Iodide sensitizes genetically modified non-small cell lung cancer cells to ionizing radiation.

While external ionizing radiation has been used for treating non-small cell lung cancer (NSCLC), improved efficacy of this modality would be an important advance. Ectopic expression of the sodium iodide symporter (NIS) and thyroperoxidase (TPO) genes in NSCLC cells facilitated concentration of iodide in NSCLC cells, which markedly induced apoptosis in vitro and in vivo. Pre-incubation of the NIS/TPO-modified NSCLC cells in iodide followed by ionizing radiation generates bystander tumoricidal effects and potently enhances tumor cell killing. This iodide-induced bystander effect is associated with enhanced gap junction intercellular communication (GJIC) activity and increased connexin-43 (Cx43) expression. Thus, iodide may serve as an enhancer to markedly improve the efficacy of radiation therapy in combined therapeutic modalities.

Ectopic expression of the thyroperoxidase gene augments radioiodide uptake and retention mediated by the sodium iodide symporter in non-small cell lung cancer.

Radioiodide is an effective therapy for thyroid cancer. This treatment modality exploits the thyroid-specific expression of the sodium iodide symporter (NIS) gene, which allows rapid internalization of iodide into thyroid cells. To test whether a similar treatment strategy could be exploited in nonthyroid malignancies, we transfected non-small cell lung cancer (NSCLC) cell lines with the NIS gene. Although the expression of NIS allowed significant radioiodide uptake in the transfected NSCLC cell lines, rapid radioiodide efflux limited tumor cell killing. Because thyroperoxidase (TPO) catalyzes iodination of proteins and subsequently causes iodide retention within thyroid cells, we hypothesized that coexpression of both NIS and TPO genes would overcome this deficiency. Our results show that transfection of NSCLC cells with both human NIS and TPO genes resulted in an increase in radioiodide uptake and retention and enhanced tumor cell apoptosis. These findings suggest that single gene therapy with only the NIS gene may have limited efficacy because of rapid efflux of radioiodide. In contrast, the combination of NIS and TPO gene transfer, with resulting TPO-mediated organification and intracellular retention of radioiodide, may lead to more effective tumor cell death. Thus, TPO could be used as a therapeutic strategy to enhance the NIS-based radioiodide concentrator gene therapy for locally advanced lung cancer.

Differential regulation of the human sodium/iodide symporter gene promoter in papillary thyroid carcinoma cell lines and normal thyroid cells.

The absence of TSH-stimulated radioiodide uptake in differentiated thyroid cancer is associated with a high recurrence rate and reduced survival. We studied regulation of the sodium/iodide symporter gene in human papillary thyroid cancer cell lines (BHP) and primary human thyroid cells. BHP cells expressed very low levels of sodium/iodide symporter mRNA and did not concentrate iodide, but iodide uptake was restored to levels seen in FRTL-5 rat thyroid cells by stable transfection of a sodium/iodide symporter cDNA. Sodium/iodide symporter gene expression, therefore, was necessary and sufficient for iodide uptake in BHP cells. We cloned the human sodium/iodide symporter gene 5'-flanking region and analyzed progressive 5'-deletions in transient transfections. We identified a region, -596 to -268, essential to confer full promoter activity in primary normal human thyroid cells. Sodium/iodide symporter promoter activity in four BHP cell lines, however, was markedly reduced, consistent with down-regulation of the endogenous sodium/iodide symporter gene. Nuclear extracts from BHP 2-7 cells had reduced or absent binding to regions of the sodium/iodide symporter promoter shown to be critical for expression, compared with nuclear extracts from FRTL-5 cells. Competition studies indicated that these nuclear proteins were not known thyroid transcription factors. Modifications of the sodium/iodide symporter promoter with demethylation or histone acetylation did not increase sodium/iodide symporter expression, and no deletions of the critical regulatory region were identified in the endogenous gene in BHP cells. Regulation of the sodium/iodide symporter 5'-flanking region in transient transfection paralleled endogenous sodium/iodide symporter expression. Reduced expression of potential novel nuclear factor(s) in these cell lines may contribute to reduced sodium/iodide symporter expression resulting in absence of iodide uptake in some papillary thyroid cancers.

Ligands for peroxisome proliferator-activated receptor gamma inhibit growth and induce apoptosis of human papillary thyroid carcinoma cells.

Ligands for peroxisome proliferator-activated receptor gamma (PPARgamma) induce apoptosis and exert antiproliferative effects on several carcinoma cell lines. The present study investigates the expression of PPARgamma and the possibility that agonists for PPARgamma also inhibit the growth of human thyroid carcinoma cells. We examined this hypothesis using six cell lines, designated BHP thyroid carcinoma cells, which originated from patients with papillary thyroid carcinoma. RT-PCR analysis revealed that the thyroid carcinoma cell lines BHP2-7, 7-13, 10-3, and 18-21 express PPARgamma. More PPARgamma was expressed in carcinoma than in adjacent normal thyroid tissue in three of six samples of human papillary carcinoma of the thyroid. PPARgamma-positive thyroid carcinoma cells were treated with agonists of PPARgamma, troglitazone, BRL 49653, and 15-deoxy-12,14-prostaglandin J2. Troglitazone (10 micromol/L), BRL 49653 (10 micromol/L), and 15-deoxy-12,14-prostaglandin J2 (1 microg/mL) decreased [(3)H]thymidine incorporation and reduced cell number, respectively, in BHP carcinoma cell lines that expressed PPARgamma. Under low serum conditions, ligands for PPARgamma induced condensation of the nucleus and fragmentation of chromatin into nucleosome ladders. These findings indicate that the death of thyroid carcinoma cells is a form of apoptosis. To investigate the molecular mechanism of the apoptosis, we assessed expression of the apoptosis-regulatory genes bcl-2, bax, and c-myc. Troglitazone significantly increased the expression of c-myc messenger RNA but had no effect on the expression of bcl-2 and bax in thyroid carcinoma cells. These results suggest that, at least in part, the induction of apoptosis in human papillary thyroid carcinoma cells may be due to an increase of c-myc. Troglitazone (500 mg/kg.day) significantly inhibited tumor growth and prevented distant metastasis of BHP18-21 tumors in nude mice in vivo. Taken together, these results suggest that PPARgamma agonist inhibit cell growth of some types of human thyroid cancer.

Induction of follicle formation in long-term cultured normal human thyroid cells treated with thyrotropin stimulates iodide uptake but not sodium/iodide symporter messenger RNA and protein expression.

Iodide uptake by the sodium/iodide symporter (NIS) in thyrocytes is essential for thyroid hormone production. Reduced NIS activity has been reported in thyroid diseases, including thyroid cancer and congenital hypothyroidism. The study of iodide uptake in thyrocytes has been limited by the availability of appropriate in vitro models. A new culture technique was recently developed that allows normal human thyroid primary culture cells to grow as monolayer cells and express differentiated functions for more than 3 months. We used this technique to study the effect of follicle formation and TSH on iodide uptake in these cells. Iodide uptake by the cells grown in monolayer was very low. Follicle formation was induced from monolayer cells, and electron micrographs demonstrated cell polarity in the follicles. No significant increase in iodide uptake was observed after TSH treatment of cells in monolayer or when follicle formation was induced without TSH. TSH stimulation of follicles, however, significantly increased iodide uptake ( approximately 4. 4-fold; P<0.001). Compared with iodide uptake in monolayers, the combination of follicle formation and TSH treatment stimulated iodide uptake synergistically to 12.0-fold (P<0.001). NIS messenger RNA (mRNA) and protein levels were almost the same in both monolayer cells and follicles. TSH treatment of monolayers and follicles produced significant (P<0.05) stimulation of mRNA ( approximately 4. 8- and approximately 4.3-fold respectively) and protein ( approximately 6.8- and 4.9-fold respectively). TSH stimulated NIS protein levels in both monolayer and follicles, however, stimulation of functional iodide uptake was only seen with TSH stimulation of follicles. The function of NIS may involve post-transcriptional events, such as intracellular sorting, membrane localization of NIS or another NIS regulatory factor. Polarized functions, such as iodide efflux into follicular lumina, may also contribute to the increased iodide concentration after follicle formation.

In vivo and in vitro regulation of thyroid leukemia inhibitory factor (LIF): marker of hypothyroidism.

Several cytokines regulate thyroid function and may be involved in the pathogenesis of thyroid disorders, including euthyroid sick syndrome. Leukemia inhibitory factor (LIF), a neuroimmune pleiotropic cytokine, was measured to assess its role in hypothalamic-pituitary-thyroid function. Mean circulating serum LIF levels in 10 hypothyroid patients [TSH, 23+/-0.5 mIU/L (mean+/-SEM); free T4, 0.77+/-0.1 ng/dL] was 0.29+/-0.04 ng/mL, 145% higher (P < 0.04) than in 20 normal subjects (LIF, 0.20+/-0.02 ng/mL; TSH, 2.23+/-0.21 mIU/L; free T4, 1.23+/-0.04 ng/dL) but was not different from those in 10 hyperthyroid patients (LIF, 0.21+/-0.03 ng/mL; TSH, 0.01+/-0.00 mIU/L; free T4, 3.63+/-0.51 ng/dL). Serum LIF concentrations linearly correlated with serum TSH in the 40 samples (r = 0.58, P < 0.001). When T4 (1-8 microg/kg x day) was administered to cynomolgus monkeys with methimazole-induced hypothyroidism, serum T4 and T3 levels increased appropriately, and TSH and LIF concentrations decreased. When methimazole was given alone, both serum TSH (146+/-30 mIU/L) and LIF (8.84+/-0.49 ng/mL) were markedly induced. When methimazole together with T4 (>2 microg/kg x day) was administered, both serum TSH (7.5+/-1.2 mIU/L) and LIF (6.22+/-0.31 ng/mL) were lowered (P < 0.01). Monkey serum LIF levels and log TSH levels also correlated (r = 0.72, P < 0.01). Cultured thyroid carcinoma cells produced LIF (9.2 ng/10(6) cells/48 h). TSH (100 mIU/mL) and interleukin (IL)-6 (10 nmol/L) stimulated in vitro LIF secretion from the cells by 170+/-12% (P < 0.05) and 261+/-8% (P < 0.05), respectively. Dexamethasone (1 micromol/L) inhibited basal LIF concentration by 83% (P < 0.05), whereas TSH and IL-6 stimulated LIF by 52% (P = 0.04) and 42% (P = 0.03), respectively. However, using Northern blot analysis, we could not observe induction of LIF mRNA by TSH, suggesting that LIF regulation by TSH may be due to stimulation of secretion. The results show that the thyroid gland is a source of LIF production; TSH, IL-6, and glucocorticoid influence thyroid cell LIF expression. The correlation between TSH and LIF suggests that LIF may participate in the physiologic regulation of hypothalamic-pituitary-thyroid function.

Human chorionic gonadotropin and the thyroid: hyperemesis gravidarum and trophoblastic tumors.

There is abundant evidence that human chorionic gonadotropin (hCG) is a weak thyrotropin (TSH) agonist. In FRTL-5 rat thyroid cells, hCG increases cyclic adenosine monophosphate (cAMP), iodide transport, and cell growth. hCG has thyroid-stimulating activity in bioassays in mice and in clinical studies in man. In cultured cells transfected with the human TSH receptor, hCG increases generation of cAMP. Molecular variants of hCG with increased thyrotropic potency include basic molecules with reduced sialic acid content, truncated molecules lacking the C-terminal tail, or molecules in which the 47-48 peptide bond in the beta-subunit loop is nicked. In normal pregnancy, when hCG levels are highest at 10 to 12 weeks gestation, there is suppression of serum TSH levels, presumably due to slight increases in free thyroxine (T4) concentration. In twin pregnancies, hCG levels tend to be higher and suppressed TSH levels are more frequent. Hyperemesis gravidarum, defined as severe vomiting in early pregnancy that causes 5% weight loss and ketonuria, is usually associated with increased hCG concentration. A high proportion of patients with hyperemesis gravidarum, about one-third to two-thirds in different series, have evidence of increased thyroid function. Only a small proportion of these patients have clinical hyperthyroidism, termed gestational thyrotoxicosis. These patients probably secrete a variant of hCG with increased thyroid-stimulating activity. Trophoblastic tumors, hydatidiform mole, and choriocarcinoma often cause hyperthyroidism because they secrete very large amounts of hCG. When the serum hCG exceeds about 200 IU/mL, hyperthyroidism is likely to be found. There is a correlation between the biochemical severity of hyperthyroidism and the serum hCG in these patients. Removal of the mole or effective chemotherapy of the choriocarcinoma cures the hyperthyroidism. In conclusion, hCG has thyroid-stimulating activity that influences thyroid function early in pregnancy when hCG levels are high. Excessive hCG secretion may cause hyperthyroidism in patients with hyperemesis gravidarum or trophoblastic tumors.

Transforming growth factor-beta1 resistance in a thyroid cancer model of tumor necrosis factor-alpha resistance.

We have shown that both tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1) inhibit the growth of the human papillary thyroid carcinoma (PTC) cell line, NPA. In previous work, we developed NPA cells that were resistant to the growth suppressive effect of TNF-alpha, called R30, R45, and R60. In this model there were alterations in the p55 and p75 TNF-alpha receptor signaling in the resistant cell lines. In the present work, we studied the action of TGF-beta1 in this PTC cell model. TGF-beta1 (111 pg/mL) inhibited the proliferation of NPA, R30, R45, and the R60 cell lines by 82.8%, 72.1%, 64.2%, and 24.2%, respectively. On Western analysis, TGF-beta1 reduced c-fos content with similar potency in the NPA and R60 cells. In contrast, TNF-alpha reduced c-fos content in the sensitive NPA cells, but failed to do so in the resistant R60 cells. TGF-beta1 reduced p53 content in the NPA but not in the R60 cells, while TNF-alpha did not affect the p53 content in these cells. Furthermore, the resistant cells had a lower baseline p53 content than the NPA cells. The resistant cells had a significantly increased growth rate. Enzyme-linked immunosorbent assay (ELISA) assays with specific antibody against human p53 showed no apparent increase in the mutant form of p53 in the resistant cells. There were also no mutant forms of Ha-Ras, Arg12p21, Val12p21, Asp12p21, and Asp13p21 detected in the resistant cells. The results showed that R30, R45, and R60 cells are partially resistant to TGFbeta1. The mechanisms of action of TNF-alpha and TGF-beta1 differ in their regulation of c-fos and p53 content. The increase in cell proliferation rate is apparently associated with a decrease of p53 content, but not with mutations of p53 or Ha-Ras.

Tumor necrosis factor, ceramide, transforming growth factor-beta1, and aging reduce Na+/I- symporter messenger ribonucleic acid levels in FRTL-5 cells.

Iodide uptake, which is necessary for thyroid hormone synthesis, can be inhibited by aging, withdrawal of TSH, or increased tumor necrosis factor (TNF) and transforming growth factor (TGF)-beta1 levels resulting from the nonthyroid illness syndrome. TNF induces receptor-mediated activation of sphingomyelinase, which converts sphingomyelin to ceramide, a mediator of TNF actions. Thyroid follicular cells transport iodide from blood into the follicular lumen against an iodide gradient by means of coupled transport of Na+ ions and I- ions via the Na+/I- symporter (NIS). An inward Na+ gradient is maintained by Na+/K+-ATPase. The recent cloning and sequencing of the rat NIS complementary DNA has made possible studies on the mechanism by which TSH, aging, and cytokines regulate I- uptake by thyroid cells. Young (<20 passages) and aged (>40 passages) FRTL-5 cells grown with or without TSH were treated with various concentrations of TNF, TGF-beta1, sphingomyelinase, or ceramide. NIS messenger RNA (mRNA) levels in aged cells were only 2% of those in young cells. Withdrawal of TSH from young cells reduced NIS mRNA levels by more than 90%. TNF reduced NIS mRNA levels in young cells grown with TSH at t1/2 = 0.62 days, a cycloheximide inhibitable effect. Similar treatments with TGF-beta1, sphingomyelinase, or ceramide reduced NIS mRNA by 70-90%. Ceramide reduced 125I(-)-uptake by 50%. The addition of TNF increased both the sphingomyelin and ceramide levels 3- to 5-fold in young and old cells. We conclude that 1) the decline in iodide uptake due to aging, a fall in serum TSH or an increase in TNF or TGF-beta1 is mediated primarily by a reduction in thyroid NIS expression; and 2) that receptor-mediated activation of sphingomyelinase is an important, protein synthesis-dependent, intracellular pathway for inhibition of NIS expression by TNF.

Growth inhibition of new human thyroid carcinoma cell lines by activation of adenylate cyclase through the beta-adrenergic receptor.

In normal thyroid cells, the TSH-adenylate cyclase system plays a pivotal role in controlling growth and differentiation. However, the role of this system in the growth of thyroid carcinoma is not well understood. To investigate this subject, we have established four new human thyroid carcinoma cell lines, designated BHP 2-7, 7-13, 10-3, and 18-21, from different patients. Northern gel analysis revealed that all of these cell lines expressed Pax-8 messenger ribonucleic acid; additionally, only BHP 18-21 cells expressed TTF-1 messenger ribonucleic acid. These cells were treated with various concentrations of 8-bromo-cAMP, forskolin, TSH, and adrenergic receptor agonist (norepinephrine, epinephrine, and isoproterenol). Cell proliferation was assessed by [3H]thymidine incorporation and cell number. In these human thyroid carcinoma cell lines, the addition of 8-bromo-cAMP reduced [3H]thymidine incorporation at a concentration of 10 mumol/L. Forskolin (0.1-10 mumol/L) significantly induced cAMP accumulation, decreased [3H]thymidine incorporation, and reduced cell number in a dose-dependent manner. Conversely, TSH (0.01-1 mU/ mL) did not affect the accumulation of cAMP or cell growth. We found that adrenergic receptor agonists induced the accumulation of cAMP and inhibited cell growth. The rank of potency was isoproterenol > epinephrine > > norepinephrine. The binding studies of [3H]CGP-12177, a specific beta-adrenergic agonist, revealed that these new thyroid carcinoma cells had beta-adrenergic receptors. These results indicate that cAMP inhibits the growth of some human thyroid carcinoma cells, and that cAMP production is regulated through beta-adrenergic receptor-mediated pathways, but not through TSH receptor-mediated pathways.

Sphingomyelinase and phospholipase A2 regulate type I deiodinase expression in FRTL-5 cells.

We have previously reported (Mol. Cell. Endocrinol. (1994) 101, R31-R35) that the proinflammatory cytokines, tumor necrosis factor-alpha (TNF), interleukin-1 beta (IL-1 beta), and interferon-gamma (IFN-gamma), have a marked inhibitory effect on the expression and activity of type I iodothyronine deiodinase (D1) in FRTL-5 rat thyroid cells, while the anti-inflammatory cytokine, transforming growth factor-beta 1 (TGF-beta 1) had no effect. These three proinflammatory cytokines utilize a number of intracellular second messenger systems including the pathways beginning with activation of sphingomyelinase and phospholipase A2. We have studied the time-dependent and dose-dependent effects of sphingomyelinase, ceramide, phospholipase A2 (PLA2), and arachidonic acid on the expression and activity of D1 in FRTL-5 cells. Sphingomyelinase (0.3 U/mL) inhibited D1 activity 55% and reduced D1 mRNA levels 70% to 90% by 8 hours. Similar treatment with 10 U/mL PLA2 inhibited D1 activity 54%. Treatment with 15 microM 5, 8, 11-eicosatriynoic acid (ETI), a nonmetabolizable analog of arachidonic acid, or 15 microM ceramide for 3 hours reduced D1 activity with a half-time of disappearance (t1/2) of 4.2 hours and 3.7 hours, respectively, but ETI and ceramide did not alter the D1 immunoreactivity or mRNA levels. Treatment for 8 hours with cycloheximide (5 or 10 micrograms/mL) had no effect on the D1 mRNA level, but blocked the TNF-induced reduction of this mRNA. We conclude that proinflammatory cytokines inhibit D1 expression and activity in FRTL-5 cells, in part, by activation of sphingomyelinase and PLA2 that results in (1) competitive inhibition of D1 activity by the enzymatic products ceramide and arachidonic acid and (2) reduction of D1 mRNA stability by protein synthesis-dependent mechanisms.

Tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta 1 (TGF-beta 1) inhibit the expression and activity of Na+/K(+)-ATPase in FRTL-5 rat thyroid cells.

We recently reported that tumor necrosis factor-alpha (TNF-alpha) induction of the synthesis and secretion of transforming growth factor (TGF)-beta 1 by FRTL-5 cells is a thyroid-stimulating hormone (TSH)-dependent and age-dependent process. TNF-alpha is only cytotoxic to aged (> 40 passages) FRTL-5 cells grown in TSH-containing medium, whereas TGF-beta induces programmed cell death (apoptosis) in epithelial cells but not in FRTL-5 cells, which otherwise retain many properties of normal thyroid follicular cells. This cell line is, therefore, a convenient model for studies on the TSH-dependent and age-dependent inhibitory effects of these cytokines on epithelial cell growth, viability, and function. One prominent effect of TNF-alpha (and TGF-beta 1) on FRTL-5 cell function is suppression of iodide uptake, which is markedly stimulated by TSH. In aged FRTL-5 cells, iodide uptake is only about 10% that of young control cells. Na+/K(+)-ATPase activity, which drives iodide uptake by thyroid cells, is inhibited by TNF-alpha and TGF-beta. The following experiments quantitate the effects of TSH, aging, TNF-alpha, and TGF-beta 1 on the expression and activity of Na+/K(+)-ATPase activity in FRTL-5 cells. Young (< 20 passages) and aged (> 40 passages) FRTL-5 cells were treated with various doses (0-100 ng/ml) of recombinant human TNF-alpha or TGF-beta 1 for various times (0-3 days) with and without 2 U/liter TSH. These treatments reduced the rate-limiting Na+/K(+)-ATPase beta 1 mRNA level and Na+/K(+)-ATPase activity in parallel in a dose-dependent and time-dependent fashion. Aged FRTL-5 cells were more sensitive to the inhibitory effects of TNF-alpha, whereas young cells were more sensitive to the suppressive effects of TGF-beta 1 on the expression and activity of Na+/K(+)-ATPase. We conclude that inhibition of Na+/K(+)-ATPase activity by TNF-alpha and TGF-beta in FRTL-5 cells is differentially affected by aging and that this inhibitory effect can be dissociated from effects on cell viability.

TNF-alpha induction of A1 expression in human cancer cells.

A1, a member of the Bcl-2 gene family, was originally identified as a hemopoietic-specific early response gene. Later it was found that A1 was overexpressed in human stomach cancer tissues and was induced by tumor necrosis factor-alpha (TNF-alpha) in human vascular endothelial cells. However, its expression in human cancer cells has not been well characterized. In the present study, we examined the expression of A1, as well as the antioxidant manganous superoxide dismutase (MnSOD), in four human thyroid carcinoma cell lines, two human pancreatic carcinoma cell lines, and two human prostate carcinoma cell lines. A1 mRNA was expressed in all four thyroid carcinoma cell lines. TNF-alpha induced A1 in a time- and dose-dependent manner. In contrast, A1 mRNA was not detectable in the pancreatic and prostate carcinoma cell lines in the presence or absence of TNF-alpha. However, TNF-alpha induced manganous superoxide dismutase (MnSOD) mRNA in all the cell lines tested. Furthermore, an agonist antibody to the p55 TNF-alpha receptor induced A1, but the agonist antibody against p75 TNF-alpha receptor did not have this effect. The results indicate that A1 is expressed in human thyroid carcinoma cells and TNF-alpha induces A1 through the p55 TNF-alpha receptor-mediated pathway.

Paracrine effect of human chorionic gonadotropin ectopically produced from papillary thyroid cancer cells on growth and function of FRTL-5 rat thyroid cells.

It is well known that human chorionic gonadotropin (hCG) is sometimes secreted from nontrophoblastic neoplasms. To elucidate the role of ectopic hCG, we investigated the effect of hCG produced from a papillary thyroid cancer cell line (B-CPAP cells) on stimulation and growth promotion of FRTL-5 rat thyroid cells. Ectopic hCG contained in the culture medium of B-CPAP cells was purified using gel filtration and bioassayed for thyrotropic activity in FRTL-5 cells. Addition of ectopic hCG (up to 5.2 x 10(4) IU/L) increased cyclic adenosine monophosphate (cAMP) accumulation and 3H-thymidine incorporation in FRTL-5 cells dose dependently. These effects were almost as potent as the stimulation induced by standard hCG CR-127. After the absorption of the ectopic hCG by anti-hCG-beta monoclonal antibody, the cAMP accumulation was significantly decreased. Analysis of ectopic hCG isoforms with different isoelectric points indicated the predominance of the acidic hCG isoform with isoelectric point (pI) 3.8-3.2 that is the major isoform of standard hCG. Basic isoforms (pI 5.7-5.3) with higher thyrotropic potency were also detected. These results indicate that the ectopic hCG secreted from papillary thyroid cancer cells possess intrinsic thyroid-stimulating and growth-promoting activity. The ectopic hCG may act as an autocrine-paracrine factor in nontrophoblastic neoplasms.

Alterations in TNF-alpha signal transduction in resistant human papillary thyroid carcinoma cells.

Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) inhibit the growth of the human papillary thyroid carcinoma (PTC) cell line, NP. Exposure of NP cells to TNF-alpha resulted in the development of several PTC cell lines (R30, R45, and R60) with graded loss to the TNF-alpha-induced antiproliferation, termed resistance. In contrast, the NP cells and the resistant cells were equally sensitive to the antiproliferative action of interferon-gamma. Utilizing TNF-alpha receptor-specific agonist monoclonal antibodies, we demonstrated that the TNF-alpha receptor p55 mediated the antiproliferative action of TNF-alpha, while the p75 receptor did not affect cell proliferation in the NP cell line. The resistant PTC cell lines, however, showed a graded loss of p55 receptor-mediated antiproliferation and a concomitant activation of a p75 receptor-mediated growth stimulation. Shedding of TNF receptors is an important mechanism of TNF-alpha receptor metabolism. The p55 receptor mediated the TNF-alpha-induced up-regulation of the shedding of the p75 TNF-alpha receptor. The p75 receptor mediated the TNF-alpha-induced down-regulation of the shedding of the p55 receptor. However, the shedding of the p75 receptor was decreased and the shedding of the p55 receptor was increased in the resistant R60 cell line compared with the NP cell line, in the presence and absence of TNF-alpha. In contrast, IFN-gamma increased shedding of both p55 and p75 TNF-alpha receptors in NP and R60 cell lines with equal potency. Furthermore, the resistant PTC cell lines have increased basal manganous superoxide dismutase (MnSOD) expression and blunted induction of MnSOD mRNA upon short-term. TNF-alpha treatment (less than 2 h of treatment). The results indicate that a decrease in signal transduction via the p55 TNF-alpha receptor and concomitant increase in signal transduction via the p75 TNF-alpha receptor are involved in the development of PTC cell resistance.

Antitumor actions of cytokines on new human papillary thyroid carcinoma cell lines.

To investigate the in vitro effects of cytokines on the growth of human papillary thyroid carcinoma (PTC) cells, we established six new PTC cell lines, designated BHP 5, 14, 15, 17, 18, and 19, from different patients. We studied the antiproliferative actions of cytokines by using BHP cells, NP cells (PTC cell line), and ARO cells (anaplastic thyroid carcinoma cell line). These cells were treated with various concentrations of tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma), interleukin-1 beta (IL-1 beta), and transforming growth factor-beta 1 (TGF beta 1), alone and in combination. Cell proliferation was assessed by [3H]thymidine incorporation and cell number measurement. In BHP cell lines, IFN gamma, IL-1 beta, and TGF beta 1 inhibited [3H]thymidine incorporation and decreased cell number, but TNF alpha stimulated [3H]thymidine incorporation. In NP cells, treatment with each cytokine decreased [3H]thymidine incorporation and cell number. In contrast, the proliferation of ARO cells was either stimulated by or resistant to TNF alpha, IL-1 beta, and TGF beta 1. The effects of these cytokines on [3H]thymidine incorporation were additive in these cell lines. The results suggest that IL-1 beta and TGF beta 1 play a pivotal role in growth inhibition of PTC cells, and the escape from negative control of IL-1 beta and TGF beta 1 may be a step toward anaplastic changes. The additive effects of these cytokines suggest that they act through different pathways.

Thyrotropic action of human chorionic gonadotropin.

Hyperthyroidism or increased thyroid function has been reported in many patients with trophoblastic tumors. In these cases, greatly increased human chorionic gonadotropin (hCG) levels and suppressed TSH levels suggest that hCG has thyrotropic activity. Recent investigations have clarified the structural homology not only in the hCG and TSH molecules but also in their receptors, and this homology suggests the basis for the reactivity of hCG with the TSH receptor. The clinical significance of the thyrotropic action of hCG is now also recognized in normal pregnancy and hyperemesis gravidarum. Highly purified hLH binds to recombinant hTSH receptor and is about 10 times as potent as purified hCG in increasing cAMP. The beta-subunits of hCG and hLH share 85% sequence identity in their first 114 amino acids but differ in the carboxy-terminal peptide because hCG beta contains a 31-amino acid extension (beta-CTP). A recombinant mutant hCG that lacks beta-CTP showed almost identical potency to LH on stimulation of recombinant hTSH receptor. If intact hCG were as potent as hLH in regard to its thyrotropic activity, most pregnant women would become thyrotoxic. One of the roles of the beta-CTP may be to prevent overt hyperthyroidism in the first trimester of pregnancy when a large amount of hCG is produced by the placenta. Nicked hCG preparations, obtained from patients with trophoblastic disease or by enzymatic digestion of intact hCG, showed approximately 1.5- to 2-fold stimulation of recombinant hTSH receptor compared with intact hCG. This suggests that the thyrotropic activity of hCG may be influenced by the metabolism of the hCG molecule itself. Deglycosylation and/or desialylation of hCG enhances its thyrotropic potency. Basic hCG isoforms with lower sialic acid content extracted from hydatidiform moles were more potent in activating adenylate cyclase, and showed high bioactivity/immunoactivity (B/I) ratio in CHO cells expressing human TSH receptors. This is consistent with the finding that the beta-CTP truncated hCG with higher thyrotropic potency is substantially deglycosylated and desialylated in the beta-subunit relative to intact hCG because all four O-linked glycosylation sites occur within the missing C-terminal extension. The desialylated hCG variant also interacts directly with recombinant hTSH receptors transfected into human thyroid cancer cells. There is thyroid-stimulating activity in sera of normal pregnant women, and this correlates with serum hCG levels. The thyroid gland of normal pregnant women may be stimulated by hCG to secrete slightly excessive quantities of T4 and induce a slight suppression of TSH, perhaps being about 1 mU/L less than nongravid levels, but not high enough to induce overt hyperthyroidism. Maternal thyroid glands may secrete more thyroid hormone during early pregnancy in response to the thyrotropic activity of hCG that overrides the normal operation of the hypothalamic-pituitary-thyroid feedback system. Biochemical hyperthyroidism associated with hyperemesis gravidarum has been attributed to hCG. In patients with hyperemesis gravidarum, thyrotropic in serum correlated with hCG immunoreactivity, and the severity of vomiting as indicated by clinical and biochemical parameters correlated with the degree of thyroid stimulation. To understand the thyrotropic action of hCG, it is necessary to know whether hCG activates the same domain of the TSH receptor as does TSH. The identification of the molecular structure of the hCG isoform with the highest thyrotropic potency will resolve the enigma of gestational thyrotoxicosis and the hyperthyroidism associated with trophoblastic disease and hCG-producing tumors.