Development of a Psychological Scale for Measuring Disruptive Clinician Behavior.

OBJECTIVES: Disruptive clinician behavior worsens communication, information transfer, and teamwork, all of which negatively affect patient safety. Improving safety in medical care requires an accurate assessment of the damage caused by disruptive clinician behavior. Psychometric scales complement case reports, but existing scales have significant limitations. Therefore, this study developed a psychometric scale based on the psychological paradigm to assess disruptive clinician behavior. METHODS: The scale was developed through a sequence of steps. First, we used an open-ended questionnaire targeting 712 nurses, content analysis, and content validity assessment by 5 experts to determine valid items for disruptive clinical behavior. Next, an Internet questionnaire survey targeting 1000 health care staff, exploratory factor analysis, and subfactor analysis was conducted to identify necessary and sufficient factors. Then, we calculated difficulty level and discriminative power. We also conducted a field questionnaire survey targeting 84 staff in a hospital. Finally, we calculated ω coefficients and then used confirmatory factor analysis to verify the fit of the hypothesized model. RESULTS: Our open-ended survey involving 478 nurses identified 47 codes in 9 categories. The questionnaire survey involving hospital 1000 medical staff identified 6 factors, with 1 factor subdivided into 4 subfactors and 1 into 2 subfactors. The goodness of fit of the hypothesized 10-factor models with factor pairs and groups was confirmed. CONCLUSIONS: We developed a psychometric scale measuring subjective assessments of harm covering various disruptive clinician behaviors. The scale complements interviews and case reports by generating valid, reliable scores for various disruptive clinician behaviors in health care institutions.

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.

The Effect of Long-Term Inorganic Iodine on Intrathyroidal Iodothyronine Content and Gene Expression in Mice with Graves' Hyperthyroidism.

Background: The main molecular mechanism underlying acute suppression of iodine organification in normal thyroids after an excessive iodine load, that is, the Wolff-Chaikoff effect, is assumed to be suppression of iodine oxidation and iodothyronine synthesis. However, the mechanism underlying chronic antithyroid action of inorganic iodine in Graves' disease is not fully understood. Using a mouse model of Graves' hyperthyroidism, we examined changes in iodothyronine content and gene expression profiles in the thyroid glands after inorganic iodine loading. Materials and Methods: Graves' hyperthyroidism was induced and maintained in BALB/c mice by repeated immunizations of recombinant adenovirus expressing the human thyrotropin (TSH) receptor A-subunit. Hyperthyroid mice were left untreated (GD-C; n = 8) or treated with inorganic iodine for 12 weeks (GD-NaI; n = 8). We used unimmunized BALB/c mice as a control group (n = 10). In each mouse, serum thyroxine (T4) levels were measured with enzyme-linked immunosorbent assay (ELISA) at 4-week intervals. The intrathyroidal iodothyronine content and gene expression levels were, respectively, evaluated by mass spectrometry and RNA sequencing (RNA-seq) at the end of the experimental period. Results: Serum T4 levels in the GD-C group remained higher than in the control group, whereas those in the GD-NaI group declined to normal levels during the experimental period. Intrathyroidal triiodothyronine (T3), reverse T3 (rT3), and T4 contents in the GD-C group were higher than the control group, and rT3 and T4 were further increased in the GD-NaI group. The observed alterations in iodothyronine levels in the thyroid and sera may be explained by altered expression levels of genes for iodothyronine biosynthetic molecules, their transporter, and deiodinases. Conclusion: In this mouse model of hyperthyroidism, higher intrathyroidal accumulation of T4 and reduced gene expression data of iodothyronine transporters in the GD-NaI group suggest that chronic antithyroid action of iodine in Graves' disease involves suppression of hormone secretion.

MIEAP and ATG5 are tumor suppressors in a mouse model of BRAFV600E-positive thyroid cancer.

Mitochondria-eating protein (MIEAP) is a molecule important for non-canonical mitophagy and thought to be a tumor suppressor. Our previous study found that MIEAP expression is defective in thyroid oncocytomas, irrespective of being benign or malignant, and also in non-oncocytic thyroid cancers. Thyroid oncocytomas are composed of large polygonal cells with eosinophilic cytoplasm that is rich in abnormal mitochondria. Thus, our data indicate that, together with increased mitochondrial biogenesis that compensates for the dysfunction of the mitochondria, MIEAP plays a critical role in the accumulation of mitochondria in thyroid oncocytic tumors, whereas a defective MIEAP expression alone is not sufficient for mitochondrial accumulation in non-oncocytic cancers with normal mitochondria. To clarify whether MIEAP is a tumor suppressor in the thyroids and whether MIEAP knockout (KO) alone is sufficient for the oncocytic phenotype and also to extend our effort toward canonical mitophagy (a selective autophagy), we here conducted mouse studies using genetically engineered mice. BrafCA/wt mice developed thyroid cancers 1 year after intrathyroidal injection of adenovirus expressing Cre, while cancer development was observed at 6 months in adenovirus-Cre-injected BrafCA/wt;MieapKO/KO and BrafCA/wt;Atg5flox/flox mice [where autophagy-related 5 (ATG5) is a component of autophagic machinery], although KO of either molecule alone was not sufficient for cancer development. These data demonstrate that MIEAP or ATG5 KO accelerated thyroid cancer development. However, cancers in adenovirus-Cre-injected BrafCA/wt ;MieapKO/KO and BrafCA/wt ;Atg5flox/flox mice were not oncocytic. In conclusion, we here show that MIEAP and ATG5 are both tumor suppressors in thyroid carcinogenesis, but as we have anticipated from our previous data, KO of either molecule does not confer the oncocytic phenotype to BRAFV600E-positive thyroid cancers. The combination of disruptive mitochondrial function and impaired mitochondrial quality control may be necessary to establish a mouse model of thyroid oncocytoma.

Re-evaluation of the role of autophagy in thyroid cancer treatment.

Numerous studies have examined the role of autophagy in thyroid cancer treatment; however there are discrepancies among the reported data, with some showing the pro-survival and others the anti-survival effects of autophagy. These discrepant results appear to be at least in part due to insufficient analyses or data misinterpretation as well as improper assessments of autophagic activity. Therefore, the present study re-evaluated the regulation of autophagic activity by various anticancer modalities and examined the role of autophagy in thyroid cancer treatment in three thyroid cancer cell lines (TPC1, ACT1 and KTC1). The immunofluorescence and DalGreen findings demonstrated that cisplatin, irradiation and sorafenib were all autophagy inducers as previously reported, but, unlike previous studies using thyroid cancer cells, doxorubicin acted as an inhibitor. KTC1 cells are unique because they only responded to cisplatin. The efficacy of anticancer therapeutics was significantly higher in chloroquine or 3-methyladenine-treated autophagy-defective cells than in autophagy-competent cells, thereby indicating the pro-survival effect of autophagy induced by anticancer therapeutics, which is partly due to inhibition of apoptosis. Thus, the present findings relating to several anticancer therapeutics and three thyroid cancer cell lines demonstrate the pro-survival effect of autophagy in thyroid cancer treatment. Although the present study only involved cell lines, it provides evidence for the beneficial combination of the anticancer therapeutic modalities with autophagy inhibitors, and proposes that autophagy inhibitors may serve as a possible adjunctive therapy for thyroid cancer.

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.

Hormonal Regulation of Autophagy in Thyroid PCCL3 Cells and the Thyroids of Male Mice.

Autophagy is an evolutionarily conserved catabolic process by which cells degrade intracellular proteins and organelles in the lysosomes and recycle their metabolites. We have recently demonstrated the crucial role for the basal level of autophagic activity in thyrocyte survival and homeostasis using the thyroid-specific autophagy knockout mice. Here, we first studied hormonal regulation of autophagy in thyrocytes in vitro using a rat thyroid cell line PCCl3 and in vivo with mice. In cultured PCCl3 cells, thyroxine decreased microtubule-associated protein 1 light chain 3 (LC3) puncta (a component of autophagosome) and increased p62 (an autophagy substrate) levels, showing thyroxine-suppression of autophagy. In contrast, TSH increased both LC3 puncta and p62 levels, but at the same time stabilized p62 protein by inhibiting p62 degradation, indicating TSH induction of autophagy. Our experiments with various inhibitors identified that both the cAMP-protein kinase (PK) A-cAMP response element binding protein/ERK and PKC signaling pathways regulates positively autophagic activity. The in vivo results obtained with wild-type mice treated with methimazole and perchlorate or thyroxine were consistent with in vitro results. Next, in thyroid-specific autophagy knockout mice treated with methimazole and perchlorate (that is, mice were placed under a stressed condition where enhanced autophagy was required) for 2 months, lower follicle sizes and lower thyroglobulin contents in thyrocytes were observed, suggesting impaired thyroglobulin production presumably from insufficient nutrient supply. We therefore conclude that TSH positively regulates autophagic activity through the cAMP-PKA-cAMP response element binding protein/ERK and PKC signaling pathways, whereas thyroid hormones inhibit its activity in thyrocytes. Metabolites produced by autophagy appear to be necessary for protein synthesis stimulated by TSH.

Causative role for defective expression of mitochondria-eating protein in accumulation of mitochondria in thyroid oncocytic cell tumors.

Oncocytic cell tumor of the thyroid is composed of large polygonal cells with eosinophilic cytoplasm that is rich in mitochondria. These tumors frequently have the mutations in mitochondrial DNA encoding the mitochondrial electron transport system complex I. However, the mechanism for accumulation of abnormal mitochondria is unknown. A noncanonical mitophagy system has recently been identified, and mitochondria-eating protein (MIEAP) plays a key role in this system. We therefore hypothesized that accumulation of abnormal mitochondria could be attributed to defective MIEAP expression in these tumors. We first show that MIEAP was expressed in all the conventional thyroid follicular adenomas (FAs)/adenomatous goiters (AGs) but not in oncocytic FAs/AGs; its expression was defective not only in oncocytic thyroid cancers but also in the majority of conventional thyroid cancers. Expression of MIEAP was not correlated with methylation status of the 5'-UTR of the gene. Our functional analysis showed that exogenously induced MIEAP, but not PARK2, reduced the amounts of abnormal mitochondria, as indicated by decreased reactive oxygen species levels, mitochondrial DNA / nuclear DNA ratios, and cytoplasmic acidification. Therefore, together with previous studies showing that impaired mitochondrial function triggers compensatory mitochondrial biogenesis that causes an increase in the amounts of mitochondria, we conclude that, in oncocytic cell tumors of the thyroid, increased abnormal mitochondria cannot be efficiently eliminated because of a loss of MIEAP expression, ie impaired MIEAP-mediated noncanonical mitophagy.

Acceleration of BRAFV600E-induced thyroid carcinogenesis by TGFß signal deficiency in mice.

PURPOSE: Transforming growth factor-ß (TGFß) has pleiotropic actions, including both anti- and pro-tumorigenic abilities. We have previously shown no tumor development in the thyroid-specific TGFß receptor type II knockout (Tgfßr2 KO) mice, indicating the insufficiency of defective TGFß signal itself for thyroid cancer initiation. In the current study, we evaluated whether defective TGFß signal accelerates BRAFV600E-mediated thyroid carcinogenesis in our mouse model, in which intrathyroidal injection of adenovirus expressing Cre under thyroglobulin (TG) promoter (Ad-TgP-Cre) into thyroid lobes of conditional BrafV600E knock-in mice (BrafCA) induces thyroid cancers 12 months later. METHODS: BrafCA/wt;Tgfbr2floxE2/floxE2 mice were generated by crossing Tgfbr2floxE2/floxE2 and BrafCA mice, and Ad-TgP-Cre was injected into the left lobes of 4-6-week-old mice. Mice were sacrificed at 6 and 12 months, and the thyroid tissues were subjected to H&E and immune-histochemistry and -fluorecence. RESULTS: Thyroid tumors were observed in 8 of 10 mice at 6 months and 4 of 7 mice at 12 months. These tumors were judged to be malignant by H&E staining, because of the presence of papillary growth of atypical follicular cells, intranuclear cytoplasmic inclusions and so on. Immunohistochemical analyses using thyroid cancer tissues obtained at 6 months demonstrated variable levels of TG but steady levels of Paired Box-8 expression and higher Ki67 positivity. The degree of epithelial-to-mesenchymal transition could not be evaluated because normal thyroid tissues and thyroid cancers developed in BrafCA and BrafCA/wt;Tgfbr2floxE2/floxE2 mice were all E-cadherin+/vimentin-, that is, epithelial type. CONCLUSION: In a mouse model, defective TGFß signaling pathway accelerates BRAFV600E-induced thyroid cancer development, which is occasionally accompanied by reduced TG expression implying dedifferentiation. The former finding is consistent with anti-tumorigenic ability of TGFß in early tumorigenic process, but the latter is contradictory to generally accepted concept for TGFß-induction of dedifferentiation.

Basal Autophagy Deficiency Causes Thyroid Follicular Epithelial Cell Death in Mice.

Autophagy is a catabolic process that involves the degradation of cellular components through the lysosomal machinery, relocating nutrients from unnecessary processes to more pivotal processes required for survival. It has been reported that systemic disruption of the Atg5 or Atg7 gene, a component of autophagy, is lethal and that its tissue-specific disruption causes tissue degeneration in several organs. However, the functional significance of autophagy in the thyroid glands remains unknown. Our preliminary data imply the possible involvement of dysfunctional autophagy in radiation-induced thyroid carcinogenesis. Therefore, we evaluated the effect of Atg5 gene knockout (KO) on thyroid morphology and function. To this end, Atg5flox/flox mice were crossed with TPO-Cre mice, yielding the thyroid follicular epithelial cell (thyrocyte)‒specific ATG5-deficient mice (Atg5thyr-KO/KO). Atg5 gene KO was confirmed by a lack of ATG5 expression, and disruption of autophagy was demonstrated by a decrease in microtubule-associated protein 1 light chain 3-II puncta and an increase in p62. Atg5thyr-KO/KO mice were born normally, and thyroid morphology, thyroid weights, and serum T4 and TSH levels were almost normal at 4 months. However, at 8 and 12 months, a decrease in the number of thyrocytes and an increase in TUNEL+-thyrocytes were observed in Atg5thyr-KO/KO mice even though thyroid function was still normal. The number of irregularly shaped (gourd-shaped) follicles was also increased. Excess oxidative stress was indicated by increased 8-hydroxy-2'-deoxyguanosine and 53BP1 foci in Atg5thyr-KO/KO mice. These data demonstrate that thyrocytes gradually undergo degradation/cell death in the absence of basal levels of autophagy, indicating that autophagy is critical for the quality control of thyrocytes.

Mouse models of sporadic thyroid cancer derived from BRAFV600E alone or in combination with PTEN haploinsufficiency under physiologic TSH levels.

The BRAFV600E mutation is the most prevalent driver mutation of sporadic papillary thyroid cancers (PTC). It was previously shown that prenatal or postnatal expression of BRAFV600E under elevated TSH levels induced thyroid cancers in several genetically engineered mouse models. In contrast, we found that postnatal expression of BRAFV600E under physiologic TSH levels failed to develop thyroid cancers in conditional transgenic Tg(LNL-BrafV600E) mice injected in the thyroid with adenovirus expressing Cre under control of the thyroglobulin promoter (Ad-TgP-Cre). In this study, we first demonstrated that BrafCA/+ mice carrying a Cre-activated allele of BrafV600E exhibited higher transformation efficiency than Tg(LNL-BrafV600E) mice when crossed with TPO-Cre mice. As a result, most BrafCA/+ mice injected with Ad-TgP-Cre developed thyroid cancers in 1 year. Histologic examination showed follicular or cribriform-like structures with positive TG and PAX staining and no colloid formation. Some tumors also had papillary structure component with lower TG expression. Concomitant PTEN haploinsufficiency in injected BrafCA/+;Ptenf/+ mice induced tumors predominantly exhibiting papillary structures and occasionally undifferentiated solid patterns with normal to low PAX expression and low to absent TG expression. Typical nuclear features of human PTC and extrathyroidal invasion were observed primarily in the latter mice. The percentages of pERK-, Ki67- and TUNEL-positive cells were all higher in the latter. In conclusion, we established novel thyroid cancer mouse models in which postnatal expression of BRAFV600E alone under physiologic TSH levels induces PTC. Simultaneous PTEN haploinsufficiency tends to promote tumor growth and de-differentiation.

Intracellular redox status controls spherogenicity, an in vitro cancer stem cell marker, in thyroid cancer cell lines.

Cancer stem cells (CSCs), a small fraction of a tumor mass, are proposed to be highly crucial for cancer initiation, recurrence and metastasis. We have recently found that aldehyde dehydrogenase (ALDH) 1A3 is a CSC marker in some thyroid cancer cell lines, whose functional activity is, however, not relevant for thyroid cancer stemness. Since previous studies on malignancies in other organs suggest that intracellular reactive oxygen species (ROS) might be a functional and targetable CSC marker, the present study was conducted to elucidate the significance of ROS as a functional CSC marker in thyroid cancer cell lines. We first found that ROS levels controlled spherogenicity; that is, ROSlow cells were more spherogenic than ROShigh cells. However, unlike typical CSCs in other cancers, CSC-like ROSlow cells in thyroid cancer cells were plastic and were not accompanied by de-differentiation status (i.e., expression of stemness markers/thyroid-specific transcription factors) or chemo-/radio-resistance. The lower levels of ROS were functionally critical because a forced increase in ROS levels by L-buthionine-S,R-sulfoximine, an inhibitor of glutathione (GSH) synthesis, and irradiation suppressed spherogenicity. ROS levels were also correlated with the number of double strand DNA breaks determined by 53BP1 staining. Lower ROS levels appear to be a result of decreased mitochondrial oxidative phosphorylation and elevated GSH contents. Given the importance of CSC-targeted therapy for achieving long-term disease eradication by exhausting self-renewal and growth potential of cancer tissues, ROS may be a good candidate for CSC-targeted therapy in thyroid cancer.

N-Acetyl-L-cysteine protects thyroid cells against DNA damage induced by external and internal irradiation.

We evaluated the effect of the antioxidant N-acetyl-L-cysteine (NAC) on the levels of reactive oxygen species (ROS), DNA double strand breaks (DSB) and micronuclei (MN) induced by internal and external irradiation using a rat thyroid cell line PCCL3. In internal irradiation experiments, ROS and DSB levels increased immediately after 131I addition and then gradually declined, resulting in very high levels of MN at 24 and 48 h. NAC administration both pre- and also post-131I addition suppressed ROS, DSB and MN. In external irradiation experiments with a low dose (0.5 Gy), ROS and DSB increased shortly and could be prevented by NAC administration pre-, but not post-irradiation. In contrast, external irradiation with a high dose (5 Gy) increased ROS and DSB in a bimodal way: ROS and DSB levels increased immediately after irradiation, quickly returned to the basal levels and gradually rose again after >24 h. The second phase was in parallel with an increase in 4-hydroxy-2-nonenal. The number of MN induced by the second wave of ROS/DSB elevations was much higher than that by the first peak. In this situation, NAC administered pre- and post-irradiation comparably suppressed MN induced by a delayed ROS elevation. In conclusion, a prolonged ROS increase during internal irradiation and a delayed ROS increase after external irradiation with a high dose caused serious DNA damage, which were efficiently prevented by NAC. Thus, NAC administration even both after internal or external irradiation prevents ROS increase and eventual DNA damage.

Aldehyde dehydrogenase activity plays no functional role in stem cell-like properties in anaplastic thyroid cancer cell lines.

Recent studies have revealed that aldehyde dehydrogenase (ALDH) is a candidate marker for thyroid cancer stem cells, although its activity is flexible. The goal of this study is to clarify the functional significance of ALDH enzymatic activity on thyroid cancer stem cells properties in anaplastic thyroid cancer cell lines. In vitro sphere formation assay was used to judge the stemness of 4 anaplastic thyroid cancer cell lines (FRO, ACT1, 8505C, and KTC3). Two well-known ALDH inhibitors, N,N-diethylaminobenzaldehyde (DEAB) and disulfiram (DS), were first used. DEAB (50 µM) almost completely suppressed ALDH activity without affecting cell proliferation or spherogenicity. Lack of effect of ALDH suppression on spherogenicity was confirmed using shRNA for ALDH1A3, an ALDH isozyme predominantly expressed in anaplastic thyroid cancer cell lines. In contrast, an ALDH2 inhibitor DS (1 µM) inhibited spherogenicity but did not inhibit ALDH1A3 activity. Based on the recent article from another group reporting the importance of sonic hedgehog (Shh) signaling in ALDH activity and spherogenicity in thyroid cancer, the effects of the Shh inhibitor cyclopamine were also studied. Like DS, cyclopamine (1 µM) decreased spherogenicity but not ALDH activity. Finally, exogenous expression of ALDH1A3 in otherwise ALDH- TPC1 cells (a papillary thyroid cancer cell line) revealed no effect on spherogenicity. In conclusion, we here show no functional role for ALDH activity in thyroid thyroid cancer stem cells properties. That is, ALDH activity and spherogenicity are clearly dissociable. Further understanding of thyroid cancer stem cells biology in thyroid cancers remains necessary for the future development of thyroid thyroid cancer stem cells-targeted therapies.

Cancer Stem Cells in the Thyroid.

The cancer stem cell (CSC) model posits that CSCs are a small, biologically distinct subpopulation of cancer cells in each tumor that have self-renewal and multi-lineage potential, and are critical for cancer initiation, metastasis, recurrence, and therapy-resistance. Numerous studies have linked CSCs to thyroid biology, but the candidate markers and signal transduction pathways that drive thyroid CSC growth are controversial, the origin(s) of thyroid CSCs remain elusive, and it is unclear whether thyroid CSC biology is consistent with the original hierarchical CSC model or the more recent dynamic CSC model. Here, we critically review the thyroid CSC literature with an emphasis on research that confirmed the presence of thyroid CSCs by in vitro sphere formation or in vivo tumor formation assays with dispersed cells from thyroid cancer tissues or bona fide thyroid cancer cell lines. Future perspectives of thyroid CSC research are also discussed.

Targeted Foxe1 Overexpression in Mouse Thyroid Causes the Development of Multinodular Goiter But Does Not Promote Carcinogenesis.

Recent genome-wide association studies have identified several single nucleotide polymorphisms in the forkhead box E1 gene (FOXE1) locus, which are strongly associated with the risk for thyroid cancer. In addition, our recent work has demonstrated FOXE1 overexpression in papillary thyroid carcinomas. To assess possible contribution of Foxe1 to thyroid carcinogenesis, transgenic mice overexpressing Foxe1 in their thyroids under thyroglobulin promoter (Tg-Foxe1) were generated. Additionally, Tg-Foxe1 mice were exposed to x-rays at the age of 5 weeks or crossed with Pten(+/-) mice to examine the combined effect of Foxe1 overexpression with radiation or activated phosphatidylinositol-3-kinase/Akt pathway, respectively. In 5- to 8-week-old Tg-Foxe1 mice, severe hypothyroidism was observed, and mouse thyroids exhibited hypoplasia of the parenchyma. Adult 48-week-old mice were almost recovered from hypothyroidism, their thyroids were enlarged, and featured colloid microcysts and multiple benign nodules of macrofollicular-papilloid growth pattern, but no malignancy was found. Exposure of transgenic mice to 1 or 8 Gy of x-rays and Pten haploinsufficiency promoted hyperplastic nodule formation also without carcinogenic effect. These results indicate that Foxe1 overexpression is not directly involved in the development of thyroid cancer and that proper Foxe1 dosage is essential for achieving normal structure and function of the thyroid.

Differences in quantification of DNA double-strand breaks assessed by 53BP1/γH2AX focus formation assays and the comet assay in mammalian cells treated with irradiation and N-acetyl-L-cysteine.

The biological effect of ionizing radiation (IR) on genomic DNA is thought to be either direct or indirect; the latter is mediated by IR induction of free radicals and reactive oxygen species (ROS). This study was designed to evaluate the effect of N-acetyl-L-cysteine (NAC), a well-known ROS-scavenging antioxidant, on IR induction of genotoxicity, cytotoxicity and ROS production in mammalian cells, and aimed to clarify the conflicting data in previous publications. Although we clearly demonstrate the beneficial effect of NAC on IR-induced genotoxicity and cytotoxicity (determined using the micronucleus assay and cell viability/clonogenic assays), the data on NAC's effect on DNA double-strand break (DSB) formation were inconsistent in different assays. Specifically, mitigation of IR-induced DSBs by NAC was readily detected by the neutral comet assay, but not by the γH2AX or 53BP1 focus assays. NAC is a glutathione precursor and exerts its effect after conversion to glutathione, and presumably it has its own biological activity. Assuming that the focus assay reflects the biological responses to DSBs (detection and repair), while the comet assay reflects the physical status of genomic DNA, our results indicate that the comet assay could readily detect the antioxidant effect of NAC on DSB formation. However, NAC's biological effect might affect the detection of DSB repair by the focus assays. Our data illustrate that multiple parameters should be carefully used to analyze DNA damage when studying potential candidates for radioprotective compounds.

Analysis of multiple markers for cancer stem-like cells in human thyroid carcinoma cell lines.

Cancer stem-like cells (CSCs) play important roles in cancer initiation and progression. CSCs have been isolated using several markers, but those for thyroid CSCs remain to be confirmed. We therefore conducted a comprehensive search for thyroid CSC markers. Expression of nine cell surface markers (CD13, CD15, CD24, CD44, CD90, CD117, CD133, CD166, and CD326) and aldehyde dehydrogenase (ALDH) activity, which are CSC markers in various solid cancers, and the ability to form spheres in vitro and tumors in vivo were investigated using eight thyroid cancer cell lines (FRO, KTC1/2/3, TPC1, WRO, ACT1, and 8505C). Among these, four cell lines (FRO, KTC3, ACT1, and 8505C) possessed the both abilities; however, common markers indicative of CSCs were not observed. The pattern of ability to form spheres was completely matched to that of tumor formation, suggesting that our sphere assay is valuable for assessment of tumor-forming ability. Next, the cells were sorted using these markers and subjected to the sphere assay. In three cell lines (FRO, KTC3, and ACT1), ALDH(pos) cells showed higher sphere forming ability than ALDH(neg) cells but not in other cells. CD326(hi) also appeared to be a candidate marker only in FRO cells. However, these subpopulations did not follow a classical hierarchical model because ALDH(neg) and CD326(low) fractions also generated ALDH(pos) and CD326(hi) cells, respectively. These data suggest that ALDH activity is probably a major candidate marker to enrich thyroid CSCs but not universal; other markers such as CD326 that regulate different CSC properties may exist.

Disruption of transforming growth factor-ß signaling in thyroid follicular epithelial cells or intrathyroidal fibroblasts does not promote thyroid carcinogenesis.

Transforming growth factor ß (TGF-ß) members, pleiotropic cytokines, play a critical role for carcinogenesis generally as a tumor suppressor in the early cancer development, but as a tumor promoter in the late stage of cancer progression. The present study was designed to clarify the role for TGF-ß signaling in early thyroid carcinogenesis using the conditional Tgfbr2(floxE2/floxE2) knock-in mice, having 2 loxP sites at introns 1 and 2 of Tgfb2r gene. When these mice were crossed with thyroid peroxidase (TPO)-Cre or fibroblast-specific protein-1 (FSP1)-Cre, the resultant mice, Tgfbr2(tpoKO) and Tgfbr2(fspKO), lost TGF-ß II receptor expression (thereby TGF-ß signaling) specifically in the thyroid follicular epithelial cells or fibroblasts, respectively. The thyroid morphology was monitored up to 52 weeks in these mice, showing no tumor development, except one Tgfbr2(tpoKO) mouse developing follicular adenoma like-lesion. Our data suggest that TGF-ß signaling in mesenchymal or follicular epithelial cells of the thyroid does not appear to function as a tumor suppressive barrier at the early stage of thyroid carcinogenesis.

Thyrotropin signaling confers more aggressive features with higher genomic instability on BRAF(V600E)-induced thyroid tumors in a mouse model.

BACKGROUND: The BRAF(V600E) mutation is the most common genetic alteration in papillary thyroid carcinomas (PTCs). Transgenic mice overexpressing BRAF(V600E) in their thyroids under control of the thyroglobulin promoter (Tg-BRAF2 mice) developed invasive PTCs with high penetrance. However, these mice showed elevated thyrotropin (TSH) levels, which also stimulate the proliferation of thyrocytes and tumorigenesis. The purpose of the present study was to investigate how TSH signaling cooperates with BRAF(V600E) in the process of thyroid carcinogenesis. METHODS: We crossed Tg-BRAF2 mice with TSH receptor knockout (TshR(-/-)) mice. Four genetically distinct mice groups-Braf(wt)/TshR(+/-) (group 1), Braf(wt)/TshR(-/-) (group 2), Tg-BRAF2/TshR(+/-) (group 3), and Tg-BRAF2/TshR(-/-) (group 4)--were sacrificed at 12 and 24 weeks of age. We performed histopathological analysis. Genomic instability was evaluated by immunofluorescence for p53-binding protein 1 (53BP1) and γH2AX. Invasiveness and genomic instability were also evaluated using thyroid PCCL3 cells expressing BRAF(V600E). RESULTS: Groups 3 and 4 developed distinct neoplasias comparable to human PTCs. Group 3 developed typically larger, more aggressive, invasive tumors compared to group 4. The frequency of 53BP1 and γH2AX foci-indicators of genomic instability--in group 3 was higher than that in group 4. TSH also enhanced invasiveness and genomic instability in PCCL3 cells with BRAF(V600E) expression. CONCLUSIONS: These data demonstrate that the TSH signaling confers more aggressive features in BRAF(V600E)-induced thyroid tumors in mice. This might be due, in part, to accelerated genomic instability.