Naofumi Ishikawa

p53 induced by ionizing radiation mediates DNA end-jointing activity, but not apoptosis of thryroid cells

To understand the effects of ionizing radiation on thyroid cells, we investigated the role of p53 in mediating apoptosis and in DNA repair following in vivo and in vitro irradiation of thyroid cells. In vitro exposure of human thyroid cells to ionizing radiation of up to 5 – 8 Gy failed to induce apoptosis in primary cells. The same results were obtained when the thyroid gland was irradiated in the intact rat. To explore the mechanism of failure of the wild-type p53 in inducing apoptosis in thyroid cells, we investigated the expression of apoptosis-related genes, bax, bcl-2 and fas/APO-1 following irradiation or induction of temperature-sensitive p53. The expression of Bax, Bcl-2 and Fas/APO-1 in human primary cultured thyroid cells did not change after irradiation. To further confirm the results, we established a clonal cell line (tsFRO) in which a temperature sensitive p53 (Val138) expression vector was stably transfected to a thyroid carcinoma cell line lacking endogenous p53. Incubation of tsFRO cells at the permissive temperature for three days, however, did not induce apoptosis although G1 arrest was noted. Although enhanced expression of the bax mRNA level was observed, the expression of Bax, Bcl-2 and Fas/APO-1 protein did not change by shifting tsFRO cells to permissive temperature as well as irradiated primary cells. Furthermore, DNA end-jointing ability was examined by transfection of linearized luciferase plasmid into tsFRO cells. Increased luciferase activity occurred when the cells were cultured at the permissive temperature, indicating that the wild-type p53 enhances DNA end-jointing activity. Our results indicate that the wild-type p53 does not lead to apoptosis but facilitates DNA end-jointing in thyroid cells. These results may reflect specific responses in thyroid cells following irradiation.

Association of the T-cell regulatory gene CTLA4 with Graves’ disease and autoimmune thyroid disease in the Japanese

AbstractAutoimmune thyroid disease (AITD) is caused by an immune response to self-thyroid antigen. The cytotoxic T-lymphocyte antigen-4 (CTLA4) gene, encoding a negative regulator of the T-lymphocyte immune response, had been reported to be associated and/or linked to AITD. Recently, AITD susceptibility in the Caucasians was mapped to the 6.1-kb 3′UTR of the CTLA4 gene, in which the three single-nucleotide polymorphisms (SNPs) CT60, JO31, and JO30 were strongly associated with AITD. In order to determine the association of the CTLA4 gene with AITD in the Japanese, case-control association analysis for the four SNPs of the CTLA4 gene using 380 AITD patients and 266 healthy controls was done. Among the SNPs examined, the SNP JO31 was most significantly associated with AITD in the Japanese, whereas the association of the JO30 with AITD was not observed. The frequency of the disease-susceptible G allele of the JO31 of the Japanese control was higher than that of the Caucasians (67.1% vs 50.2%); however, the G allele of the JO31 was associated with Graves′ disease (GD) (67.1% vs 76.3%, P=0.0013) and AITD in the Japanese (67.1% vs 74.2%, P=0.0055). Furthermore, the G allele of the JO31 was associated with the increased risk for GD [ P=0.0051, odds ratio (OR)=1.7] and AITD (P=0.016, OR=1.5) in a dominant model. These results suggested that the CTLA4 gene is involved in the susceptibility for GD and AITD in the Japanese.

CD4+ T cell-mediated cytotoxicity toward thyrocytes: the importance of Fas/Fas ligand interaction inducing apoptosis of thyrocytes and the inhibitory effect of thyroid-stimulating hormone.

The accumulation of activated CD4+ T cells and antigen (Ag)-dependent cellular interactions between thyrocytes and CD4+ T cells have been determined in thyroid gland from patients with Graves’ disease. The Fas/Fas ligand (FasL) interaction between antigen-presenting cells and T cells regulates the apoptosis of the former cells triggered by the latter cells. The inhibition of Fas-mediated apoptosis in thyrocytes could be a underlying mechanism of hyperplasia of thyrocytes in patients with Graves’ disease. We investigated the potential role of Fas/FasL interaction between thyrocytes and CD4+ T cells in the induction of Fas-mediated apoptosis of the former cells induced by the latter cells. The presence of only a few specific T cells responsive to a putative autoantigen has hampered the investigation of specific T cell activation toward antigen-presenting cells (APCs). Therefore, we used a superantigen, staphylococcal enterotoxin B (SEB), to examine specific T cell activation toward thyrocytes in vitro since it stimulates a large proportion of T cells with particular Vβ elements. Spontaneous apoptosis of thyrocytes in culture was not found even in the presence of various kinds of cytokines. In contrast, a clear induction of Fas-mediated apoptosis by anti-Fas IgM was determined in interferon-γ (IFN-γ)-stimulated thyrocytes. In addition, a significant cytotoxicity of purified CD4+ T cells toward IFN-γ-stimulated thyrocytes in the presence of SEB was induced, and the addition of anti-HLA-DR and -DQ monoclonal antibodies (mAbs) or blockade of the Fas/FasL interaction reduced this cytotoxicity. FasL expression of CD4+ T cells cocultured with IFN-γ-stimulated thyrocytes in the presence of SEB was clearly induced. Furthermore, the addition of mAbs against CD54 and CD58 inhibited both cytotoxicity and FasL expression of CD4+ T cells. The cytotoxicity of CD4+ T cells toward IFN-γ-stimulated, SEB-pulsed thyrocytes was markedly inhibited when we used thyrocytes cultured with IFN-γ in the presence of thyroid-stimulating hormone (TSH) as target cells. Our results suggest that 1) CD4+ T cells were activated by thyrocytes expressing MHC class II molecules in an SEB-dependent manner and then expressed FasL. 2) These activated FasL+ CD4+ T cells killed thyrocytes by interacting with Fas on thyrocytes and FasL on activated CD4+ T cells. The presence of costimulating molecules such as CD54 and CD58 on thyrocytes was also necessary to generate activated FasL+ CD4+ T cells. 3) Since the actions of thyroid stimulating antibody (TSAb) toward thyrocytes are similar to those of TSH, one goitrogenic activity of TSAb may, in part, be due to the inhibitory effect on Fas-mediated apoptosis of thyrocytes triggered by activated CD4+ T cells.

Cytokine regulation of HLA on thyroid epithelial cells

The regulation of class I and class II HLA expression in human thyroid follicular cells was studied in vitro. Tumour necrosis factor‐alpha (TNF‐α) enhanced the expression of class I antigen on thyrocytes, but these cytokines had little effect on the expression of class II antigen. Interleukin‐lβ (IL‐ lβ) and interleukin‐6(IL‐6) did not affect class I and class II antigen expression. The combination of interferon‐gamma (IFN‐γ) with TNF‐α or IL‐1β enhanced the induction of class I and class II antigens, compared with the effect of IFN‐β alone. Neither class I nor class II expression was induced by lL‐6 alone or in combination with IFN‐γ. These findings suggest that TNF‐α and IL‐1β may have an important role in inappropriate expression of HLA antigens on thyrocytes in thyroid gland.

Toxic multinodular goitre in a patient with generalized resistance to thyroid hormone who harbours the R429Q mutation in the thyroid hormone receptor β gene

The association of resistance to thyroid hormone (RTH) due to a receptor defect with toxic multinodular goitre or with carcinoma of the thyroid has not been previously reported. Previous histopathological studies of the thyroid gland in patients with RTH have revealed changes similar to multinodular goitre, probably due to continuous stimulation by TSH. We report here a case of generalized resistance to thyroid hormone associated with a multinodular goitre, which became toxic. The patient was a 46‐year‐old Japanese woman who noticed a goitre although she had no symptoms of thyrotoxicosis. Initial examination revealed elevated serum thyroid hormone levels and a normal TSH level. Ultrasonography disclosed a multinodular goitre with cystic lesions. Three years later, the patient complained that the goitre had become larger and that she had developed symptoms of thyrotoxicosis such as palpitation and hyperhydrosis. Progressive hyperthyroxinaemia with relatively suppressed TSH, increased radioiodine uptake and negative anti‐TSH receptor antibodies led to the diagnosis of toxic multinodular goitre. Subtotal thyroidectomy was performed, and pathological examination revealed a micropapillary carcinoma within the multinodular goitre. Occurrence of thyroid carcinoma should be considered in RTH because its incidence is high in multinodular goitre. Molecular examination revealed the R429Q mutation in the thyroid hormone receptor β gene, which is one of the mutations usually manifesting as the pituitary resistance phenotype. That thyrotoxic manifestations appeared only during toxic stage of multinodular goitre in this case suggests that the phenotype of this type of mutation can be dependent on the amount of thyroid hormone.

Retinole acid inhibits human thyroid peroxidase and thyroglobulin gene expression in cultured human thyrocytes

The effect of retinoic acid (RA) on thyroid peroxidase (TPO) and thyroglobulin (Tg) gene expression was investigated in cultured human thyrocytes. Thyrocytes dispersed from Graves’ thyroid tissues were incubated with TSH 5mU/ml and RA 0, 0.01, 0.1,1.0 μM for 72 h respectively. The samples were then subjected to Northern gel analysis. Northern gel analysis using the specific cDNA probes showed that RA suppressed the accumulation of TPO and Tg mRNA stimulated by TSH in a time- and dose-responsive manner. Furthermore, RA inhibited forskolin and 8-Bromo-cyclic-AMP-induced TPO and Tg gene expression, suggesting a distal action site for these cAMP mediated gene expressions. Immunoprecipitation analysis using the specific monoclonal antibodies showed that TSH increased newly synthesized 100, 75, 36-kDa [35S] TPO. The increased de novo TPO was markedly inhibited by RA. Tg secretion from monolayer cultures was measured by radioimmunoassay. RA also inhibited TSH-in-duced Tg secretion in a dose dependent manner. RA did not affect [3H] thymidine uptake into primary cultured human thyrocytes. In conclusion, RA inhibits the synthesis of TPO and Tg via the suppression of thyroid-specific gene expression although the exact site of RA action on these genes in human thyroids remains to be further elucidated. These results suggest that RA may play a regulatory role in Tg and TPO gene expression, subsequently resulting in the suppression of thyroid hormone synthesis.

Stimulation of thyroid‐stimulating hormone (TSH) receptor antibody production following painless thyroiditis

objective  Development or recurrence of Graves’ disease (GD) following painless thyroiditis (PT) has been documented. Therefore, we measured titres of TSH receptor antibodies (TSHR Ab) using a novel sensitive TSHR Ab assay in patients with PT to determine whether PT enhances TSHR Ab production, possibly triggering the development or recurrence of GD.

Identification of independent risk loci for Graves' disease within the MHC in the Japanese population.

To identify genetic variants that confer the risk of Graves’ disease (GD) in the Japanese population, we conducted a two-stage genome-wide association study (GWAS) using 1119 Japanese individuals with GD and 2718 unrelated controls, and a subsequent replication study using independent 432 GD cases and 1157 controls. We identified 34 single nucleotide polymorphisms (SNPs) to be significantly associated with GD in the GWAS phase. Twenty-two out of 34 SNPs remained positive in the replication study. All 22 SNPs were located within the major histocompatibility complex (MHC) locus on chromosome 6p21. No strong long-range linkage disequilibrium (LD) was observed among the 22 SNPs, indicating independent involvement of multiple loci within the MHC with the risk of GD. Multivariate stepwise logistic regression analysis selected rs3893464, rs4313034, rs3132613, rs4248154, rs2273017, rs9394159 and rs4713693, as markers for independent risk loci for GD. The analysis of LD between these seven SNPs and tagging SNPs for GD-associated human leukocyte antigen (HLA) alleles in the Japanese population (HLA-DPB1*0501 and HLA-A*0206) demonstrated that all of and five of seven SNPs were not in strong LD with HLA-DPB1*0501 and HLA-A*0206, respectively. Although causal variants remain to be identified, our results demonstrate the existence of multiple GD susceptibility loci within the MHC region.

Elevation of Serum Pro-Gastrin - Releasing Peptide in Patients with Medullary Thyroid Carcinoma and Small Cell Lung Carcinoma

Medullary thyroid carcinoma (MTC) arises from parafollicular or C cells of the thyroid gland and produces a variety of peptides such as calcitonin (CT) and gastrin-releasing peptide (GRP). Here we measured serum levels of pro-gastrin-releasing peptide (Pro-GRP), a more stable precursor of GRP, in 15 patients with MTC (4 males, 11 females) who did not show any clinical or radiologic signs of small cell lung cancer. Serum Pro-GRP levels were elevated in 80% (12/15) patients. Significant correlation was observed between serum Pro-GRP and CT (r = 0.52) and carcinoembryonic antigen (CEA) (r = 0.56). Serum Pro-GRP levels also correlated with tumor size (r = 0.70). Serum Pro-GRP levels also decreased below the cut-off range in one patient after surgical resection. Our data suggest that Pro-GRP, which is considered to be a specific marker for small cell lung carcinoma, seems to be also helpful and additional marker for the diagnosis and monitoring the response to therapy in patients with MTC in addition to calcitonin as the main tumor marker.

High maintenance rate of sinus rhythm after cardioversion in post-thyrotoxic chronic atrial fibrillation.

We performed rhythm conversion on 33 euthyroid patients with post-thyrotoxic atrial fibrillation who do not revert spontaneously to sinus rhythm from atrial fibrillation. The duration of atrial fibrillation ranged from 9 to 59 months (mean 25 +/- 14). The protocol of rhythm conversion was to first attempt pharmacological conversion with disopyramide and then to perform electrical cardioversion on the non-converters. Of 33 patients, 25 were converted to sinus rhythm (6 by disopyramide and 19 by electrical cardioversion), resulting in 8 non-converters. However, it was later discovered that cardioversion had been applied to 4 of the 8 non-converters when these patients had had a recurrence of thyrotoxicosis. These 4 patients were subjected to a second electrical cardioversion after attaining the euthyroid state. Sinus rhythm was restored in all 4 patients, giving a cardioversion rate of 88%. The sinus rhythm was maintained in 25 of the total 29 converters (86%) at the time of follow-up (10-68 months, mean 35 +/- 19 months). Our studies suggest that cardioversion should be encouraged for thyrotoxic atrial fibrillation, even if the duration of atrial fibrillation is long-standing, since there is excellent maintenance of sinus rhythm and even delayed application of cardioversion may improve its success rate.