Reevaluation of the Effect of Iodine on Thyroid Cell Survival and Function Using PCCL3 and Nthy-ori 3-1 Cells

The appropriate amount of iodine is critical for normal function of thyroid cells synthesizing thyroid hormones. Although normal thyroid cell lines such as rat PCCL3 and FRTL5 and human Nthy-ori 3-1 have been widely used for in vitro studies on physiological and pathophysiological effects of iodine on thyroid cells, we have recently pointed out the critical differences between FRTL5/PCCL3 cells and Nthy-ori 3-1 cells. Therefore, we here directly compared some of the cellular characteristics?iodine uptake, differentiated status, iodine-induced cytotoxicity, and iodine-regulation of autophagy?between PCCL3 and Nthy-ori 3-1 cells. PCCL3 cells express messenger RNAs for thyrotropin receptor and sodium/iodine symporter and incorporate iodine in a thyrotropin-dependent manner, whereas Nthy-ori 3-1 cells do not either. Nevertheless, both cells were comparably resistant to iodine cytotoxicity: Only far excess iodine (5 × 10?2 M) killed 20% to 40% cells in 24 hours with perchlorate exhibiting no effect, suggesting this cytotoxic effect is due to extracellular iodine. In contrast, a wide range of iodine (5 × 10?9 to 5 × 10?2 M) induced autophagy in PCCL3 cells, which was abolished by perchlorate, indicating intracellular iodine-induction of autophagy, but this effect was not observed in Nthy-ori 3-1 cells. In conclusion,it is critical to discriminate the effect of iodine incorporated into cells from that of extracellular iodine on thyroid cells. Iodine-uptake competent thyroid cells such as PCCL3 and FRTL5 cells, not Nthy-ori 3-1 cells, should be used for studies on iodine effect on thyroid cells

Characterization of metabolic reprogramming by metabolomics in the oncocytic thyroid cancer cell line XTC.UC1

Oncocytic thyroid cancer is characterized by the aberrant accumulation of abnormal mitochondria in the cytoplasm and a defect in oxidative phosphorylation. We performed metabolomics analysis to compare metabolic reprogramming among the oncocytic and non-oncocytic thyroid cancer cell lines XTC.UC1 and TPC1, respectively, and a normal thyroid cell line Nthy-ori 3-1. We found that although XTC.UC1 cells exhibit higher glucose uptake than TPC1 cells, the glycolytic intermediates are not only utilized to generate end-products of glycolysis, but also diverted to branching pathways such as lipid metabolism and the serine synthesis pathway. Glutamine is preferentially used to produce glutathione to reduce oxidative stress in XTC.UC1 cells, rather than to generate α-ketoglutarate for anaplerotic flux into the TCA cycle. Thus, growth, survival and redox homeostasis of XTC.UC1 cells rely more on both glucose and glutamine than do TPC1 cells. Furthermore, XTC.UC1 cells contained higher amounts of intracellular amino acids which is due to higher expression of the amino acid transporter ASCT2 and enhanced autophagy, thus providing the building blocks for macromolecules and energy production. These metabolic alterations are required for oncocytic cancer cells to compensate their defective mitochondrial function and to alleviate excess oxidative stress

Defective killer cell activity in patients with chronic active Epstein-Barr virus infection.

Natural killer (NK) cell activity, lymphokine activated killer (LAK) activity and Epstein-Barr virus specific cytotoxic T lymphocyte (EBV-CTL) activity were examined in 10 children with chronic active EB-virus infection and an adult with persistently positive early antigen-antibody to EB-virus. NK cell activity against erythroleukemia cell line K-562 was significantly (p less than 0.005) lower in the patients (22.3 +/- 8.5%, mean +/- SD) than in normal controls (40.4 +/- 15.9%). Spontaneous cytotoxicity against an EB-virus transformed autologous lymphoblastoid cell line was 15.0 +/- 7.6% in the patients, and was comparable to spontaneous cytotoxicity activity in normal controls (11.7 +/- 4.3%). LAK activity against Raji cells was significantly (p less than 0.02) lower in the patients (14.6 +/- 11.4%) than in normal controls (29.2 +/- 15.9%). EBV-CTL activity against an EB-virus transformed autologous lymphoblastoid cell line was significantly (p less than 0.005) lower in the patients (11.8 +/- 5.5%) than in seropositive normal controls (33.7 +/- 14.7%). No regression of the lymphoblastoid cell line was observed when EBV-CTL activity of the patients was tested by regression assay. It is conceivable that defects in both EB-virus specific and nonspecific killer cell activities play important roles in the pathogenetic abnormalities which allow EB-virus infection to progress to a chronic active state.</p

Hyperreactivity of lymphocytes to streptolysin O and lack of plasma inhibitory factor (s) in patients with mucocutaneous lymphnode syndrome.

Lymphocyte activation by streptolysin O (SLO) and factors in the plasma which inhibit the response to SLO were examined in 19 patients with mucocutaneous lymphnode syndrome (MCLS), 54 age-matched (6 months-6 years) normal children, 41 normal children older than 6 years and 10 normal adults. In normal children younger than 6 years, the response to SLO was weak and in many cases no response was seen. On the other hand, in the patients with MCLS, the response of lymphocytes to SLO was high and comparable to the response in adults and children older than 6 years. The DNA synthesis of lymphocytes stimulated by SLO was inhibited almost completely by autologous or allogeneic plasma of many of the normal children and adults. The plasma of patients with MCLS did not inhibit, but rather enhanced the response to SLO. These results suggest that the increased response of lymphocytes to SLO and the lack of plasma inhibitory factors in patients with MCLS may be due to the immune response to the pathogen of MCLS, as yet undiscovered.</p

Minor contribution of cytotoxic T lymphocyte antigen 4 and programmed cell death 1 ligand 1 in immune tolerance against mouse thyrotropin receptor in mice

We have previously shown that wild type (wt) mice exhibit susceptibility to immunization with human (h) thyrotropin receptor (TSHR), but resistance to mouse (m) TSHR, while TSHR knockout (KO) mice are susceptible to mTSHR, indicating the existence of robust immune tolerance against the mTSHR in wt mice. This tolerance may be mediated by either centrally or peripherally. We here explored the contribution of a peripheral arm of immune tolerance against the mTSHR by using antibodies to deplete regulatory T cells (Tregs), to antagonize co-inhibitory molecules and/or to stimulate co-stimulatory molecules. Antagonistic anti-co-inhibitory molecules, cytotoxic T lymphocyte antigen 4 (CTLA4) and programmed cell death 1 ligand 1 (PDL1), induced only low levels of anti-TSHR antibodies without induction of hyperthyroidism in a mouse Graves\u27model. In this experimental setting, antibody levels were significantly higher in THSR+/- mice than wt mice. However, agonistic anti-co-stimulatory molecules, CD40 and CD137, and Treg-depleting anti-CD25 antibodies showed no effect. All these data suggest that peripheral immune tolerance against the mTSHR may play a minor role, and imply the importance of central tolerance, in immune tolerance against mTSHR in mice. Additional studies on central tolerance to the mTSHR will be necessary for completely delineating the mechanisms for immune tolerance against mTSHR in mice

Studies on Expression of Aldehyde Dehydrogenase in Normal and Cancerous Tissues of Thyroids

Recently published articles have reported the controversial data regarding expression of aldehyde dehydrogenase isozyme 1A1 (ALDH1A1), a potential candidate marker for normal and cancer stem cells (CSCs), in thyroid tissues. These data prompted us to re-evaluate expression of ALDH1A1 in normal and cancerous thyroid tissues by 2 different means. The first method was immunohistochemistry with 2 different anti-ALDH1A1 antibodies from distinct companies. Following validating the integrity of these 2 antibodies by Western blotting with ALDH-expressing and nonexpressing cancer cell lines and immunohistochemistry with breast and colon tissues, we report here significant and comparable expression of ALDH1A1 in both normal and cancerous thyroid tissues with both antibodies. Next, relative expression levels of ALDH isozymes were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), revealing that ALDH1A1 was the most highly expressed isozyme followed by ALDH9A1 and relative expression patterns of isozymes were very similar in normal and cancerous tissues. All these data demonstrate that thyroid cells of normal and cancer origins do express ALDH1A1 and to a lesser extent 9A1. Further study will be necessary to study functional significance of ALDH1A1 in the function and behaviors of thyroid normal and cancer stem cells

Age-dependent effects on radiation-induced carcinogenesis in the rat thyroid

Childhood radiation exposure is a known thyroid cancer risk factor. This study evaluated the effects of age on radiation-induced thyroid carcinogenesis in rats irradiated with 8 Gy X-rays. We analyzed cell proliferation, cell death, DNA damage response, and autophagy-related markers in 4-week-old (4W) and 7-month-old (7M) rats and the incidence of thyroid tumors in 4W, 4-month-old (4M), and 7M rats 18 months after irradiation. Cell death and DNA damage response were increased in 4W rats compared to those in controls at 1 month post-irradiation. More Ki-67-positive cells were observed in 4W rats at 12 months post-irradiation. Thyroid tumors were confirmed in 61.9% (13/21), 63.6% (7/11), and 33.3% (2/6) of irradiated 4W, 4M, and 7M rats, respectively, compared to 0%, 14.3% (1/7), and 16.7% (1/6) in the respective nonirradiated controls. There were 29, 9, and 2 tumors in irradiated 4W, 4M, and 7M rats, respectively. The expression of several autophagy components was downregulated in the area surrounding radiation-induced thyroid carcinomas in 4W and 7M rats. LC3 and p62 expression levels decreased in radiation-induced follicular carcinoma in 4W rats. Radiosensitive cells causing thyroid tumors may be more prevalent in young rats, and abrogation of autophagy may be associated with radiation-induced thyroid carcinogenesis

Cell-mediated cytotoxicity-supporting activity of various human gammaglobulin preparations.

Antibody activity, especially that involved in the reaction of antibody-dependent cell-mediated cytotoxicity (ADCC), of five commercially available human gammaglobulin preparations (standard, pepsin-treated, plasmin-treated, polyethylene glycol-fractionated and S-sulfonated gammaglobulin) was measured. All these gammaglobulin preparations had high titers of hemagglutination inhibition and neutralizing antibody against measles virus. In ADCC reaction, the pepsin-treated gammaglobulin preparation showed no antibody activity. The standard gammaglobulin preparation showed weak activity only when highly diluted. The remaining three preparations showed high activity. Though the S-sulfonated gammaglobulin preparation showed no activity in ADCC reaction, it showed high activity after reconversion by means of oxidation and reduction in vitro. The plasmin-treated gammaglobulin preparation showed greater activity than the polyethylene glycol-fractionated preparation of the optimal concentration. In ADCC tests using the plasmin-treated gammaglobulin preparation, K cell activity was strongly inhibited by Hg (thimerosal), while, in those using the standard gammaglobulin preparation, the activity was hardly influenced by Hg, suggesting that the low ADCC activity of the standard gammaglobulin preparation of high concentrations was due to the inhibitory effect of aggregated immunoglobulin G molecules.</p

甲状腺刺激ホルモンシグナルは、BRAFV600E甲状腺癌マウスモデルにおけるゲノム不安定性を伴った高度悪性化に関与している

長崎大学学位論文 [学位記番号]博（医歯薬）甲第636号 [学位授与年月日]平成25年10月2