Reexpression of thyroid peroxidase in a derivative of an undifferentiated thyroid carcinoma cell line by introduction of wild-type p53.

Loss of function of p53 is believed to result in transformation through impairment of its properties as a transcription factor, which interferes with the regulation of the cell cycle and under certain conditions, with programmed cell death. We report that stable transfection of clonal undifferentiated thyroid carcinoma cell lines harboring endogenous p53 mutations with a wild-type p53 expression vector only rarely yields transfectants expressing authentic wild-type p53. Among these, most exhibited an increase in doubling time and an impairment of colony formation in soft agar. Only one clonal wild-type p53-overexpressing derivative of the NPA papillary carcinoma cell line was obtained, and these cells were found to reexpress thyroid peroxidase (TPO). This clone also demonstrated reexpression of the paired box domain transcription factor Pax-8, which specifically activates transcription of TPO. Wild-type p53 did not directly stimulate transcriptional activity of a TPO promoter construct. Although the low frequency of authentic wild-type p53 stable transfectants limits the power of this analysis, these data suggest that in addition to its role in malignant transformation, p53 may be significant in the determination or maintenance of cell differentiation in thyroid neoplasms.

Expression of the neoplastic phenotype by human thyroid carcinoma cell lines requires NFkappaB p65 protein expression.

We have investigated the role of the NFkappaB complex in the process of thyroid carcinogenesis by analysing thyroid carcinoma cell lines. A significant increase in p65 NFkappaB mRNA and protein expression, compared to normal thyroid cultures or tissue, was found in all of the cancer cell lines. Conversely, only a modest increase in the p50 NFkappaB mRNA and protein was found in most, but not all carcinoma cell lines. The block of p65 protein synthesis with specific antisense oligonucleotides greatly reduced the ability of two undifferentiated carcinoma cell lines to form colonies in agar and reduced their growth rate. On the other hand, no effect was observed in the same cell lines when treated with p50 specific antisense oligonucleotides. These inhibitory effects seem to be mediated by the suppression of c-myc gene expression, since treatment with antisense oligonucleotides for p65 gene interfered negatively with c-myc gene expression. Our results indicate that activation of the NFkappaB complex by overexpression of p65 plays a critical role in the process of thyroid cell transformation.

Parathyroid hormone-related peptide-(1-34) [PTHrP-(1-34)] induces vasopressin release from the rat supraoptic nucleus in vitro through a novel receptor distinct from a type I or type II PTH/PTHrP receptor.

PTH and PTH-related peptide (PTHrP) bind to a type I PTH/PTHrP receptor expressed in bone and kidney or a type II receptor in nonclassical target tissue with equal affinity and similar bioactivities. PTHrP is abundant in the central nervous system, but its physiological role remains unknown. Herein, we examined the role of PTHrP-(1-34) on arginine vasopressin (AVP) release from the rat supraoptic nucleus (SON). Application of PTHrP-(1-34) to SON slices caused an increase in AVP release in a concentration-dependent manner. Neither PTHrP-(7-34) nor PTH-(1-34) had any effect on AVP release from the SON. PTHrP-(1-34)-induced AVP release was antagonized by a large excess of PTHrP-(7-34) and by H89, an inhibitor of cAMP-dependent protein kinase (A kinase), but not by PTH-(1-34) or PTH-(13-34). PTHrP-(1-34), but not PTH-(1-34), also dose-dependently increased the levels of cAMP in the SON. 125I-Labeled PTHrP-(1-34) bound specifically to crude membranes isolated from the SON. Scatchard analysis showed a single class of binding sites for PTHrP-(1-34) with a Kd of 36.4 nM and a maximum binding capacity of 3.94 pmol/mg protein. No specific binding for 125I-labeled PTH-(1-34) was noted. The binding of 125I-labeled PTHrP-(1-34) was displaced by unlabeled PTHrP-(1-34) and unlabeled PTHrP-(7-34), but not by unlabeled PTH-(1-34). These findings suggest that PTHrP-(1-34), but not PTH-(1-34), causes the release of AVP from the SON through a novel receptor distinct from type I or II PTH/PTHrP receptors.

Centrally administered parathyroid hormone (PTH)-related protein(1-34) but not PTH(1-34) stimulates arginine-vasopressin secretion and its messenger ribonucleic acid expression in supraoptic nucleus of the conscious rats.

It has been suggested that PTH-related protein (PTHrP) is an endogenous modulator of cardiovascular systems. We have reported that PTHrP(1-34), but not PTH(1-34), causes the release of arginine-vasopressin (AVP) from the supraoptic nucleus (SON) of the hypothalamus in vitro through a novel receptor distinct from the PTH/PTHrP receptors (type I or type II) described previously. In this study, we have investigated the in vivo effects of PTHrP(1-34) on AVP secretion and its, messenger RNA (mRNA) expression in the SON in conscious rats. Intracerebroventricular (i.c.v.) administration of PTHrP(1-34) resulted in an increase in plasma AVP concentration in a dose-dependent manner (0-400 pmol/rat). The maximal effect was obtained at 15 min after i.c.v. administration of PTHrP(1-34). Neither PTHrP(7-34) nor PTH(1-34) had any effect on plasma AVP levels. PTHrP(1-34)-induced AVP secretion was antagonized by pretreatment with PTHrP(7-34) but not by that with PTH(1-34). In addition, in situ hybridization study revealed that AVP mRNA expression in the SON and paraventricular nucleus was significantly increased 30 min after i.c.v. administration of PTHrP(1-34) and reached a maximum at 180 min. Furthermore, in Northern blot analyses, AVP mRNA expression in the SON was increased to approximately a 2-fold of basal level by PTHrP(1-34). On the other hand, neither PTHrP(7-34) or PTH(1-34) had any effect on the mRNA expression. The PTHrP(1-34)-stimulated AVP mRNA expression was eliminated by pretreatment with PTHrP(7-34) but not with PTH(1-34). These results suggest that, in the central nervous system, PTHrP(1-34) is involved in AVP secretion through a novel receptor distinct from the PTH/PTHrP receptors reported previously, playing a role in the body water and electrolyte homeostasis.

Mutations of the adenomatous polyposis coli gene in sporadic thyroid neoplasms.

Several epidemiological studies have demonstrated an association between familial adenomatous polyposis coli (FAP) and thyroid neoplasms. Predisposition to FAP is conferred by mutations in the APC gene, located on chromosome 5q21. Somatic mutations of APC are also observed in about 60% of sporadic colorectal adenomas and carcinomas, suggesting that disruption of this putative tumor suppressor gene may play a role in both familial as well as acquired colorectal tumorigenesis. The APC gene is expressed in normal human thyroid, thyroid adenomas, and differentiated carcinoma tissues as well as in four clonal human thyroid carcinoma cell lines, as demonstrated by reverse transcriptase-polymerase chain reaction of a 388-base APC messenger ribonucleic acid fragment spanning exons 14 and 15, followed by hybridization to an exon 15-specific complementary DNA probe. Eighty human thyroid neoplasms were examined for loss of heterozygosity of the APC locus, using primers flanking a hypervariable dinucleotide (CA) repeat (CB26) immediately adjacent to the APC gene. Of 71% informative samples, 2 showed allelic loss: a follicular adenoma (FA) and a nodule from a multinodular goiter (MNG). The DNA of 83 benign and malignant thyroid neoplasms and 4 thyroid carcinoma cell lines was examined for mutations within a 1200-basepair stretch of exon 15 by single strand conformation polymorphism. Five sets of overlapping primers were used for PCR. The anaplastic thyroid carcinoma cell line (ARO) had 1 APC allele with an adenine insertion at codon 1556 (ACTA to AACTA), leading to a premature stop codon at 1558. An anaplastic carcinoma had a mutation of codon 1346 (TCA-CCA; Ser to Pro). In summary, the APC gene is expressed in normal and neoplastic human thyroid tissue and is a target for inactivating mutations in some thyroid tumors.

Aberrant deoxyribonucleic acid methylation in human thyroid tumors.

DNA methylation is a covalent modification of cytosine residues that occurs at the dinucleotide sequence CpG in vertebrates. Abnormal patterns of DNA methylation are observed consistently in human tumors, including widespread areas of genomic hypomethylation as well as regional sites of hypermethylation. We examined the DNA of benign and malignant human thyroid tumors for changes in the methylation state of the genes for human GH, platelet-derived growth factor B-chain, and H-ras. The human GH gene was aberrantly methylated in 6 of 22 (27%) nodules from multinodular goiters (MNG), 21 of 33 (64%) follicular adenomas (FA), and 10 of 16 (63%) papillary carcinomas (PC). Platelet-derived growth factor B-chain was also abnormally methylated in 4 of 13 (31%) MNG, 17 of 24 (71%) FA, and 9 of 13 (69%) PC. The H-ras gene, located within a region on chromosome 11p known to be a hot spot for hypermethylation in other tumors types, showed complex patterns of methylation (mainly hypermethylation) in 6 of 22 (27%) MNG, 22 of 35 (63%) FA, and 10 of 16 (63%) PC. Those tumors with methylation abnormalities tended to be affected at multiple loci (i.e. aberrant patterns with all 3 probes), whereas those that were negative were usually normal at all sites. Benign and malignant thyroid neoplasms show a high prevalence of aberrant methylation patterns of selected genes. Adenomatous nodules from multinodular goiters, consisting largely of hyperplastic tissue, have a lower frequency of these events. Aberrant DNA methylation may contribute to subsequent cell transformation through changes in DNA conformation, transcriptional activity, and/or increased fragile site instability. This suggests that widespread changes in DNA methylation may occur as a relatively early step in thyroid tumor formation.

Autocrine stimulation of interleukin-1 in the growth of human thyroid carcinoma cell line NIM 1.

We reported first in this study that human thyroid cell line NIM 1 established from a patient with papillary adenocarcinoma of the thyroid associated with hypercalcemia and peripheral neutrocytosis produced interleukin (IL)-1 alpha and IL-1 beta in the culture supernatant and cell lysate as detected by murine thymocyte proliferative response and enzyme-linked immunosorbent assay. Production of IL-1 alpha and IL-1 beta was further confirmed by the demonstration of IL-1 alpha and IL-1 beta messenger ribonucleic acid expression with Northern blot hybridization analysis. The in vitro growth of NIM 1 cells was inhibited by the addition of anti-IL-1 alpha and IL-1 beta antibody. The growth of NIM 1 cells was further enhanced by the addition of recombinant human IL-1 alpha and IL-1 beta, whereas this enhancement was also inhibited by the addition of anti-IL-1 antibody. IL-1 receptors were expressed on NIM 1 cells. These results suggest that IL-1 plays a regulatory role in the growth of NIM 1 cells by an autocrine mechanism.

Thyrocyte proliferation by cellular adhesion to infiltrating lymphocytes through the intercellular adhesion molecule-1/lymphocyte function-associated antigen-1 pathway in Graves' disease.

Graves' disease (GD) is an autoimmune thyroid disease characterized by infiltration of lymphocytes into the thyroid, and intrathyroid lymphocytes are known to play an important role in the pathogenesis of GD. However, it remains to be understood how lymphocytes adhere to thyrocytes and regulate the thyrocyte function through cellular adhesion. We studied the mechanisms of T cell adhesion to thyrocytes using intrathyroid mononuclear cells (ITMC) and thyrocytes purified from the thyroids of patients with GD. The following novel features of cellular adhesion of ITMC to thyrocytes in the regulation of the thyrocyte function in GD were observed: 1) GD-ITMC expressed lymphocyte function-associated antigen (LFA)-1, which became an active adhesive configuration much higher than peripheral blood mononuclear cells (PBMC) from normal volunteers and GD patients; 2) GD-thyrocytes expressed a high quantity of intercellular adhesion molecule (ICAM)-1; 3) GD-ITMC adhered to GD-thyrocytes, whereas normal PBMC required activation stimuli by phorbol myriacetate, a pharmacological integrin-trigger, to adhere to GD- thyrocytes; 4) monoclonal antibody-blocking studies showed that the adhesion of the activated PBMC and ITMC to thyrocytes was mainly mediated by the LFA-1/ICAM-1 pathway; 5) the adhesion of GD-thyrocytes to the activated-PBMC or ITMC induced the proliferation of the thyrocytes, which was blocked by the addition of ICAM-1 and/or LFA-1 monoclonal antibodies; and 6) in GD thyrocytes of early cultures, ICAM-1 expression on GD-thyrocytes and its adhesion to LFA-1 on phorbol myriacetate-activated PBMC or ITMC were not modulated by the addition of interleukin-1beta or interferon-gamma, and proliferation of thyrocytes by the cellular adhesion via the ICAM-1/LFA-1 pathway was independent of the proliferative response of these cytokines. Taken together, these results suggest that lymphocytes infiltrating GD thyroid induce proliferation of GD-thyrocyte by the cellular adhesion to thyrocytes via ICAM-1/LFA-1, which may lead to the development of a goiter.

Macrophage-colony stimulating factor inhibits the growth of human ovarian cancer cells in vitro.

The effect of macrophage-colony stimulating factor (M-CSF), which regulates the growth and differentiation of haematopoietic progenitor cells on the growth of ovarian cancer cells was investigated in three ovarian cancer cell lines in vitro. The spontaneous growth of these cells was significantly inhibited by the addition of M-CSF in a concentration-dependent manner over 96 h of culturing. The maximum response was obtained with 10 ng/ml (3857 U/ml) of M-CSF by counting the viable cell number using the trypan blue exclusion assay. [(3)H]-thymidine incorporation by these cells was also suppressed following a 96-h incubation with M-CSF. The inhibitory effect of M-CSF was reversed by the addition of anti-M-CSF monoclonal antibody. Flow cytometric analysis revealed that the treated ovarian cancer cells arrested at the G0/G1 phase of the cell cycle. These cells expressed M-CSF receptors on their surface as detected by Scatchard plot analysis using (125)I-labelled M-CSF. These results indicate that M-CSF has an antitumour activity for ovarian cancer cells and suggest that it can be applied for the treatment of this disease.

Interleukin-1alpha regulates G1 cell cycle progression and arrest in thyroid carcinoma cell lines NIM1 and NPA.

This study provides the first report that the same cytokine (interleukin-1 (IL-1)) can induce opposite effects on cyclin-dependent kinases (Cdks) and Cdk inhibitors (Cdkis) in the G1 phase even in the same type of cancer cells (papillary thyroid carcinoma cells). Cell cycle analysis revealed an increase in NIM1 cells and a decrease in NPA cells in the S and G2+M phases after treatment with IL-1alpha. The addition of IL-1alpha to NIM1 cells reduced the expression of p16 and p21 protein and induced the expression of Cdk2 and Cdk4 protein, which leads to the phosphorylation of retinoblastoma protein. The addition of IL-1alpha to NPA cells induced the expression of p27 protein and reduced the expression of Cdk2 protein, which leads to induction of p107 protein expression. It is of interest that p21 protein expression was not observed in NPA cells. These results suggest that several Cdks and Cdkis play a regulatory role in the G1 cell cycle progression and arrest induced by IL-1alpha in thyroid carcinoma cell lines.

Inhibition of angiogenesis and tumorigenesis, and induction of dormancy by p53 in a p53-null thyroid carcinoma cell line in vivo.

Our recent in vitro findings for suppression of thrombospondin-1 (TSP1; an antiangiogenic factor) expression by wild-type (wt) p53 in a p53-null thyroid carcinoma cell line, FRO, prompted us to investigate the in vivo effect of exogenous wt-p53 and TSP1 expression on tumor growth and angiogenesis of FRO xenografts in nude mice. Overexpression of TSP1, which did not affect the in vitro cell growth, significantly inhibited the in vivo tumor growth and neovascularization but not tumorigenesis; all the mice inoculated with FRO cells expressing TSP1 developed tumors, which were smaller and less vascularized than those derived from FRO cells. In contrast, restoration of wt-p53 expression, which reduced the in vitro cell growth rate, inhibited tumorigenesis and induced a state of "dormancy". Thus, approximately 40% of mice inoculated with FRO cells expressing wt-p53 (FRO-p53) were tumor free and the remaining mice developed hypovascular tumors which remained small (< or = 5 mm in size) for up to 60 days. Of interest, the phenotype of FRO-p53 tumors reverted to a well vascularized, progressively expanding tumor by exogenous expression of vascular endothelial growth factor (a proangiogenic factor). Our data demonstrated wt-p53 inhibition of tumorigenesis and induction of dormancy by suppression of neovascularization in FRO cells. The results suggest that p53 gene therapy for thyroid carcinoma harboring p53 mutation may be more efficacious than we had expected from previous in vitro data.

Involvement of wild-type p53 in radiation-induced c-Jun N-terminal kinase activation in human thyroid cells.

c-jun-N-terminal kinases (JNKs) play an important role in defense against external stresses including ionizing radiation (IR). We have previously shown that sensitivity to IR is influenced by p53 status in human thyroid cells. In this study, we investigated the effect of p53 status on IR-induced JNK activation in human thyroid cells. Our results showed high basal JNK activity in the p53-null thyroid cancer cell line, FRO. In contrast, primary cultured thyroid cells (PT), which harbor wild-type p53, had low basal JNK activity. IR increased JNK activity in PT, however, no such increase was noted in FRO cells. Introduction of the wild-type p53 into FRO cells reduced JNK activity to a low basal level and rendered it responsive to IR. There was no difference in IR-induced ceramide production between PT and FRO cells. Our results provide clear evidence that p53 status influences, directly or indirectly, radiation-induced JNK activation in human thyroid cells, suggesting that a feedback or interaction pathway between p53 and JNK regulates radiation-induced cell fate.

Siblings of 21-hydroxylase deficiency (non-salt-losing) with aldosterone hypersecretion.

We describe siblings with the non-salt-losing form of 21-hydroxylase deficiency who had hypersecretion of aldosterone and plasma renin activity (PRA). Blood pressure and serum electrolytes in both cases were normal despite the aldosterone hypersecretion. Aldosterone secretion was elevated markedly with ACTH administration and with sodium deprivation and/or volume depletion during ACTH suppression by dexamethasone. With suppression by dexamethasone, aldosterone hypersecretion was decreased with lowering of the steroids proximal to the block in the biosynthetic pathway. However, urinary sodium excretion was decreased. These results suggest that the biosynthetic pathway for aldosterone production was preserved. Furthermore, aldosterone hypersecretion and high PRA may serve to compensate for the sodium loss which results in turn from the overproduction of the sodium-losing steroids, such as progesterone and 17 alpha-hydroxyprogesterone which are aldosterone antagonists.

[Clinical experiences of LAK therapy in patients with hepatocellular carcinoma].

A patient with hepatocellular carcinoma (HCC) was treated with newly established adoptive immunotherapy using LAK cells (LAK therapy) together with transcatheter arterial embolization therapy (TAE). This patient responded well, and the therapeutic efficacy still continues 6 months after the therapy. Since the efficacy of LAK therapy does not last long, it is recommended that LAK therapy should be employed in combination with such therapeutic maneuvers as TAE or anticancer drugs in patients with HCC.

[Lymphokine-activated killer (LAK) cells and interleukin-2 (IL-2) therapy that improved lymphadenopathy in a patient with B cell non-Hodgkin's lymphoma].

A 70-year-old man was admitted to our hospital on March 9, 1989 because of fever, superficial generalized lymphadenopathy, upper abdominal mass and right pleural effusion. The diagnosis of non-Hodgkin's lymphoma (follicular medium sized cell type, B cell) was made by a biopsy of the neck lymph node. Peripheral blood mononuclear cells were obtained from the patient by cytopheresis. The cells were cultured for 8 days with interleukin-2 (IL-2) to generate Lymphokine-activated killer (LAK) cells. The patient received a total of 7.7 x 10(9) LAK cells intravenously over a period of 3 weeks. He also received continuous intravenous infusion of IL-2 for 17 days, starting 2 days before the first infusion of LAK cells. After this therapy, although his superficial generalized lymphadenopathy disappeared or decreased in size, the size of the upper abdominal mass did not decrease. Therefore, it is suggested that adoptive immunotherapy is a beneficial treatments for B cell lymphoma. However, LAK cells should be generated in much larger quantities for a more successful therapeutic result.

[Autoantibody against Na+/I- symporter (NaIS)].

Na+/I- symporter (NaIS) cDNA was first cloned in 1996. Endo et al. reported that autoantibody against NaIS was found in 84% sera from patients with Graves' disease and 12% sera from patients with Hashimoto's thyroiditis. These IgGs, purified from sera from patients with Hashimoto's thyroiditis, caused 14 to 62% inhibition of iodide uptake. Furthermore, Ajjan et al. reported that 30.7% sera from patients with Graves' disease inhibited 7 to 44% of iodide uptake. In addition, Morris et al. suggested that NaIS represents an important autoantigen in autoimmune thyroid disease. In conclusion, it is suggested that the incidence of the antibodies against NaIS is higher in Graves' disease than Hashimoto's thyroiditis, and these antibodies inhibit iodide uptake.

Inhibitory effect of IL-1 on the TSH dependent growth of rat thyroid cells (FRTL-5).

Using a functioning rat thyroid cell line (FRTL-5), we examined the effects of some cytokines, particularly interleukin-1 (IL-1) on the growth of thyroid cells. In 5H medium, namely Coon's modified Ham's F-12 medium supplemented with 5% calf serum and a five-hormone preparation consisting of insulin, hydrocortisone, transferrin, glycyl-L-histidyl-L-lysine acetate and somatostatin, IL-1 enhanced the growth of FRTL-5 cells detected by [3H]TdR incorporation. However, in 6H medium (5H medium plus bovine TSH), IL-1 inhibited the growth of FRTL-5 cells. Both effects were neutralized by the addition of anti-IL-1 antibody. Furthermore, IL-1 inhibited the growth of FRTL-5 cells induced by forskolin which is known as an adenylate cyclase activator. FRTL-5 cells have specific IL-1 receptors detected by the binding of 125I-labeled IL-1 alpha. By Scatchard plot analysis, the numbers and the dissociation constants of IL-1 receptors on FRTL-5 cells were shown to be 5225/cell and 8.69 x 10(-10) M. Interleukin-2, interleukin-6 and interferon-gamma (IFN-gamma) had no significant effects on the cell growth in 6H medium, while IFN-gamma and insulin-like growth factor I stimulated cell growth somewhat in 5H medium. These results suggest that IL-1 plays a regulatory role in the growth of thyroid cells through binding to the IL-1 receptors.

Evidence of bone resorption-stimulating factor in adult T-cell leukemia.

Adult T-cell leukemia (ATL) is known to be frequently accompanied by hypercalcemia, but the mechanisms responsible for hypercalcemia in this disorder are not fully understood. We have recently experienced two male patients (25 and 36 yr old) with ATL diagnosed from typical leukemic cells with grooved and folded nucleus, surface marker, anti-ATLA antibody etc. Serum calcium levels of these patients were 16.4 and 21.4 mg/dl, respectively, with no radiological evidence of bone destruction. Peripheral blood leukemic lymphocytes from these patients were purified by the Ficoll-Hypaque method and cultured at a concentration of 1.5 X 10(6) cells/ml for 3 days on F-10 medium supplemented with 10% fetal calf serum. The supernatant fluids from the cell cultures were bioassayed for bone resorption-stimulating activity (BRSA) by an assay based on the release of 45Ca from prelabeled fetal mouse forearm bones in organ culture according to Raisz's method. The supernatant fluid of cultures from both patients which showed marked BRSA was nondialyzable through a dialysis membrane with a molecular weight cutoff of 3500. Parathyroid hormone and prostaglandins were not detectable in the supernatant fluids of the leukemic cell cultures. In one patient, BRSA was measured twice and found to be decreased to a normal level when the patient was in hematological remission with a normal calcium level (8.3 mg/dl). These results suggest that the hypercalcemia observed in patients with ATL is due, in part, to a bone resorption-stimulating factor which is produced by leukemic T-cell lymphocytes.

Primary malignant lymphoma of the thyroid in a patient with long-standing Graves' disease.

We have recently encountered a patient with rapidly enlarging thyroid masses histologically diagnosed as diffuse histiocytic lymphoma which developed in the active course of Graves' disease. The primary thyroid lymphoma has been in complete remission after local radiation therapy. The association of Hashimoto's thyroiditis and thyroid lymphoma has well been recognized. Meanwhile, data have accumulated to demonstrate that Hashimoto's thyroiditis and Graves' disease share possible similar causal immunological abnormalities and are closely related entities. However, the association of Graves' disease and primary thyroid lymphoma has never been reported, as far as we know. Therefore, this case may be the first one that supports the natural concept that thyroid lymphoma develops from pre-existing Graves' disease secondary to the similar immunological abnormalities in Hashimoto's thyroiditis.