. While two affected members of A-H, as expected, had nearly equal amounts of normal and mutant hTR beta mRNA, two other members had mutant mRNA levels that accounted for at least 70% of the hTR beta mRNA. Phenotypic variability within and between kindreds with generalized resistance to thyroid hormone GRTH may be due to this differential expression of the mutant and wild type mRNA. Furthermore, when several clinical parameters of THR were compared in several affected members from two kindreds with GRTH, we found that two cases in one kindred exhibited a high mutant-to-normal hTR beta ratio and had considerably more bone resistance during their development. In certain kindreds with THR, differing ratios of normal and mutant hTR receptors may be age and growth related and may account for the reported attenuation of phenotypic symptoms with age.

Role of a pituitary-specific transcription factor (pit-1/GHF-1) or a closely related protein in cAMP regulation of human thyrotropin-beta subunit gene expression.

cAMP regulation of the human thyrotropin-beta (TSH beta) gene cAMP was studied in two heterologous cell lines, a human embryonal kidney cell line (293) and a rat pituitary cell line (GH3). In 293 cells, human TSH beta gene expression was not stimulated by the adenylate cyclase activator forskolin or the cAMP analogue 8-bromo-cAMP (8-Br-cAMP). On the other hand, these agents induced human TSH beta gene expression 4-12-fold in GH3 cells. Deletion analysis demonstrated that the regions from +3 to +8 bp and from -128 to -61 bp were both necessary for cAMP stimulation. The latter region contains three DNA sequences homologous to a pituitary-specific transcription factor, Pit-1/GHF-1, DNA-binding site. Gel-mobility assays demonstrated that a radiolabeled human TSH beta probe (-128 to -61 bp) formed five specific DNA-protein complexes with mouse thyrotropic tumor (MTT) nuclear extract and two specific complexes with in vitro translated Pit-1/GHF-1. Four of the five MTT complexes and both in vitro Pit-1/GHF-1 complexes were reduced or eliminated by excess of an unlabeled Pit-1/GHF-1 DNA-binding site from the rat growth hormone gene, but not a mutated version of the same DNA fragment, suggesting that Pit-1/GHF-1 or a closely related thyrotroph protein binds to these DNA sequences. In 293 cells, co-transfection of an expression vector containing the Pit-1/GHF-1 cDNA restored cAMP-responsiveness to the human TSH beta promoter (5.2- and 6.6-fold maximal stimulation by 8-Br-cAMP and forskolin, respectively) but not the herpes virus thymidine kinase promoter (1.2-fold maximal stimulation by either agent). Thus we conclude that the human TSH beta gene is positively regulated by cAMP in GH3 but not 293 cells. Since the human TSH beta gene contains at least one high-affinity binding site for Pit-1/GHF-1 in a region necessary for cAMP stimulation and cAMP stimulation could be restored to the human TSH beta promoter in a previously nonresponsive cell line by the addition of Pit-1/GHF-1, this suggests that Pit-1/GHF-1, or a closely related protein in the thyrotroph, may be a trans-acting factor for cAMP stimulation of the TSH beta gene.

Intrinsic bioactivity of thyrotropin in human serum is inversely correlated with thyroid hormone concentrations. Application of a new bioassay using the FRTL-5 rat thyroid cell strain.

We have developed a new bioassay for thyrotropin (TSH) in human serum to evaluate bioactivity in normal individuals and patients with different degrees of primary hypothyroidism. Unpurified TSH in serum showed no stimulation of cyclic AMP production in cultured FRTL-5 rat thyroid cells, but after immunopurification showed potent stimulatory activity. Immunoaffinity purification permitted up to 400-fold concentration of serum TSH, allowing bioactivity measurements even in certain normal sera. The limit of detection in the FRTL-5 bioassay was 10 microU of human TSH per 0.5 ml incubate, and half-maximal responses for standard human TSH was 102 +/- 26 (+/- SE) microU/0.5 ml. Immunoaffinity-purified serum TSH varied in bioactivity-to-immunoactivity (B/I) ratios from less than 0.25 to 1.21 among four euthyroid subjects and eight primary hypothyroid patients. An inverse correlation was found between B/I ratios of immunopurified basal TSH and the serum-free T4 (r = -0.7237, P less than 0.01), T4 (r = -0.6650, P less than 0.05), and T3 (r = -0.6382, P less than 0.05). B/I ratios of immunopurified TSH from three hypothyroid patients before and after acute stimulation by thyrotropin-releasing hormone showed no significant change, despite major changes in serum TSH. In summary, the present study shows an inverse relationship between the metabolic status of an individual and the intrinsic bioactivity of TSH.

Suppression of pituitary TSH secretion in the patient with a hyperfunctioning thyroid nodule.

10 patients with a single hyperfunctioning thyroid nodule each were studied for pituitary thyrotropin (TSH) suppression. They were judged to be euthyroid on clinical grounds. The total thyroxine (T(4)D), free thyroxine (FT(4)), total triiodothyronine (T(3)D), and free triiodothyronine (FT(3)) were normal in most of the patients. Incorporation of (131)I into the hyperfunctioning thyroid nodules was not suppressed by the administration of physiological doses of T(3). Basal serum TSH concentrations were undetectable (<0.5 - 1.0 muU/ml) in all patients. The metabolic clearance of TSH in one patient before and after excision of the thyroid nodule was unchanged (40 vs. 42 ml/min) whereas the calculated production rate was undetectable before the operation (<29 mU/day) and normal after (103 mU/day). These data, in one patient, suggest that the undetectable concentration of TSH in these patients is a result of suppressed TSH secretion rather than accelerated TSH clearance. In eight patients, basal serum TSH concentrations failed to increase after the intravenous administration of 200 mug of thyrotropin-releasing hormone (TRH); minimal increases in serum TSH concentrations were observed in two patients. The suppression of TSH was evident despite "normal" concentrations of circulating thyroid hormones. The observation that normal serum concentrations of T(4)D, FT(4), T(3)D, and FT(3) may be associated with undetectable basal serum TSH concentrations and suppressed TSH response to TRH was also found in four hypothyroid patients given increasing doses of L-thyroxine and sequential TRH stimulation tests.

Characterization of seven novel mutations of the c-erbA beta gene in unrelated kindreds with generalized thyroid hormone resistance. Evidence for two "hot spot" regions of the ligand binding domain.

Genetic analysis in our laboratory of families with generalized thyroid hormone resistance (GTHR) has demonstrated tight linkage with a locus, c-erbA beta, encoding a nuclear T3 receptor. Three point mutations and two deletions in this locus have previously been reported in affected individuals in unrelated families as potential molecular bases for this disorder. In the present study, we have used direct sequencing of polymerase chain reaction-amplified exons of the c-erbA beta gene to rapidly identify novel point mutations from seven previously uncharacterized kindreds with GTHR. Six single base substitutions and one single base insertion were identified and found to be clustered in two regions of exons 9 and 10 in the ligand binding domain of the receptor: in the distal ligand-binding subdomain L2 and across the juncture of the taui and dimerization subdomains. Reduction of T3-binding affinity in each of four mutations tested as well as segregation of all mutations to clinically affected individuals strongly supports the hypothesis that these changes are the cause of GTHR in these kindreds. In view of the diversity of clinical phenotypes manifested, the distinct topographic clustering of the mutations provides an invaluable genetic tool for the molecular dissection of thyroid receptor function.

Metabolic clearance and secretion rates of subunits of human thyrotropin.

Metabolic clearance rates (MCR) of the alpha and beta subunits of human thyrotropin (hTSH-alpha and hTSH-beta) were determined by a constant infusion to equilibrium method. In 15 normal individuals (six men, six premenopausal women, and three post-menopausal women), the mean MCR of hTSH-alpha (68 ml/min per m2) was significantly faster than that of hTSH-beta (48 ml/min per m2) was significantly faster than that of hTSH-beta (48 ml/min per m2); both were two to three times more rapid than the previously determined MCR of hTSH. In patients with primary hypothyroidism, MCR were significantly slower with a mean value of 55 ml/min per m2 for hTSH-alpha and 37 ml/min per m2 for hTSH-beta. However, MCR of subunits were not significantly faster than normal in hyperthyroid patients. Serum concentrations of alpha subunits and hTSH-beta were measured by radioimmunoassay, and secretion rates of alpha and hTSH-beta from the pituitary were calculated using hTSH-alpha and hTSH-beta MCR, respectively. In the normal individuals, alpha secretion rates averaged 91 mug/day per m2, greater than those previously determined for hTSH and human follicle-stimulating hormone. Alpha secretion rates were significantly elevated in the normal postmenopausal women (211 mug/day per m2) and in the premenopausal hypothyroid women (202 mug/day per m2); they were also elevated in the postmenopausal hypothyroid women (277 mug/day per m2). Alpha secretion rates were significantly decreased in the premenopausal hyperthyroid women (66 mug/day per m2). Usually, the secretion rates of hTSH-beta could not be calculated in normal individuals, and the rates in hyperthyroid patients could never be calculated because serum hTSH-beta was not detected. Six normals had detectable hTSH-beta secretion rates (17 mug/day per m2); hTSH-beta secretion rates were significantly increased in patients with primary hypothyroidism (28 mug/day per m2). Although we had previously demonstrated a 50-fold increase in hTSH secretion rates in primary hypothyroidism, there was only a 2-fold increase in alpha and hTSH-beta secretion rates. Thus, increased subunit synthesis appears to be utilized predominantly for production of complete hTSH.

Evidence for direct estrogen regulation of the human gonadotropin-releasing hormone gene.

This study is an attempt to determine whether estrogen could directly regulate human gonadotropin-releasing hormone (GnRH) gene expression. Human GnRH expression vectors were constructed by fusing various 5' flanking regions of the human GnRH gene upstream of the luciferase reporter gene (LUC) or the thymidine kinase promoter linked to the chloramphenicol acetyltransferase reporter gene (CAT). These constructs were transiently transfected into a human choriocarcinoma cell line (JEG-3) and LUC or CAT activity was measured after either no treatment or treatment with various concentrations of estradiol. A stimulatory estrogen response element (ERE) was localized to a 32-bp region between -547 and -516 bp. To determine whether estrogen receptor bound to this region of the gene, we performed DNase I footprinting using purified calf uterine estrogen receptor. DNase I footprinting demonstrates a strong footprint between -567 and -514 bp of the human GnRH gene. In addition, an avidin-biotin complex DNA-binding assay demonstrated that a biotinylated DNA fragment containing -541 to -517 bp of the human GnRH gene bound 35S-labeled estrogen receptor as well as a biotinylated DNA fragment containing the xenopus vitellogenin ERE. On the other hand, the negative control biotinylated DNA fragment derived from adenovirus 5 bound insignificant amounts of 35S-labeled estrogen receptor. Both the GnRH ERE and vitellogenin ERE bound 35S-labeled estrogen receptor with high affinity (approximately 1 nM). These data indicate that the human GnRH gene contains an ERE sufficient to mediate a stimulatory response to estrogen in heterologous cells. Based upon these data we hypothesize that the human GnRH gene might also be directly regulated by estrogen in the hypothalamus, and that this regulation may explain the GnRH hypersecretion observed at the time of the preovulatory luteinizing hormone (LH) surge.

Interaction of human beta 1 thyroid hormone receptor and its mutants with DNA and retinoid X receptor beta. T3 response element-dependent dominant negative potency.

Mutations in the human beta thyroid hormone receptor (h-TR beta) gene are associated with the syndrome of generalized resistance to thyroid hormone. We investigated the interaction of three h-TR beta 1 mutants representing different types of functional impairment (kindreds ED, OK, and PV) with different response elements for 3,3',5-triiodothyronine (T3) and with retinoid X receptor beta (RXR beta). The mutant receptors showed an increased tendency to form homodimers on a palindromic T3-response element (TREpal), a direct repeat (DR + 4), and an inverted palindrome (TRElap). On TRElap, wild type TR binding was decreased by T3, while the mutant receptors showed a variably decreased degree of dissociation from TRElap in response to T3. The extent of dissociation was proportional to their T3 binding affinities. RXR beta induced the formation of h-TR beta 1:RXR beta heterodimers equally well for mutants and the wild type h-TR beta 1 on these T3 response elements. However, the T3-dependent increase in heterodimerization with RXR beta was absent or reduced for the mutant TRs. Transient transfection studies indicated that the dominant negative potency was several-fold more pronounced on the TRElap as compared to TREpal or DR + 4. In CV-1 and HeLa cells, transfection of RXR beta could not reverse the dominant negative action. These results demonstrate that the binding of mutant h-TRs to DNA, as well as their dominant negative potency, are TRE dependent. In addition, competition for DNA binding, rather than for limiting amounts of RXR beta, is likely to mediate the dominant negative action.

A base mutation of the C-erbA beta thyroid hormone receptor in a kindred with generalized thyroid hormone resistance. Molecular heterogeneity in two other kindreds.

Generalized thyroid hormone resistance (GTHR) is a disorder of thyroid hormone action that we have previously shown to be tightly linked to one of the two thyroid hormone receptor genes, c-erbA beta, in a single kindred, A. We now show that in two other kindreds, B and D, with differing phenotypes, there is also linkage between c-erbA beta and GTHR. The combined maximum logarithm of the odds score for all three kindreds at a recombination fraction of 0 was 5.77. In vivo studies had shown a triiodothyronine (T3)-binding affinity abnormality in nuclear receptors of kindred A, and we therefore investigated the defect in c-erbA beta in this kindred by sequencing a major portion of the T3-binding domain in the 3'-region of fibroblast c-erbA beta cDNA and leukocyte c-erbA beta genomic DNA. A base substitution, cytosine to adenine, was found at cDNA position 1643 which altered the proline codon at position 448 to a histidine. By allelic-specific hybridization, this base substitution was found in only one allele of seven affected members, and not found in 10 unaffected members of kindred A, as expected for a dominant disease. Also, this altered base was not found in kindreds B or D, or in 92 random c-erbA beta alleles. These results and the fact that the mutation is predicted to alter the secondary structure of the crucial T3-binding domain of the c-erbA beta receptor suggest this mutation is an excellent candidate for the genetic cause of GTHR in kindred A. Different mutations in the c-erbA beta gene are likely responsible for the variant phenotypes of thyroid hormone resistance in kindreds B and D.

A soluble transcription factor, Oct-1, is also found in the insoluble nuclear matrix and possesses silencing activity in its alanine-rich domain.

Expression of the human thyrotropin beta (hTSHbeta) gene is restricted to thyrotrophs, at least in part, by silencing. Using transient-transfection assays, we have localized a silencer element to a region between -128 and -480 bp upstream of the transcription initiation site. The silencing activity was overcome in a thyrotroph-specific manner by an unknown enhancer located in the sequences at -approximately 10000 to -1200 bp. The ubiquitous POU homeodomain protein Oct-1 recognized the A/T-rich silencer element at multiple sites in gel mobility shift assays and in vitro footprinting analyses. The silencing activity of Oct-1 was localized in its C-terminal alanine-rich domain, suggesting that Oct-1 plays a role in silencing of the hTSHbeta promoter. Further, a significant fraction of Oct-1 was shown to be associated with the nuclear matrix, and the hTSHbeta silencer region was tethered to a nuclear matrix of human cells in vivo, suggesting a possible role of the Oct-1-hTSHbeta silencer region interaction in chromatin organization.

A rational design strategy for protein hormone superagonists.

By combining evolutionary considerations, sequence comparisons and homology modeling we have designed recombinant human thyroid-stimulating hormone (hTSH) analogs with increased receptor binding and activity. The introduction of seven basic residues into the peripheral loops of hTSH resulted in up to a 50,000-fold increase in receptor binding affinity and 1300-fold increase in intrinsic activity. Such analogs are not only of potential clinical interest but can be tools to explore molecular aspects of conventional as well as nonclassical actions of glycoprotein hormones. These design strategies should be applicable to the development of novel analogs of other related hormones and growth factors with a variety of therapeutic and basic science applications, particularly for proteins that have undergone evolutionary decrease in bioactivity.

Engineering human glycoprotein hormone superactive analogues.

We report the generation of superactive analogues of human glycoprotein hormones, with potential applications in thyroid and reproductive disorders. Current biological and structural data were used to rationalize mutagenesis. The 11-20 region in the alpha-subunit with a cluster of lysine residues forms a previously unrecognized domain critical for receptor binding and signal transduction, as well as an important motif in the evolution of glycoprotein hormone activities. The gradual elimination of basic residues in the alpha-subunit coincided with the evolutionary divergence of the hominids from the Old World monkeys. By selective reconstitution of certain critical residues present in homologous nonhuman hormones we have developed human thyroid stimulating hormone and chorionic gonadotropin analogues with substantial increases in receptor binding affinity and bioactivity, thus providing a paradigm for the design of novel therapeutic protein analogues.

Influence of chemotherapeutic agents on chorionic gonadotropin-alpha subunit secretion in a human lung cancer cell line (ChaGo): discordance of cytotoxic and secretory effects.

Cells from cultures of ChaGo, a cell line of a human lung cancer that ectopically produces chorionic gonadotropin (hCG) and its alpha subunit (hCG-alpha) were exposed to five different cancer chemotherapeutic agents in vitro in separate experiments (one drug/expt). The control doubling time averaged 4 days, with molar biphasic secretory rates of hCG-alpha ranging from a high of 58.1 to a low of 10.5 pmoles/10(6) cells/24 hours. Drug concentrations were chosen to induce a 30-60% inhibition of cell replication over a period of 8-10 days. Neither methotrexate nor vincristine demonstrated major effects on extracellular hCG-alpha production, but each agent moderately depressed cell number and each produced major inhibition of intracellular protein synthesis. Procarbazine inhibited marker production only in slight excess of inhibition of cell growth and cell protein. Actinomycin D and mechlorethamine, however, had profound effects on inhibition of hCG-alpha production in excess of cell growth. Our results indicated that cancer chemotherapeutic agents have specific and differing effects on cell growth and cell protein on the one hand and marker production on the other. These data suggested a mechanism for certain cases of discordance between hormone production and clinical status.

Ultrastructural concomitants of sodium butyrate-enhanced ectopic production of chorionic gonadotropin and its alpha subunit human bronchogenic carcinoma (ChaGo) cells.

Sodium butyrate treatment of cultures of ChaGo (human lung cancer) cells resulted in increased production of human chorionic gonadotropin (hCG) and its alpha subunit (hCG-alpha) and induced a variety of morphologic changes. Elongation and flattening of cells were seen by light microscopy. Immunocytochemistry with antisera against hCG and against hCG-alpha showed an increase in cells containing stainable hCG-alpha. Scanning electron microscopy demonstrated enhanced adhesion of cells to glass cover slips, with elongation, flattening, and decreased cytoplasmic blebs. Ultrastructural changes were examined by transmission electron microscopy and evaluated quantitatively by an unbiased observer. Significant findings included increases in perinuclear tonofilaments, smooth endoplasmic reticulum vesicles, dense mitochondrial inclusions, and lipid granules, as well as decreases in intercellular desmosomes, free polyribosomes, mitochondrial dense granules, and Golgi complexes. The most notable change, a marked decrease in condensed chromatin clumps, may have reflected a butyrate-induced biochemical modification of chromatin leading to enhanced accessibility of certain genes for transcription.