Agrin is a novel oncogenic protein in thyroid cancer.

Agrin (AGRN) is a matricellular glycoprotein involved in extracellular signal transduction. AGRN is involved in tumorigenesis and cancer progression; however, the role of AGRN in thyroid cancer (TC) remains unclear. In the present study, using cell lines derived from various subtypes of TC including CGTH, FTC-133 and BcPAP and transcriptomic data from patients with TC, the role of AGRN in TC was analyzed by migration, invasion, viability and proliferation assays as well as Western blot with EMT markers. AGRN expression was significantly increased in thyroid tumors and cell lines derived from various TC subtypes. The highest AGRN expression was found in follicular and papillary thyroid carcinoma subtypes. Immunocytochemistry revealed nuclear AGRN localization in normal (NTHY) and TC cells. Silencing of AGRN decreased viability, proliferation, migration and invasion of TC cell lines by upregulating vimentin and downregulating N-cadherin and E-cadherin. Furthermore, the expression of AGRN was associated with neutrophil infiltration in thyroid tumors. In conclusion, the present results indicated that increased AGRN expression promoted tumorigenic phenotypes of TC cells, while AGRN expression was associated with immune infiltration in thyroid tumors. AGRN may represent a target for future cancer therapy and requires further evaluation.

Expression of pendrin and NIS iodide transporters in human breast tumor and peri-tumoral tissue.

Introduction: Thyroid iodide transporters, Na+/I- symporter (NIS) and pendrin (PDS), are responsible for supplying this vital micronutrient for thyroid hormone synthesis by thyroid peroxidase (TPO). Both proteins were shown to be expressed, apart from the thyroid, also in other human tissues, including lactating mammary gland. NIS expression in human breast cancers has been widely studied. On the other hand, until now PDS mRNA levels in breast tumor tissue have been estimated only in high throughput analyses. Previously, we have observed that TPO is expressed in normal and cancerous human breast tissues and shows enzymatic activity. However, biochemical activity of TPO in human breast cancer cells requires iodide transport by NIS and PDS. Therefore, to extend our previous study on TPO expression and function in human breast tumors we performed analysis of NIS and PDS levels in the same group of patients. Material and methods: The study involved detection of NIS and PDS protein levels by immunohistochemistry and Western blotting, as well as mRNA levels by real-time quantitative polymerase chain reaction. Results: Here we provide direct evidence that NIS and PDS are expressed in human breast cancer tissue, with NIS levels being increased and PDS levels decreased in tumor tissue. Interestingly, PDS mRNA levels in breast cancer tissue seem to be influenced by the estrogen receptor status and age of the patients, while NIS mRNA levels were dependent on histological type of the tumor. Conclusions: This study provides valuable information important for consideration in diagnostic or therapeutic application of radioiodine in breast cancer management.

Extracellular Vesicles as Signal Carriers in Malignant Thyroid Tumors?

Extracellular vesicles (EVs) are small, membranous structures involved in intercellular communication. Here, we analyzed the effects of thyroid cancer-derived EVs on the properties of normal thyroid cells and cells contributing to the tumor microenvironment. EVs isolated from thyroid cancer cell lines (CGTH, FTC-133, 8505c, TPC-1 and BcPAP) were used for treatment of normal thyroid cells (NTHY), as well as monocytes and endothelial cells (HUVEC). EVs' size/number were analyzed by flow cytometry and confocal microscopy. Gene expression, protein level and localization were investigated by qRT-PCR, WB and ICC/IF, respectively. Proliferation, migration and tube formation were analyzed. When compared with NTHY, CGTH and BcPAP secreted significantly more EVs. Treatment of NTHY with cancer-derived EVs changed the expression of tetraspanin genes, but did not affect proliferation and migration. Cancer-derived EVs suppressed tube formation by endothelial cells and did not affect the phagocytic index of monocytes. The number of 6 µm size fraction of cancer-derived EVs correlated negatively with the CD63 and CD81 expression in NTHY cells, as well as positively with angiogenesis in vitro. Thyroid cancer-derived EVs can affect the expression of tetraspanins in normal thyroid cells. It is possible that 6 µm EVs contribute to the regulation of NTHY gene expression and angiogenesis.

hMTH1 and GPX1 expression in human thyroid tissue is interrelated to prevent oxidative DNA damage.

Oxidative stress (OS) is recognized as disturbance of cellular equilibrium between reactive oxygen species (ROS) formation and their elimination by antioxidant defense systems. One example of ROS-mediated damage is generation of potentially mutagenic DNA precursor, 8-oxodGTP. In human cells genomic 8-oxodGTP incorporation is prevented by the MutT homologue 1 (MTH1 or hMTH1 for human MTH1) protein. It is well established that malignant cells, including thyroid cancer cells, require hMTH1 for maintaining proliferation and cancerous transformation phenotype. Above observations led to the development of hMTH1 inhibitors as novel anticancer therapeutics. In the current study we present extensive analysis of oxidative stress responses determining sensitivity to hMTH1 deficiency in cultured thyroid cells. We observe here that hMTH1 depletion results in downregulation of several glutathione-dependent OS defense system factors, including GPX1 and GCLM, making some of the tested thyroid cell lines highly dependent on glutathione levels. This is evidenced by the increased ROS burden and enhanced proliferation defect after combination of hMTH1 siRNA and glutathione synthesis inhibition. Moreover, due to the lack of data on hMTH1 expression in human thyroid tumor specimens we decided to perform detailed analysis of hMTH1 expression in thyroid tumor and peri-tumoral tissues from human patients. Our results allow us to propose here that anticancer activity of hMTH1 suppression may be boosted by combination with agents modulating glutathione pool, but further studies are necessary to precisely identify backgrounds susceptible to such combination treatment.

The Impact of Transcription Factor Prospero Homeobox 1 on the Regulation of Thyroid Cancer Malignancy.

Transcription factor Prospero homeobox 1 (PROX1) is continuously expressed in the lymphatic endothelial cells, playing an essential role in their differentiation. Many reports have shown that PROX1 is implicated in cancer development and acts as an oncoprotein or suppressor in a tissue-dependent manner. Additionally, the PROX1 expression in many types of tumors has prognostic significance and is associated with patient outcomes. In our previous experimental studies, we showed that PROX1 is present in the thyroid cancer (THC) cells of different origins and has a high impact on follicular thyroid cancer (FTC) phenotypes, regulating migration, invasion, focal adhesion, cytoskeleton reorganization, and angiogenesis. Herein, we discuss the PROX1 transcript and protein structures, the expression pattern of PROX1 in THC specimens, and its epigenetic regulation. Next, we emphasize the biological processes and genes regulated by PROX1 in CGTH-W-1 cells, derived from squamous cell carcinoma of the thyroid gland. Finally, we discuss the interaction of PROX1 with other lymphatic factors. In our review, we aimed to highlight the importance of vascular molecules in cancer development and provide an update on the functionality of PROX1 in THC biology regulation.

Vitamin D deficiency and thyroid autoantibody fluctuations in patients with Graves' disease - A mere coincidence or a real relationship?

PURPOSE: The aim of this study was to evaluate the association between vitamin D (vitD) and changes in the titers of anti-TSH receptor (TSHR-Abs), antithyroglobulin (Tg-Abs), and antiperoxidase (TPO-Abs) autoantibodies. MATERIALS/METHODS: The study involved 269 patients with Graves' disease (GD), divided into four subgroups (1-4), i.e. 65 smokers treated with vitD(+) (1), 76 smokers not treated with vitD(-) (2), 61 non-smokers treated with vitD(+) (3) and 67 non-smokers with vitD(-) (4). All thyroid parameters were analyzed at entry and 1, 3, 6, 9 and 12 months later. RESULTS: The titer of TSHR-Abs in group 3 was significantly lower than in groups 1 and 2 across all time points. At 3, 6 and 12 months, the titers of TSHR-Abs were also lower in group 4 compared to groups 1 and 2. At 9 months, the titers in group 3 were lower than in all other groups. There was a significant inverse correlation between baseline levels of vitD and baseline titers of Tg-Abs (in group 1 only), Tg-Abs after 12 months (in group 1 only), TPO-Abs after 12 months (in groups 1 and 3), fT4 (in group 4 only), and a significant positive correlation with TPO-Abs (in group 2 only). VitD levels at 12 months were inversely correlated with Tg-Abs in group 1. CONCLUSIONS: VitD measurements in patients with GD, especially smokers with an increased TSHR-Ab titers before 131I therapy, are recommended. Immunological remission is more likely in patients with GD who receive vitD, particularly smokers.

Transcription Factor Prospero Homeobox 1 (PROX1) as a Potential Angiogenic Regulator of Follicular Thyroid Cancer Dissemination.

It is well known that Prospero homeobox 1 (PROX1) is a crucial regulator of lymphangiogenesis, that reprograms blood endothelial cells to lymphatic phenotype. However, the role of PROX1 in tumor progression, especially in angiogenesis remains controversial. Herein, we studied the role of PROX1 in angiogenesis in cell lines derived from follicular thyroid cancer (FTC: FTC-133) and squamous cell carcinoma of the thyroid gland (SCT: CGTH-W-1) upon PROX1 knockdown. The genes involved in angiogenesis were selected by RNA-seq, and the impact of PROX1 on vascularization potential was investigated using human umbilical vein endothelial cells (HUVECs) cultured in conditioned medium collected from FTC- or SCT-derived cancer cell lines after PROX1 silencing. The angiogenic phenotype was examined in connection with the analysis of focal adhesion and correlated with fibroblast growth factor 2 (FGF2) levels. Additionally, the expression of selected genes involved in angiogenesis was detected in human FTC tissues. As a result, we demonstrated that PROX1 knockdown resulted in upregulation of factors associated with vascularization, such as metalloproteinases (MMP1 and 3), FGF2, vascular endothelial growth factors C (VEGFC), BAI1 associated protein 2 (BAIAP2), nudix hydrolase 6 (NUDT6), angiopoietin 1 (ANGPT1), and vascular endothelial growth factor receptor 2 (KDR). The observed molecular changes resulted in the enhanced formation of capillary-like structures by HUVECs and upregulated focal adhesion in FTC-133 and CGTH-W-1 cells. The signature of selected angiogenic genes' expression in a series of FTC specimens varied depending on the case. Interestingly, PROX1 and FGF2 showed opposing expression levels in FTC tissues and seven thyroid tumor-derived cell lines. In summary, our data revealed that PROX1 is involved in the spreading of thyroid cancer cells by regulation of angiogenesis.

Molecular Signature of Prospero Homeobox 1 (PROX1) in Follicular Thyroid Carcinoma Cells.

The prospero homeobox 1 (PROX1) transcription factor is a product of one of the lymphangiogenesis master genes. It has also been suggested to play a role in carcinogenesis, although its precise role in tumour development and metastasis remains unclear. The aim of this study was to gain more knowledge on the PROX1 function in thyroid tumorigenesis. Follicular thyroid cancer-derived cells-CGTH-W-1-were transfected with PROX1-siRNA (small interfering RNA) and their proliferation, cell cycle, apoptosis and motility were then analysed. The transcriptional signature of PROX1 depletion was determined using RNA-Sequencing (RNA-Seq) and the expression of relevant genes was further validated using reverse transcriptase quantitative PCR (RT-qPCR), Western blot and immunocytochemistry. PROX1 depletion resulted in a decreased cell motility, with both migratory and invasive potential being significantly reduced. The cell morphology was also affected, while the other studied cancer-related cell characteristics were not significantly altered. RNA-seq analysis revealed significant changes in the expression of transcripts encoding genes involved in both motility and cytoskeleton organization. Our transcriptional analysis of PROX1-depleted follicular thyroid carcinoma cells followed by functional and phenotypical analyses provide, for the first time, evidence that PROX1 plays an important role in the metastasis of thyroid cancer cells by regulating genes involved in focal adhesion and cytoskeleton organization in tumour cells.

Podoplanin (PDPN) affects the invasiveness of thyroid carcinoma cells by inducing ezrin, radixin and moesin (E/R/M) phosphorylation in association with matrix metalloproteinases.

BACKGROUND: Podoplanin (PDPN) is a mucin-type transmembrane glycoprotein specific to the lymphatic system. PDPN expression has been found in various human tumors and is considered to be a marker of cancer. We had previously shown that PDPN expression contributes to carcinogenesis in the TPC1 papillary thyroid cancer-derived cell line by enhancing cell migration and invasiveness. The aim of this study was to determine the effect of PDPN down-regulation in another thyroid cancer-derived cell line: BcPAP. METHODS: In order to determine the effects of PDPN on malignant features of BcPAP cells (harboring the BRAFV600E mutated allele) and TPC1 cells (carrying the RET/PTC1 rearrangement), we silenced PDPN in these cells using small interfering RNA (siRNA). The efficacy of PDPN silencing was confirmed by qRT-PCR and Western blotting. Then, we tested the motility and invasiveness of these cells (using scratch test and Transwell assay), their growth capacities F(cell cycle analysis, viability, clonogenic activity) and apoptosis assays), adhesion-independent colony-formation capacities, as well as the effect of PDPN silencing on MMPs expression and activity (zymography). RESULTS: We found that PDPN-induced cell phenotype depended on the genetic background of thyroid tumor cells. PDPN down-regulation in BcPAP cells was negatively correlated with the migration and invasion, in contrast to TPC1 cells in which PDPN depletion resulted in enhanced migration and invasiveness. Moreover, our results suggest that in BcPAP cells, PDPN may be involved in the epithelial-mesenchymal transition (EMT) through regulating the expression of the ezrin, radixin and moesin (E/R/M) proteins, MMPs 9 and MMP2, remodeling of actin cytoskeleton and cellular protrusions. We also demonstrated that PDPN expression is associated with the MAPK signaling pathway. The inhibition of the MAPK pathway resulted in a decreased PDPN expression, increased E/R/M phosphorylation and reduced cell migration. Additionally, PDPN depleted BcPAP cells treated with inhibitors of MEK1/2 kinases (U0126) or of the BRAF V600E protein (PLX4720) had reduced motility, similar to that previously observed in TPC1 cells after PDPN knock-down. CONCLUSIONS: Altogether, our data suggest that PDPN may play an important role in the control of invasion and migration of papillary thyroid carcinoma cells in association with the E/R/M, MMPs and MAPK kinases.

hMTH1 is required for maintaining migration and invasion potential of human thyroid cancer cells.

Cancer cells, including thyroid cancer cells, suffer from oxidative stress damaging multiple cellular targets, such as DNA and the nucleotide pool. The human MutT homologue 1 (hMTH1) controls the oxidative DNA damage load by sanitizing the nucleotide pool from the oxidized DNA precursor, 8-oxodGTP. It has previously been shown that hMTH1 is essential for cancer cell proliferation and survival, therefore hMTH1 inhibition has been proposed as a novel anticancer therapeutic strategy. Here we show that thyroid cancer cells respond to siRNA mediated hMTH1 depletion with increased DNA damage load and moderately reduced proliferation rates, but without detectable apoptosis, cell-cycle arrest or senescence. Importantly, however, hMTH1 depletion significantly reduced migration and invasion potential of the thyroid cancer cells. Accordingly, our results allow us to propose that hMTH1 may be a therapeutic target in thyroid malignancy, especially for controlling metastasis.

Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines.

Thyroid peroxidase (TPO) is an enzyme and autoantigen expressed in thyroid and breast tissues. Thyroid TPO undergoes a complex maturation process however, nothing is known about post-translational modifications of breast-expressed TPO. In this study, we have investigated the biochemical properties of TPO expressed in normal and cancerous human breast tissues, and the maturation process and antigenicity of TPO present in a panel of human breast tissue-derived cell lines. We found that the molecular weight of breast TPO was slightly lower than that of thyroid TPO due to decreased glycosylation and as suggest results of Western blot also shorter amino acid chain. Breast TPO exhibit enzymatic activity and isoelectric point comparable to that of thyroid TPO. The biochemical properties of TPO expressed in mammary cell lines and normal thyrocytes are similar regarding glycan content, molecular weight and isoelectric point. However, no peroxidase activity and dimer formation was detected in any of these cell lines since the majority of TPO protein was localized in the cytoplasmic compartment, and the TPO expression at the cell surface was too low to detect its enzymatic activity. Lactoperoxidase, a protein highly homologous to TPO expressed also in breast tissues, does not influence the obtained data. TPO expressed in the cell lines was recognized by a broad panel of TPO-specific antibodies. Although some differences in biochemical properties between thyroid and breast TPO were observed, they do not seem to be critical for the overall three-dimensional structure. This conclusion is supported by the fact that TPO expressed in breast tissues and cell lines reacts well with conformation-sensitive antibodies. Taking into account a close resemblance between both proteins, especially high antigenicity, future studies should investigate the potential immunotherapies directed against breast-expressed TPO and its specific epitopes.

Thyroid peroxidase (TPO) expressed in thyroid and breast tissues shows similar antigenic properties.

BACKGROUND: Thyroid peroxidase (TPO) is essential for physiological function of the thyroid gland. The high prevalence of thyroid peroxidase antibodies (TPOAbs) in patients with breast cancer and their protective role had previously been demonstrated, indicating a link between breast cancer and thyroid autoimmunity. Recently, TPO was shown to be present in breast cancer tissue samples but its antigenicity has not been analyzed. METHODS: In this study, we investigated TPO expression levels in a series of fifty-six breast cancer samples paired with normal (peri-tumoral) tissue and its antigenic activity using a panel of well-characterized murine anti-human TPOAbs. RESULTS: We have shown that TPO transcripts were present in both normal and cancer tissue samples, although the amounts in the latter were reduced. Additionally, we observed that TPO levels are lower in more advanced cancers. TPO protein expression was confirmed in all tissue samples, both normal and cancerous. We also found that the antigenicity of the immunodominant regions (IDRs) in breast TPO resembles that of thyroid TPO, which is crucial for effective interactions with human TPOAbs. CONCLUSIONS: Expression of TPO in breast cancer together with its antigenic activity may have beneficial effects in TPOAb-positive breast cancer patients. However, further studies are needed to confirm the beneficial role of TPOAbs and to better understand the underlying mechanism.

The role of prospero homeobox 1 (PROX1) expression in follicular thyroid carcinoma cells.

The prospero homeobox 1 (Prox1) transcription factor is a key player during embryogenesis and lymphangiogenesis. Altered Prox1 expression has been found in a variety of human cancers, including papillary thyroid carcinoma (PTC). Interestingly, Prox1 may exert tumor suppressive or tumor promoting effect, depending on the tissue context. In this study, we have analyzed Prox1 expression in normal and malignant human thyroid carcinoma cell lines. Moreover, we determined the effect of Prox1 silencing and overexpression on the cellular processes associated with the metastatic potential of tumor cells: proliferation, migration, invasion, apoptosis and anchorage-independent growth, in the follicular thyroid carcinoma (FTC) FTC-133 cell line. We found that Prox1 expression was significantly higher in FTC-derived cells than in PTC-derived cells and normal thyroid, and it was associated with the PI3K/Akt signaling pathway. In the FTC-133 cells, it was associated with cell invasive potential, motility and wound closure capacities, but not with proliferation or apoptosis. Modifying Prox1 expression also induced substantial changes in the cytoskeleton structure and cell morphology. In conclusion, we have shown that Prox1 plays an important role in the development of FTC and that its suppression prevents, whereas its overexpression promotes, the malignant behavior of thyroid follicular cancer cells.

Does TSH Trigger the Anti-thyroid Autoimmune Processes? Observation on a Large Cohort of Naive Patients with Thyroid Hemiagenesis.

Thyroid hemiagenesis (THA) is a rare abnormality characterized by the absence of one thyroid lobe. Elevated thyroid stimulating hormone (TSH) level and higher incidence of thyroid diseases were reported in THA. The aim of the study is to evaluate the thyroid autoimmunity incidence in patients with THA and influence of higher than average TSH level on thyroid volume (TV) and its change with age. The study included a group of naive patients with THA and a control group of subjects with bilobate thyroid. All patients underwent clinical examination, thyroid ultrasound, scintiscan and laboratory tests. In the studied and control group the presence of thyroid autoantibodies (TAb) was evaluated. The THA group consisted of 65 patients. In THA group 53.85 % of patients were positive for TAb. Patients with positive TAb were older (46.0 ± 18.3 years) than those with negative (35.0 ± 19.8 years); p = 0.02. The incidence of TAb was lower in controls (13.85 %, p < 0.0001). In the study group, positive correlation between the age and TV (r = 0.46, p = 0.0001), and negative correlations between the age and TSH level (r = -0.31, p = 0.01), and TSH concentration and TV (r = -0.35, p = 0.004) were found. In a subgroup of 30 patients with THA negative for TAb, even stronger correlations were observed. The median single lobe volume and median TSH level were higher in patients with THA when compared to controls (13.60 vs 8.20 ml, p < 0.0001; 3.23 vs 1.48 µU/ml, p < 0.0001, respectively). Patients with THA constitute an in vivo model of long-term thyroid TSH overstimulation. Further studies are needed to reveal, whether TSH overstimulation may be the trigger for thyroid autoimmunity.

Increased expression of the epithelial anion transporter pendrin/SLC26A4 in nasal polyps of patients with chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is a multifactorial disease of unknown cause characterized by sinonasal inflammation, increased mucus production, and defective mucociliary clearance. Expression of Pendrin, an epithelial anion transporter, is increased in asthma and chronic obstructive pulmonary disease. Pendrin increases mucus production and regulates mucociliary clearance. OBJECTIVES: We sought to investigate the expression of pendrin and the mucus-related protein Muc5AC in sinonasal tissues of control subjects and patients with CRS and to evaluate the regulation of pendrin expression in nasal epithelial cells (NECs) in vitro. METHODS: The expression and distribution of pendrin in sinonasal tissues was analyzed by using real-time PCR, immunoblot analysis, and immunohistochemistry. Differentiated NECs were used to study the regulation of pendrin expression. RESULTS: Increased pendrin expression was observed in nasal polyp (NP) tissue of patients with CRS. Immunohistochemistry analysis revealed that pendrin was largely restricted to the epithelial layer. Pendrin expression significantly correlated with inflammatory cell markers, suggesting that the factors made by these cells might induce pendrin expression. Furthermore, both pendrin and periostin levels (a biomarker in asthma) correlated with IL-13 levels, suggesting that pendrin can be induced by this cytokine in sinonasal tissues. Expression of the mucus component protein Muc5AC correlated weakly with pendrin expression, indicating that pendrin might modulate mucus production in NPs. In cultured NECs pendrin expression was induced by TH2 cytokines and induced synergistically when TH2 cytokines were combined with IL-17A. Interestingly, human rhinovirus had a potentiating effect on IL-13-induced pendrin expression. Dexamethasone suppressed pendrin expression, suggesting that the therapeutic benefit of dexamethasone in asthmatic patients and those with CRS might involve regulation of pendrin expression. CONCLUSIONS: TH2-mediated pendrin expression is increased in NPs of patients with CRS and might lead to increased inflammation, mucus production, and decreased mucociliary clearance.

The role of podoplanin in the biology of differentiated thyroid cancers.

Podoplanin (PDPN), a mucin-type transmembrane glycoprotein specific to the lymphatic system is expressed in a variety of human cancers, and is regarded as a factor promoting tumor progression. The purpose of this study was to elucidate the molecular role of PDPN in the biology of thyroid cancer cells. PDPN expression was evaluated in primary thyroid carcinomas and thyroid carcinoma cell lines by RT-qPCR, Western blotting, IF and IHC. To examine the role of podoplanin in determining a cell's malignant potential (cellular migration, invasion, proliferation, adhesion, motility, apoptosis), a thyroid cancer cell line with silenced PDPN expression was used. We observed that PDPN was solely expressed in the cancer cells of 40% of papillary thyroid carcinoma (PTC) tissues. Moreover, PDPN mRNA and protein were highly expressed in PTC-derived TPC1 and BcPAP cell lines but were not detected in follicular thyroid cancer derived cell lines. PDPN knock-down significantly decreased cellular invasion, and modestly reduced cell migration, while proliferation and adhesion were not affected. Our results demonstrate that PDPN mediates the invasive properties of cells derived from papillary thyroid carcinomas, suggesting that podoplanin might promote PTC progression.

A redundant role of human thyroid peroxidase propeptide for cellular, enzymatic, and immunological activity.

BACKGROUND: Thyroid peroxidase (TPO) is a dimeric membrane-bound enzyme of thyroid follicular cells, responsible for thyroid hormone biosynthesis. TPO is also a common target antigen in autoimmune thyroid disease (AITD). With two active sites, TPO is an unusual enzyme, and thus there is much interest in understanding its structure and role in AITD. Homology modeling has shown TPO to be composed of different structural modules, as well as a propeptide sequence. During the course of studies to obtain homogeneous preparations of recombinant TPO for structural studies, we investigated the role of the large propeptide sequence in TPO. METHODS: An engineered recombinant human TPO preparation expressed in Chinese hamster ovary (CHO) cells lacking the propeptide (TPOΔpro; amino acid residues 21-108) was characterized and its properties compared to wild-type TPO. Plasma membrane localization was determined by cell surface protein biotinylation, and biochemical studies were performed to evaluate enzymatic activity and the effect of deglycosylation. Immunological investigations using autoantibodies from AITD patients and other epitope-specific antibodies that recognize conformational determinants on TPO were evaluated for binding to TPOΔpro by flow cytometry, immunocytochemistry, and capture enzyme-linked immunosorbent assay. Molecular modeling and dynamics simulation of TPOΔpro comprising a dimer of myeloperoxidase-like domains was performed in order to investigate the impact of propeptide removal and the role of glycosylation. RESULTS: The TPOΔpro was expressed on the cell surface at comparable levels to wild-type TPO. The TPOΔpro was enzymatically active and recognized by patients' autoantibodies and a panel of epitope-specific antibodies, confirming structural integrity of the two major conformational determinants recognized by autoantibodies. Faithful intracellular trafficking and N-glycosylation of TPOΔpro was also maintained. Molecular modeling and dynamics simulations were consistent with these observations. CONCLUSIONS: Our results point to a redundant role for the propeptide sequence in TPO. The successful expression of TPOΔpro in a membrane-anchored, enzymatically active form that is insensitive to intramolecular proteolysis, and importantly is recognized by patients' autoantibodies, is a key advance for purification of substantial quantities of homogeneous preparation of TPO for crystallization, structural, and immunological studies.

Humanized medium (h7H) allows long-term primary follicular thyroid cultures from human normal thyroid, benign neoplasm, and cancer.

CONTEXT: Mechanisms of thyroid physiology and cancer are principally studied in follicular cell lines. However, human thyroid cancer lines were found to be heavily contaminated by other sources, and only one supposedly normal-thyroid cell line, immortalized with SV40 antigen, is available. In primary culture, human follicular cultures lose their phenotype after passage. We hypothesized that the loss of the thyroid phenotype could be related to culture conditions in which human cells are grown in medium optimized for rodent culture, including hormones with marked differences in its affinity for the relevant rodent/human receptor. OBJECTIVE: The objective of the study was to define conditions that allow the proliferation of primary human follicular thyrocytes for many passages without losing phenotype. METHODS: Concentrations of hormones, transferrin, iodine, oligoelements, antioxidants, metabolites, and ethanol were adjusted within normal homeostatic human serum ranges. Single cultures were identified by short tandem repeats. Human-rodent interspecies contamination was assessed. RESULTS: We defined an humanized 7 homeostatic additives medium enabling growth of human thyroid cultures for more than 20 passages maintaining thyrocyte phenotype. Thyrocytes proliferated and were grouped as follicle-like structures; expressed Na+/I- symporter, pendrin, cytokeratins, thyroglobulin, and thyroperoxidase showed iodine-uptake and secreted thyroglobulin and free T3. Using these conditions, we generated a bank of thyroid tumors in culture from normal thyroids, Grave's hyperplasias, benign neoplasms (goiter, adenomas), and carcinomas. CONCLUSIONS: Using appropriate culture conditions is essential for phenotype maintenance in human thyrocytes. The bank of thyroid tumors in culture generated under humanized humanized 7 homeostatic additives culture conditions will provide a much-needed tool to compare similarly growing cells from normal vs pathological origins and thus to elucidate the molecular basis of thyroid disease.

Anti-natrium/iodide symporter antibodies and other anti-thyroid antibodies in children with Turner's syndrome.

Antibodies against the Na/I symporter (anti-NIS ab) have been found in adult patients with autoimmune thyroid diseases. As easily available for the immune system, NIS can play a role in the initial stage of autoimmune thyroid diseases. Children with Turner's syndrome (TS) being at high risk of autoimmune thyroid disease development seem a valuable group for the investigation of the early autoimmune process. The aim of the study was to investigate the presence of anti-NIS ab and its potential clinical significance in TS children. Fifty four girls with TS were examined (age 11.9 ± 2.46 years), and 23 healthy girls with normal thyroid function, free of autoimmune diseases. Anti-NIS antibodies were measured by the in-house ELISA method and the Western blotting. Sera considered positive for anti-NIS ab were used for the iodide uptake bioassay using COS7 cells stably transfected with hNIS. In all patients the thyroid function, antithyroid antibodies presence and thyroid ultrasonography were evaluated. In 20% of the patients a subclinical hypothyroidism was diagnosed and 70.4% had antithyroid antibodies (anti-TPO - 64.8% and Anti-Tg - 24%). Anti-NISab were present in 14.8% girls with TS and in none of the control group. Their presence was unrelated to other antithyroid antibodies titre or patients' age. A positive correlation between the anti-NIS ab presence and the hypothyroidism was found (p < 0.04). Anti-NIS ab-positive sera did not suppress iodine uptake. In conclusion, anti-NIS antibodies were present in 14.8% of children with TS and they were related to the presence of hypothyroidism.