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Characterization of a human follicular thyroid carcinoma cell line (UCLA RO 82 W-1)
Abstract
A thyroid tumor cell line has been established from the metastases of a follicular carcinoma in a female patient. Although the primary tumor released thyroglobulin (Tg) into the circulation (greater than 10,000 ng/ml), the uptake of I131 was less than 2%. After 37 replications the doubling time was 4 days and confluency was reached after 7 days from inoculation of 3 x 10(7) cells. This human thyroid tumor cell line has now been growing in culture for several years. An aneuploid chromosomal pattern was observed (62-82 chromosomes). A pair of X chromosomes was present but no Y chromosome was found which is compatible with the female origin of the cell line. EM studies revealed the presence of microvilli. Immunoperoxidase staining using specific anti-human Tg antisera indicated the presence of Tg within the cells. Nude mice developed solid-cystic tumors within 6 months after injection of the cells. The basal release of immunodetectable Tg, as measured in a perifusion system, increased in response to thyroid stimulating hormone (TSH) (P less than 0.025) or TSH combined with theophylline (P less than 0.001). Unusual isoenzyme patterns for galactose-1-phosphate-uridyltransferase (GALT) and phosphoglucomutase1 (PGM1) were detected in the tumor, compared with normal human fibroblasts and blood cells and isoenzyme patterns from the patient's lymphocytes. Because this malignant human thyroid follicular cell line has retained the ability to synthesize Tg it represents a valuable model for the study of human follicular carcinomas.
... It has been reported that the BRAF mutation occurs exclusively in PTC (40-50% of all PTC) and PTC-derived ATC (about 24%) and that it does not occur in follicular thyroid carcinoma (FTC) or other types of thyroid tumors [Xing, 2005;Li et al., 2012;Kleiman et al., 2013;Rothenberg et al., 2015]. Thus, the behavior of WRO cell line is somehow intriguing because, on the contrary, it is derived from human differentiated FTC [Estour et al., 1989] and WRO cells are known to harbor a mutated BRAF V600E [Saiselet et al., 2012]. ...
... Despite these advances, little information is available about the cellular responses of thyroid tumor cell lines of human origin in vitro, particularly of cells harboring mutated BRAF (BRAF V600E ). The first characterization of WRO cell was documented by Estour et al. [1989] who evaluated the effect of external radiation by 60 Co in a single dose of 10 Gy. Reeb et al. [2016] demonstrated that they were the most tumorigenic and metastatic of the three human FTC cell lines (WRO, FTC-238, TT 2609-CO2) analyzed in xenotransplantation assays in immunodeficient mice. ...
... The Environmental and Molecular Mutagenesis. DOI 10.1002/em doubling time was 96.3 hr, calculated from the exponential equation (Y 5 Y 0 e (kX) ), which indicates a relatively slow cell cycle for these thyroid cells, confirming the data of Estour et al. [1989], who reported a doubling time of 4 days. Figure 2 shows the profile of cell viability as a function of the concentration of radioactive 131 I, with and without rhTSH at 96 after 24 hr exposure to radioiodine. ...
Normally, differentiated thyroid cancer (DTC) tends to be biologically indolent, highly curable and has an excellent prognosis. However, the treatment may fail when the cancer has lost radioiodine avidity. The present study was carried out in order to evaluate the cytotoxic and genotoxic effects of (131) I and (60) Co and radioiodine uptake in WRO cells, derived from DTC, harboring the BRAF(V600E) mutation. WRO cells showed a relatively slow cell cycle of 96.3 h with an unstable karyotype containing various double minutes. The genotoxicity assay (micronucleus test) showed a relative high radioresistance to (131) I (0.07-3.70 MBq/mL), independent of treatment with recombinant human thyroid-stimulating hormone (rhTSH). For the cytotoxicity assay, WRO cells were also relatively resistant to (60) Co (range: 0.2-8.3 Gy), but with a gradual decrease of viability as a function of time for higher doses (20 and 40 Gy, starting from the fifth to sixth day). For internal irradiation with (131) I, WRO cells showed a decline in viability at radioactive concentration higher than 1.85 MBq/mL; this was even more effective at 3.70 MBq/mL, but only when preceded by rhTSH, in coincidence with the highest level of (131) I uptake. These data show promising results, since the loss of the ability of thyroid cells to concentrate radioiodine is considered to be one of the main factors responsible for the failure of (131) I therapy in patients with DTC. The use of tumor-derived cell lines as a model for in vivo tumor requires, however, further investigations and deep evaluation of the corresponding in vivo effects. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc.
... BHP18-21v cells, that express Pax-8, but do not express either the thyroglobulin or the thyroid transcription factor-1 gene, were isolated from BHP18-21v cells [9]. FRO and WRO cells, which were reported in citation [10,11] were kindly provided by Dr. Shunichi Yamashita, University of Nagasaki. These cell lines are not de novo cell lines but have already been reported [8,10,11] and were kindly provided as gifts by our collaborators. ...
... FRO and WRO cells, which were reported in citation [10,11] were kindly provided by Dr. Shunichi Yamashita, University of Nagasaki. These cell lines are not de novo cell lines but have already been reported [8,10,11] and were kindly provided as gifts by our collaborators. All cells were grown in RPMI 1640 medium with 10% (v/v) fetal bovine serum (FBS) in a humidified incubator under a 5% CO 2 atmosphere. ...
... Marlow et al. reported that reactivation of suppressed RhoB is a critical step for inhibition of the proliferation of anaplastic thyroid cancers [29]. In the current study, we analyzed 3 thyroid cancer cell lines, BHP18-21v; papillary thyroid carcinoma, FRO; anaplastic thyroid carcinoma, and WRO; follicular thyroid carcinoma [11] that had lost the expression of endogenous TR and in which RhoB protein expression was also not observed by Western blotting analysis. These findings suggested the possibility that endogenous TR, when present, might function as a tumor suppressor by inducing RhoB protein expression. ...
Thyroid hormone receptor (TR) mediates the crucial effects of the thyroid hormone (T3) on cellular growth, development, and differentiation. Decreased expression or inactivating somatic mutations of TRs have been found in human cancers of the liver, breast, lung, and thyroid. The mechanisms of TR-associated carcinogenesis are still not clear. To establish the function of TRβ in thyroid cancer cell proliferation, we constructed a recombinant adenovirus vector, AdTRβ, which expresses human TRβ1 cDNA. Thyroid cancer cell lines in which TRβ protein levels were significantly decreased as compared to intact thyroid tissues were infected with AdTRβ and the function of TRβ on cell proliferation and migration was analyzed. Ligand-bound TRβ induced HDAC1 and HDAC3 dissociation from, and histone acetylation associated with the RhoB promoter and enhanced the expression of RhoB mRNA and protein. In AdTRβ-infected cells, T3 and farnesyl transferase inhibitor (FTI)-treatment induced the distribution of RhoB on the cell membrane and enhanced the abundance of active GTP-bound RhoB. This RhoB protein led to p21-associated cell-cycle arrest in the G0/G1 phase, following inhibition of cell proliferation and invasion. Conversely, lowering cellular RhoB by small interfering RNA knockdown in AdTRβ-infected cells led to downregulation of p21 and inhibited cell-cycle arrest. The growth of BHP18-21v tumor xenografts in
vivo was significantly inhibited by AdTRβ injection with FTIs-treatment, as compared to control virus-injected tumors. This novel signaling pathway triggered by ligand-bound TRβ provides insight into possible mechanisms of proliferation and invasion of thyroid cancer and may provide new therapeutic targets for thyroid cancers.
... Among TTFs, PAX8 was found in FTC133, WRO82-1, BCPAP, and TPC1 cells, TTF-1 was expressed in all the cell lines and TTF-2 was faintly detected in WRO82-1, TPC1, and BCPAP cell lines. Their inability to detect TSH-R expression in all the cell lines differed from previous studies by other investigators (20)(21)(22)(23)(24)(25). One can attribute the above discrepancies to different experimental conditions such as varying numbers of polymerase chain reaction cycles used and=or different durations of exposure of immunoblots (25). ...
... Structural and numerical karyotype abnormalities have been described in follicular carcinomas. An extensive karyo-type description has been reported only for the FTC133 and WRO82-1 cell lines (12,21,28). In particular, the FTC133 cells revealed significant gains in part or whole of chromosomes 1,11,6,7,8,14,15,19, and 20, and losses in chromosomes 16, 21, and 22 (28). ...
... The hyperdiploid karyograms in the FTC133 and WRO82-1 cells (62-71 and 68-77 chromosomes, respectively) have also been reported (12). Divergence between present and previously reported karyotypes concerned numerical and structural alterations; in particular, the WRO82-1 chromosomal pattern (62-82 chromosomes) described by Klandorf and coworkers (21) has not yet been confirmed. This again suggests an in vitro evolution of changes in the cell line, a finding similar to that noted for the PTC-derived BCPAP cell line. ...
Tumor-derived cell lines are widely used to study the mechanisms involved in thyroid carcinogenesis but recent studies have reported redundancy among thyroid cancer cell lines and identification of some "thyroid cell lines" that are likely not of thyroid origin.
In this review, we have summarized the uses, the limitations, and the existing problems associated with the available follicular cell-derived thyroid cancer cell lines. There are some limitations to the use of cell lines as a model to "mimic" in vivo tumors. Based on the gene expression profiles of thyroid cell lines originating from tumors of different types it has become apparent that some of the cell lines are closely related to each other and to those of undifferentiated carcinomas. Further, many cell lines have lost the expression of thyroid-specific genes and have altered karyotypes, while they exhibit activation of several oncogenes (BRAF, v-raf murine sarcoma viral oncogene homolog B1; RAS, rat sarcoma; and RET/PTC, rearranged in transformation/papillary thyroid carcinoma) and inactivation of tumor suppressor gene (TP53) which is known to be important for thyroid tumorigenesis.
A careful selection of thyroid cancer cell lines that reflect the major characteristics of a particular type of thyroid cancer being investigated could be used as a good model system to analyze the signaling pathways that may be important in thyroid carcinogenesis. Further, the review of literature also suggests that some of the limitations can be overcome by using multiple cell lines derived from the same type of tumor.
... This cell line derived from the metastases of a follicular carcinoma in a female patient. Although the primary tumor of RO82-W-1 released thyroglobulin (Tg) into the circulation, the uptake of I131 was less than 2% [26]. RO82-W-1 cells are Tg-positive and are also tumorigenic in nude mice [26]. ...
... Although the primary tumor of RO82-W-1 released thyroglobulin (Tg) into the circulation, the uptake of I131 was less than 2% [26]. RO82-W-1 cells are Tg-positive and are also tumorigenic in nude mice [26]. ...
In this study we focused on gravity-sensitive proteins of two human thyroid cancer cell lines (ML-1; RO82-W-1), which were exposed to a 2D clinostat (CLINO), a random positioning machine (RPM) and to normal 1g-conditions. After a three (3d)- or seven-day-culture (7d) on the two devices, we found both cell types growing three-dimensionally within multicellular spheroids (MCS) and also cells remaining adherent (AD) to the culture flask, while 1g-control cultures only formed adherent monolayers, unless the bottom of the culture dish was covered by agarose. In this case, the cytokines IL-6 and IL-8 facilitated the formation of MCS in both cell lines using the liquid-overlay technique at 1g. ML-1 cells grown on the RPM or the CLINO released amounts of IL-6 and MCP-1 into the supernatant, which were significantly elevated as compared to 1g-controls. Release of IL-4, IL-7, IL-8, IL-17, eotaxin-1 and VEGF increased time-dependently, but was not significantly influenced by the gravity conditions. After 3d on the RPM or the CLINO, an accumulation of F-actin around the cellular membrane was detectable in AD cells of both cell lines. IL-6 and IL-8 stimulation of ML-1 cells for 3d and 7d influenced the protein contents of ß1-integrin, talin-1, Ki-67, and beta-actin dose-dependently in adherent cells. The ß1-integrin content was significantly decreased in AD and MCS samples compared with 1g, while talin-1 was higher expressed in MCS than AD populations. The proliferation marker Ki-67 was elevated in AD samples compared with 1g and MCS samples. The ß-actin content of R082-W-1 cells remained unchanged. ML-1 cells exhibited no change in ß-actin in RPM cultures, but a reduction in CLINO samples. Thus, we concluded that simulated microgravity influences the release of cytokines in follicular thyroid cancer cells, and the production of ß1-integrin and talin-1 and predicts an identical effect under real microgravity conditions.
... However, thyroid cancer cells used in most zero gravity-related studies are follicular thyroid cancer (FTC) cells, whereas studies with papillary thyroid cancer (PTC) cells are rare 3,8,10 . Most microgravity studies using thyroid cells have been conducted using cells obtained from Westerns (Nthy-ori 3-1, HTU 5, FTC-133, UCLA RO82-W-1, and ONCO-DG1), while no such study exists using thyroid cells derived from Asians [11][12][13][14][15] . In 2007, Koh et al. established SNU-790 and SNU-80 cells derived from Korean breast cancer and papillary thyroid cancer, respectively. ...
Microgravity in space impacts human health. In particular, thyroid cancer, which has a high incidence rate, has been the subject of numerous studies with respect to microgravity. However, most studies have focused on Western follicular thyroid cancer cell lines, while data regarding the effects of microgravity on Asian cell lines are lacking. Therefore, we aimed to investigate the effect of simulated ground-based microgravity on two Korean thyroid cancer cell lines, namely SNU-790 and SNU-80. We found that both cell lines formed multicellular spheroids under simulated microgravity. Gene expression analysis revealed that in SNU-790 cells, histone-related genes were upregulated and microRNA-related genes were downregulated. Meanwhile, in SNU-80 cells, genes related to the cellular response to hypoxia were downregulated. These findings contribute to a better understanding of the effects of microgravity on thyroid cancer cells. Further validation studies and clinical significance analyses are needed to fully understand the implications of these findings.
... For example, 1F6 human melanoma cells (Fontijn et al., 2009;Van Muijen et al., 1991) grown in slide flasks and 2D-clinorotated at 60 rpm for 24 h showed significantly reduced guanylyl cyclase A mRNA levels limited to samples taken from the inner 6 mm wide axial area (Eiermann et al., 2013). Moreover, ML-1 (Schönberger et al., 2000) and RO82-W-1 (Estour et al., 1989) follicular thyroid cancer cells grown in slide flasks and 2D-clinorotated at 60 rpm for either 3 or 7 days showed marked formation of actin stress fibres and subplasmalemmal enrichment of F-actin within the inner 6 mm wide axial area (Svejgaard et al., 2015). Another comprehensive study in spontaneously beating cardiomyocytes derived from human-induced pluripotent stem cells grown in modified slide flasks and 2D-clinorotated at 60 rpm for 2 days showed multiple mitochondria-, contraction-, and senescence-related changes and dysfunctions in samples taken from the inner 3 mm of the axial region. ...
To study processes related to weightlessness in ground-based cell biological research, a theoretically assumed microgravity environment is typically simulated using a clinostat – a small laboratory device that rotates cell culture vessels with the aim of averaging out the vector of gravitational forces. Here, we report that the rotational movement during fast clinorotation induces complex fluid motions in the cell culture vessel, which can trigger unintended cellular responses. Specifically, we demonstrate that suppression of myotube formation by 2D-clinorotation at 60 rpm is not an effect of the assumed microgravity but instead is a consequence of fluid motion. Therefore, cell biological results from fast clinorotation cannot be attributed to microgravity unless alternative explanations have been rigorously tested and ruled out. We consider two control experiments mandatory, i) a static, non-rotating control, and ii) a control for fluid motion. These control experiments are also highly recommended for other rotation speed settings and experimental conditions. Finally, we discuss strategies to minimize fluid motion in clinorotation experiments.
... Briefly, this cell line was derived from metastatic tissue from a patient with a microfollicular carcinoma of the thyroid. The tumor retained its capacity to secrete thyroglobulin (Tg) in vivo (plasma Tg > 10 000 ng/ml) but lost its ability to concentrate I-131 (Estour et al., 1989). RT-PCR (qRT-PCR) Total RNA was extracted by TRIzol reagent and cDNA was synthesized using the Superscript II reverse transcriptase (Invitrogen). ...
Purpose
The present study analyzed different protocols of administration of boronophenylalanine (BPA) and sodium butyrate (NaB) to increase the BNCT efficacy for poorly differentiated thyroid cancer (PDTC).
Materials and methods
Nude mice implanted with human PDTC cells (WRO) were distributed into four protocols: 1) BPA; 2) BPA + ip NaB; 3) BPA + oral NaB; 4) Control. Biodistribution and histologic studies were performed. LAT (BPA transporter) isoforms gene expression was assessed by RT-PCR.
Results
Tumor growth delay was observed in animals of the Protocol #3 (p < 0.05). NaB (Protocol #2) increased tumor boron uptake 2-h post BPA injection (p˂0.05). On the other hand, NaB upregulated the expression of all the isoforms of the LAT transporter in vitro. Histologic studies showed a significant decrease of mitotic activity and an increase of vacuoles in tumors of Protocol #3. Neutrons alone or combined with NaB caused some tumor growth delay (p < 0.05), while in the BNCT and BNCT + NaB groups, there was a halt in tumor growth in 70 and 80% of the animals, respectively.
Conclusions
intraperitoneally administration of NaB increased boron uptake while oral administration for a longer period of time induced tumor growth delay previous to BPA administration. The use of NaB via ip would optimize the irradiation results.
... To investigate the spatial arrangement of vinculin and talin-1 incubated either at 1 g or exposed to the RPM (Airbus Defense and Space, former Dutch Space, Leiden, Netherlands) for 24 h, the follicular thyroid cancer cells of the UCLA RO82-W-1 cell line [23] were purchased from Sigma-Aldrich Chemie (Munich, Germany) and cultured in RPMI-1640 medium containing 2 mM L-glutamine, supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 µg/mL streptomycin (all Invitrogen, Eggenstein, Germany). Prior to the experiment, the cells were seeded into slide flasks (BD, Heidelberg, Germany) and placed in an incubator (37°C, 5% CO 2 ) overnight, until they attached to the slides. ...
If monolayers of cancer cells are exposed to microgravity, some of the cells cease adhering to the bottom of a culture flask and join three-dimensional aggregates floating in the culture medium. Searching reasons for this change in phenotype, we performed proteome analyses and learnt that accumulation and posttranslational modification of proteins involved in cell-matrix and cell-cell adhesion are affected. To further investigate these proteins, we developed a methodology to find histological images about focal adhesion complex (FA) proteins. Selecting proteins expressed by human FTC-133 and MCF-7 cancer cells and known to be incorporated in FA, we transformed the experimental data to RDF to establish a core semantic knowledgebase. Applying iterative SPARQL queries to Linked Open Databases, we augmented these data with additional functional, transformation- and aggregation-related relationships. Using reasoning, we retrieved publications with images about the spatial arrangement of proteins incorporated in FA. Contextualizing those images enabled us to gain insights about FA of cells changing their site of growth, and to independently validate our experimental results. This new way to link experimental proteome data to biomedical knowledge from various sources via searching images may generally be applied in science when images are a tool of knowledge dissemination.
... [19][20][21] The role of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway is a wellknown mediator of tumorigenesis in different tumors, and is associated with migration and invasion of cancers by increasing expression of MMP2 and MMP9. [22][23][24] Phosphorylation of STAT3 on Tyr705 is the major mechanism of STAT3 activation, which is mediated by JAK upon stimulation of the heterodimeric gp130/cytokine-specific receptor complex by the IL-6 family of cytokines, including IL-6. 25 The present study was undertaken to explore the effects of nevirapine on cell migration and invasion/metastasis and the underlying mechanism in WRO 82-1 cells, which was derived from a metastatic lesion of a patient with follicular thyroid carcinoma [26][27][28] and in athymic mouse xenografts. ...
Background:
Metastatic or recurrent thyroid cancer often behaves aggressively, and approximately two-thirds of patients present with radioiodine resistance. Effective therapies to suppress thyroid cancer metastasis are urgently needed. Nevirapine has been proved to suppress tumor growth and induce differentiation in several tumor cells, but has not previously been evaluated in metastasis of thyroid cancer. The present study aimed to investigate the effect of nevirapine on migration and invasion in dedifferentiated thyroid cancer cells.
Methods:
Human dedifferentiated thyroid cancer cell line (WRO 82-1) was subject to real-time quantitative PCR, western blot and transwell migration/invasion assays. The liver metastasis in tumor xenografts of nude mice was subject to hematoxylin-eosin (HE) staining.
Results:
Nevirapine significantly repressed cell migration and invasion in WRO 82-1 cells, and surprisingly significantly decreased liver metastatic tumor in the nude mouse model of dedifferentiated thyroid cancer compared with that of the control. Moreover, nevirapine significantly decreased the expression of IL-6 mRNA and phosphorylation of JAK2 (Y1007+Y1008) and STAT3 (Tyr 705) in WRO 82-1 cells compared with those in control cells.
Conclusion:
Our findings suggest that nevirapine significantly repressed migration and invasion/metastasis in WRO 82-1 cells and tumor xenografts, which may be related to inhibition of IL-6/STAT3 signaling pathway. It promises great potential as a novel therapy for thyroid cancer, especially for those patients with metastasis.
... Even when cell lines have been characterized with unique short tandem repeat (STR) DNA profiles, there is no guarantee that they are descendent from a particular tumor, as those have often not been characterized in the first place. For example, the FTC origin of WRO (13) is questionable because one version of the cell line in distribution harbors a BRAF T1799A (V600E) mutation, which is specific to PTC (3,14), whereas another version is BRAF wild-type. Both WRO versions have unique STR profiles (14), but the question remains: which version is the real WRO? ...
Objective
In order to investigate the molecular underpinnings of thyroid cancer, preclinical cell line models are crucial; however, ∼40% of these have been proven to be either duplicates of existing thyroid lines or even non-thyroid derived lines or are not derived from human at all. Therefore, we set out to establish procedures and guidelines that should proactively avoid these problems, which facilitated the creation of criteria to make valid pre-clinical models for thyroid cancer research.
Design
Based upon our recommendations, we systematically characterized all new cell lines that we generated by a standardized approach that included (i) determination of human origin, (ii) exclusion of lymphoma, (iii) DNA fingerprinting and histological comparisons to establish linkage to presumed tissue of origin, (iv) examining thyroid differentiation by screening 2-3 thyroid markers, (v) examination of biological behavior (growth rate, tumorigenicity) and (vi) presence of common thyroid cancer genetic changes (TP53, BRAF, PTEN, PIK3CA, RAS, TERT promoter, RET/PTC, PAX8/PPARγ, NF1 and EIF1AX).
Results
We established 7 new thyroid cell lines (LAM136, EAM306, SDAR1, SDAR2, JEM493, THJ529, and THJ560) out of 294 primary culture attempts, and 10 patient-derived tumor xenografts (PDTX; MC-Th-95, MC-Th-374, MC-Th-467, MC-Th-491, MC-Th-493, MC-Th-504, MC-Th-524, MC-Th-529, MC-Th-560, MC-Th-562) out of 67 attempts. All were successfully validated by our protocols.
Conclusions
This standardized approach for cell line and PDTX characterization should prevent (or detect) future cross-contamination and ensure that only valid preclinical models are used for thyroid cancer research.
... Moreover, in deprived medium, TGF-b1 induces apoptosis in normal thyrocyte cultures and all neoplastic cultures except in those cultures derived from metastatic tumors (Bravo et al., 2003). Interestingly, the WRO cell line has its origin in the metastasis of a follicular human thyroid carcinoma (Estour et al., 1989). ...
Introduction:
Transforming growth factor beta (TGF-β) regulates thyroid function and growth. However, tumoral thyroid cells became resistant to this factor as they undifferentiated. Little is known about the effects of TGF-β isoforms. We compared the role of redox metabolism in the response to TGF-β isoforms between non tumoral and tumoral thyroid cells.
Methodology and results:
Differentiated rat thyroid cells (FRTL-5) and human thyroid follicular carcinoma cells (WRO) were treated with the three isoforms of TGF-β. TGF-β isoforms stopped cell cycle at different steps; G1 for FRTL-5 and G2/M for WRO. The three isoforms decreased cell viability and increased ROS accumulation in both cell lines. These effects were more pronounced in FRTL-5 than in WRO, and the isoform β1 was more potent in ROS production than the other two. TGF-β isoforms decreased total glutathione, catalase expression and it activity in both cell lines. Only in FRTL-5 the lipid peroxidation was demonstrated. Moreover, TGF-β1 decreased glutathione peroxidase and mitochondrial superoxide dismutase mRNA expression and increased mitochondrial ROS in FRTL-5, but no in WRO. Pretreatment with selenium increased glutathione peroxidase activity and decreased ROS production in WRO treated with TGF-β isoforms. Furthermore, selenium partially reversed the effect of TGF-β isoforms on cell viability only in WRO cells. The knockdown of endogenous NOX4 significantly reduced the TGF-β1 effect on cell viability in WRO but no in FRTL-5.
Conclusion:
TGF-β disrupted the redox balance and increased ROS accumulation in both cell lines. FRTL-5 cells showed reduced antioxidant capacity and had a greater sensitivity to TGF-β isoforms, while WRO cells were more resistant. This observation provides new insights into the potential role of TGF-β in the redox regulation of thyroid cells.
... The ARO cells expressed thyroglobulin (Tg), suggesting that ARO is a thyroid cancer cell line [27]. The WRO cells were originally established by Prof. Klandorf's group; the cells responded to thyroid stimulating hormone and released Tg [28]. The passage ranges for the cell lines used in this study were p13-p26 for the ARO cells, p17-p31 for the WRO cells, and p10-p25 for the SW579 cells. ...
Thyroid cancer is the most common endocrine malignancy, the global incidence rate of which is rapidly rising. Surgery and radioiodine therapies are common and effective treatments only for nonmetastasized primary tumors. Therefore, effective treatment modalities are imperative for patients with radioiodine-resistant thyroid cancer. Honokiol, a biophenolic compound derived from Magnolia spp., has been shown have diverse biological and pharmacological activities, including anti-inflammatory, antioxidative, antiangiogenic, and anticancer properties. In the present study, three human thyroid cancer cell lines, namely anaplastic, follicular, and poorly differentiated thyroid cancer cells, were used to evaluate the chemotherapeutic activity of honokiol. Cell viability, cell cycle, apoptosis, and autophagy induction were determined through flow cytometry and western blot analysis. We found that honokiol treatment can suppress cell growth, induce cell cycle arrest, and enhance the induction of caspase-dependent apoptosis and autophagy in cancer cells. Moreover, honokiol treatment modulated signaling pathways including Akt/mTOR, ERK, JNK, and p38 in the studied cells. In addition, the antitumorigenic activity of honokiol was also confirmed in vitro and in vivo. Our data provide evidence that honokiol has a unique application in chemotherapy for human thyroid cancers.
... Four human thyroid carcinoma cell lines with defective or mutant p53 (ARO, FRO, NPA, and WRO cells) were obtained from Prof. J. A. Fagin (University of Cincinnati College of Medicine) and were grown in RPMI 1640 medium supplemented with 10% FBS and ampicillin/streptomycin. ARO and FRO cells are both derived from anaplastic carcinomas and harbor defective and mutant (R27 3 H) p53, respectively (3). WRO (20) and NPA cells, which are however from papillary and follicular carcinomas, respectively, were also used in this study to provide the proof-of-principle for feasibility of our approach because these cells also contain mutant p53 (G266E in NPA and P223L in WRO) (3). ...
The present study was designed to evaluate the therapeutic efficacy of adenovirus-mediated wild-type (wt) tumor suppressor p53 expression in four human thyroid carcinoma cell lines harboring p53 mutations (ARO, FRO, NPA, and WRO) and normal human thyroid follicular cells with wt-p53 in vitro and in vivo. In vitro infection of replication-deficient recombinant adenovirus vector expressing wt-p53 led to a dose-dependent cell killing in both normal and carcinoma cells. In contrast, adenovirus expressing Escherichia coli β-galactosidase showed little effect. The sensitivity to p53-mediated cell killing varied among the cells used. It was, at least partly, dependent on their adenovirus infectivity in carcinoma cells, whereas normal thyroid cells were relatively resistant to p53-mediated cell death despite its highest adenovirus infectivity. The mechanism of cell killing by wt-p53 was shown, by flow cytometric analysis, to be apoptosis. Furthermore, wt-p53 expression renders two out of four carcinoma cell lines (FRO and NPA) more sensitive to doxorubicin and one (FRO) to 5-fluorouracil, independent of treatment schedule. In vivo experiments, using FRO and NPA cells, showed that growth of sc tumors in nude mice was nearly completely inhibited by direct injection of adenovirus expressing wt-p53 [1 × 10⁹ plaque-forming units/tumor]. This effect was augmented by its combination with doxorubicin treatment (4 mg/kg, thrice a week), which led to tumor regression. Our results therefore indicate that adenovirus-mediated wt-p53 gene introduction seems to be a potential clinical utility in gene therapy for anaplastic thyroid carcinomas, particularly when combined with chemotherapy.
The present study was designed to evaluate the therapeutic efficacy of adenovirus-mediated wild-type (wt) tumor suppressor p53 expression in four human thyroid carcinoma cell lines harboring p53 mutations (ARO, FRO, NPA, and WRO) and normal human thyroid follicular cells with wt-p53 in vitro and in vivo. In vitro infection of replication-deficient recombinant adenovirus vector expressing wt-p53 led to a dose-dependent cell killing in both normal and carcinoma cells. In contrast, adenovirus expressing Escherichia coli β-galactosidase showed little effect. The sensitivity to p53-mediated cell killing varied among the cells used. It was, at least partly, dependent on their adenovirus infectivity in carcinoma cells, whereas normal thyroid cells were relatively resistant to p53-mediated cell death despite its highest adenovirus infectivity. The mechanism of cell killing by wt-p53 was shown, by flow cytometric analysis, to be apoptosis. Furthermore, wt-p53 expression renders two out of four carcinoma cell lines (FRO and NPA) more sensitive to doxorubicin and one (FRO) to 5-fluorouracil, independent of treatment schedule. In vivo experiments, using FRO and NPA cells, showed that growth of sc tumors in nude mice was nearly completely inhibited by direct injection of adenovirus expressing wt-p53 [1 × 10⁹ plaque-forming units/tumor]. This effect was augmented by its combination with doxorubicin treatment (4 mg/kg, thrice a week), which led to tumor regression. Our results therefore indicate that adenovirus-mediated wt-p53 gene introduction seems to be a potential clinical utility in gene therapy for anaplastic thyroid carcinomas, particularly when combined with chemotherapy.
... Immunoperoxidase staining revealed thyroglobulin-positivity within the cells. The cell line was tumorigenous in nude mice [74]. The cells were cultured in RPMI-1640 medium containing 100 µM sodium pyruvate and 2 mM¨L-glutamine, supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 µg/mL streptomycin (all Invitrogen, Eggenstein, Germany). ...
Microgravity induces three-dimensional (3D) growth in numerous cell types. Despite substantial efforts to clarify the underlying mechanisms for spheroid formation, the precise molecular pathways are still not known. The principal aim of this paper is to compare static 1g-control cells with spheroid forming (MCS) and spheroid non-forming (AD) thyroid cancer cells cultured in the same flask under simulated microgravity conditions. We investigated the morphology and gene expression patterns in human follicular thyroid cancer cells (UCLA RO82-W-1 cell line) after a 24 h-exposure on the Random Positioning Machine (RPM) and focused on 3D growth signaling processes. After 24 h, spheroid formation was observed in RPM-cultures together with alterations in the F-actin cytoskeleton. qPCR indicated more changes in gene expression in MCS than in AD cells. Of the 24 genes analyzed VEGFA, VEGFD, MSN, and MMP3 were upregulated in MCS compared to 1g-controls, whereas ACTB, ACTA2, KRT8, TUBB, EZR, RDX, PRKCA, CAV1, MMP9, PAI1, CTGF, MCP1 were downregulated. A pathway analysis revealed that the upregulated genes code for proteins, which promote 3D growth (angiogenesis) and prevent excessive accumulation of extracellular proteins, while genes coding for structural proteins are downregulated. Pathways regulating the strength/rigidity of cytoskeletal proteins, the amount of extracellular proteins, and 3D growth may be involved in MCS formation.
... Three immortalised cell lines derived from human thyroid tumours of follicular origin, including NPA cells originated from a papillary carcinoma (Pang et al. 1989), WRO cells from a follicular carcinoma (Estour et al. 1989) and ARO cells from an anaplastic carcinoma (Pang et al. 1989), were generously supplied by Drs A Fusco and M Santoro (University of Naples). All the cell lines were grown in Dulbecco's modified Eagle's medium (DMEM)-F12 medium supplemented with 10% foetal calf serum and penicillin/streptomycin in a 5% CO 2 -humidified atmosphere at 37 C and used to establish the effect of GHS on cell proliferation. ...
The presence of specific receptors for synthetic growth hormone secretagogues (GHSs) has been investigated in non-tumoral and neoplastic human thyroid tissue using a radio-iodinated peptidyl GHS ((125)I-labelled Tyr-Ala-hexarelin) as ligand. Specific binding sites for Tyr-Ala-hexarelin were detected in membranes from non-tumoral and follicular-derived neoplastic thyroid tissue, but not in thyroid tumours (medullary carcinomas) of parafollicular (C cell) origin. The binding activity was greatest in well differentiated neoplasms (papillary and follicular carcinomas), followed by poorly differentiated carcinomas, non-tumoral thyroid parenchyma, follicular adenomas and anaplastic carcinomas. Both peptidyl (Tyr-Ala-hexarelin, hexarelin, growth hormone releasing peptide (GHRP6) and non-peptidyl (MK 0677) GHSs completely displaced the radioligand from binding sites of non-tumoral thyroid gland, but MK 0677 was significantly less potent. The IC(50) values were (1. 9+/-0.3)x10(-8) mol/l for Tyr-Ala-hexarelin, (2.1+/-0.2)x10(-8) mol/l for hexarelin, (2.4+/- 0.3)x10(-8) mol/l for GHRP6 and only (1. 5+/-0.4)x 10(-7) mol/l for MK 0677. Similar IC(50) values were found in neoplastic thyroid tissue. Scatchard analysis of the binding revealed a finite number of binding sites in non-tumoral (B(max): 1232+/-32 fmol/mg protein, n=3) and neoplastic (papillary carcinomas) thyroid tissue (B(max): 2483+/-380 fmol/mg protein, n=5), with dissociation constants (K(d)) of (3.8+/-0.3)x10(-9) and (4. 4+/-0.6)x 10(-9) mol/l, respectively. On the basis of this evidence, we investigated the effects of some GHS on the proliferation of three different human follicular thyroid carcinoma cell lines (NPA, WRO and ARO) in which the presence of specific GHS receptors was also demonstrated. Tyr-Ala-hexarelin, GHRP6 and MK 0677 were able to inhibit serum-stimulated [(3)H]thymidine incorporation in NPA cells at concentrations close to their binding affinity. These substances also caused a significant inhibition of cell proliferation, which was evident at the earliest time of treatment (24 h) in all the cell lines, and at the latest time (96 h) in NPA cells only. In conclusion, this paper confirms the existence of specific binding sites for GHS in normal thyroid tissue and demonstrates, for the first time, that these binding sites are present in papillary and follicular carcinomas, low in anaplastic carcinomas and absent in medullary carcinomas of the thyroid. This work also provides evidence of a growth-inhibitory effect of GHS on cell lines derived from follicular thyroid cancers.
... Thena cells exhibited a short lag phase, with rapid tumor growth in nude mice as compared with other well-differentiated thyroid carcinoma cell lines described in the literature. Thena cells produced measurable tumors in nude mice (.3 mm in diameter) within 2 weeks, whereas WRO, a follicular thyroid carcinoma cell line, produced tumors 4 months after the injection (Estour et al. 1989). B-CPAP, a papillary thyroid carcinoma cell line, developed tumors of 0·5 mm in diameter 8 weeks after the inoculation (Fabien et al. 1994). ...
An anaplastic thyroid cancer cell line, Thena, was recently established in our laboratory following radical thyroidectomy of a patient with anaplastic thyroid cancer. Microscopically, Thena cells were spindle-shaped or small round cells. Thena cells were reactive with cytokeratin AE1/ AE3 antibodies, epithelial membrane antigen, interleukin (IL)-6, epithelial growth factor receptor, transforming growth factor (TGF)-, vascular endothelial growth factor, and vimentin. Thena cells secreted high levels of IL-6, leukemia inhibitor factor (LIF), tumor necrosis factor (TNF)-, and TGF-1 in the culture supernatants, as determined by enzyme-linked immunosorbent assay. When subcutaneously injected with Thena cells, athymic nude mice developed tumor masses in the skin within 2 weeks. Furthermore, Thena cells induced cachexia in these tumor-bearing mice. High levels of human IL-6, LIF and TGF-1 were detected in the mouse sera. To our knowledge, the Thena cell line is the first thyroid cancer cell line reported to induce cachexia in nude mice. This cachectic animal model is worthy of further study to explore the treatment of thyroid cancer-induced cachexia.
... Tumour cell lines were derived from FTC WRO, 27 PTC BCPAP 28 and ATC 8505C. 29 All cell lines were cultured as monolayers in a humidified atmosphere (5% CO 2 ) at 37°C according to the instructions of their providers. ...
CD133 expression in cancer is frequently associated with poor outcome. Thyroid carcinomas are rare in childhood and adolescence and are associated with a higher risk of recurrence and more metastases than the adult tumours. The aim of the study was to assess whether the expression of CD133 in thyroid carcinomas of children, adolescents and young adults was correlated with clinical prognostic factors.
Tissue microarrays were constructed with 235 tumours coming from 208 young adults with a median age of 28 years and 27 children with a median age of 13 years. An immunohistochemical study was performed with anti-CD133 antibody. CD133 expression was evaluated, using a semiquantitative score based on the percentage of positive cells. The mutation status of tumours was evaluated by reverse transcriptase PCR. Three cell lines were used to confirm CD133 expression by western blot.
CD133 expression was found in 43% of adult and 37% of child tumours and was confirmed by western blot in cell lines. In young adults, the expression of CD133 was significantly more frequent in patients with tumours >3 cm (p=0.04) and in patients with lymph node metastases (p=0.02). The expression of CD133 was more frequent in patients in whom the tumour presented a BRAF mutation (p=0.03).
CD133 expression is correlated with tumour size, lymph nodes metastases and BRAF mutations in young adults. The presence of these cancer stem cells could offer new therapeutic alternatives for aggressive thyroid cancers.
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... A well-differentiated (follicular) human thyroid cancer cell line (UCLA RO 82 W-1 (WRO); Estour et al. 1989) and an undifferentiated cell line (UCLA RO-81A-1 (ARO)) were kindly provided by Dr GJF Juillard (UCLA, Los Angeles, CA, USA). Both cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM -glutamine, 100 U/ml penicillin and 0·1 mg/ml streptomycin, at 37 C in a CO 2 incubator. ...
To correlate the differentiation phenotype of two human thyroid cancer cell lines with their expression of various molecular markers, we analyzed the mRNA levels of four thyroid-specific genes, including thyrotropin receptor (TSHR), thyroglobulin (Tg), thyroid transcription factor-1 (TTF-1), and paired-box containing transcription factor-8 (PAX-8) genes. The results showed a differentiation-status-related pattern in which a well-differentiated cell line (WRO) expressed all the four genes, in contrast to an anaplastic cell line (ARO) that expressed TTF-1 and reduced levels of TSHR, but no Tg or PAX-8 genes. Furthermore, to verify the finding of concomitant loss of beta subtype thyroid hormone receptor (TRbeta) and TSHR gene expression in neoplastic thyroid tumors (Bronnegard et al. 1994), we examined the expression levels of TRbeta1 gene in these cell lines. Whereas the WRO cells produced an abundant amount of TRbeta1 protein detectable by immunoprecipitation, the ARO cells produced none. This new observation prompted us to investigate whether overexpression of TRbeta1 protein in ARO cells might produce changes in the differentiation phenotypes. We found that the level of expression of the TSHR gene and the proliferative index of ARO cells were significantly upregulated in the cells stably transfected with wild-type TRbeta1. These findings suggest that TRbeta1 protein overexpression can affect the differentiation phenotypes and induce more efficient cell proliferation of the anaplastic ARO cells.
... Functional Assays Cell culture. Cell lines were kindly donated by Dr. Heldin (SW1736, Hth7 and Hth83 [50]), Dr. Fagin (WRO [51]) and Dr. Fusco and Dr. Santoro (BCPAP [52] and TPC-1 [53]), or obtained from the German Collection of Microorganism and Cells Culture (Cal62, 8505c) and the European collection of cell culture (NthyORI, FTC133). All cell lines were genetically fingerprinted and verified to be unique and of thyroid origin [54]. ...
Papillary Thyroid Cancer (PTC) is a heterogeneous and complex disease; susceptibility to PTC is influenced by the joint effects of multiple common, low-penetrance genes, although relatively few have been identified to date. Here we applied a rigorous combined approach to assess both the individual and epistatic contributions of genetic factors to PTC susceptibility, based on one of the largest series of thyroid cancer cases described to date. In addition to identifying the involvement of TSHR variation in classic PTC, our pioneer study of epistasis revealed a significant interaction between variants in STK17B and PAX8. The interaction was detected by MD-MBR (p = 0.00010) and confirmed by other methods, and then replicated in a second independent series of patients (MD-MBR p = 0.017). Furthermore, we demonstrated an inverse correlation between expression of PAX8 and STK17B in a set of cell lines derived from human thyroid carcinomas. Overall, our work sheds additional light on the genetic basis of thyroid cancer susceptibility, and suggests a new direction for the exploration of the inherited genetic contribution to disease using association studies.
... B-CPAP, TPC1, and WRO human cell lines were obtained from Dr. Massimo Santoro, University of Naples (Naples, Italy) [18,19,20,21]. Nthy.ori 3.1 and FTC133 cell lines were purchased from Sigma-Aldrich (Milan, Italy). ...
Modifications in adhesion molecules profile may change the way tumor cells interact with the surrounding microenvironment. The Cadherin family is a large group of transmembrane proteins that dictate the specificity of the cellular interactions. The Cadherin switch that takes place during epithelial-mesenchymal transition (EMT) contributes to loosening the rigid organization of epithelial tissues and to enhancing motility and invasiveness of tumor cells. Recently, we found Cadherin-6 (CDH6, also known as K-CAD) highly expressed in thyroid tumor cells that display mesenchymal features and aggressive phenotype, following the overexpression of the transcriptional regulator Id1. In this work, we explored the possibility that CDH6 is part of the EMT program in thyroid tumors. We demonstrate that CDH6 is a new transforming growth factor-β (TGF-β) target and that its expression is modulated similarly to other EMT mesenchymal markers, both in vitro and in thyroid tumor patients. We show for the first time that CDH6 is expressed in human thyroid carcinomas and that its expression is enhanced at the invasive front of the tumor. Finally, we show that CDH6 is under the control of the transcription factor RUNX2, which we previously described as a crucial mediator of the Id1 pro-invasive function in thyroid tumor cells. Overall, these observations provide novel information on the mechanism of the EMT program in tumor progression and indicate CDH6 as a potential regulator of invasiveness in thyroid tumors.
... In this study, we used six different human cancer cell lines derived from different types of thyroid cancers . The WRO (Estour et al., 1989) and FTC133 (Goretzki et al., 1990) cell lines are derived from FTC; the BCPAP (Fabien et al., 1994) commonly known as a PTC cell line is derived from a poorly differentiated PTC; TPC1 (Tanaka et al., 1987) and K1 (Challeton et al., 1997 ) cell lines are derived, respectively , from PTC and from a metastasis of a well-differentiated PTC (Ribeiro et al., 2008); the 8505C (Ito et al., 1993) cell line from ATC. These six cell lines are among those mostly used for thyroid cancer research. ...
Human thyroid cancer cell lines are the most used models for thyroid cancer studies. They must be used with detailed knowledge of their characteristics. These in vitro cell lines originate from differentiated and dedifferentiated in vivo human thyroid tumors. However, it has been shown that mRNA expression profiles of these cell lines were closer to dedifferentiated in vivo thyroid tumors (anaplastic thyroid carcinoma, ATC) than to differentiated ones. Here an overview of the knowledge of these models was made. The mutational status of six human thyroid cancer cell lines (WRO, FTC133, BCPAP, TPC1, K1, and 8505C) was in line with previously reported findings for 10 genes frequently mutated in thyroid cancer. However, the presence of a BRAF mutation (T1799A: V600E) in WRO questions the use of this cell line as a model for follicular thyroid carcinoma (FTC). Next, to investigate the biological meaning of the modulated mRNAs in these cells, a pathway analysis on previously obtained mRNA profiles was performed on five cell lines. In five cell lines, the MHC class II pathway was down-regulated and in four of them, ribosome biosynthesis and translation pathways were up-regulated. mRNA expression profiles of the cell lines were also compared to those of the different types of thyroid cancers. Three datasets originating from different microarray platforms and derived from distinct laboratories were used. This meta-analysis showed a significant higher correlation between the profiles of the thyroid cancer cell lines and ATC, than to differentiated thyroid tumors (i.e., PTC or FTC) specifically for DNA replication. This already observed higher correlation was obtained here with an increased number of in vivo tumors and using different platforms. In summary, this would suggest that some papillary thyroid carcinoma or follicular thyroid carcinoma (PTC or FTC) cell lines (i.e., TPC-1) might have partially lost their original DNA synthesis/replication regulation mechanisms during their in vitro cell adaptation/evolution.
... The human WRO cell line from FTC, the normal and transformed rat thyroid cell lines, and their culture conditions have been described elsewhere (19, 20). For stable transfections of WRO and PC Cl 3 cells, Arrest-in Transfection reagent (Open Biosystems, Huntsville, AL) was used, following the manufacturer's instructions. ...
Context:
Thyroid neoplasias of the follicular histotype include the benign follicular adenomas and the malignant follicular carcinomas. Although several genetic lesions have already been described in human thyroid follicular neoplasias, the mechanisms underlying their development are still far from being completely elucidated. MicroRNAs (miRs or miRNAs) have recently emerged as important regulators of gene expression, also playing a key role in the process of carcinogenesis.
Objective:
The aim of our work has been to identify the miRNAs differentially expressed in human thyroid follicular neoplasias and define their role in thyroid carcinogenesis.
Design:
The miRNA expression profile of 10 human thyroid follicular adenomas was compared to that of 10 normal thyroid tissues.
Results:
The miRNA expression profiles revealed the down-regulation of let-7a in thyroid follicular adenomas compared to normal thyroid. Then, quantitative RT-PCR analyses validated the microarray data and showed a significantly higher decrease in let-7a expression in follicular carcinomas. Enforced let-7a expression in the follicular thyroid carcinoma cell line WRO induces an epithelial-like phenotype, increases cell adhesion, and decreases cell migration. Conversely, silencing of let-7a in the normal rat thyroid cell line PC Cl 3 has opposite effects. We identified dysadherin (FXYD5), a cell membrane glycoprotein, correlated with tumor progression and invasiveness, as a target of let-7a. Consistently, an inverse correlation between dysadherin and let-7a expression levels was found in human thyroid follicular adenomas and carcinomas.
Conclusions:
These results suggest a role of let-7a down-regulation in the development of thyroid neoplasias of the follicular histotype, likely regulating dysadherin protein expression levels.
... A recent DNA profiling analysis has revealed that ARO cells match the HT-29 colon cancer cell line and NPA cells match the M14/MDA-MB-435S melanoma cell line [29]. WRO cells derive from a follicular thyroid carcinoma [30]. All cells were maintained in DMEM medium supplemented with 10% FCS, 1% penicillin and 1% streptomycin at 37uC in a humidified atmosphere with 5% CO 2 . ...
Cyclic AMP (cAMP) inhibits the proliferation of several tumor cells. We previously reported an antiproliferative effect of PKA I-selective cAMP analogs (8-PIP-cAMP and 8-HA-cAMP) on two human cancer cell lines of different origin. 8-Cl-cAMP, another cAMP analog with known antiproliferative properties, has been investigated as a potential anticancer drug. Here, we compared the antiproliferative effect of 8-Cl-cAMP and the PKA I-selective cAMP analogs in three human cancer cell lines (ARO, NPA and WRO). 8-Cl-cAMP and the PKA I-selective cAMP analogs had similarly potent antiproliferative effects on the BRAF-positive ARO and NPA cells, but not on the BRAF-negative WRO cells, in which only 8-Cl-cAMP consistently inhibited cell growth. While treatment with the PKA I-selective cAMP analogs was associated with growth arrest, 8-Cl-cAMP induced apoptosis. To further investigate the actions of 8-Cl-cAMP and the PKA I-selective cAMP analogs, we analyzed their effects on signaling pathways involved in cell proliferation and apoptosis. Interestingly, the PKA I-selective cAMP analogs, but not 8-Cl-cAMP, inhibited ERK phosphorylation, whereas 8-Cl-cAMP alone induced a progressive phosphorylation of the p38 mitogen-activated protein kinase (MAPK), via activation of AMPK by its metabolite 8-Cl-adenosine. Importantly, the pro-apoptotic effect of 8-Cl-cAMP could be largely prevented by pharmacological inhibition of the p38 MAPK. Altogether, these data suggest that 8-Cl-cAMP and the PKA I-selective cAMP analogs, though of comparable antiproliferative potency, act through different mechanisms. PKA I-selective cAMP analogs induce growth arrest in cells carrying the BRAF oncogene, whereas 8-Cl-cAMP induce apoptosis, apparently through activation of the p38 MAPK pathway.
... 14 human cell lines and one sample of fresh tissue were examined in this study: MCF 7 (mamma carcinoma) [33], MM 31 (myometrium); MRI-H215, MRI-H196 and MRI-H186 (cervical carcinoma) (provided by H. Löhrke, German Cancer Research Center, Heidelberg); Ad 211 (pleomorphic adenoma) [34]; NB-4 (promyelocytic leukemia) [35]; FTC133 and FTC238 (follicular thyroid carcinoma) [36]; TPC-1 (papillary thyroid carcinoma) [37]; Li14 (lipoma) [38]; FRO (anaplastic thyroid carcinoma) [39]; WRO (follicular carcinoma) [40]; supT1 (T cell lymphoblastic lymphoma) [41]; HCT116 (colon carcinoma) [42]. They were cultured in RPMI 1640, TC 199 or McCoy's 5A medium supplemented with 10% or 20% fetal bovine serum and 2% penicillin/streptomycin (all Invitrogen, Karlsruhe, Germany). ...
HMGA2 is an architectonic transcription factor abundantly expressed during embryonic and fetal development and it is associated with the progression of malignant tumors. The protein harbours three basically charged DNA binding domains and an acidic protein binding C-terminal domain. DNA binding induces changes of DNA conformation and hence results in global overall change of gene expression patterns. Recently, using a PCR-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure two consensus sequences for HMGA2 binding have been identified.
In this investigation chromatin immunoprecipitation (ChIP) experiments and bioinformatic methods were used to analyze if these binding sequences can be verified on chromatin of living cells as well.
After quantification of HMGA2 protein in different cell lines the colon cancer derived cell line HCT116 was chosen for further ChIP experiments because of its 3.4-fold higher HMGA2 protein level. 49 DNA fragments were obtained by ChIP. These fragments containing HMGA2 binding sites have been analyzed for their AT-content, location in the human genome and similarities to sequences generated by a SELEX study. The sequences show a significantly higher AT-content than the average of the human genome. The artificially generated SELEX sequences and short BLAST alignments (11 and 12 bp) of the ChIP fragments from living cells show similarities in their organization. The flanking regions are AT-rich, whereas a lower conservation is present in the center of the sequences.
... Human anaplastic thyroid cancer cell lines SW1736 and C643 (Mark et al, 1987) were kindly provided by Dr NE Heldin, Denmark, and human follicular thyroid cancer cell lines ML1 ( Schonberger et al, 2000) and WRO ( Estour et al, 1989) were from J Schonberger. The human papillary thyroid cancer cell line NPA was a gift from Professor Santoro, Italy (Fagin et al, 1993). ...
Thyroid cancers are difficult to treat due to their limited responsiveness to chemo- and radiotherapy. There is thus a great interest in and a need for alternative therapeutic approaches.
We studied the cytotoxic activity of anti-thyroperoxidase autoantibodies (anti-TPO aAbs, expressed in baculovirus/insect cell (B4) and CHO cells (B4') or purified from patients' sera) against a papillary thyroid cancer (NPA) cell line. Anti-TPO aAbs from patients' sera led to a partial destruction of NPA cell line by complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) and exhibited an anti-proliferative activity. Comparison of the cytotoxic activity of anti-TPO aAbs shows that B4' induced an anti-proliferative effect and a better ADCC than B4, but a lower one than anti-TPO aAbs from patients' sera. Antibody-dependent cell-mediated cytotoxicity was increased when human peripheral blood mononuclear cells were used as effector cells, suggesting that FcgammaRs, CD64, CD32 and CD16 are involved. Indeed, anti-TPO aAbs from patients' sera, but not B4 and B4', exhibited CDC activity.
These data indicate that anti-TPO aAbs display moderate ADCC and anti-proliferative activities on NPA cells; IgG glycosylation appears to be important for cytotoxic activity and ADCC efficiency depends on FcgammaR-bearing cells. Finally, recombinant human anti-TPO aAbs cannot yet be considered as an optimal tool for the development of a novel therapeutic approach for thyroid cancer.
... Although it is tempting to speculate when or how these events occurred, it is difficult to recreate the past. Several of these original cell lines have been characterized as having evidence of thyroid-specific gene expression by Northern analysis and TSH receptor binding by several groups (13)(14)(15). The use of RT-PCR to detect gene expression may lead to false-positive results depending on the precise method used and the number of amplification cycles in the reaction, as noted by Schweppe et al. (12). ...
... So far rat thyroid tumor cell lines e.g. FRTL-5 (Mulcahy et al. 1985), the continuous cell line MTC-SK (Pfragner et al. 1990) and other cell lines derived from human medullary thyroid carcinomas as well as the follicular thyroid cell line UCLA RO 82 W1 (Estour et al. 1989) are established. However, a papillary thyroid carcinoma cell line with oxyphilic cell differentiation has not been available previously. ...
In the present study the establishment and characterization of a new oxyphilic papillary thyroid carcinoma cell line--ONCO-DG1- is given. With immunohistological, histochemical and flow cytometric methods, ONCO-DG 1 cells revealed features of epithelial differentiation. Furthermore the cells formed von Kossa-positive deposits resembling psammoma bodies in monolayer and spheroid culture until late passages. The tumor cell line is now in the 40th subculture. Because of the ability to form multicellular tumor spheroids (MCTS), this cell line is a good model for examining the interaction between thyroid tumor cells and confluent human endothelial cells on extracellular matrix in vitro. It is also suitable for xenotransplantation studies, because it is tumorigenic in NMRI nude mice in vivo.
To study processes related to weightlessness in ground-based cell biological research, a theoretically assumed microgravity environment is typically simulated using a clinostat – a small laboratory device that rotates cell culture vessels with the aim of averaging out the vector of gravitational forces. Here, we report that the rotational movement during fast clinorotation induces complex fluid motions in the cell culture vessel, which can trigger unintended cellular responses. Specifically, we demonstrate that suppression of myotube formation by 2D-clinorotation at 60 rpm is not an effect of the assumed microgravity but instead is a consequence of fluid motion. Therefore, cell biological results from fast clinorotation cannot be attributed to microgravity unless alternative explanations have been rigorously tested and ruled out. We consider two control experiments mandatory, i) a static, non-rotating control, and ii) a control for fluid motion. These control experiments are also highly recommended for other rotation speed settings and experimental conditions. Finally, we discuss strategies to minimize fluid motion in clinorotation experiments.
Background:
Telomerase reverse transcriptase (TERT) promoter mutations play a role in carcinogenesis and are found in both tumors and cancer cell lines. TERT promoter methylation, transcription factor binding, chromatin remodeling, and alternative splicing are also known to play an integral role in TERT regulation.
Methodology:
Using nanopore Cas9 Targeted-Sequencing, we characterized allele-specific methylation in thyroid cancer cell lines heterozygous for the TERT promoter mutation. Furthermore, using chromatin immunoprecipitation followed by Sanger sequencing, we probed allele-specific binding of the transcription factors GABPA and MYC, as well as the chromatin marks H3K4me3 and H3K27me3. Lastly, utilizing coding SNPs and the long read sequencing, we examined cDNA for monoallelic expression.
Results:
We found the mutant TERT promoter allele to be significantly less methylated than wildtype, while more methylated in the gene body in heterozygous TERT mutant cell lines. We demonstrated the transcriptional activators GABPA and MYC bind only to the mutant TERT allele. In addition, the activating and repressive chromatin marks H3K4me3 and H3K27me3, respectively bind mutant and wildtype alleles exclusively. Lastly, in heterozygous mutant cell lines, TERT exhibits monoallelic expression from the mutant allele only.
Conclusions:
In summary, by employing new long read sequencing methods, we were able to definitively demonstrate allele-specific DNA methylation, histone modifications, transcription factor binding, and the resulting mono-allelic transcription in cell lines with heterozygous TERT mutations.
Context
The interleukin-13 receptor alpha2 (IL13RA2), which is known to be overexpressed in glioblastoma multiforme, plays a role in various cellular processes such as cell migration that may contribute to tumor progression. Studies have attributed IL13RA2 to invasion and metastasis in cancers of the ovary, breast, and pancreas but the pathological role of IL13RA2 in thyroid cancer is still unclear.
Objective
This study aims to evaluate IL13RA2 expression in thyroid carcinomas and to examine the role of IL13RA2 in the progression of papillary thyroid cancer (PTC).
Methods
IL13RA2 immunochemical staining was performed on tissue microarrays of 137 thyroid carcinomas from patients and the differential profile of IL13RA2 was validated in thyroid cancer cell lines. In PTC cell lines, we functionally assessed the effects of IL13RA2-under and -overexpression on cell proliferation, cell migration and epithelial-mesenchymal transition (EMT) using CCK-8, transwell migration assay, qRT-PCR and western blot analysis.
Results
IL13RA2 expression was significantly correlated with advanced tumor T stage (pT3 / pT4; p=0.001) and regional lymph node metastasis (pN1; p<0.001). The staining scores of IL13RA2 were significantly higher in PTC compared to follicular subtypes (p<0.001) and correlated with advanced tumor stage amongst PTC samples (pT3 / pT4; p=0.028). Knockdown of IL13RA2 in B-CPAP cells significantly reduced cell viability, cell migration and EMT markers including N-cadherin, Vimentin and Snail. Exogenous overexpression of IL13RA2 in K1 cells increased cell migration and EMT although cell proliferation was not affected.
Conclusion
IL13RA2 is differentially regulated in PTC and is involved in cell migration via enhancing EMT.
A subtractive thyroid cDNA library was constructed from two human thyroid carcinoma cell lines originating from an anaplastic carcinoma and a papillary thyroid carcinoma. The library was used to identify genes correlated with the progression to a highly malignant phenotype. The thymosin ß-10gene was isolated and found to be expressed at much higher levels in the anaplastic cell line than in the papillary cells. The thymosin ß-10gene was overexpressed in five carcinoma cell lines com pared with normal thyroid tissue and normal thyroid primary culture cells. The highest expression occurred in the most malignant cell lines. Thymosin ß-10 gene expression was also increased in surgically removed human thyroid carcinomas and was highest in the anaplastic carcinomas. Thymosin ß-10gene expression was correlated with the degree of the malignant phenotype also in rat thyroid cells transfected with cellular and viral oncogenes of different tumorigenicity. These results show that thy mosin ß-10Overexpression is a general event of thyroid cell neoplastic transformation and suggest that the gene is involved in the progression of thyroid carcinogenesis. Finally, the thymosin ß-10gene was located on chromosome 2q37 by fluorescence in situ hybridization analysis.
The expression and cell-membrane distribution of the β1 family of integrins (very-late-activation antigens, VLA) were investigated in benign and malignant human thyroid tumors. We compared tissue samples of normal glands, nodular goiters, adenomas and carcinomas. We also examined 3 thyroidcarcinoma cell lines cultured in vitro. The expression of subunits of the β1 family of integrins was assessed by flow cytometry and specific antibodies in dispersed single-cell suspensions and by immunofluorescence on frozen tissue sections. In contrast to the heterogeneity of the expression of β1 integrins observed in other tumors, thyroid neoplastic lesions showed a remarkably constant VLA profile. In all tumors, benign as well as malignant, and in carcinoma cell lines, all sub-units of β1 integrins were expressed at high levels. While sub-units α1, α3, α5, α6 and occasionally α2 were also present in a cell sub-set of normal glands and nodular goiters, expression of α4 was restricted to neoplastic lesions; this integrin can be therefore considered an antigen associated with thyroid tumors. It has been reported that in normal glands and in nodular goiters, the expression of β1 integrins is restricted to the basal-cell membrane. Immunofluorescence on tissue sections showed instead that, in adenomas and carcinomas, the polarized distribution of these integrins on the cell membrane is lost.
The establishment of cell lines from thyroid carcinomas can provide an in vitro model of oncogenesis. B-CPAP is a new cell line that has been obtained from a differentiated papillary thyroid carcinoma. The data presented give a broader characterization and expression of tumoral markers of this cell line and identify the differentiated functions that are preserved.
An ultrastructural study was performed to confirm the thyroid nature of the new cell line. The cellular markers (thyroglobulin, S100, neuron-specific enolase [NSE]) and the oncogenes (mutated p53, H-ras, c-myc, PTC, trk) were studied by immunohistochemistry, Southern blot, or in situ hybridization.
The cells were of a differentiated ultrastructural thyroid type. All of the cells proved immunoreactive with antibodies specific to thyroglobulin, S100 proteins, NSE, and mutant p53 protein. Mutations of H-ras, PTC, and trk were not observed. The c-myc gene was not amplified.
The cell line described in these data provides a suitable model for the study of thyroid carcinogenesis, given that the cells present thyroid characteristics, and metabolic disorders not previously found in such cell lines. In addition, the coexpression of S100 proteins and mutant p53 proteins in the cells should permit the study of the interaction between these two proteins.
Background
Our aim was to establish Chinese human thyroid cancer cell lines and to provide the material for thyroid cancer research.Methods
We collected thyroid cancer tissues from 93 patients with thyroid cancer for developing the primary culture. Thyroid cancer tissues were verified by frozen sections during the operations, then subjected to primary cell culture. During the first several passages, fibroblasts were removed by selective attachment.ResultsFrom the 93 cancer tissues used, two follicular cancer tissues from the metastatic area (CGTH W-1, CGTH W-2) and one papillary thyroid cancer tissue (CGTH W-3) could be passed over 50 times. Cellular transformation with loss of contact inhibition occurred during passages 5–8. Electron microscopic studies of the CGTH W-1 cell line showed the presence of an abundance of mitochondria and Golgi complex. Presence of microvilli with interdigitations between neighboring cells were found in CGTH W-2 and CGTH W-3 cell lines. Severe combined immunodeficient (SCID) mice were used to determine whether these cells were tumorigenic. Two months after transplantation of 1 × 107 CGTH W-1, 2, and 3 cells into SCID mice, subcutaneous tumors ∼ 2–2.5 cm in size were clearly visible. After affinity cross-linking, an IGF-I with insulin-like growth factor binding proteins complex corresponding to a molecular size of 41 kDa was observed in culture media collected from CGTH W-1, SW 579, and RO82 W-1 cells.Conclusions
Three thyroid cancer cell lines were established, which may provide material for future thyroid cancer research. © 1996 Wiley-Liss, Inc.
The present study focuses on the establishment and characterization of a new follicular thyroid carcinoma cell line. The human cell line ML-1 was derived from a dedifferentiated follicular thyroid carcinoma relapse, which progressed despite preceding surgery followed by two radioiodine therapies. More than 90% of the cells of this line express thyroglobulin, chondroitin sulfate, and vimentin antigens, but only about 70% show cytokeratin filaments and a negative surface charge density such as human erythrocytes. More importantly, cells of this line are able to take up iodine and/or glucose both in vitro and in vivo and to secrete thyroglobulin, chondroitin sulfate, and fibronectin into the interstitial space. In addition, triiodothyronine is released constitutively into culture supernatants. Moreover, it is also suitable for xenotransplantation studies because it is tumorigenic in NMRI nude mice in vivo. The cell line forms tumors with follicular structures when transplanted to nude mice. Due to these unique features the ML-1 cell line can be considered as a very suitable test model for pharmacological and cell biological studies. Since chemicals may interfere with the production of thyroid hormones, this cell line represents also a tool for toxicological investigations.
We studied the urokinase-type plasminogen activator (uPA) receptor (uPA-R) in normal and neoplastic human thyroid cells. It has recently been shown that cleaved forms of uPA-R display an extremely strong chemotactic activity. Normal human thyroid TAD-2 cells express the intact form of the uPA-R and a truncated form lacking the uPA-binding domain on their surface, in a similar manner to tumor thyroid cell lines. However, in tumor thyroid cell lines, the amount of the truncated form is variable: high in papillary carcinoma cells, very low in follicular carcinoma cells, and not detectable in anaplastic carcinoma cells. Similar studies on primary cell cultures confirm the presence of the truncated form of uPA-R in normal and in papillary carcinoma cells and its partial or total loss in follicular carcinoma cells. The presence of truncated uPA-R correlates to uPA secretion, except in papillary carcinoma cells, which express the truncated form of uPA-R but do not release uPA. uPA-R is also able to act as an adhesion receptor by binding vitronectin (VTN) and interacting with integrins. We observe that removal of uPA-R from the surface of normal thyroid and anaplastic carcinoma cells by phosphatidylinositol-specific phospholipase C or treatment with anti-uPA-R antibodies decreases the adhesion of both cell types to VTN and, less efficiently, to fibronectin or collagen. On the other hand, uPA treatment strongly increases the adhesion of anaplastic carcinoma cells specifically to VTN.
Anaplastic thyroid carcinomas are deadly tumors that are highly invasive, particularly into the bones. Although oncogenic Ras can transform thyroid cells into a severely malignant phenotype, thyroid carcinomas do not usually harbor ras gene mutations. Therefore, it is not known whether chronically active Ras contributes to thyroid carcinoma cell proliferation, although galectin-3 (Gal-3), which is strongly expressed in thyroid carcinomas but not in benign tumors or normal glands, is known to act as a K-Ras chaperone that stabilizes and drives K-Ras.GTP nanoclustering and signal robustness. Here, we examined the possibility that thyroid carcinomas expressing high levels of Gal-3 exhibit chronically active K-Ras. Using cell lines representing three types of malignant thyroid tumors--papillary, follicular, and anaplastic--we investigated the possible correlation between Gal-3 expression and active Ras content, and then examined the therapeutic potential of the Ras inhibitor S-trans, trans-farnesylthiosalicylic acid (FTS; Salirasib) for thyroid carcinoma. Thyroid carcinoma cells strongly expressing Gal-3 showed high levels of K-Ras.GTP expression, and K-Ras.GTP transmitted strong signals to extracellular signal-regulated kinase. FTS disrupted interactions between Gal-3 and K.Ras, strongly reduced K-Ras.GTP and phospho-extracellular signal-regulated kinase expression, and enhanced the expression of the cell cycle inhibitor p21 as well as of the thyroid transcription factor 1, which is involved in thyroid cell differentiation. FTS also inhibited anaplastic thyroid carcinoma cell proliferation in vitro and tumor growth in nude mice. We conclude that wild-type K-Ras.GTP in association with Gal-3 contributes to thyroid carcinoma malignancy and that Ras inhibition might be a useful treatment strategy against these deadly tumors.
The identification of follicular thyroid adenoma-associated transcripts will lead to a better understanding of the events involved in pathogenesis and progression of follicular tumours. Using Serial Analysis of Gene Expression, we identified five genes that are absent in a malignant follicular thyroid carcinoma (FTC) library, but expressed in follicular adenoma (FTA) and normal thyroid libraries.
NR4A1, one of the five genes, was validated in a set of 27 normal thyroid tissues, 10 FTAs and 14 FTCs and three thyroid carcinoma cell lines by real time PCR. NR4A1 can be transiently increased by a variety of stimuli, including lithium, which is used as adjuvant therapy of thyroid carcinoma with (131)I. We tested if lithium could restore NR4A1 expression. The expression of other genes potentially involved in the same signalling pathway was tested. To this end, lithium was used at different concentration (10 mm or 20 mm) and time (2 h and 24 h) and the level of expression was tested by quantitative PCR. We next tested if Lithium could affect cell growth and apoptosis.
We observed that NR4A1 expression was under-expressed in most of the FTCs investigated, compared with expression in normal thyroid tissues and FTAs. We also found a positive correlation between NR4A1 and FOSB gene expression. Lithium induced NR4A1 and FOSB expression, reduced CCDN1 expression, inhibited cell growth and triggered apoptosis in a FTC cell line.
NR4A1 is under-expressed in most of FTCs. The loss of expression of both NR4A1 and the Wnt pathway gene FOSB was correlated with malignancy. This is consistent with the hypothesis that its loss of expression is part of the transformation process of FTCs, either as a direct or indirect consequence of Wnt pathway alterations. Lithium restores NR4A1 expression, induces apoptosis and reduces cell growth. These findings may explain a possible molecular mechanism of lithium's therapeutic action.
Cell lines derived from human cancers provide critical tools to study disease mechanisms and develop novel therapies. Recent reports indicate that up to 36% of cell lines are cross- contaminated.
We evaluated 40 reported thyroid cancer-derived cell lines using short tandem repeat and single nucleotide polymorphism array analysis.
Only 23 of 40 cell lines tested have unique genetic profiles. The following groups of cell lines are likely derivatives of the same cell line: BHP5-16, BHP17-10, BHP14-9, and NPA87; BHP2-7, BHP10-3, BHP7-13, and TPC1; KAT5, KAT10, KAT4, KAT7, KAT50, KAK1, ARO81-1, and MRO87-1; and K1 and K2. The unique cell lines include BCPAP, KTC1, TT2609-C02, FTC133, ML1, WRO82-1, 8505C, SW1736, Cal-62, T235, T238, Uhth-104, ACT-1, HTh74, KAT18, TTA1, FRO81-2, HTh7, C643, BHT101, and KTC-2. The misidentified cell lines included the DRO90-1, which matched the melanoma-derived cell line, A-375. The ARO81-1 and its derivatives matched the HT-29 colon cancer cell line, and the NPA87 and its derivatives matched the M14/MDA-MB-435S melanoma cell line. TTF-1 and Pax-8 mRNA levels were determined in the unique cell lines.
Many of these human cell lines have been widely used in the thyroid cancer field for the past 20 yr and are not only redundant, but not of thyroid origin. These results emphasize the importance of cell line integrity, and provide the short tandem repeat profiles for a panel of thyroid cancer cell lines that can be used as a reference for comparison of cell lines from other laboratories.
In order to further evaluate the role of TSH in the proliferation and the differentiation of human thyroid carcinoma cells, we have analyzed the function of the TSH receptor in the established thyroid carcinoma cell lines NPA and WRO. The TSH signal transduction system in the carcinoma cells was also compared with that in normal thyroid cells. Although unresponsiveness to bovine and human TSH was demonstrated by measurement of cAMP production and [3H]thymidine incorporation after treatment of TSH, cAMP production was induced after stimulation of these cells by forskolin, cholera toxin, and isoproterenol. Specific binding to 125I-TSH was demonstrated in both NPA and WRO cells in addition to the existence of a TSH receptor mRNA and thyroglobulin mRNA species, although thyroid-specific gene expression in these cells was not regulated by TSH. These findings suggest that the unresponsiveness to TSH in these cells may be due to an abnormality of TSH receptor-G protein coupling rather than to a decreased level of TSH-receptor expression or a Gs protein abnormality.
A continuous cell line, named SMC R86 F1, was established from a surgically resected primary thyroid lesion. The cell grew as an adhering monolayer with a doubling time of about 25 hours in modified Eagle's medium supplemented with fetal bovine serum. When the cells were transplanted into athymic nude mice, tumors developed at the site of inoculation. The cells not only showed epithelial origin upon light and electron microscopic examination but also possessed a biosynthetic marker human thyroglobulin (hTg). In order to examine the iodide trapping ability of the xenografts, radioiodine at doses of 3.7 MBq was injected into the peritoneum of 131I treated nude mice bearing xenografts at about 4 weeks after the cell inoculation. Judging from the results of scintigraphic, autoradiographic and biodistribution studies, viable tissue of the xenografts in the treated mice had the ability to trap radioiodine. Histological sections of the xenografts resected from the treated mice consisted of follicle-like and trabecular growing structures, and immunohistochemically the cytoplasm of the tissues was hTg positive. The cells possessed the ability to trap radioactive iodine in vitro under the control of TSH. In addition, the expression of iodinated 19S Tg in the cell cytoplasms in the monolayer cultures was revealed by immunoblotting and autoradiographic assays. These observations provide strong evidence that the SMC R86 F1 cell line possesses well-differentiated properties of the malignant thyroid follicular epithelial cells.
To understand the inconsistent immunodetection of tumors in vivo, a labelled monoclonal antibody (MAb) against a human follicular cancer cell line (UCLA RO 82 W-1) was used as a model for in vitro and in vivo studies. The 131I labelled MAb x RO 82 W-1 bound to its target cells (10% to 70%) mainly because of the generation of immunoglobulin aggregates. Aggregates generated by the freezing process were shown by polyacrylamide gel electrophoresis (PAGE) and their removal by filtration. When the aggregated 131I MAb x RO 82 W-1 was injected into BALB/c mice bearing UCLA RO 82 W-1 tumors, a high tumor/blood ratio was found in the large tumors. The tracer concentrated in the macroscopically visible necrotic part of the tumor was largely responsible for the scintigraphic detection. Irrelevant 131I-IgG also concentrated in necrotic regions of tumors. Scintigraphic detection of thyroid tumors in this model was related to the degree to which labeled aggregates of IgG, regardless of their specificity, localized in necrotic regions of the tumors.
The receptor for the stem cell factor encoded by the c-kit proto-oncogene is expressed by a number of epithelial cells including thyrocytes. Since malignant transformation may be associated with loss of this receptor (melanoma and breast cancer), we have analyzed its expression in benign (38 cases) and malignant (31 cases) thyroid lesions. While low levels of c-kit are expressed in normal thyroids and in 60% of benign lesions, the receptor is undetectable in 60 and 90% of the follicular and papillary carcinomas, respectively. Northern blot analysis from surgical specimens of carcinomas and from carcinoma cell lines has demonstrated a lack of specific c-kit transcripts. These findings indicate that the c-kit receptor may be involved in the growth control of thyroid epithelium and that this function may be lost following malignant transformation.
We investigated the sensitivity and sequential events that take place in thyroid epithelial cells after irradiation. Cell survival ratios at a dose of 2 Gy were 18 +/- 2.5%, 58 +/- 1.0%, 59 +/- 1.5%, and 98 +/- 1.8% in primary thyroid cells, papillary thyroid carcinoma cells, follicular thyroid carcinoma cells, and anaplastic thyroid carcinoma cells, respectively. Thyroid carcinoma cell lines carrying mutations in the p53 gene were resistant to ionizing radiation. Although irradiated cells were accumulated at G1 in primary thyroid cells even after low-dose irradiation (0.2 Gy), this phenomenon was not observed in the thyroid carcinoma cell lines. Wild-type p53 expression in primary thyroid cell was increased following irradiation, but mutated p53 in the thyroid carcinoma cell lines was unchanged. To clarify the signal transduction in the G1 arrest following irradiation, levels of expression of the p53 putative downstream effectors GADD45 and WAF1/Cip1 were examined. Despite the consistent level of GADD45 mRNA, the level of WAF1/Cip1 transcripts was increased in a radiation dose-dependent manner in primary thyroid cells. This increase in the WAF1/Cip1 mRNA level was observed 30 min after irradiation and continued for at least 48 h. A mobility shift assay performed using the sequence of the putative p53 DNA binding site on the WAF1/Cip1 and GADD45 genes as a probe showed that nuclear protein extracted from primary thyroid cells, anti-p53 antibody, and probe oligonucleotide-bound complex was clearly shifted. An increase in binding activity of the p53/antibody/DNA complex was observed following irradiation. In contrast, the nuclear extract from thyroid carcinoma cells could not bind the specific DNA site, suggesting that mutant p53 has lost its binding ability. Actinomycin D inhibited WAF1/Cip1 and GADD45 mRNA levels and cycloheximide stimulated up-regulation of both basal mRNA levels, but an additional increase of the mRNA expression following irradiation was observed only in the WAF1/Cip1 gene. These data suggest that p53 in postradiation acts at a transcriptional level on WAF1/Cip1 gene expression and that de novo protein synthesis is not required for this effect. These results suggest that the p53-WAF1/Cip1 pathway may play a central role in induction of G1 arrest following irradiation in human thyroid epithelial cells.
Antiproliferative effects of somatostatin (SRIH) analogs were investigated in human thyroid carcinoma cell lines. Membrane preparations from six human thyroid cell lines, including follicular (RO 87-M-1 and RO 82-W-1), papillary (NPA87), and anaplastic (RO 90-D-1) carcinomas, a follicular adenoma, as well as benign and malignant human thyroid tissues, contained high affinity SRIH-binding sites. Ligand-binding studies ([125I]Tyr11-SRIH) demonstrated mean dissociation constants ranging from 114-224 pmol/L, and 20-154 fmol/mg membrane protein mean receptor sites, in cell lines. Four cell lines were grown for 3 days in monolayers with SRIH analogs (octreotide or MK0678) at concentrations from 0.05-100 nmol/L in serum-free medium to assess changes in cell numbers. At the highest dose, MK0678 produced dose-dependent inhibition of growth in RO 87-M-1 and NPA87, each to about 70% of that in control cells. Octreotide produced dose-dependent stimulation of growth in RO 87-M-1 cells, but caused growth inhibition in NPA87 cells, with loss of effect at the highest dose. RO 82-W-1 did not respond to MK0678, yet caused biphasic inhibition with octreotide (75% and 45% of control cell numbers at 0.05 and 100 nmol/L doses, respectively). Anaplastic cells, RO 90-D-1, did not respond to either analog despite similar ligand binding. Addition of epidermal growth factor (100 micrograms/L) or TSH (200 mU/L) increased the sensitivity of RO 87-M-1 cells to growth inhibition by the lowest dose of MK0678, producing biphasic dose-response curves. In conclusion, the present data demonstrate specific SRIH binding to membranes of thyroid carcinoma cells and tissues as well as discordant growth effects of different SRIH analogs on the same cell lines. This may be a result of differential stimulation and regulation of distinct SRIH receptor subtypes.
The expression and cell-membrane distribution of the beta 1 family of integrins (very-late-activation antigens, VLA) were investigated in benign and malignant human thyroid tumors. We compared tissue samples of normal glands, nodular goiters, adenomas and carcinomas. We also examined 3 thyroid-carcinoma cell lines cultured in vitro. The expression of subunits of the beta 1 family of integrins was assessed by flow cytometry and specific antibodies in dispersed single-cell suspensions and by immunofluorescence on frozen tissue sections. In contrast to the heterogeneity of the expression of beta 1 integrins observed in other tumors, thyroid neoplastic lesions showed a remarkably constant VLA profile. In all tumors, benign as well as malignant, and in carcinoma cell lines, all sub-units of beta 1 integrins were expressed at high levels. While sub-units alpha 1, alpha 3, alpha 5, alpha 6 and occasionally alpha 2 were also present in a cell sub-set of normal glands and nodular goiters, expression of alpha 4 was restricted to neoplastic lesions; this integrin can be therefore considered an antigen associated with thyroid tumors. It has been reported that in normal glands and in nodular goiters, the expression of beta 1 integrins is restricted to the basal-cell membrane. Immunofluorescence on tissue sections showed instead that, in adenomas and carcinomas, the polarized distribution of these integrins on the cell membrane is lost.
A thyroid carcinoma cell line, BHT-101, has been established in vitro from a metastatic lymph node deposit in a female patient with a non-hormone producing anaplastic, partly thyroglobulin- and thyroxine (T4)-positive papillary thyroid cancer. The cell population is heterogeneous, containing epithelial-like and fibroblast-like cells, and has a doubling time of 24 h. The cell line is polyploid with hypertetraploid predominance and the karyotype showed trisomies, tetrasomies, pentasomies as well as many marker chromosomes. The majority of the cells are negative or weakly positive for thyroglobulin and thyroxine and estrogen and progesterone receptors are present in the cells. BHT-101 cells produce tumours when injected into immunosuppressed CBA/Ca mice. The cells are sensitive to adriamycin, methotrexate and tamoxifen but not to methimazole (Favistan). The epithelial-like clone 1 and the fibroblast-like clone 3, isolated from the parental line, differed in drug sensitivity. This new cell line is suitable for studying the biology of thyroid carcinoma and for parallel in vivo and in vitro studies of drug activity against thyroid cancer.
A new permanent cell line (GLAG-66) has been established from the metastases of a papillary thyroid carcinoma in a male patient. Herein are reported the cytogenetic characteristics of this new cell line, which is tumorigenic in athymic mice. An aneuploid chromosomal pattern was observed (48 chromosomes) with various chromosomal abnormalities. The karyotype was: 48,XY,der(1)t(9;1;9), +der(8)t(1;8),der(9)t(1;9),der(9)t(1;9), +14. This cell line should prove to be of great value in the study of the biology of human papillary thyroid carcinomas.
In vitro culture of a human breast cancer biopsy fragment gave rise to two permanent cell lines, CAL 18 A and CAL 18 B, which were differentiated by both morphological and ultrastructural analysis. The karyotypic and growth properties of these two cell lines also differed, providing further evidence of cell heterogeneity within a given tumor. Both cell lines lost their hormone receptors in vitro. CAL 18 A cells grew in agar and were tumorigenic after inoculation into nude mice; neither of these properties was observed in CAL 18 B cells. The chemosensitivity of 12 antineoplastic drugs was assessed by a short-term assay, using inhibition of tritiated thymidine incorporation by the cells after contact with the drugs as the end point. Only a few drugs were active at moderate concentrations. The overall responses of both cell lines were similar. The cell survival curves, established by the colony method following a single dose of radiation, were also very similar, despite the greater heterogeneity of CAL 18 B cells. The two cell lines appear to be interrelated, since CAL 18 B cells were occasionally observed to emerge from CAL 18 A clones, suggesting that malignant cell redifferentiation may occur spontaneously in vitro.
Using a chemically defined medium containing hydrocortisone, insulin, transferrin, 17 beta-estradiol and selenium, with or without serum supplementation (2.5% v/v), continuous cell lines can be established from 72% of all fresh biopsy specimens of small cell lung cancer (SCLC) containing tumor cells. No differences were observed in the rate of establishing cell lines from newly diagnosed untreated patients, or from patients who have relapsed from prior therapy, or from a variety of different organ sites. Biochemical characterization of 50 SCLC cell lines for the expression of L-dopa decarboxylase; bombesin-like immunoreactivity; neuron-specific enolase, and the brain isozyme of creatine kinase, revealed that SCLC cell lines can be subdivided into two distinct classes: classic SCLC cell lines (35 lines), which express elevated levels of all four biomarkers; and variant SCLC cell lines (15 lines) which have undetectable levels of L-dopa-decarboxylase and bombesin-like immunoreactivity, but continue to express neuron-specific enolase and the brain isozyme of creatine kinase. The presence of the latter two markers distinguishes variant lines fron non-SCLC cell lines. In addition, four distinct classes were identified morphologically. The biomedical differences among established SCLC cell lines may account for the differences in response rates to cytotoxic therapy observed in newly diagnosed SCLC patients. A prospective study of biomarker characterization of SCLC tumors will determine if clinical differences exist between classic and variant SCLC tumors.
Tissue was taken from 16 patients with benign thyroid lesions (10 nontoxic nodular colloid goiter, two follicular adenoma, one autonomous adenoma, one iodine-induced thyrotoxicosis, 2 Graves' disease) and 18 patients with malignant thyroid tumors [seven papillary, five follicular, five undifferentiated (anaplastic), and one medullalry carcinoma] and was xenotransplanted into the flanks of 124 syngeneic female BALB/c-nu/nu mice 6 weeks of age. Subsequently, without any further treatment, serum levels of thyroglobulin (TG), T3, T4, and thyroid-stimulating hormone were determined by radioimmunoassay at 4 or 5 weeks posttransplantation and at the end of the experimental time period of 4 months. All animals were autopsied. The grafts were examined by light microscopy and TG immunohistochemistry. Morphologically, the grafts of benign and malignant thyroid tumors showed features overall identical to the original tissue. Conversely, nontoxic nodular colloid goiter and Graves' disease grafts revealed a transformation to normofollicular structures. All benign thyroid grafts showed a stationary growth, as did most differentiated thyroid carcinomas. Tumor take rates in differentiated and in medullary carcinoma were 15%, and in undifferentiated carcinomas, 100%. In the cancer grafts, a correlation between resting phase (period until progressive tumor growth) and survival time of the corresponding patients was disclosed. All patients whose tumors were not taken by nude mice are still alive and show no signs of progressive tumor growth at 9 to 34 months after surgery. All but one patient with tumors revealing positive tumor take died within 3 months (resting phase, 3 weeks) or one year (resting phase, 7 to 14 weeks) after surgery. Integrity of hormonal function in benign and malignant xenografts at 4 months posttransplantation could be shown by significantly higher T3 and T4 serum concentrations in animals with benign thyroid tissues (T3, 1.69 +/- 0.13 nmol/liter; T4, 45.69 +/- 2.09 nmol/liter; S.E.) as compared to controls without grafted tissue [T3, 1.29 +/- 0.10 nmol/liter (p less than 0.05); T4, 33.39 +/- 2.71 nmol/liter (p less than 0.05)] and by increased TG serum concentrations in animals receiving benign (TG, 2.70 +/- 1.39 ng/ml) or malignant (e.g., TG in follicular carcinoma, 34.44 +/- 13.83 ng/ml; controls, 0.30 +/- 0.02 ng/ml) thyroid tissue. Thus, we conclude that benign and malignant thyroid xenografts in the nude mouse maintain full morphological and, regarding T3, T4, and TG serum levels, functional integrity for at least 4 months after transplantation.
Subcellular distribution of hexokinase (HK) isoenzymes in 22 human breast cancers (21 primary cancers and 1 axillary metastatic growth) and 7 non-pathological human mammary gland tissue samples was studied with starch gel electrophoresis on isolated cell fractions obtained by differential centrifugation. Fractions used were cytosol, mitochondria and microsomes. A comparison of two methods for detecting HK activity was made, yielding different results regarding HK II and HK III. A method based on the ability of NADPH to fluoresce in UV gave a constant pattern of HK isoenzymes. In non-pathological breast tissue, only HK I was seen, i.e. in the cytosol and the microsomal fractions. HK I was also seen in all fractions of the cancers, but another more anodal band of HK I, as well as HK II and HK III, consistently appeared in the cytosol fractions. The more commonly used staining technique with tetrazolium dye revealed HK II in 45% and HK III in 50% of the samples. The pattern of HK isoenzymes in the cancers was the same irrespective of estrogen and progesterone cytosolic receptor contents and the histology of the tumors. The fluorescence method is, therefore, much more sensitive than the tetrazolium technique for detecting HK activity after electrophoresis and could explain difference in results obtained by various laboratories.
The BJC is owned by Cancer Research UK, a charity dedicated to understanding the causes, prevention and treatment of cancer and to making sure that the best new treatments reach patients in the clinic as quickly as possible. The journal reflects these aims. It was founded more than fifty years ago and, from the start, its far-sighted mission was to encourage communication of the very best cancer research from laboratories and clinics in all countries. The breadth of its coverage, its editorial independence and it consistent high standards, have made BJC one of the world's premier general cancer journals. Its increasing popularity is reflected by a steadily rising impact factor.
Basal and stimulated adenylate cyclase and protein phosphokinase activities were measured in normal and diseased human thyroids. Seven specimens of thyroid carcinomas (2 papillary, 2 mixed papillo-follicular or trabecular, 1 follicular and 2 medullary), and 6 hyperfunctional autonomous nodules were obtained at thyroidectomy. Nine specimens of normal tissue, taken from the opposite lobe of the nodular thyroids and from entirely normal glands, constituted the control group.Biochemical observations were the following: adenylate cyclase: basal activity and stimulation by TSH were rather similar in all cases. Hyperfunctional nodules were characterized by a significant increase of stimulation, as compared to the normal, by sodium fluoride (13·0 vs 7·8 fold) and prostaglandin E1 (2·6 vs 1·3 fold). Adrenalin had no significant effect. Protein phosphokinase: basal activity was significantly higher in carcinomas by a factor of up to 4-fold. Activation by cyclic AMP was similar in normal and pathological homogenates.These data show that adenylate cyclase activity is not unequivocally related to the degree of malignancy in human thyroid tumors and that protein phosphokinase activity is increased in such tissues.The data are discussed within the framework of current hypotheses derived from studies of transformed cells in vitro.
A comprehensive listing of putative human breast carcinoma cell lines and the extent to which each has been characterized is presented. Criteria used to certify the human, mammary, and malignant origin of a cell line include: (a) a reliable histopathological diagnosis; (b) interspecies specificity established by human karyotype, isoenzyme profiles, and/or cell surface antigenicity; (c) intraspecies specificity, demonstrated by genetic evidence of a unique, human donor distinct from other cells including HeLa cells; and (d) organ specficity, supported by morphological evidence of epithelial structure and secretory activity, and especially by the expression of differentiated functions; these include presence of receptors for sex steroid hormones, hormone responsiveness, and production of milk proteins, fatty acids, or milk-specific antigens. Of the 47 cell lines for which data are here reported, 22 have been shown to be derived from human non-HeLa donors and to have epithelial morphology as revealed by light or electron microscopy. Differentiated function has been recorded for 19 cell lines. Additional human breast cancer cell lines have been reported, but characterization of some of these has been insufficient to judge the legitimacy of their predigrees. For others mammary origin is questionable. Six purported breast cell lines are in reality HeLa cells, and one is of nonhuman origin.
A newly developed electrophoretic technique for human galactose-1-phosphate uridyl transferase confirms the multiple band patterns for the Duarte and Los Angeles variants. This represents the first confirmation for the Los Angeles variant. The observed frequencies of N, D, and LA types are similar to earlier reports for these variants.
In this study, the authors try to specify the concept of the dependence of thyroid cancers on thyroid-stimulating hormone (TSH) in 7 cases of differentiated orthoplasic cancer and 2 cases of undifferentiated anaplasic cancer. In each case, the basal activity and the stimulability of the cyclase adenylate (TSH, PGE1,NaF) contained in the 12,000 g residuum of the homogenate of thyroid tissue has been measured and the results compared with those obtained with similar preparations of normal tissue. Although highly dispersed, the basal activity per mg of proteins is more increased in the orthoplasic cancers (177 + 50 pmoles of AMPc generated per mg of proteins against 66.2 + 18.0) but not in the anaplasics (47.2). The stimulability of the adenylate cyclase by TSH is on average normal for the orthoplasics but nil for the anaplasics. Studied in terms of the subgroups of orthoplasic cancers, the sensibility of the adenylate cyclase to TSH is very feeble in a case of papillary cancer, normal in two cases of vesicular cancer and increased in four cases of mixed form. The response to PGE1 is normal. Stimulation by NaF is normal in all cases except the papillary one. The preliminary findings seem to indicate that the anomalies of the adenylate cyclase lie at the level of the TSH receptor.
The determination of phosphoglucomutase (PGM1) phenotypes was performed by isoelectric focusing on samples from 1678 unrelated individuals from Hessen, Germany. Ten common phenotypes are considered as gene products of four alleles at the PGM1 locus with the following frequencies: PGM
1a1=0.6305, PGM
1a2=0.1844, PGM
1a3=0.1320, and PGM
1a4=0.0530. Twenty-two different mating types were observed in 113 families with 202 children. The segregation of the phenotypes in the offspring supports the assumed way of autosomal codominant inheritance. The example of a silent allele (PGM
10) as well as a rare variant (PGM
17) is reported.
The presence of human thyroglobulin (HTg) in serum of patients was identical by immunological criteria to the serum standard used in the radioimmunoassay. The serum thyroglobulin levels in untreated patients with differentiated thyroid carcinoma ranged from 22.0 to 445.0 ng/ml with a mean of 144.3 +/- 46.5 ng/ml (SEM) (n = 10). The mean serum thyroglobulin measured postoperatively in seven of these patients was 6.4 +/- 1.5 ng/ml, not statistacally different from the mean level of 5.1 +/- 0.49 ng/ml (range 0-20.7 ng/ml) observed in 71 out of 95 control subjects with detectable HTg levels. By contrast serum HTg levels were normal or undetectable in subjects with medullary carcinoma of the thyroid. HTg levels were within normal limits in sera of patients who had previously undergone successful therapy for a differentiated thyroid carcinoma and in whom no metastases could be documented. The mean level for this group was 4.9 +/- 0.51 ng/ml (n = 43). In contrast, patients with documented metastases had a mean serum thyroglobulin level of 464.9 +/- 155.6 ng/ml (n = 6). The data support the thesis that in differentiated thyroid carcinoma serum thyroglobulin levels are elevated when metastases develop after initial treatment. It is proposed that the measurement of thyroglobulin in the serum represents a simple and valuable adjunct in the posttreatment follow-up of patients with differentiated thyroid cancer.
Hurthle cell neoplasms are though to arise from the follicular cells of the thyroid gland, although some studies suggest that they originate from the parafollicular cells. We studied tissue from five patients (three men and two women, aged 33-72 yr) with Hurthle cell neoplasms (four adenomas and one carcinoma) to determine whether Hurthle cell neoplasms have an intact TSH receptor-adenylate cyclase (AC) system and, if so, whether it differs in benign and malignant Hurthle cell and neoplasms. Binding of [125I]bovine (b) TSH and an AC response to TSH occurred in all four Hurthle cell adenomas. In three of these tumors, there was good binding and a relatively good correlation between the concentration of bTSH producing half-maximal inhibition of [125I]bTSH binding (4.8 mU/ml) and the TSH concentration causing half-maximal stimulation of AC (2.2 mU/ml). There was also a 2- to 5-fold increase in AC activity in response to bTSH (300 mU/ml), a value comparable to that which occurs in follicular thyroid neoplasms and differentiated thyroid cancers. In the fourth Hurthle cell adenoma, however, binding was low, the apparent Kd (170 mU/ml) and Km (20 mU/ml) were high, and there was only a 1.4-fold increase in AC activity in response to bTSH (300 mU/ml). In the one metastatic Hurthle cell carcinoma, there was no high affinity TSH binding or AC response to TSH. Thus, Hurthle cell neoplasms are of follicular cell origin, since benign Hurthle cell tumors have an intact TSH receptor-AC system. Malignant Hurthle cell neoplasms, like undifferentiated and medullary thyroid cancer, lack a functional TSH receptor.
"Cold" thyroid nodules do not concentrate (131)I before or after thyrotropin (TSH) administration. In an attempt to elucidate the reason for this TSH unresponsiveness, the effect of TSH in vitro on several metabolic parameters was studied in 11 "cold" thyroid adenomas, 2 medullary carcinomas, and in the surrounding normal thyroid tissue. Basal adenyl cyclase activity, glucose-1-(14)C oxidation, and (32)P incorporation into phospholipids were significantly greater in the adenomas than in the adjacent normal thyroid; basal cyclic 3',5'-adenosine monophosphate (cyclic AMP) concentration and adenine-(3)H incorporation into (3)H-labeled cyclic AMP were not different. In adenomas as well as normal thyroid, all parameters responded significantly to in vitro TSH stimulation. The response to TSH of adenyl cyclase activity and (32)P incorporation was enhanced in adenomas compared with that of the adjacent normal thyroid. These differences were not explained by an increased cellularity of the adenomas. Medullary carcinomas did not respond to TSH in any of the above parameters. The studies demonstrate an intact, TSH-responsive adenyl cyclase-cyclic AMP system in the adenomas and, accordingly, imply the presence of receptor sites for TSH on the cells of the adenoma. The failure of such nodules to concentrate (131)I may be owing to a subsequent impairment in the expression of cyclic AMP action on iodine metabolism.
A specific and reproducible double antibody radioimmunoassay for the measurement of thyroglobulin (HTg) in human serum has been developed. Since antithyroglobulin autoantibodies combine with the [(131)I] HTg tracer, antibody-positive sera were rejected for measurement. Specificity is demonstrated in that thyroid analogous such as thyroxine (T(4)), triiodothyronine (T(2)) monoiodotyrosine (MIT) and diiodotyrosine (DIT) did not crossreact. Sera previously reacted with anti-HTg-Sepharose contained no immunoassayable HTg. Finally, sera obtained from patients after total thyroid ablation for thyroid carcinoma did not contain demonstrable HTg. The sensitivity of the assay is 1.6 ng/ml, and HTg was detectable in 74% of 95 normal subjects. The mean concentration was 5.1 ng/ml +/-0.49 SEM (range <1.6-20.7 ng/ml). Day to day variation in HTg levels is large in some euthyroid subjects and nearly absent in others. HTg was detectable in 90% of the sera obtained in 23 pregnant women at delivery in whom a mean concentration of 10.1 ng/ml +/-1.3 SEM was observed. The mean level for the corresponding newborn infants at birth was 29.3 ng/ml +/-4.7 SEM a value significantly higher than the mean maternal HTg concentration (P <0.01). A group of 17 thyrotoxic individuals all had elevated HTg levels; the mean for this group was 344.8 ng/ml +/-90.7 SEM. In the acute phase of subacute thyroiditis HTg was also elevated in all of 12 patients, and the mean for this group was 136.8 ng/ml +/-74.6 SEM.
A simple method is described for obtaining relatively pure fields of leucocytes from peripheral blood for electron microscopic study. The procedure does not require any unusual skill or equipment and exposes the cells to a minimum of handling before they are fixed. The basis of the method is that glutaraldehyde causes plasma to solidify into a firm substance that can be cut into small pieces and handled as blocks of tissue. By fixing centrifuged blood in situ, this phenomenon is used to circumvent the difficulty of recovering the usual thin buffy coat with a pipette without also picking up a large number of erythrocytes. The use of glutaraldehyde offers the additional advantages of good preservation and the opportunity to store the specimen in fixative for an indefinite period of time until a convenient time for further processing.
In order to define the roles of intracellular Ca++ and cAMP in the mechanisms governing GH release, we have carried out perifusion studies using a purified preparation of acutely dispersed somatotrophs obtained from rat adenohypophyses. Two groups of secretagogues were used: those that act by increasing intracellular cAMP [(Bu)2cAMP], the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, and prostaglandin E2; and those thought to act primarily by increasing Ca++ influx into the cells (high extracellular K+ and the Ca++ ionophore A23187). The release of GH induced by the three secretagogues that increase cAMP levels is instantaneous and maintained during perifusion, whereas the release induced by high K+ and A23187, unlike the cAMP-induced release, reaches a peak within 2 min and then falls rapidly back to baseline levels, even though the secretagogues are still present. After GH release has returned to baseline levels but while high K+ is maintained, the somatotrophs respond briskly to (Bu)2cAMP, 3...
The action of thyrotropin (TSH) on plasma membranes was studied to elucidate the mechanism of hormonal regulation of malignant versus normal human thyroid tissue. Thyroid plasma membranes of six specimens of papillary or follicular carcinoma and six of adenoma, as well as adjacent normal tissue obtained from these patients, were evaluated with respect to binding of 125I-labeled TSH and stimulation of adenylate cyclase. Scatchard analysis of TSH binding revealed the presence of two species of binding sites in normal thyroid of different affinities and capacities. In 11 of 12 tumors studied, the high-affinity binding site remained intact; however, the total number of low-affinity sites was markedly lower than normal tissue. Other parameters of binding were not altered in neoplastic thyroid. In each of these tissues, the hormone responsiveness and kinetics of adenylate cyclase activation were essentially identical to those observed in normal tissue, although basal activity was typically greater in the neoplasm. One carcinoma was totally deficient in both 125I-labeled TSH binding and TSH-stimulatable adenylate cyclase, although basal activity was detected. Furthermore, adenylate cyclase of this specimen was not activated by prostaglandin, in contrast to normal thyroid and other thyroid tumors. These results suggest that: (a) clinical behavior of thyroid carcinomas may not be reflected by TSH receptor-adenylate cyclase function; (b) lack of clinical response as manifest by tumor regression cannot be ascribed to the absence of functional TSH receptors or adenylate cyclase; and (c) decreased low-affinity binding present in tumors is not correlated with altered hormone responsiveness of adenylate cyclase but may reflect more general cancer-induced changes in membrane structure or composition.
A new sensitive in vitro assay for human thyroid stimulator (HTS) was developed using human thyroid adenoma cells in monolayer culture. After being cultured for 2 days, the cells were incubated in 0.3 ml Hank's solution without 0.8% NaCl (medium 1) and with thyroid stimulator (bovine TSH or 3 mg patient serum immunoglobulin G) at 37 C for 2 h. The cAMP generated in the cells and the medium during the incubation was measured by RIA. The assay was sensitive enough to elicit a 1.7- to 7.9-fold increase in cAMP at a TSH concentration of 10 microU/ml. HTS was detected in 33 (82.5%) of the 40 patients with untreated graves' disease using this assay system. In Hank's solution (medium 2), however, HTS was detected in only 5 (23.8%) of the 21 patients with untreated GRaves' disease. cAMP increment upon stimulation by either TSH or HTS in medium 1 was larger than that in medium 2, and the difference in the response to HTS using the two media was much greater than that in the response to TSH. Therefore, all HTS-immunoglobulin G studies showed higher activity using medium 1 than using medium 2 when expressed as bovine TSH equivalent. Analysis by the Lineweaver-Burk plot of dose-response curves of the effect of TSH and HTS stimulation on cAMP increment showed an increase in the Km upon the addition of NaCl to the medium. A similar inhibitory effect of NaCl (150 mM) was also observed in the assay system of human thyroid adenylate cyclase stimulator using crude plasma membrane fractions. In summary: 1) an assay for HTS measuring cAMP production in cultured thyroid adenoma cells was developed and the assay using low NaCL medium was found to be the most sensitive, and 2) the inhibitory effect of NaCl on the response to HTS was much greater than that on the response to TSH. These data suggest different behaviors of these two stimulators at their receptor sites.
Experiments were performed to determine whether the TSH receptor-adenylate cyclase (AC) system in benign and malignant thyroid neoplasms differs from the TSH receptor-AC system in normal thyroid tissue removed from the same patients. TSH binding and AC assays were performed using the same in vitro conditions. TSH binding was rapid, reversible, saturable, and hormone specific in particulate fractions from both normal and neoplastic thyroid tissue. A positive correlation existed between the equilibrium constants for [125I]bovine ([125I]bTSH) TSH binding and the concentration of TSH required to activate AC, suggesting that binding sites were coupled to AC in neoplastic thyroid tissue. Mean values for dissociation constants (Kd1 and Kd2), capacity (site 2), as determined by Scatchard analysis, and nonspecific binding (NSB) for the TSH receptors were lower in neoplastic thyroid. Some normal thyroid tissue appeared to lack a high affinity site, and some tumors lacked a low affinity binding site. Hormone specificities (bTSH, human (h) TSH, hLH, hFSH, hGH, hACTH, and glucagon) in normal thyroid and neoplastic tissue were virtually identical. hFSH, hACTH, hGH, and glucagon failed to inhibit [125I]bTSH binding or stimulate AC in either normal or neoplastic thyroid tissue, whereas hLH inhibited [125I]bTSH binding and stimulated AC, but required 10- to 100-fold higher concentrations than hTSH or bTSH. The specific binding and NSB of [125I]bTSH in both normal and neoplastic thyroid tissue was highest at pH 7.0 and lowest at pH 8.3. In contrast to bTSH binding, TSH stimulation of AC was lowest at pH 7.0 in both normal and neoplastic tissues and highest at pH levels of 7.5-8.0. TSH binding and TSH stimulation of AC activity were highest in the absence of NaCl and decreased progressively as the salt concentration was increased in both normal and neoplastic thyroid tissues. Increasing the sucrose concentration and, thus, the osmolarity of the system had a minimal effect on the binding of [125I]bTSH. Preincubation with ammonium sulfate did not significantly influence binding. Basal AC activity and the AC response to TSH were greater in neoplastic thyroid than in normal tissues. These studies demonstrate that changes in salt concentration and pH affect the TSH receptor-cyclase system in a comparable fashion in normal and neoplastic thyroid tissues. The discriminatory properties of the TSH receptor are also maintained in thyroid neoplasms. Thyroid tumors, however, have a higher affinity for TSH and display a greater AC response to TSH than normal thyroid tissue.
Four human bladder carcinoma cell lines have been characterized by G-banding, and the chromosomal patterns correlated to growth in agar and tumorigenicity in nude mice. Each cell line was shown to be chromosomally unique and although numerical and structural anomalies were present, none were common to all four cell lines. However, one or more copies of a structurally altered chromosome 8 were present in all four cell lines and may be associated with tumorigenicity in nude mice. A combination of three marker chromosomes was found in the more anaplastic cell lines, but not in the two well-differentiated tumour cell lines. Growth in agar may be associated with the presence of the three marker chromosomes but was not correlated with tumorigenicity in nude mice.
Bone marrow (about 0.5 ml) from au erythropoietic region is freed of blood clots by washing 1-3 min in 1 μg/ml colchicine solution (2-3 ml) and then soaking 1-2 hr at 20-30° C in a second change. For mammalian or avian marrows, the colchicine is made up in phosphate-buffered (pH 7) physiological NaCl solution; for amphibian, Ringer's A solution. Next the specimens are soaked about 20 min in a hypotonic solution as follows: for mammalian, 1% Na-citrate; for avian, a 1:4 dilution of the buffered NaCl solution by distilled water; and for amphibian, Ringer's A-distilled water, 1:1. Then they are heated in a mixture of 2% orcein in 45% acetic acid and 1 N HCl, 9:1. Immediately after heating, squash preparations are made with 2% acetic-orcein in the usual manner. An alternative method is to dissociate the marrow cells by agitating after colchicine treatment. Then, recovering the cells between changes by low-speed centrifugation, to carry out the hypotonic treatment and subsequent fixation in Carnoy's solution I (alcohol acetic, 3:1) before drying the cells onto slides from the fixative. After thorough drying the slides may be stained 10-20 min in acetic orcein, or by other suitable technics.
Strategy of thyroid cell culture in defined media and the isolation of the ovnis and porthos B. Estour et al. : A human follicular tumor cell line cell strains Endocrine re-search and therapy
- G Fayet
- Hovsepian
Fayet G, Hovsepian S (1985) Strategy of thyroid cell culture in defined media and the isolation of the ovnis and porthos B. Estour et al. : A human follicular tumor cell line cell strains. In: Eggo MC, Burrow GN (eds) Endocrine re-search and therapy, Raven Press, New York, pp 211
Chromosome preparations of bone marrow cells with prior in vitro culture or in vivo colchicine administration Elevated serum thyroglobulin: A marker of metastases in differentiated thyroid carcinoma
- Tj Tjio
- Whang
Tjio TJ, Whang T (1962) Chromosome preparations of bone marrow cells with prior in vitro culture or in vivo colchicine administration. Stain Technol 37:17-20 Van Herle AJ, Uller RP (1975) Elevated serum thyroglobulin: A marker of metastases in differentiated thyroid carcinoma. J Clin Invest 56:272-277
Investigations on the PGM Poly-morphism (phosphoglucomutase-EC
- P Kuhnl
- Spielmann
Kuhnl P, Spielmann W (1978) Investigations on the PGM Poly-morphism (phosphoglucomutase-EC
Handbook of enzymes electro-phoresis in human genetics Chromosome pattern, growth in agar and tumorigenicity in nude mice of four human bladder carcinoma cell lines
- Harris H Hopkinson
- Da
Harris H, Hopkinson DA (1976) Handbook of enzymes electro-phoresis in human genetics. American Elsevier Publ Co, Inc, New York Hastings RJ, Franks LM (1981) Chromosome pattern, growth in agar and tumorigenicity in nude mice of four human bladder carcinoma cell lines. Int J Cancer 27:15-21
Human breast carcinoma cells in continuous culture. A review
- L W Engel
- N A Young
- LW Engel
Handbook of enzymes electrophoresis in human genetics
- H Harris
- Da Hopkinson
Establishment and identification of small lung cancer cell lines having classic and variant features
- D N Carney
- E F Gazdar
- G Bepler
- J G Guccion
- P J Marangos
- T W Moody
- M H Zweig
- J D Minna
- DN Carney