ABI3 ectopic expression reduces in vitro and in vivo cell growth properties while inducing senescence

<p>Abstract</p> <p>Background</p> <p>Mounting evidence has indicated that <it>ABI3 </it>(ABI family member 3) function as a tumor suppressor gene, although the molecular mechanism by which ABI3 acts remains largely unknown.</p> <p>Methods</p> <p>The present study investigated <it>ABI3 </it>expression in a large panel of benign and malignant thyroid tumors and explored a correlation between the expression of ABI3 and its potential partner ABI3-binding protein (ABI3BP). We next explored the biological effects of <it>ABI3 </it>ectopic expression in thyroid and colon carcinoma cell lines, in which its expression was reduced or absent.</p> <p>Results</p> <p>We not only observed that <it>ABI3 </it>expression is reduced or lost in most carcinomas but also that there is a positive correlation between <it>ABI3 </it>and <it>ABI3BP </it>expression. Ectopic expression of <it>ABI3 </it>was sufficient to lead to a lower transforming activity, reduced tumor <it>in vitro </it>growth properties, suppressed <it>in vitro </it>anchorage-independent growth and <it>in vivo </it>tumor formation while, cellular senescence increased. These responses were accompanied by the up-regulation of the cell cycle inhibitor <it>p21 </it>^WAF1and reduced ERK phosphorylation and <it>E2F1 </it>expression.</p> <p>Conclusions</p> <p>Our result links <it>ABI3 </it>to the pathogenesis and progression of some cancers and suggests that ABI3 or its pathway might have interest as therapeutic target. These results also suggest that the pathways through which <it>ABI3 </it>works should be further characterized.</p

Short tandem repeat profiling: part of an overall strategy for reducing the frequency of cell misidentification

The role of cell authentication in biomedical science has received considerable attention, especially within the past decade. This quality control attribute is now beginning to be given the emphasis it deserves by granting agencies and by scientific journals. Short tandem repeat (STR) profiling, one of a few DNA profiling technologies now available, is being proposed for routine identification (authentication) of human cell lines, stem cells, and tissues. The advantage of this technique over methods such as isoenzyme analysis, karyotyping, human leukocyte antigen typing, etc., is that STR profiling can establish identity to the individual level, provided that the appropriate number and types of loci are evaluated. To best employ this technology, a standardized protocol and a data-driven, quality-controlled, and publically searchable database will be necessary. This public STR database (currently under development) will enable investigators to rapidly authenticate human-based cultures to the individual from whom the cells were sourced. Use of similar approaches for non-human animal cells will require developing other suitable loci sets. While implementing STR analysis on a more routine basis should significantly reduce the frequency of cell misidentification, additional technologies may be needed as part of an overall authentication paradigm. For instance, isoenzyme analysis, PCR-based DNA amplification, and sequence-based barcoding methods enable rapid confirmation of a cell line’s species of origin while screening against cross-contaminations, especially when the cells present are not recognized by the species-specific STR method. Karyotyping may also be needed as a supporting tool during establishment of an STR database. Finally, good cell culture practices must always remain a major component of any effort to reduce the frequency of cell misidentification

Conventional and molecular cytogenetics of human non-medullary thyroid carcinoma: characterization of eight cell line models and review of the literature on clinical samples

<p>Abstract</p> <p>Background</p> <p>Cell lines are often poorly characterized from a genetic point of view, reducing their usefulness as tumor models. Our purpose was to assess the genetic background of eight commonly used human thyroid carcinoma models and to compare the findings with those reported for primary tumors of the gland.</p> <p>Methods</p> <p>We used chromosome banding analysis and comparative genomic hybridization to profile eight non-medullary thyroid carcinoma cell lines of papillary (TPC-1, FB2, K1 and B-CPAP), follicular (XTC-1) or anaplastic origin (8505C, C643 and HTH74). To assess the representativeness of the findings, we additionally performed a thorough review of cytogenetic (n = 125) and DNA copy number information (n = 270) available in the literature on clinical samples of thyroid carcinoma.</p> <p>Results</p> <p>The detailed characterization of chromosomal markers specific for each cell line revealed two cases of mistaken identities: FB2 was shown to derive from TPC-1 cells, whereas K1 cells have their origin in cell line GLAG-66. All cellular models displayed genomic aberrations of varying complexity, and recurrent gains at 5p, 5q, 8q, and 20q (6/7 cell lines) and losses at 8p, 13q, 18q, and Xp (4/7 cell lines) were seen. Importantly, the genomic profiles were compatible with those of the respective primary tumors, as seen in the meta-analysis of the existing literature data.</p> <p>Conclusion</p> <p>We provide the genomic background of seven independent thyroid carcinoma models representative of the clinical tumors of the corresponding histotypes, and highlight regions of recurrent aberrations that may guide future studies aimed at identifying target genes. Our findings further support the importance of routinely performing cytogenetic studies on cell lines, to detect cross-contamination mishaps such as those identified here.</p

Role of oxidative stress and intracellular glutathione in the sensitivity to apoptosis induced by proteasome inhibitor in thyroid cancer cells

<p>Abstract</p> <p>Background</p> <p>The proteasome inhibitor bortezomib has shown impressive clinical activity alone and in combination with conventional and other novel agents for the treatment of multiple myeloma (MM) and some solid cancers. Although bortezomib is known to be a selective proteasome inhibitor, the downstream mechanisms of cytotoxicity and drug resistance are poorly understood.</p> <p>Methods</p> <p>Proteasome activity, intracellular glutathione (GSH) and ROS levels, as well as activities of GSH synthesis enzymes were measured using spectrophotometric methods. Cell death was analyzed using flow cytometry and caspase activity assay. The expression level of GSH synthesis enzymes were measured using real-time RT-PCR.</p> <p>Results</p> <p>At concentrations that effectively inhibited proteasome activity, bortezomib induced apoptosis in FRO cells, but not in ARO cells. Bortezomib elevated the amount of glutathione (GSH) and the treatment with bortezomib increased the level of mRNA for GCL, a rate-limiting enzyme in glutathione synthesis. Furthermore, depletion of GSH increases apoptosis induced by bortezomib, in contrast, repletion of GSH decreases bortezomib-mediated cell death.</p> <p>Conclusion</p> <p>GSH protects cells from proteasome inhibition-induced oxidative stress and glutathione-dependent redox system might play an important role in the sensitivity to proteasome inhibition-induced apoptosis.</p

Dedifferentiation of Human Primary Thyrocytes into Multilineage Progenitor Cells without Gene Introduction

While identification and isolation of adult stem cells have potentially important implications, recent reports regarding dedifferentiation/reprogramming from differentiated cells have provided another clue to gain insight into source of tissue stem/progenitor cells. In this study, we developed a novel culture system to obtain dedifferentiated progenitor cells from normal human thyroid tissues. After enzymatic digestion, primary thyrocytes, expressing thyroglobulin, vimentin and cytokeratin-18, were cultured in a serum-free medium called SAGM. Although the vast majority of cells died, a small proportion (∼0.5%) survived and proliferated. During initial cell expansion, thyroglobulin/cytokeratin-18 expression was gradually declined in the proliferating cells. Moreover, sorted cells expressing thyroid peroxidase gave rise to proliferating clones in SAGM. These data suggest that those cells are derived from thyroid follicular cells or at least thyroid-committed cells. The SAGM-grown cells did not express any thyroid-specific genes. However, after four-week incubation with FBS and TSH, cytokeratin-18, thyroglobulin, TSH receptor, PAX8 and TTF1 expressions re-emerged. Moreover, surprisingly, the cells were capable of differentiating into neuronal or adipogenic lineage depending on differentiating conditions. In summary, we have developed a novel system to generate multilineage progenitor cells from normal human thyroid tissues. This seems to be achieved by dedifferentiation of thyroid follicular cells. The presently described culture system may be useful for regenerative medicine, but the primary importance will be as a tool to elucidate the mechanisms of thyroid diseases

Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues

Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues

Growth inhibition of thyroid follicular cell-derived cancers by the opioid growth factor (OGF) - opioid growth factor receptor (OGFr) axis

<p>Abstract</p> <p>Background</p> <p>Carcinoma of the thyroid gland is an uncommon cancer, but the most frequent malignancy of the endocrine system. Most thyroid cancers are derived from the follicular cell. Follicular carcinoma (FTC) is considered more malignant than papillary thyroid carcinoma (PTC), and anaplastic thyroid cancer (ATC) is one of the most lethal human cancers. Opioid Growth Factor (OGF; chemical term - [Met⁵]-enkephalin) and its receptor, OGFr, form an inhibitory axis regulating cell proliferation. Both the peptide and receptor have been detected in a wide variety of cancers, and OGF is currently used clinically as a biotherapy for some non-thyroid neoplasias. This study addressed the question of whether the OGF-OGFr axis is present and functional in human thyroid follicular cell - derived cancer.</p> <p>Methods</p> <p>Utilizing human ATC (KAT-18), PTC (KTC-1), and FTC (WRO 82-1) cell lines, immunohistochemistry was employed to ascertain the presence and location of OGF and OGFr. The growth characteristics in the presence of OGF or the opioid antagonist naltrexone (NTX), and the specificity of opioid peptides for proliferation of ATC, were established in KAT-18 cells. Dependence on peptide and receptor were investigated using neutralization studies with antibodies and siRNA experiments, respectively. The mechanism of peptide action on DNA synthesis and cell survival was ascertained. The ubiquity of the OGF-OGFr axis in thyroid follicular cell-derived cancer was assessed in KTC-1 (PTC) and WRO 82-1 (FTC) tumor cells.</p> <p>Results</p> <p>OGF and OGFr were present in KAT-18 cells. Concentrations of 10^-6M OGF inhibited cell replication up to 30%, whereas NTX increased cell growth up to 35% relative to cultures treated with sterile water. OGF treatment reduced cell number by as much as 38% in KAT-18 ATC in a dose-dependent and receptor-mediated manner. OGF antibodies neutralized the inhibitory effects of OGF, and siRNA knockdown of OGFr negated growth inhibition by OGF. Cell survival was not altered by OGF, but DNA synthesis as recorded by BrdU incorporation was depressed by 28% in OGF-treated cultures compared to those exposed to sterile water. The OGF-OGFr axis was detected and functional in PTC (KTC-1) and FTC (WRO 82-1) cell lines.</p> <p>Conclusion</p> <p>These data suggest that OGF and OGFr are present in follicular-derived thyroid cancers, and that OGF serves in a tonically active inhibitory manner to maintain homeostasis of cell proliferation. These results may provide a biotherapeutic strategy in the treatment of these cancers.</p

Thyroid Cancer Imaging In Vivo by Targeting the Anti-Apoptotic Molecule Galectin-3

Background The prevalence of thyroid nodules increases with age, average 4-7% for the U.S.A. adult population, but it is much higher (19-67%) when sub-clinical nodules are considered. About 90% of these lesions are benign and a reliable approach to their preoperative characterization is necessary. Unfortunately conventional thyroid scintigraphy does not allow the distinction among benign and malignant thyroid proliferations but it provides only functional information (cold or hot nodules). The expression of the anti-apoptotic molecule galectin-3 is restricted to cancer cells and this feature has potential diagnostic and therapeutic implications. We show here the possibility to obtain thyroid cancer imaging in vivo by targeting galectin-3. Methods The galectin-3 based thyroid immuno-scintigraphy uses as radiotracer a specific 99mTc-radiolabeled mAb. A position-sensitive high-resolution mini-gamma camera was used as imaging capture device. Human galectin-3 positive thyroid cancer xenografts (ARO) and galectin-3 knockout tumors were used as targets in different experiments in vivo. 38 mice with tumor mass of about 1 gm were injected in the tail vein with 100 ?Ci of 99mTc-labeled mAb to galectin-3 (30 ?g protein/in 100 ?l saline solution). Tumor images were acquired at 1 hr, 3 hrs, 6 hrs, 9 hrs and 24 hrs post injection by using the mini-gamma camera. Findings Results from different consecutive experiments show an optimal visualization of thyroid cancer xenografts between 6 and 9 hours from injection of the radiotracer. Galectin-3 negative tumors were not detected at all. At 6 hrs post-injection galectin-3 expressing tumors were correctly visualized, while the whole-body activity had essentially cleared. Conclusions These results demonstrate the possibility to distinguish preoperatively benign from malignant thyroid nodules by using a specific galectin-3 radio-immunotargeting. In vivo imaging of thyroid cancer may allow a better selection of patients referred to surgery. The possibility to apply this method for imaging and treatment of other galectin-3 expressing tumors is also discussed

CD133+ Anaplastic Thyroid Cancer Cells Initiate Tumors in Immunodeficient Mice and Are Regulated by Thyrotropin

Anaplastic thyroid cancer (ATC) is one of the most lethal human malignancies. Its rapid onset and resistance to conventional therapeutics contribute to a mean survival of six months after diagnosis and make the identification of thyroid-cancer-initiating cells increasingly important.In prior studies of ATC cell lines, CD133(+) cells exhibited stem-cell-like features such as high proliferation, self-renewal and colony-forming ability in vitro. Here we show that transplantation of CD133(+) cells, but not CD133(-) cells, into immunodeficient NOD/SCID mice is sufficient to induce growth of tumors in vivo. We also describe how the proportion of ATC cells that are CD133(+) increases dramatically over three months of culture, from 7% to more than 80% of the total. This CD133(+) cell pool can be further separated by flow cytometry into two distinct populations: CD133(+/high) and CD133(+/low). Although both subsets are capable of long-term tumorigenesis, the rapidly proliferating CD133(+/high) cells are by far the most efficient. They also express high levels of the stem cell antigen Oct4 and the receptor for thyroid stimulating hormone, TSHR. Treating ATC cells with TSH causes a three-fold increase in the numbers of CD133(+) cells and elicits a dose-dependent up-regulation of the expression of TSHR and Oct4 in these cells. More importantly, immunohistochemical analysis of tissue specimens from ATC patients indicates that CD133 is highly expressed on tumor cells but not on neighboring normal thyroid cells.To our knowledge, this is the first report indicating that CD133(+) ATC cells are solely responsible for tumor growth in immunodeficient mice. Our data also give a unique insight into the regulation of CD133 by TSH. These highly tumorigenic CD133(+) cells and the activated TSH signaling pathway may be useful targets for future ATC therapies

Proliferation and survival molecules implicated in the inhibition of BRAF pathway in thyroid cancer cells harbouring different genetic mutations

<p>Abstract</p> <p>Background</p> <p>Thyroid carcinomas show a high prevalence of mutations in the oncogene BRAF which are inversely associated with RAS or RET/PTC oncogenic activation. The possibility of using inhibitors on the BRAF pathway as became an interesting therapeutic approach. In thyroid cancer cells the target molecules, implicated on the cellular effects, mediated by inhibition of BRAF are not well established. In order to fill this lack of knowledge we studied the proliferation and survival pathways and associated molecules induced by BRAF inhibition in thyroid carcinoma cell lines harbouring distinct genetic backgrounds.</p> <p>Methods</p> <p>Suppression of BRAF pathway in thyroid cancer cell lines (8505C, TPC1 and C643) was achieved using RNA interference (RNAi) for BRAF and the kinase inhibitor, sorafenib. Proliferation analysis was performed by BrdU incorporation and apoptosis was accessed by TUNEL assay. Levels of protein expression were analysed by western-blot.</p> <p>Results</p> <p>Both BRAF RNAi and sorafenib inhibited proliferation in all the cell lines independently of the genetic background, mostly in cells with BRAF^V600Emutation. In BRAF^V600Emutated cells inhibition of BRAF pathway lead to a decrease in ERK1/2 phosphorylation and cyclin D1 levels and an increase in p27^Kip1. Specific inhibition of BRAF by RNAi in cells with BRAF^V600Emutation had no effect on apoptosis. In the case of sorafenib treatment, cells harbouring BRAF^V600Emutation showed increase levels of apoptosis due to a balance of the anti-apoptotic proteins Mcl-1 and Bcl-2.</p> <p>Conclusion</p> <p>Our results in thyroid cancer cells, namely those harbouring BRAF^V600Emutation showed that BRAF signalling pathway provides important proliferation signals. We have shown that in thyroid cancer cells sorafenib induces apoptosis by affecting Mcl-1 and Bcl-2 in BRAF^V600Emutated cells which was independent of BRAF. These results suggest that sorafenib may prove useful in the treatment of thyroid carcinomas, particularly those refractory to conventional treatment and harbouring BRAF mutations.</p