TR Expression Research Articles

A novel fusion protein TBLR1-RAR\u03b1 acts as an oncogene to induce murine promyelocytic leukemia: identification and treatment strategies

Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation involving RARα and its fusion partners. For decades, the advent of all-trans retinoic acid (ATRA) synergized with arsenic trioxide (As2O3) has turned most APL from highly fatal to highly curable. TBLR1-RARα (TR) is the tenth fusion gene of APL identified in our previous study, with its oncogenic role in the pathogenesis of APL not wholly unraveled. In this study, we found the expression of TR in mouse hematopoietic progenitors induces blockade of differentiation with enhanced proliferative capacity in vitro. A novel murine transplantable leukemia model was then established by expressing TR fusion gene in lineage-negative bone marrow mononuclear cells. Characteristics of primary TR mice revealed a rapid onset of aggressive leukemia with bleeding diathesis, which recapitulates human APL more accurately than other models. Despite the in vitro sensitivity to ATRA-induced cell differentiation, neither ATRA monotherapy nor combination with As2O3 confers survival benefit to TR mice, consistent with poor clinical outcome of APL patients with TR fusion gene. Based on histone deacetylation phenotypes implied by bioinformatic analysis, HDAC inhibitors demonstrated significant survival superiority in the survival of TR mice, yielding insights into clinical efficacy against rare types of APL.

Protective effect of Shaoyao Decoction against colorectal cancer via the Keap1–Nrf2–ARE signaling pathway

Ethnopharmacological relevanceUlcerative colitis is one of the three high risk factors for colorectal cancer. Studies have found that about 20% of cancers are caused by repeated chronic inflammatory stimuli over a long period of time. Ulcer-related colorectal cancer is one of the main causes of death in patients with ulcerative colitis. At present, surgery is the first choice for the treatment of colorectal cancer, combined with radiotherapy and chemotherapy, which have serious side effects. However, reportedly, a compound prescription of Chinese traditional medicine Shaoyao Decoction (SYD) commonly used to treat damp-heat dysentery has anti-colorectal cancer effect. Thus this study described the effect of SYD to AOM/DSS-induced colon cancer model. Aim of the studyIn this study, modern biomedical approaches were employed for investigating the protective/preventive effects of SYD in mice with azoxymethane (AOM)/DSS-induced CRC. Materials and methodsThe mice pretreated with AOM/DSS were randomly allocated to SYDL, SYDM, SYDH group and SASP (sulfasalazine) group. Mice without AOM/DSS treatment were randomly divided into PBS control group and SYD control group. ResultsIt was found that SYD inhibited the production of inflammatory cytokines, TNF-α, IL-1β, superoxide dismutase (SOD), and malonaldehyde (MDA), and increased the antioxidant indices, as measured by the mRNA expression of GR, TR, HO-1, γ-GCSc, γ-GCSm, NQO-1, UGT1A1, and UGT1A10 in AOM-treated mice. Particularly, the expressions rates of NF-κB and Ki-67 in the SYD-treated experimental groups were significantly lower than those in the model group, indicating that the proliferative ability of the CRC tissues was weaker in the SYD-treated experimental groups. Moreover, the positive levels of Nrf2 in the SYD-treated experimental groups were slightly higher than those in the model group, suggesting that SYD exhibited antioxidant activity. ConclusionsTo sum up, our results suggest that SYD inhibits the development of acute/chronic colitis and prevents colitis-associated CRC by suppressing inflammation and preventing oxidative stress-induced cellular damage.

MP51-07 TRANSGELIN PROMOTES BLADDER CANCER INVASION THROUGH REGULATING INVADOPODIA FORMATION

INTRODUCTION AND OBJECTIVES:Transgelin, a well-known actin-binding protein, implicates in different aspects of cancer development with series of controversial studies. The aberrant expression of tr...

Induction of nuclear protein-1 by thyroid hormone enhances platelet-derived growth factor A mediated angiogenesis in liver cancer.

Background & Aims: Hepatocellular carcinoma (HCC) is among the leading causes of cancer deaths worldwide. Many studies indicate that disruption of cellular thyroid hormone signaling promotes HCC progression. However, the mechanisms underlying the regulation of genes downstream of thyroid hormone actions in HCC have remained elusive. In the current study, we identified NUPR1 (nuclear protein-1), a stress-induced protein that overexpresses in various neoplasia, is upregulated by triiodothyronine/thyroid hormone receptor (T3/TR) signaling and aimed to elucidate its role in angiogenesis in cancer progression.Methods: Quantitative reverse transcription-PCR, luciferase promoter and chromatin immunoprecipitation assays were performed to identify the NUPR1 regulatory mechanism by T3/TR. In vitro and In vivo vascular formations were performed to detect the angiogenic function of NUPR1. Human angiogenesis arrays were performed to identify the downstream angiogenic pathway. The sorafenib resistant ability of TR/NUPR1 was further examined in vitro and in vivo. Clinical relevance of TR, NUPR1 and platelet-derived growth factor A (PDGFA) were investigate in HCC samples using qRT-PCR and western blot.Results: Our experiments disclosed positive regulation of NUPR1 expression by T3/TR through direct binding to the -2066 to -1910 region of the NUPR1 promoter. Elevated NUPR1 and TR expression link to poor survival in clinical HCC specimens. An analysis of clinicopathological parameters showed that expression of NUPR1 is associated with vascular invasion and pathology stage. Functional studies revealed that NUPR1 induced endothelial cell angiogenesis in vitro and in vivo. Using a human angiogenesis array, we identified PDGFA as a target of NUPR1 in the downstream angiogenic pathway. NUPR1 induced transcription of PDGFA through direct binding to the corresponding promoter region, and inhibition of the PDGFA signaling pathway impaired angiogenesis in human umbilical vein endothelial cells (HUVECs). Notably, the angiogenic effects of NUPR1/PDGFA were mediated by the MEK/ERK signaling pathway. TR/NUPR1 expression increased cell viability and resistance to sorafenib treatment. Moreover NUPR1 expression was positively correlated with TRα, TRβ, and PDGFA expression.Conclusions: We propose that the T3/TR/NUPR1/PDGFA/MEK/ERK axis has a vital role in hepatocarcinogenesis and suggest NUPR1 as a potential therapeutic target in HCC.

Saturation loadings on 13X (Faujasite) zeolite above and below the critical conditions. Part IV: inorganic multi-atomic species, halocarbons and oxygenated hydrocarbons data evaluation and modeling

The saturation loadings for subcritical adsorption of multi-atomic inorganic species, halocarbons and oxygenated hydrocarbons on 13X zeolite are modeled using the modified Rackett model of Spencer and Danner (J. Chem. Eng. Data 17(2):236–240, 1972) for the saturated liquid densities combined with crystallographic data for the 13X zeolite. A similar equation is used for supercritical adsorption involving supercritical adsorbate densities and crystallographic data for the 13X zeolite employing a different f(Tr) expression than used by Spencer and Danner. Adsorption data from the literature are first critically evaluated and then compared to the model. Log–log plots are used to determine whether each isotherm is near saturation; isotherms that exhibit a \(\left( {\partial \ln q} \right)/\left( {\partial \ln p} \right)\) slope of zero at the maximum pressure point are assumed to be saturated (capillary condensation points are deleted). The highest loading is used from each isotherm that approaches saturation. Unsaturated isotherms are not considered further. The theoretical equation satisfactorily models the available experimental data for the data that is subcritical except for water and methanol. However, steric factors are required in the model for tetrafluoromethane, sulfur hexafluoride and the aldehydes. The adsorption data for ethyl acetate is questionable. A significant amount of data in the supercritical region (tetrafluouromethane, and hexafluoroethane) revealed a decreasing trend with increasing Tr. For this data a f(Tr) is modeled using TCAR and the slope of the decreasing linear plot against Tr. The physical phenomenom causing this effect is attributed to increasing molecular vibration in the cavity reducing the total molecular loading with temperature rise.

Thyroid hormone protects hepatocytes from HBx-induced carcinogenesis by enhancing mitochondrial turnover.

Infection by hepatitis B virus (HBV) accounts for 50-80% of hepatocellular carcinoma (HCC) development worldwide, in which the HBV-encoded X protein (HBx) has critical role in the induction of carcinogenesis. Several studies have shown that thyroid hormone (TH) suppresses HCC development and protects hepatocytes from HBx-induced damage, thus it is of interest to examine whether TH can protect hepatocytes from HBx-induced carcinogenesis. By treating HBx- transgenic mice with or without TH, we confirmed the protective effects of TH on HBx-induced hepatocarcinogenesis, which was achieved via reduction of reactive oxygen species (ROS) inflicted DNA damage. We further found that TH induced biogenesis of mitochondria (MITO) and autophagy of HBx-targeted MITO simultaneously, consequently leading to suppression of HBx-promoted ROS and carcinogenesis. Using microarray data analysis, this protective effect of TH was found to be mediated via activation of PTEN-induced kinase 1 (PINK1) in hepatocytes. PINK1, in turn, activated and recruited Parkin, an E3 ligase, to ubiquitinate MITO-associated HBx protein and trigger selective mitophagy. The pathological significance of the TH/PINK1 pathway in liver protection was confirmed by the concomitant decrease in expression of both TR and PINK1 in matched HCC tumor tissues and negatively correlated with aggressive progression of cancer and poor prognosis. Our data indicate that TH/PINK1/Parkin pathway has a critical role in protecting hepatocytes from HBx-induced carcinogenesis. Notably, several liver-targeting therapeutic derivatives of TH facilitating prevention or therapy of steatosis have been identified. Furthermore, our proof-of-concept experiments suggest that application of T3 constitutes an effective novel therapeutic or preventive option for HCC. Thus, the utilization of the agonists of TRs could be the meaningful strategy in liver relative diseases, ranging from simple hepatic steatosis to HCC.

Luteal Expression of Thyroid Hormone Receptors During Gestation and Postpartum in the Rat

Progesterone (P4) is the main steroid secreted by the corpora lutea (CL) and is required for successful implantation and maintenance of pregnancy. Although adequate circulating levels of thyroid hormone (TH) are needed to support formation and maintenance of CL during pregnancy, TH signaling had not been described in this gland. We determined luteal thyroid hormone receptor isoforms (TR) expression and regulation throughout pregnancy and under the influence of thyroid status, and in vitro effects of triiodothyronine (T3) exposure on luteal P4 synthesis. Euthyroid female Wistar rats were sacrificed by decapitation on gestational day (G) 5, G10, G15, G19, or G21 of pregnancy or on day 2 postpartum (L2). Hyperthyroidism and hypothyroidism were induced in female Wistar rats by daily administration of thyroxine (T4; 0.25 mg/kg subcutaneously) or 6-propyl-2-thiouracil (PTU; 0.1 g/L in drinking water), respectively. Luteal TR expression of mRNA was determined using real-time reverse-transcription quantitative polymerase chain reaction, and of protein using Western blot and immunohistochemistry. Primary cultures of luteal cells and of luteinized granulosa cells were used to study in vitro effects of T3 on P4 synthesis. In addition, the effect of T3 on P4 synthesis under basal conditions and under stimulation with luteinizing hormone (LH), prolactin (PRL), and prostaglandin E2 (PGE2) was evaluated. TRα1, TRα2, and TRβ1 mRNA were present in CL, increasing during the first half and decreasing during the second half of pregnancy. At the protein level, TRβ1 was abundantly expressed during gestation reaching a peak at G19 and decreasing afterwards. TRα1 was barely expressed during early gestation, peaked at G19, and diminished thereafter. Expression of TRβ1 and TRα1 at the protein and mRNA level were not influenced by thyroid status. T3 neither modified P4 secretion from CL of pregnancy nor its synthesis in luteinized granulosa cells in culture. This study confirms for the first time the presence of TR isoforms in the CL during pregnancy and postpartum, identifying this gland as a TH target during gestation. TR expression is modulated in this tissue in accordance with the regulation of P4 metabolism, and the abrupt peripartum changes suggest a role of TH during luteolysis. However, TH actions on the CL do not seem to be related to a direct regulation of P4 synthesis.

Reduced effects of thyroid hormone on gene expression and metamorphosis in a paedomorphic plethodontid salamander.

It has been over a century since Gudernatsch (1912, Wilhelm Roux Arch Entwickl Mech Org 35:457-483) demonstrated that mammalian thyroid gland extracts can stimulate tadpole metamorphosis. Despite the tremendous developmental diversity of amphibians, mechanisms of metamorphosis have mostly been studied in a few model systems. This limits our understanding of the processes that influence the evolution of developmental aberrations. Here we isolated thyroid hormone receptors alpha (TRα) and beta (TRβ) from Oklahoma salamanders (Eurycea tynerensis), which exhibit permanently aquatic (paedomorphic) or biphasic (metamorphic) developmental modes in different populations. We found that TRα and TRβ were upregulated by thyroid hormone (T3 ) in tail tissues of larvae from metamorphic populations, but basal levels of TR expression and T3 responsiveness were reduced in larvae from paedomorphic populations. Likewise, we found that T3 treatment resulted in complete loss of larval epibranchials in larvae from metamorphic populations, but little to no epibranchial remodeling occurred in larvae from paedomorphic populations over the same duration. This is the first study to directly demonstrate reduced gene expression and metamorphic responses to T3 in a paedomorphic plethodontid compared to metamorphic conspecifics, and the first salamander system to show differential expression of thyroid hormone receptors associated with alternative developmental patterns.

Thyroid hormones and their nuclear receptors: new players in intestinal epithelium stem cell biology?

Thyroid hormones participate in the development and homeostasis of several organs and tissues. It is well documented that they act via nuclear receptors, the TRs, which are transcription factors whose function is modulated by the hormone T3. Importantly, T3-induced physiological response within a cell depends on the specific TR expression and on the T3 bioavailability. However, in addition to this T3-dependent control of TR functionality, increasing data show that the action of TRs is coordinated and integrated with other signaling pathways, specifically at the level of stem/progenitor cell populations. By focusing on the intestinal epithelium of both amphibians and mammals we summarize here new data in support of a role for thyroid hormones and the TR nuclear receptors in stem cell biology. This new concept may be extended to other organs and have biological relevance in therapeutic approaches aimed to target stem cells such as tissue engineering and cancer.

Changes in Thyroid Hormone Receptors After Permanent Cerebral Ischemia in Male Rats

Thyroid hormones (TH) and receptors (TRs) may play an important role in the pathophysiology of acute cerebral ischemia. In the present study, we sought to determine whether serum triodothyronine (T3)/thyroxine (T4) and brain TRs (TRα1, TRβ1) might change after experimental stroke. Male adult Wistar rats were subjected to permanent middle cerebral artery occlusion (group P) and compared to sham-operated controls (group S). Animals were followed clinically for 14 days until brain collection for Western blot (WB) or neuropathological analysis of TRs in three different brain areas (infarcted tissue, E1; noninfarcted ipsilateral hemisphere, E2; and contralateral hemisphere, E3). Analysis of serum TH levels showed a reduction of T4 in group P (p = 0.002) at days 2 to 14, while half of the animals also displayed "low T3" values (p = 0.012) on day 14. This T4 reduction was inversely correlated to the clinical severity of stroke and the concomitant body weight loss (p < 0.005). WB analysis of TRα1 and TRβ1 protein expression showed heterogenic responses at day 14: total and nuclear TRα1 were similar between the two groups, while total TRβ1 decreased 7.5-fold within E1 (p ≤ 0.001) with a concomitant 1.8-fold increase of nuclear TRβ1 in E2 area (p = 0.03); TRβ1 expression did not differ in E3. Neuropathological analysis revealed that activated macrophages/microglia exclusively expressed nuclear TRα1 within the infarct core. Astrocytes mildly expressed nuclear TRα1 in and around the infarct, along with a prominent TRβ nuclear signal restricted in the astrocytic scar. Neurons around the infarct expressed mainly TRα1 and, to a milder degree, TRβ. Surprisingly enough, we detected for the first time a TRβ expression in the paranodal region of Ranvier nodes, of unknown significance so far. Our data support that cerebral ischemia induces a low TH response, associated with significant and heterogenic changes in brain TR expression. These findings could imply an important role of TH signaling in cerebral ischemia.

Thyroid Hormones Maintain The CTCL Malignant Phenotype Through Membrane- and Nuclear-Initiated Transcriptional Programs

Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of T-cell non-Hodgkin lymphomas (T-NHL) derived from skin-homing mature T cells. Thyroid hormones (THs) are crucial regulators of cellular differentiation, growth and metabolism. We recently found that THs stimulate the proliferation and metabolism of T cells and that this pathway is co-opted by T-NHL cells to maintain their malignant phenotype. Accordingly, a decrease in TH levels affects the proliferation of T-NHL cells. We also found that THs modulate these functions by activating both canonical nuclear (TR) and membrane receptors (mTR, that for most cells is represented by integrin αvβ3). We now hypothesized that the characterization of the relative contribution of these receptors to the malignant phenotype of T-NHLs will allow for a more precise therapeutic intervention. The study presented here was conducted to determine (i) whether THs are pro-survival factors in CTCL and (ii) the relative contribution of TR and mTR to the effect of THs in CTCLs.To test this hypothesis, we first analyzed the expression of TR and mTR in CTLC cells (HuT 78, MJ and HuT102) vs. normal T cells (from peripheral blood) by qRT-PCR and immunoblotting. We found that CTCL expressed 2 to 10 fold more THRA, ITGAV and ITGB3 mRNA and protein than their normal counterpart (p<0.05, T test). Moreover, in a tissue microarray of 52 CTCLs, the integrin avβ3 was overexpressed in 92% of the cases. To differentiate between nuclear vs. membrane-initiated effects, CTCL cells were treated with physiological concentrations of free and cell impermeable agarose-bound T3/T4 (TH-ag). After 24h of treatment, cell proliferation increased 20-38% in HuT-78 cells and 18-32% in MJ cells with both forms of THs (p<0.05 for both comparisons vs. control). Long-term exposure (96h) to free TH and TH-ag accelerated HuT-78 doubling time by 17-25% and 16-22%, respectively (p<0.05 for both comparisons). By time course analysis of mRNA abundance (1, 2, 3, 6, 12 and 24 h post TH), we found that TH-induced phenotypic changes were associated with 1.5 to 3 fold up-regulation of proliferation markers such as PCNA, CCND1, CCND2, CCND3, CCNE2 and CCNB1 in addition to a concomitant decrease of the tumor suppressor genes TP53 and CDKN1A.To determine the specific transcriptional programs regulated by mTR and TR, T-NHLs were treated with free T3/T4 (for total TR activity) and agarose-coupled T3/T4 (for mTR activity) for 24h and analyzed by mRNA-sequencing. Differentially regulated genes were further analyzed using Ingenuity Pathway Analysis. We found the TNFR and interleukin-2 pathways (e.g. TNFRSF19, TNFRSF9, IL2RA) among the top programs regulated by free THs through canonical nuclear TR activation whereas mTR activation upregulated genes involved in angiogenesis induction (e.g. PDGFR, VEGFA, VEFGB), lymphocyte proliferation/differentiation and DNA replication (e.g. IL4, DOK2). We independently validated these genes in HuT-78 and MJ cell lines by qRT-PCR. Taken together, these data suggest that TR and mTR regulate distinct transcriptional programs that are complimentary towards CTCL proliferation.Considering that (i) mTR are over-expressed in CTCL vs. normal T cells (in contrast to canonical nuclear TR that are broadly expressed) and (ii) mTR regulate pro-survival pathways in CTLC, we wished to determine whether mTR inhibition will be sufficient to decrease CTCL proliferation. We therefore tested the effect of mTR abrogation by using siRNA to ITGAV and ITGB3 as well as the effect of pharmacological mTR inhibitors (i.e: RGD peptide and Tetrac) in CTCL proliferation. We found that mTR inhibition with siRNA or compounds affected mTR-induced transcriptional programs. In fact, the TH-ag mediated upregulation of VEGFB, VEGFA, DOK2 and IL4 mRNA levels were totally abrogated in CTCL cells transfected with the siRNA to ITGAV and ITGB3. Moreover, almost 100% of the TH-ag-induced and 80% of the free-TH-induced CTCL proliferative effect was abrogated by ITGAV- and/or ITGB3-targeting siRNA.Taken together these data suggest that THs are important inducers of CTCL proliferation by transcriptionally regulating major survival pathways, and inhibition of mTR could constitute a selective chemotherapy-free treatment for CTCLs. Disclosures:No relevant conflicts of interest to declare.

FX enzyme and GDP‐L‐Fuc transporter expression in colorectal cancer

Fucosylation is regulated by fucosyltransferases, the guanosine diphosphate-L-fucose (GDP-L-Fuc) synthetic pathway, and the GDP-L-fucose transporter (GDP-L-Fuc Tr). We have reported previously an increased level of α(1,6)fucosyltransferase activity and expression in colorectal cancer (CRC). The present study aimed to analyse the expression profiles of the FX enzyme and GDP-L-Fuc Tr in a cohort of operated CRC patients to elucidate their role in α(1,6)fucosylation in this neoplasm. We assessed the immunohistochemical expression of FX and GDP-L-Fuc Tr in a series of tumour samples and healthy tissues from CRC specimens. FX expression was observed in 58 of 91 (63.7%) tumours and 23 of 28 (82.1%) corresponding healthy samples. GDP-L-Fuc Tr expression was detected in 86 of 102 (84.3%) colorectal tumours, and 13 of 27 (48.1%) healthy tissue specimens. The expression of GDP-L-Fuc Tr was statistically higher in tumours than in healthy tissues (P < 0.001). A correlation was found between FX and GDP-L-Fuc Tr expression in tumour samples (P = 0.003). GDP-L-Fuc Tr overexpression in the tumour tissue of CRC patients suggests that GDP-L-Fuc transport to the Golgi apparatus may be an important factor associated with increased α(1,6)fucosylation in CRC.

Ligand-induced changes in Oestrogen and thyroid hormone receptor expression in the developing rat cerebellum: A comparative quantitative PCR and Western blot study

Oestrogen (E2) and thyroid hormones (THs) are key regulators of cerebellar development. Recent reports implicate a complex mechanism through which E2 and THs influence the expression levels of each other's receptors (ERs and TRs) to precisely mediate developmental signals and modulate signal strength. We examined the modulating effects of E2 and THs on the expression levels of their receptor mRNAs and proteins in cultured cerebellar cells obtained from 7-day-old rat pups. Cerebellar granule cell cultures were treated with either E2, THs or a combination of these hormones, and resulting receptor expression levels were determined by quantitative PCR and Western blot techniques. The results were compared to non-treated controls and to samples obtained from 14-day-old in situ cerebella. Additionally, we determined the glial effects on the regulation of ER-TR expression levels. The results show that (i) ER and TR expression depends on the combined presence of E2 and THs; (ii) glial cells mediate the hormonal regulation of neuronal ER-TR expression and (iii) loss of tissue integrity results in characteristic changes in ER-TR expression levels. These observations suggest that both E2 and THs, in adequate amounts, are required for the precise orchestration of cerebellar development and that alterations in the ratio of E2/THs may influence signalling mechanisms involved in neurodevelopment. Comparison of data from in vitro and in situ samples revealed a shift in receptor expression levels after loss of tissue integrity, suggesting that such adjusting/regenerative mechanisms may function after cerebellar tissue injury as well.

Association of decreased expressions of α1,6-fucosyltransferase (α1,6-FT), GDP-mannose-4,6-dehydratase (GMD), and GDP-fucose transporter (GDP-Fuc Tr) with squamous histology in non-small cell lung cancers (NSCLCs).

e17520 Background: α1,6-FT, a key enzyme for the core fucosylation of N-glycans, has been shown to be needed for the function of EGF and TGF-β1 receptors. α1,6-FT expression is found in human normal bronchial epithelial cells and bronchial gland cells. However, α1,6-FT expression has not been previously examined in NSCLCs. GMD generates GDP-fucose from GDP-mannose, and is imperative for the synthesis of all fucosylated oligosaccharides. GDP-fucose is transported into the Golgi apparatus by GDP-Fuc Tr to serve as a substrate of FTs. Methods: We examined expressions of α1,6-FT, GMD and GDP-Fuc Tr by immunohistochemistry in 156 surgically resected NSCLCs. Results: High, moderate and low expression of α1,6-FT was found in 25 (16.0%), 35 (22.4%) and 96 (61.6%) NSCLCs, respectively. Multivariate logistic regression analysis for the correlation between α1,6-FT expression and various characteristics revealed a significant association between low α1,6-FT expression and squamous cell carcinomas, as compared with non-squamous cell carcinomas (low vs. moderate, p = 0.0005; low vs. high, p = 0.005). High, moderate and low expression of GMD was found in 32 (20.5%), 32 (20.5%) and 92 (59.0%) NSCLCs, respectively. Multivariate logistic regression analysis for the correlation between GMD expression and various characteristics revealed a significant association between low GMD expression and squamous cell carcinomas, as compared with non-squamous cell carcinomas (low vs. moderate, p = 0.005; low vs. high, p = 0.02). Low expression of α1,6-FT was more prevalent in tumors with low GMD expression than in those with moderate or high GMD expression (p = 0.002). High/moderate, low and no expression of GDP-Fuc Tr was found in 7 (4.5%), 23 (14.7%) and 126 (80.8%) NSCLCs, respectively. Low GDP-Fuc Tr expression was significantly more prevalent in squamous cell carcinomas compared with non-squamous cell carcinomas (p = 0.003). Conclusions: These results indicate that α1,6-FT, GMD and GDP-Fuc Tr may be new markers of NSCLCs with specificity for histology.

Abstract 241: A novel function of thyroid hormone receptors in promoting metastasis of human hepatoma cells via regulation of TRAIL

Abstract Thyroid hormone (T3) and its binding receptors (TRs) are potent regulators mediating cell metabolism, proliferation, and differentiation. Accumulating evidence has confirmed an important role of TRs in tumor progression. However, the specific functions of TRs in carcinogenesis have yet to be clarified. Experiments from the present study have shown that tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is upregulated by T3 in TR-overexpressing hepatoma cell lines. The T3-responsive element of the TRAIL promoter was localized to nucleotides at positions −1967 to −1977 upstream of the transcription start site. Interestingly, TRAIL acts as an apoptosis inducer in several cancer cell types, but also triggers non-apoptotic signals favoring tumorigenesis in apoptosis-resistant cancer cells. In our experiments, TR-overexpressing hepatoma cells treated with T3 were apoptosis-resistant, even under conditions where TRAIL was upregulated. This finding may be attributed to the simultaneous upregulation of Bcl-xL, an anti-apoptosis gene, by T3. Thus, overexpression of Bcl-xL in hepatoma cells possibly protects against apoptotic death triggered by TRAIL, consequently leading to TRAIL-promoted metastasis in hepatoma cells. Moreover, T3-enhanced metastasis was repressed by the treatment of TRAIL-blocking antibody. Notably, TRAIL was highly expressed in tumor cells of HCC patients, and this high expression was significantly correlated with the high TR expression as examined in 65 HCC tissues. Collectively, our findings indicate that TR directly binds to the TRAIL promoter to induce TRAIL expression, which acts in concert with the simultaneously activated anti-apoptotic factor, Bcl-xL, to promote metastasis, rather than apoptosis. Our findings provide a novel mechanistic link for increased TR and TRAIL in HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 241. doi:1538-7445.AM2012-241

085 Thyroid hormone receptor alpha1 is down regulated during the progression to congestive heart failure after myocardial infarction

Thyroid hormone (TH) signaling is altered in response to various stresses including myocardial ischaemia. The present study investigated potential implication of TH in the pathophysiology of postischaemic remodeling. Acute myocardial infarction was induced in rats by coronary artery ligation (AMI). After 34 wk, n = 6 animals were on congestive heart failure (CHF) as indicated by measurements in lung and right ventricular weight. N = 7 animals were in compensated state (non-CHF) and n = 8 SHAM operated animals served as controls (SHAM). Progression to congestive heart failure was characterized by marked decrease in EF% and all other functional echocardiographic parameters. Furthermore, β-MHC expression was significantly increased in CHF. A distinct pattern of TR expression was observed in the course of postischaemic remodeling; TRα1 was up-regulated and TRβ1 was down-regulated in non-CHF and TRα1 expression was markedly decreased during the transition from non-CHF to CHF resulting in tissue hypothyroidism. Circulating T3 and T4 remained unchanged. This response was associated with marked decrease in ERK and mTOR activation. A potential link between ERK, mTOR and TRα1 expression was shown in a neonatal cardiomyocytes model of PE (phenylephrine) induced pathological growth. PE increased the expression of TRα1 in nucleus and this response was abrogated after inhibition of mTOR or ERK. Progression to congestive heart failure after myocardial infarction is associated with suppressed expression of TRα1 and results in tissue hypothyroidism. This process may at least in part be mTOR and ERK dependent.

Combining siRN A-mediated hTR and hTERT expression in bladder uroepithelium cancer BIU-87 cell lines

Objective To perform a DNA microarray analysis of combining small interfering RNA (siRNA) of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) gene in bladder transitional cell carcinoma cell line BIU-87 telomere Inhibition of enzyme activity. Methods According to the siRNA design principles and TERT.TR gene mRNA coding sequence,respectively,were designed and synthesized three pairs of different and can interfere with TR and TERT gene expression in siRNA sequence and filter out the most efficient suppression sequence ( pRNAT-TERT-Ⅲ: GTACAG-GTTTCACGCATGT gene No. 3229, pRNAT-TR- Ⅲ: CGAAGGTGCCTTGGAATAT gene No. 306). Constructing the hTR,hTERT gene as a target of the joint RNA interference ( RNAi) device pRNAT-TR-Ⅲ+ pRNAT-TERT-Ⅲ(liposome) and transfected into bladder transitional cell carcinoma of BIU-87 cell line, using fluorescent quantitative polymerase chain reaction (PCR) analysis of hTR and hTERT mRNA expression (in the target gene and GAPDH copy number ratios for the relative quantitative analysis) , TRAP-ELJSA detection of telomerase activity (measured A value) ,MTT assay BIU-87 cell growth inhibition (by MTT method Detection of the survival rate of transfected cells) ,and before and after co-RNAi gene chip a-nalysis,using RNA extraction,RNA quality control,cRNA marking and synthesis,chip hybridization, chemi-luminescence detection,image acquisition and data analysis,and further use of part of the reverse transcription (RT)-PCR validation of differentially expressed genes. Results The combined RNA interference hTR and hTERT gene, pRNAT-TERT-Ⅲ+ pRNAT-TR-Ⅲcombined RNAi can specifically inhibit the bladder transitional cell carcinoma cell line BIU-87 in TERT and TR expression (pRNAT-TERT- Ⅲ+ pRNAT-TR-I TERT:0.300 ±0.033 ,P <0.05;TR:0.430 ±0.028,P <0.05). After the combined RNAi and bladder transitional cell carcinoma of BIU-87 cell line growth and telomerase activity were significantly inhibited (pRNAT-TERT-Ⅲ+ pRNAT-TR- Ⅲ: 1.250 ± 0.012, P < 0.05 ). Before and after the joint siRNA gene chip analysis found 21 genes down ( ATM, BAX, BCL2, BCL2L1, BIRC5, CD44, CTNNB1, E2F1, JUN, MCAM, MTA1, MYC, NFKB1, NFKBIA, NME4, PNN, PRKDC, SERP1NE1, THBS1, TNFRSF1A, UCCl). Conclusion The combined RNA interference with hTERT and hTR gene can significantly inhibit bladder cancer BIU-87 cell line growth;joint siRNA inhibited bladder transitional cell carcinoma of BIU-87 cell line causes growth of 21 genes were cut. Key words: Telomerase; siRNA; Bladder transitional cell carcinoma

Evaluation of Two Internalizing Carcinoembryonic Antigen Reporter Genes for Molecular Imaging

PurposeThe objective of this article is to develop internalizing positron emission tomography (PET) reporter genes for tracking genetically modified T cells in vivo.ProceduresThe transmembrane and cytoplasmic domains of the human transferrin receptor (TfR) and CD5 were each fused to the carcinoembryonic (CEA) minigene N-A3 and expressed in Jurkat T cells. Internalization was evaluated by confocal microscopy or by intracellular uptake of 125I-labeled anti-CEA scFv-Fc. Reporter gene-transfected Jurkat xenografts in mice were analyzed by immunohistochemistry (IHC) and imaged by PET using 124I- or 64Cu-scFv-Fc as tracers.ResultsSurface expression of TR(1–99)-NA3 was lower than that of NA3-CD5. Both reporter genes were internalized following binding of the anti-CEA antibody fragment. IHC of tumors showed strong staining of NA3-CD5, whereas TR(1–99)-NA3 stained weakly. Specific targeting of TR(1–99)-NA3 or NA3-CD5 was shown by PET in xenografted mice.ConclusionsThe in vivo imaging studies suggest a potential application of the internalizing form of CEA (N-A3) as a PET reporter gene.

Protective Effect of Sulforaphane on Human Vascular Endothelial Cells Against Lipopolysaccharide-Induced Inflammatory Damage

Sulforaphane (SFN), mainly derived from cruciferous vegetables, has received much attention for its cancer chemopreventive property. Though there have been a few epidemiological studies supporting its beneficial effect on cardiovascular diseases, much experimental evidence are still required to understand its mechanism. In this study, human vascular endothelial cell, a barrier of blood, was used as an in vitro model to investigate the protective effect of sulforaphane on inflammatory damage induced by lipopolysaccharide (LPS). The results showed that sulforaphane inhibited the expression of COX-2 and iNOS stimulated by lipopolysaccharide in a dose- and time-dependent manner. Moreover, sulforaphane suppressed the phosphorylation of ERK1/2, JNK, and p38 activated by lipopolysaccharide. Pretreatment with SB202190, the specific inhibitor of p38, abolished the expression of COX-2 induced by LPS. Likewise, SP600125, inhibitor of JNK, abrogated iNOS expression stimulated by LPS. Moreover, pretreatment with anisomycin (AM), an activator of p38 and JNK, instead of LPS, the expression of COX-2 and iNOS is still inhibited by sulforaphane. Interestingly, SFN significantly induced HO-1 and TR expression down-regulated by LPS. Taken together, these data indicated that sulforaphane exhibited the protective role against the inflammatory injury in vascular endothelia cells, through inactivating p38 MAPK and JNK, as well as inducing phase 2 enzymes.

Ontogenetic Profile of the Expression of Thyroid Hormone Receptors in Rat and Human Corpora Cavernosa of the Penis

IntroductionIn the last few years, various studies have underlined a correlation between thyroid function and male sexual function, hypothesizing a direct action of thyroid hormones on the penis.AimTo study the spatiotemporal distribution of mRNA for the thyroid hormone nuclear receptors (TR) α1, α2 and β in the penis and smooth muscle cells (SMCs) of the corpora cavernosa of rats and humans during development.MethodsWe used several molecular biology techniques to study the TR expression in whole tissues or primary cultures from human and rodent penile tissues of different ages.Main Outcome MeasureWe measured our data by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) amplification, Northern blot and immunohistochemistry.ResultsWe found that TRα1 and TRα2 are both expressed in the penis and in SMCs during ontogenesis without development-dependent changes. However, in the rodent model, TRβ shows an increase from 3 to 6 days post natum (dpn) to 20 dpn, remaining high in adulthood. The same expression profile was observed in humans. While the expression of TRβ is strictly regulated by development, TRα1 is the principal isoform present in corpora cavernosa, suggesting its importance in SMC function. These results have been confirmed by immunohistochemistry localization in SMCs and endothelial cells of the corpora cavernosa.ConclusionsThe presence of TRs in the penis provides the biological basis for the direct action of thyroid hormones on this organ. Given this evidence, physicians would be advised to investigate sexual function in men with thyroid disorders. Carosa E, Di Sante S, Rossi S, Castri A, D'Adamo F, Gravina GL, Ronchi P, Kostrouch Z, Dolci S, Lenzi A, and Jannini EA. Ontogenetic profile of the expression of thyroid hormone receptors in rat and human corpora cavernosa of the penis. J Sex Med 2010;7:1381–1390.