No association was found between drug resistance and the expression of EWS/ETS regulated genes in the EFT cell lines. No consistent association was observed between drug sensitivity and p53 functionality or between drug sensitivity and p16/14 functionality across the cell lines. Exposure to chemotherapy prior to cell line initiation correlated with drug resistance of EFT cell lines in 5/8 tested agents at clinically achievable concentrations (CAC) or the lower tested concentration (LTC): (cyclophosphamide (as 4-HC) and doxorubicin at CAC, etoposide, irinotecan (as SN-38) and melphalan at LTC; P<0.1 for one agent, and P<0.05 for four agents. This panel of well-characterized drug-sensitive and drug-resistant cell lines will facilitate in vitro preclinical testing of new agents for EFT.Despite intensive treatment with chemotherapy, radiotherapy and surgery, over 70% of patients with metastatic Ewing's Sarcoma Family of Tumors (EFT) will die of their disease. We hypothesize that properly characterized laboratory models reflecting the drug resistance of clinical tumors will facilitate the application of new therapeutic agents to EFT. To determine resistance patterns, we studied newly established EFT cell lines derived from different points in therapy: two established at diagnosis , two after chemotherapy and progressive disease , and two at relapse after myeloablative therapy and autologous bone marrow transplantation (post-ABMT) . The new lines were compared to widely studied EFT lines TC-71, TC-32, SK-N-MC, and A-673. These lines were extensively characterized with regard to identity (short tandem repeat (STR) analysis), p53, p16/14 status, and EWS/ETS breakpoint and target gene expression profile. The DIMSCAN cytotoxicity assay was used to assess  Ewing's Family of Tumors (EFT) (Ewing's sarcoma (ES) and peripheral primitive neuroectodermal tumors (PNET)) are aggressive malignancies occurring in the childhood through adolescent/young adult years TP53 mutations and alterations in p16/p14 function have been shown to influence therapeutic responsiveness in a variety of tumors and may be another cause of innate chemotherapy resistance. While most primary EFT have wild-type TP53, some have noted a higher incidence of treatment resistance in p53-non-functional EFT One dichotomy in EFT is between the dramatic chemoresponsiveness of primary tumors and the chemoresistance observed in most patients with metastases at diagnosis and in patients with localized disease which recurs. Though the mechanisms responsible for chemotherapy resistance in EFT have not been systematically studied, some disease-specific hypotheses may be entertained. A distinguishing feature of EFT is the universal presence of EWS/FLI1 (and related EWS/ETS) fusion transcription factors To address these potential mechanisms of resistance, we established a panel of cell lines from patients with EFT at various points during the course of their disease. Cell line genomic identity was validated and each was characterized with regard to their EWS/ETS fusion gene product, p53 and p16/14 status, and expression of EWS/ETS target genes of known or suspected biologic significance. We then characterized these lines with regard to their sensitivity to a number of common therapeutic agents employed in the treatment of clinical EFT.This was determined by nested PCR amplification of cell line derived cDNA. Primers are listed in Determination of p53 functionality was made by treating cell lines with etoposide (5 µg/ml) for 16 hours after which immunoblot and qPCR for p53 and p21 was conducted as previously described Real time quantitative PCR was performed as previously described Melphalan (L-PAM), carboplatin (CBDCA), topotecan (TPT), etoposide (ETOP), vincristine (VINC), and doxorubicin (DOX) were obtained from the Drug Synthesis & Chemistry Branch, Developmental Therapeutics Program, National Cancer Institute . The active metabolite of irinotecan (SN-38) was from ABATRA Technology Co, Ltd,  and the active metabolite of cyclophosphamide (4-hydroperoxycyclophosphamide = 4-HC) was synthesized at Duke University, North Carolina and was a kind gift of Susan M. Ludeman and O. Michael Colvin. Fluorescein diacetate (FDA) was from the Eastman Kodak Company . Iscove's modified Dulbecco's medium (IMDM) was from BioWhittaker . ITS™ Premix culture supplement  was from Collaborative Biomedical Products  and Eosin Y was from Sigma Chemical Co .in vivo exposure to drugs in patients, the sites from which the specimens were obtained, the stage of the disease, the patient's age at diagnosis, and the doubling time (DT). For reference, A673 2 atmosphere. Cell lines were cultured without antibiotics so that Mycoplasma infection would not be masked and were tested and shown to be Mycoplasma negative. All cell lines used for this study except for A673 (which was not tested) were tested for viral pathogens by Research Animal Diagnostic Laboratory at the University of Missouri  and were negative for the following viruses: HIV1, HIV2, hepatitis A, hepatitis B, hepatitis C, Hantaan, Seoul, Sin Nombre, and lymphocytic choriomenengitis. Microscopic images of live EFT cell lines were captured using the Olympus IX71 Inverted Research Microscope, and visualized with QCapture Pro software from Qimaging The human EFT cell lines used in this study are listed in The cell lines SK-N-MC and A-673 were obtained from the American Type Culture Collection. All other cell lines were established in the laboratories of the authors (CPR or TJT) under protocols approved by the appropriate institutional Committee for Protection of Human Subjects (IRB). The COG-E-352 sample was obtained with written family consent from a post-mortem sample and was thus not established under an IRB-approved protocol as it was not human subject's research.2 with room air), various concentrations of chemotherapeutic drugs in 100 µl of complete medium were added to each well. The final drug concentrations were selected to include clinically achievable concentrations for each drug: 4-HC: 0–8 µg/ml, L-PAM: 0–10 µg/ml, CBDCA: 0–10 µg/ml, ETOP: 0–10 µg/ml, TPT: 0–100 ng/ml, SN-38: 0–24 ng/ml, VINC: 0–200 ng/ml, and DOX: 0–30 ng/ml The cytotoxicity of 4-HC, L-PAM, CBDCA, TPT, ETOP, SN-38, VINC, and DOX was determined in 96-well plates using the semi-automated Digital Image Microscopy (DIMSCAN) system. DIMSCAN is a digital imaging system that measures the total fluorescence per well using digital thresholding with eosin Y quenching to eliminate background fluorescence and has a dynamic range of greater than 4 logs of cell kill 3 flasks with a hemocytometer (using trypan blue) every 2–3 days and then calculated with Microsoft Excel 2000 software, using the Y = Xo*exp(Bc) formula, where C is the time after seeding the cells, Y is the number cells after a period of time C, and X is the number of cells seeded at the time zero.Doubling time (DT)  was dete99)  and clinically achievable concentration (CAC) for each of the eight tested agents . The ass99)  was calcwww.COGcell.org/clid.php). The kit provides primers for the following microsatelite loci: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA, and includes the non-microsatellite, gender-specific amelogenin locus Avant Genetic Analyzer  using POP-4 polymer and default conditions. Results were then analyzed using Genemapper ID version 3.2 software . Results were visualized on electropherograms. CHLA-9 and CHLA-10 showed matching fingerprints and were considered validated since they were established from samples from the same patient acquired at separate times. CHLA-258 and COG-E-352 STR profiles matched corresponding patient material and the remaining cell line STR data showed unique signatures relative to all other cell lines in the COG cell line repository (http://strdb.cogcell.com) (Short-tandem-repeat (STR) genotyping was performed to confirm the molecular identity of each cell line  by polymell.com) .www.COGcell.org).This research and consent process was reviewed and approved by the Human Subjects Protection Committees of Children's Hospital Los Angeles and Texas Tech University Health Sciences Center. Except for SK-N-MC and A-673 (obtained from the American Type Culture Collection) written informed consent was documented in the patient medical records. Cell lines TC-71 and TC-32 were established at the National Cancer Institute from biopsies taken to confirm the diagnosis. Both lines were established prior to HIPAA legislation and the consent utilized at the time allowed for unrestricted use of the lines for research and diagnosis. The cell lines CHLA-9, CHLA-10, CHLA-25, CHLA-32, CHLA-258 were established from samples obtained via written informed consent from parents and/or patients (as appropriate for age) and COG-E-352 was from a post-mortem blood sample obtained with written informed consent from the family of the patient in the laboratory of CP Reynolds, which is also the COG Cell Culture & Xenograft Repository . Of note, the CHLA-9 cell line has functional p53, while the CHLA-10 line, established from a persistent focus of viable tumor after neo-adjuvant chemotherapy, is p53 non-functional. The p16/14 status of the cell lines  seemed to be more effective in p16/p14 null cell lines compared to wild type lines . DoxorubP<0.1 for one agent, and P<0.05 for four agents)  with a total of 5/8 tested agents at CAC or LTC  .All drugs in this study except ETOP and VINC showed significantly more cell kill in average at the CAC than at the LTC. The high resistance to ETOP , VINC  in the cell lines exposed to these drugs in patients suggest that etoposide and vincristine may have limited activity in recurrent EFT. There was no apparent correlation observed in the cytotoxicity of the drugs and expression of EWS/FLI1 regulated genes.Over the past two decades, chemotherapy intensity of EFT treatment protocols has steadily increased. The standard chemotherapy for localized EFT includes vincristine, ifosfamide, doxorubicin and etoposide (VIDE) in Europe or vincristine, doxorubicin, cyclophosphamide, ifosfamide and etoposide (VDC-IE) in North America www.PPTPinvitro.org; http://pptp.nchresearch.org/). The cell line panel characterized in this paper can be readily obtained from the Children's Oncology Group (COG) Cell Line and Xenograft Repository (www.COGcell.org). Such a public repository and its associated STR database facilitates biological and preclinical therapeutic studies by helping to avoid discrepancies that may result from differences in cell lines acquired as a result of prolonged passage or contamination. The comprehensive data presented in this paper on EWS/ETS breakpoint, on p53 and p16/14 status, and on expression of EWS/ETS target loci improves the utility of these readily available cell lines for EFT translational studies.The findings reported in this paper provide an important resource in this regard. A subset of the cell lines are in use by the National Cancer Institute Pediatric Preclinical Testing Program Importantly, we have also characterized this cell line panel with regard to baseline chemosensitivity. These data reveal several important aspects of common EFT cell lines. First, many of the commonly employed EFT cell lines are relatively drug sensitive. While most EFT are drug sensitive at diagnosis, resistant disease emerges in 30% of patients with localized disease and in 70–80% of patients with metastases. As chemotherapy-resistant EFT patients are rarely salvaged with any known therapy, the key to improving outcome is to understand the mechanisms present in resistant cells that lead to treatment failure and to employ models of such resistant cells in laboratory studies seeking novel therapies. To simply study the same long-established drug-sensitive lines does not adequately address the problem of resistant disease. Therefore, the newer, multi-drug resistant lines in this panel will provide an important new resource for preclinical therapeutic studies in EFT.We have also observed that there appears to be no obvious correlation between the EWS/FLI1 or EWS/ETS fusion genes, their gene target expression, and drug resistance. Certainly, one cannot conclude from this limited study that an EWS/FLI1 target gene set predicting resistance does not exist. However, the identification of such a predictive gene expression pattern will depend on more extensive gene expression profiling studies and will likely require an even larger panel of well-characterized cell lines. Furthermore, since the majority of EFT at presentation are drug-sensitive, profiling strategies of primary tumors may fail to identify the roots of chemoresistance that are present in a small subpopulation of the tumor.TP53 wild-type, loss of function for p53 does not explain the majority of treatment failures, unless p53 loss-of-function emerges during treatment as was the case for the CHLA-9/10 pair.Our data did not demonstrate a consistent pattern in the association between drug sensitivity and p53 or p16/14 status, which is not consistent with the previously cited observations based on clinical outcomes Finally, we observe that a history of chemotherapy exposure prior to cell line establishment correlated with drug resistance. This is consistent with clinical observations and has also been observed in neuroblastoma cell lines www.COGcell.org. They should prove helpful in conducting preclinical studies to support the design of novel clinical strategies to overcome drug resistance developed during the course of treatment by identifying agents active against drug-resistant tumor cells.The cell lines described here provide a well characterized panel of cell lines for preclinical studies aimed at identifying new active agents in EFT that can be obtained at Figure S1Fractional cytotoxicity of EFT cell lines treated with irinotecan as SN-38 (0–24 ng/ml) and vincristine (VINC: 0–200 ng/ml).(TIF)Click here for additional data file.Figure S2Fractional cytotoxicity of EFT cell lines treated with melphalan (L-PAM: 0–10 µg/ml) and etoposide ETOP (0–10 µg/ml).(TIF)Click here for additional data file.Figure S3Fractional cytotoxicity of EFT cell lines treated with topotecan (TPT: 0–100 ng/ml) and doxorubicin (DOX: 0–30 ng/ml).(TIF)Click here for additional data file.Table S1PCR Primers. A. Primers used for gene expression. B. Primers used for p53 testing.(DOC)Click here for additional data file.Table S2Short tandem repeat (STR) profiles of EFT cell lines.(DOC)Click here for additional data file.


This is a case of a 71-year-old woman, who presented with a large abdominal mass. Microscopic examination revealed metastatic renal cell carcinoma in an otherwise massively enlarged benign ovarian tumour of mixed mucinous cystadenoma and Brenner tumour. Clinicopathological and radiological assessment together with a thorough evaluation of gross and histological features, including the use of immunohistochemical stains, is essential to confirm the diagnosis and rule out the possibility of primary clear cell carcinoma of ovary, as this has important prognostic and therapeutic implications. Renal cell carcinoma (RCC) is the most common malignant tumour of the kidney. It constitutes 75% of renal neoplasms in adults and 3% of all adult malignancies . Approxi2.A 71-year-old woman presented clinically with abdominal distention and weight loss. On physical examination, the patient had a large abdominal mass. Blood biochemistry showed Urea 6.5 mmol/L, Creatinine 77 umol/L, Sodium: 136 mmol/L, Potassium 5.2 mmol/L, and GFR 79 mL/min/1.7 mμg/L . The abdominal/pelvic ultrasound revealed a right ovarian mass which was confirmed on CT scan.The tumour markers showed CA125 was 7.4 KU/L , CA19.9 = 19 KU/L , and CEA = 1.2 Laparotomy was performed, and the ovarian mass together with right fallopian tube were excised. The mass weighed 9.5 kg and measured 290 × 115 × 120 mm. Sectioning of the mass showed a multilocular cystic areas containing amber coloured, thick mucoid material alternating with multiple, solid firm nodules within the fibrous stroma of the walls. Histological examination showed a multilocular cystic ovarian tumour which consisted of mixed benign mucinous cystadenoma and Brenner tumour Figures  and 2. TThe grading and nature of this tumor was difficult to assess on conventional H&E sections. The differential diagnoses were clear cell carcinoma of the ovary, carcinoid, and Sertoli cell tumor.The clear cells were positive for immunostains CD10, Pan CK, Cam5.2, Vimentin, and EMA. The cells were negative for CK7, CK20, GP200, ER/PR, and S100. The histology and the immunostaining profile were suggestive of a metastatic renal cell carcinoma—clear cell type which is present in a benign mixed mucinous cystadenoma and Brenner tumour Figures . Further investigation of the patient revealed the presence of a tumour in the right kidney which was confirmed as renal cell carcinoma.Renal cell carcinoma (RCC) is the most common malignant tumor of the kidney. It constitutes 85% of renal neoplasms in adults and 3% of all adult malignancies . It usuaSometimes, mucinous cystadenomas are associated with other types of primary ovarian tumours such as Brenner tumour. The latter constitutes about only 2% of all ovarian tumours and has been reported in 20% of cases in association with other tumours such as benign serous, mucinous cystadenoma, or benign cystic teratoma , 8. Secondary tumours of the ovary constitute only around 6% of ovarian tumors. The usual cancers that metastasize to the ovaries are those arising from stomach, colon, breast, and lymphomas . Renal cell carcinomas rarely metastasize to the ovaries because these tumors usually occur during the 6th and 7th decades of life, where sclerosis of the ovarian vessels had usually occurred. Therefore, due to its rarity, clear cell carcinoma of the kidney when metastasizes to the ovaries is usually misdiagnosed as primary clear cell carcinoma of the ovary . Sometimes, it can be difficult to differentiate between renal cell carcinoma—clear cell type and clear cell carcinoma of the ovary. However, the presence of certain histological criteria can help pathologists to differentiate between the two tumors. Tubulocystic pattern with the presence of “Hobnail cells” and extracellular mucin are more in favour of clear cell carcinoma of the ovary, whereas the presence of solid sheets of clear cells and thin wall blood vessels surrounded by a pseudocapsule formed by the compressed parenchyma and fibrous tissue are all in favour of renal cell carcinoma—clear cell type . The differential diagnosis of primary clear cell tumour of the ovary includes primary clear cell carcinoma, Sertoli cell tumour, dysgerminoma, Lipoid cell tumour, Hilus cell and Leydig cell tumours . DespiteOur case highlights an unusual presentation of renal cell carcinoma that has metastasized to the ovary which was extensively involved by a mucinous cystadenoma associated with Brenner tumour.In conclusion, a thorough clinical history, with careful evaluation of gross and microscopic features of the clear cell ovarian tumour, and use of immunohistochemical stains are extremely important in the diagnosis of rare metastatic renal cell carcinoma to the ovary. It is also important to consider the possibility of renal cell carcinoma in the differential diagnosis of any suspicious abdominal mass, in view of the unpredictable biological behaviour of this tumour .


Myeloid derived suppressor cells (MDSC) are detected at elevated levels in the peripheral blood mononuclear cells (PBMC) of human renal cell carcinoma (RCC) patients and have been shown to mediate tumor growth by promoting angiogenesis and immune suppression . Granulocytic-MDSCs (G-MDSCs), the dominant population in RCC patients, have been shown to suppress T-cell function in RCC patients . However, less is known regarding the role of neutrophils in promoting tumor growth in RCC patients and their relationship to G-MDSC. In this study we compared the suppressive and angiogenic profile of neutrophils from RCC patients to those of normal donors and the impact that RCC conditioned media has in altering neutrophils normal function. We showed that IFNγ production of T-cells stimulated with anti-CD3/antiCD28 antibodies (n=4) was suppressed by neutrophils pretreated with SK-RC-26b tumor supernatant (TCM) when compared to neutrophils not exposed to TCM. Further experiments showed that neutrophils from RCC patients had increased expression compared to normal healthy donor neutrophils of genes Vegfa (15.28 fold increase), Vegfb (10.84 fold increase), Tgfb1 (3.31 fold increase), IL10 (23.45 fold increase), IDO1 (4.60 fold increase), Nox1 (9.33 fold increase), and Prokr2 (4.14 fold increase) utilizing custom pro-angiogenic and immunosuppressive RT-PCR arrays (n=4). Furthermore, human angiogenesis Proteome Profile Arrays (n=2) were performed comparing RCC patient and normal healthy donor neutrophils and exhibited an increased level of Activin A (94.5% increase), Angiogenin (70% increase), Angiostatin/ plasminogen (56% increase), IGFBP-1 (188% increase), IGFBP-2 (263% increase),TGF-B1 (157.5% increase), Pentraxin 3 (140% increase), PD-ECGF (184.5% increase), Serpin E1 (196% increase), and Serpin F1 (114.5% increase) in RCC patient neutrophils. Normal neutrophils cultured with ACHN tumor supernatant for 18 hours (n=2) had up-regulation of proteins CXCL16 (111% increase), FDF-7 (82% increase), Serpin E1 (470.5% increase), and uPA (198.5% increase) when compared to normal healthy donor’s neutrophils cultured 18 hours in media. In summary, we observed up-regulation of several pro-angiogenic/ immunosuppressive genes in RCC patient neutrophils, suggesting RCC patient neutrophils may promote tumor escape. In addition, the normal healthy donor neutrophils cultured with ACHN tumor supernatant produces several angiogenic proteins suggesting the tumor microenvironment could be providing factors to mediate these angiogenic functions in RCC patient’s neutrophils.


Sequencing of PCR products amplified using pan-16S rRNA primers revealed the presence of DNA sequences from bacterial endosymbionts in several cell lines derived from Amblyomma and Dermacentor spp. ticks. Identification to species level was attempted using Rickettsia- and Francisella-specific primers. Pan-Nairovirus primers amplified PCR products of uncertain specificity in cell lines derived from Rhipicephalus, Hyalomma, Ixodes, Carios, and Ornithodoros spp. ticks. Further characterisation attempted with primers specific for Crimean-Congo haemorrhagic fever virus segments confirmed the absence of this arbovirus in the cells. A set of pan-Flavivirus primers did not detect endogenous viruses in any of the cell lines. Transmission electron microscopy revealed the presence of endogenous reovirus-like viruses in many of the cell lines; only 4 of these lines gave positive results with primers specific for the tick Orbivirus St Croix River virus, indicating that there may be additional, as yet undescribed ‘tick-only’ viruses inhabiting tick cell lines.As well as being vectors of many viral, bacterial, and protozoan pathogens of medical and veterinary importance, ticks harbour a variety of microorganisms which are not known to be pathogenic for vertebrate hosts. Continuous cell lines established from ixodid and argasid ticks could be infected with such endosymbiotic bacteria and endogenous viruses, but to date very few cell lines have been examined for their presence. DNA and RNA extracted from over 50 tick cell lines deposited in the Roslin Wellcome Trust Tick Cell Biobank ( Borrelia spp. spirochaetes, the obligatory intracellular genera Rickettsia and Ehrlichia, and the protozoan parasite genera Babesia and Theileria. Others, such as the obligatory intracellular bacterial genera Coxiella, Francisella, and some species of Anaplasma, can be naturally transmitted between vertebrates both by ticks and by other routes   . A thirdg flies) . A fourtp. ticks .Dermacentor andersoni embryo-derived cell line DAE100 was found to harbour the non-pathogenic endosymbiont Rickettsia peacockii  decoloratus cell lines. A previously undescribed Orbivirus was found to infect the IDE2 cell line derived from embryonic Ixodes scapularis  for presence of intracellular bacteria and viruses.Amblyomma, Dermacentor, Hyalomma, Ixodes, and Rhipicephalus, and the argasid genera Carios and Ornithodoros (2 flasks (Nunc), with weekly medium changes. The incubation temperatures used for each cell line are given in g for 5 min or from cryopreserved stabilates which were thawed rapidly, diluted 1 in 10 in appropriate complete medium and centrifuged as above. Cell pellets were resuspended in PBS. For RNA extraction, cells were harvested from actively growing cultures as described above; RNA extraction was not attempted from cryopreserved stabilates.Fifty continuous tick cell lines derived from species of the ixodid genera thodoros , were cuTotal genomic DNA was prepared from harvested tick cells using the DNeasy Blood and Tissue Kit  following the manufacturer's protocol. Purified DNA was eluted from the spin column with 400 μl TE buffer (two successive 200-μl elutions) and stored at −20 °C until use. RNA was extracted using an RNeasy Midi Kit  following the manufacturer's instructions. Purified RNA was eluted using 300 μl (2 × 150 μl) RNase-free water stored at −80 °C until use.First-strand cDNA synthesis was carried out using the SuperScript III System . Five hundred nanograms of total RNA and 1 μl of a 50-μM solution of random hexamers in a total volume of 13 μl were incubated for 10 min at 65 °C and chilled on ice. After adding 4 μl of 1st Strand Buffer, 1 μl of DTT (0.1 M), 2 μl of deoxynucleotide triphosphate mix (10 mM), and 1 μl of SuperScript reverse transcriptase III (200 units/μl), the reaction was incubated for 5 min at 25 °C, 1 h at 50 °C, and, finally, for 15 min at 70 °C. The resulting cDNA was used directly as a template for PCR amplification.DNA and cDNA were amplified by PCR using primers listed in www.dnaseq.co.uk) using Applied Biosystems Big-Dye Ver 3.1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer. Homology searches were performed in the NCBI database using the BLAST search programme  following the manufacturer's recommendations. DNA sequencing in the forward and reverse directions was performed by DNA Sequencing & Services, MRCPPU, College of Life Sciences, University of Dundee, Scotland , stained in uranyl acetate and lead citrate, and viewed in a Phillips CM120 transmission electron microscope. Images were taken on a Gatan Orius CCD camera.Amblyomma and Dermacentor yielded PCR products in the expected size range of 528 bp (Rickettsia (from the 2 A. variegatum cell lines) and Francisella (from 3 Dermacentor spp. cell lines). Further analysis with genus-specific primers  decoloratus , R. appendiculatus , H. anatolicum (HAE/CTVM8 and 9), I. scapularis (IDE12), C. capensis (CCE1) and O. moubata  yielded PCR products of, or near the expected 400 bp size with the pan-Nairovirus primers from both of 2 cDNA samples generated on separate occasions. When sequenced, most were found to contain a short segment which was identical to nucleotides 431–463 of the nucleocapsid gene of the S genome segment of CCHFV. However, the remainder of each of the tick cell line-derived sequences did not match well with any other part of the genomes of CCHFV or other published nairoviruses, and it was not clear whether the amplification was specific or non-specific. In some cases, the sequence obtained with the forward primer differed from that obtained with the reverse primer. Considering the sequences obtained from both the forward and reverse primers, all the sequences were different from each other (R. (B.) decoloratus and the 2 H. anatolicum cell lines which yielded consensus sequences from both primers which were, respectively, 98% and 100% identical within tick species, but only 40% identical between species and with less than 38% identity to the published Nairovirus sequences (Thirteen of the cell lines derived from the tick species ch other  apart frequences . PCR usiRickettsia (AVL/CTVM13 and AVL/CTVM17) or Francisella-like endosymbionts  were included in this study, but no convincing evidence of intracellular bacteria was seen in any of the 5 lines.Samples of 35 of the ixodid tick cell lines were examined by TEM , and 25 Rickettsia  or electron microscopy. We have attempted to infect Amblyomma americanum (AAE2) and R. (B.) microplus (BME/CTVM2) cells with the putative Rickettsia from the A. variegatum cell lines, also without success (data not shown). Therefore, we cannot say with certainty that these endosymbiotic bacteria are growing in the host cell lines. A similar problem arose in the study of Candidatus M. mitochondrii gene in 2 tick cell lines (BDE/CTVM14 and IRE/CTVM19), but failed to amplify a larger section of a different Candidatus M. mitochondrii gene from the same samples. Incorporation of segments of Wolbachia genes in invertebrate host genomes has been reported by Although we detected the presence of Amblyomma and Dermacentor spp. cell lines. The availability of systems for propagation of such endosymbionts in tick cell lines will aid in their characterisation, providing a useful model system for study of the interaction between the bacteria and their arthropod vectors. Understanding the mechanisms by which ticks and endosymbionts interact might reveal ways in which the non-pathogenic endosymbionts can be manipulated for control of both the ticks and the pathogens that they also harbour, as demonstrated for mosquito cell-adapted Wolbachia pipientis which, when transfected into Aedes aegypti mosquitoes, interfered with replication of the human pathogens Dengue virus, Chikungunya virus, and Plasmodium . Further study is required to establish the origin and identity of the virus(es) in RA243 and RA257, and their relationship to the SCRV in IDE2 and IDE8.es, IDE2  and IDE8es, IDE2 . InteresI. scapularis cell lines examined except IDE12, but did not observe any in the RA243 cells, suggesting that if SCRV is replicating in this cell line, it is present at a low level compared to the viruses in the I. scapularis cells, and/or it only infects a small proportion of the total cell population. We cannot definitely say that the reovirus-like particles observed in IDE2 and IDE8 cells are in fact SCRV, and the viruses in ISE6, ISE18, and the 21 other cell lines in which we saw reovirus-like particles remain at present unidentified. We could not find any reports of TEM detection of reoviruses or reovirus-like particles in tick cell lines, apart that from R. appendiculatus ticks infected with Theileria parva by R. (B.) microplus , Flaviviridae (genus Flavivirus), and Reoviridae (genus Orbivirus). Therefore we carried out a preliminary screening of the tick cell lines using one set each of pan-Nairovirus and pan-Flavivirus primers. Due to time restrictions, we were unable to include additional primer sets such as those of R. (B.) decoloratus cell lines, which were all derived from the same pool of 4 egg batches, suggesting that the same virus may be present in all 3 lines. Similarly, almost identical sequences were amplified from the 2 H. anatolicum lines suggesting that they may also harbour the same virus, although they were derived from different egg batches (O. moubata lines and 2 of the R. appendiculatus lines (RAE/CTVM1 and RAN/CTVM3), suggesting that these lines could each harbour more than one virus. The negative results obtained using the CCHFV-specific primer sets confirmed the absence of this particular Nairovirus in the tick cell lines. Further analysis is needed to identify and characterise these new putative nairoviruses and to determine whether they are arboviruses with the potential to infect vertebrate hosts, or tick-only viruses. The primers targeting the L polymerase-encoding region of the Nairovirus genome, used by  batches . In contRhipicephalus spp. ticks using such a PCR-based method (Rhipicephalus sp., suggesting the possibility that it might be another ‘tick virus’. Moreover, the recent detection of ASFV-like gene sequences in human serum (The pan-Flavivirus primers which we used are reported to consistently amplify several flaviviruses ; howeverd method . This vian serum  suggestsOver half of the known tick-borne viruses have been propagated in tick cells, along with many insect-borne arboviruses . Infecti


We have previously found that the novel tetramethylnaphthalene derivative TMNAA selectively inhibits the proliferation of HTLV-1-infected T-cell lines but not HTLV-1-uninfected T-cell lines. Although its target molecule is still unknown, TMNAA did not affect NF-κB activity. Therefore, we further examined the anti-proliferative activity of TMNAA against various T-cell lines in combination with cepharanthine (CEP), which is known to inhibit NF-κB.HTLV-1-infected and uninfected T-cell lines were cultured in the presence of various concentrations of TMNAA and CEP, and their proliferation and viability were determined by a tetrazolium dye method. The mode of cell death was also examined by flow cytometry and Western blot analysis.The 50% inhibitory concentrations (IC50s) of TMNAA and CEP for the ATL cell line (S1T) were 1.65 ± 0.03 and 1.97 ± 0.29 µM, respectively. On the other hand, the IC50 of TMNAA and CEP combination resulted in 0.93 ± 0.13 µM, indicating that the combination synergistically inhibited the proliferation of S1T cells. Such synergism was observed for another infected cell line (MT-2) but not for HTLV-1-uninfected cell lines. Moreover, TMNAA did not induce apoptosis of S1T cells, but CEP did. Interestingly, TMNAA significantly enhanced the CEP-induced apoptosis of S1T and MT-2 cells.The combination of TMNAA and CEP selectively inhibits the proliferation of HTLV-1-infected cell lines through the induction of apoptosis. Therefore, TMNAA and CEP may have potential for chemotherapy of ATL.


These findings will enlighten the biomedical application of manganese-based metal complexes as anti-leukemic agents.Transitional metals and metal compounds have been used in versatile platforms for biomedical applications and therapeutic intervention. Severe side effects of anticancer drugs produce an urgent urge to develop new classes of anticancer agents with great potency as well as selectivity. In this background, recent studies demonstrate that monomeric manganese (MnII) thiocyanate complex (MMTC) holds great promise to exert effective antileukemic effects. MMTC was developed by a simple chemical reaction and characterized by elemental analyses, thermal analyses, and Fourier transform infrared (FTIR) spectroscopy. Anti-leukemic efficacy of the developed MMTC was estimated in KG-1A (AML) and K562 (CML) cell lines. Cell viability study, drug uptake assay, cellular redox balance (GSH and GSSG level), nitric oxide (NO) release level, reactive oxygen species (ROS) formation, alteration of mitochondrial membrane potential (MMP), and DNA fragmentation revealed that MMTC was able to produce significant antiproliferative effects on both cell lines at 25  Leukemia is a type of cancer of the blood or bone marrow, characterized by an abnormal increase of immature white blood cells called “blasts” . Acute m2CO3), sucrose, Hanks balanced salt solution (HBSS), HEPES-Na+ buffer, and 3--2,5-diphenyltetrazolium bromide (MTT) were purchased from Himedia, India. Tris-HCl, Tris buffer, KH2PO4, K2HPO4, HCl, formaldehyde, alcohol, Triton X-100, sodium dodecyl sulphate (SDS), phenol, chloroform, isoamyl alcohol, ethidium bromide (EtBr), 2-vinylpyridine and other chemicals were procured from Merck Ltd., SRL Pvt. Ltd., Mumbai, India. Commercially available dimethyl sulfoxide (DMSO) was procured from Hi-media, India, and was purified by vacuum distillation over KOH. All other chemicals were from Merck Ltd., SRL Pvt. Ltd., Mumbai, India, and were of the highest purity grade available. Solvents were dried according to the standard procedure and distilled prior to use  (MMTC) was prepared according to our previously reported method -2,5-diphenyltetrazolium bromide (MTT). Briefly, MTT was dissolved in phosphate-buffered saline at 5 mg mLectively . The perIn vitro MMTC uptake assay was performed according to standard methods  by chemical reactions. The single crystals of MMTC suitable for X-ray data collection were obtained from the filtrate after a few days. Yield: 90. Anal. Calcd for C26O2N4S2H18Mn: C, 58.05; H, 3.35; N, 10.42 (%). Found: C, 58.13; H, 3.28; N, 10.37 (%). IR: v(SCN) = 2076 cm−1, ν(C=O) = 1625 cm−1, ν = 1579 cm−1.Leukemia is a life threatening hematological malignancy characterized by uncontrolled cell growth. Transition metals have an important role in biomedical application. Research has shown significant progress in the utilization of transition metal complexes as drugs to treat several human diseases like carcinomas, lymphomas, infection control, anti-inflammatory, diabetes, and neurological disorders . In the α radiation, λ = 0.71073 Å) equipped with a CCD. Cell refinement, indexing, and scaling of the data set were carried out using Bruker Smart Apex packages, and Bruker Saint Package [F2 with all observed reflections  understand the thermal decomposition patterns of the complexes, (ii) to verify the molecular composition of the complexes, and (iii) to synthesize the thermally stable end products. Critical analyses suggest that MMTC yields corresponding metal sulfides as the end products. The thermogram as well as the calculated and experimental weight losses, temperature range of decomposition are reported here .−1 which corresponds to the stretching frequency of SCN moiety. The complex exhibits IR stretching frequencies corresponding to the carbonyl group of the hemilabile legend, phpyk at around 1640 cm−1 and skeletal vibrations at ~1430 and ~1580 cm−1  cytotoxic effects in PBL. As the dose increased to 50 μg mL−1, cell viability of PBL was decreased significantly by 47.77% due to MMTC exposure. MMTC exposure was significant (P < 0.05) and caused a reduction in cell viability in leukemic cell lines by dose dependent fashion. In KG-1A cell line, the viability was significantly decreased by 58.82% and 61.38% at 25 and 50 μg mL−1 doses, respectively, whereas in K562 cell line this metal complex was able to reduced cell viability by 56.97% and 63.31% at 25 and 50 μg mL−1 doses, respectively. Simultaneously, doxorubicin showed potential toxic effects on normal PBL. It decreased the viability of PBL significantly (P < 0.05) by 46.93%, 72.69%, and 81.08% at 10, 25, and 50 μg mL−1 doses, respectively. In leukemic cell lines DOX significantly (P < 0.05) decreased cell viability from 10 μg mL−1 dose.Selectivity and good antileukemic efficacy are one of the basic requirements of an anticancer agent. In our study, two types of leukemic cell lines (KG-1A and K562) and normal peripheral blood lymphocytes (PBL) were exposed to DOX and MMTC at the concentrations of 0, 1, 10, 25, and 50  Figures . ResultsP < 0.05) toxic effects toward healthy cells up to 25 μg mL−1 dose. Collectively, these results demonstrate that this dose can be taken as an effective dose for antileukemic therapy using MMTC. So, further experiments were done using this dose only.From this result, it can be stated that though DOX exerted more potent antileukemic potential, side by side, it showed severe toxic effects on PBL. The cytotoxic effects of DOX toward normal cells have already been reported by many researchers , 22. In in vitro cultures  decreased by 78.97%, and in the case of K562 cell, lines it was decreased by 74.35% at 25 μg mL−1 dose (P < 0.05) in MMTC treated leukemic cells by 49.46% (in KG-1A cell line), and 78.78% (in K562 cell line) at the same dose , which has an important microbicidal and tumoricidal function. In our study, the elevated level of NO in leukemic cells may be due to severe oxidative injury which in turn helped in cell killing ability due to MMTC exposure [NO reacts with superoxide to form a more toxic peroxynitrite (ONOOexposure .P < 0.05) increased ROS generation level by 232% in KG-1A cell line and by 282% in K562 cell line at the effective dose (Leukemic cells (KG-1A and K562 cell lines) exposed to MMTC in 24 hours showed increased ROS formation as evidenced by the increased DCF fluorescence intensity. In our study, MMTC exposure significantly (Reactive oxygen species (ROS) are molecules and ions containing unpaired valence shell electrons, and being free radicals they are highly active and show an important role in cell signaling, leading to oxidative cell damage . In the One of the possible mechanisms involved in the apoptosis of KG-1A cell line and K562 cell line induced by MMTC was the loss of mitochondrial membrane potential. Mitochondrial membrane potential was determined in terms of Rhodamine 123 fluorescence intensity. In this study, MMTC caused depletion of MMP in both cell lines. In KG-1A cell line, MMP was decreased by 68.27% and in K562 cell line by 71.75% .Loss of MMP and increased DNA fragmentations are the most common targets for apoptosis induced cell death . Alteratμg mL−1 dose of MMTC for 24 h was capable of fragmenting the DNA strands of KG-1A and K562 cell lines, indicating the involvement of oxidative injury and apoptosis.DNA fragmentation study in terms of agarose gel electrophoresis revealed that MMTC was able to interact with the DNA of leukemic cells. Qualitative gel documentation image  suggesteThe DNA fragmentation is caused by the activation of an endogenous nuclear endonuclease, which selectively and distinctively cleaves the double-stranded nuclear DNA at sites located between nucleosomal units (linker DNA), generating mono- and oligonucleosomal DNA fragments . In our In this study, we have demonstrated that MMTC exhibited antileukemic activity in AML and CML cells by dose-dependent manner, whereas it appeared to be less cytotoxic in human normal lymphocytes up to a certain dose. Involvement of oxidative stress mediated by RNS and ROS, alteration of cellular redox status, loss of mitochondrial membrane potential, and genotoxic effects were observed due to MMTC exposure. Based on these results, it can be concluded that oxidative stress is the possible antileukemic mechanism underlying the etiology of cytotoxicity and apoptosis induced by MMTC. However, further studies are necessary to reduce the cytotoxic effects of MMTC in normal cells and to understand the mode of action of such a complex in leukemic cells.


The correct definition of the acronym ACECR used in affiliations 1, 2, and 4 is "Academic Center for Education, Culture and Research." The corrected affiliations are:1 Department of Molecular Systems Biology, Cell Science Research Centre, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research(ACECR), Tehran, Iran2 Department of Stem Cells and Developmental Biology, Cell Science Research Centre, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research(ACECR), Tehran, Iran4 Department of Developmental Biology, University of Science and Culture, Academic Center for Education, Culture and Research, Tehran, Iran


P) by the deliberate inhibition of cell growth (controlled proliferation). Three methods commonly used to control proliferation are use of (i) chemical agents, (ii) hypothermic conditions and (iii) genetic manipulation. In this study, we focused on increasing volumetric productivity by manipulating QP; in particular, by the use of chemical agents.After selection of a recombinant cell line for the production of a therapeutic protein, attempts are often made to increase volumetric productivity. Various techniques have been employed during the production process when trying to increase product concentration. Some aim to increase the time integral of viable cell concentration  by increasing the maximum viable cell concentration and maintaining high viabilities. Techniques include (i) optimization of media and feeds, (ii) optimization of feeding strategies and (iii) genetic manipulation. Others aim to increase specific production rate ? It could be that the cell line with the highest product concentration is typically a non-responder.The work described looks to answer the questions: (1) To what extent does the response to such methods vary in a large panel of cell lines producing the same antibody? and (2) Would their use in an earlier stage of cell line development enable the selection of a ‘better’ manufacturing cell line?et al, 2010, Biotechnol Prog 26: 1455-1464).Cell lines: A panel of 148 GS-CHO cell lines was generated by transfecting the host cell line CHOK1SV with the GS vector pEE12.4 containing the gene for the model antibody cB72.3  of Lonza Biologics’ final production bioreactor process, using CDACF medium and feeds. Three cultures were initiated for each cell line. The first was a control culture, in the second the culture medium was supplemented with 1 mM Sodium Butyrate (NaBu), and in the third the culture medium was supplemented with 7.5 mM Sodium Acetate (NaAc). The cell concentration was determined using a Vi-CELL automated cell counter. Product concentration was determined using Protein A HPLC.P and product concentration were investigated for each condition . For IVC, the distribution of the values for both the NaBu and NaAc conditions are lower than that of the control. For QP, the distribution of values for the NaBu condition are higher that that of the control. The NaAc condition shows no improvement. For product concentration, the distribution of values for the NaBu condition are lower than that of the control. Little difference is observed between the control and the NaAc condition. Data were analyzed by one-way ANOVA and Tukey’s multiple comparison test at a 5% significance test. The analysis reveals that there is a significant difference between the control and NaBu conditions for IVC, QP and product concentration. In addition, there is a significant difference between the control and NaAc conditions for IVC but not for QP and product concentration.The distribution of the values for the parameters IVC, QReview of individual cell lines Figure  reveals ■ The majority of cell lines did not achieve a higher product concentration compared to the control, when either NaBu or NaAc was added■ The use of NaBu resulted in an increase in productivity, compared to the control, for 27% of the cell lines■ The use of NaAc resulted in an increase in productivity, compared to the control, for 41% of the cell lines■ An increase in productivity was approximately twice as likely to be observed with the use of NaAc compared to the use of NaBu■ For both NaBu and NaAC, an increase in product concentration compared to the control was more likely if the cell line was a low producer■ For those cell lines exhibiting an increase in product concentration compared to the control when NaBu or NaAc was added, 80% and 62% respectively were in the lower 50% of producers when ranked by control product concentation■ The highest product concentration was achieved using control conditions■ No advantage of NaAc or NaBu if looking to identify a more productive cell line from the populationP is ‘cell line specific’. On average, no increase in mean product concentration was seen and only ~25-50% cell lines exhibited a benefit. The question ‘Would the use of productivity enhancers in an earlier stage of cell line development enable the selection of a ‘better’ manufacturing cell line?’ has been answered: There is no advantage in their use. If the selection strategy has identified a high producing cell line, the productivity enhancers are unlikely to be effective. Also, the highest product concentration was achieved with control conditions.The data generated in the rigorous testing support the anecdote that the ability of productivity enhancers to increase Q


MDM2 gene and overexpression of MDM2 protein. However, the majority of human sarcomas have normal level of MDM2 and the therapeutic potential of MDM2 antagonists in this group is still unclear. We have investigated if Nutlin-3a could be employed to augment the response to traditional therapy and/or reduce the genotoxic burden of chemotherapy.Frequent failure and severe side effects of current sarcoma therapy warrants new therapeutic approaches. The small-molecule MDM2 antagonist Nutlin-3a activates the p53 pathway and efficiently induces apoptosis in tumours with amplified TP53 and MDM2 status were treated with Nutlin-3a combined with Doxorubicin, Methotrexate or Cisplatin, and their combination index determined.A panel of sarcoma cell lines with different TP53 and amplified MDM2, or with Methotrexate in both MDM2 normal and amplified sarcoma cell lines, allowing for up to tenfold reduction of cytotoxic drug dose. Interestingly, Nutlin-3a seemed to potentiate the effect of classical drugs as Doxorubicin and Cisplatin in cell lines with mutated TP53, but inhibited the effect of Methotrexate.Clear synergism was observed when Doxorubicin and Nutlin-3a were combined in cell lines with wild-type The use of Nutlin in combination with classical sarcoma chemotherapy shows promising preclinical potential, but since clear biomarkers are still lacking, clinical trials should be followed up with detailed tumour profiling. TP53 gene, coding for the transcription factor p53, is thought to be the most frequently mutated gene in cancer, inactivated in about 50% of all tumours. However, aberrations of this pathway are probably even more wide-spread, as tumours retaining wild-type p53 (WtTP53) might have defects in other parts of the pathway [MDM2 (murine double minute 2) is relatively common (20%) in tumours having WtTP53, resulting in disabled p53 function because overexpressed MDM2 protein binds to and inactivates p53 [MutTP53, 11-31% depending on subtype), or other changes in the downstream pathway that do not affect the level of MDM2  [The  pathway . In sarcsubtype) -6.Sarcomas are among the more frequent cancers among children , and botin vitro and reducing tumour burden in vivo [MDM2 gene (AmplMDM2) are most responsive to Nutlin, most likely due to otherwise intact downstream p53 signalling [MDM4  gene has also been proposed to explain the responses to Nutlin [Nutlin-3a, a novel small-molecule inhibitor of the p53-MDM2 interaction, has been shown to be highly effective in killing osteosarcoma cells  in vivo ,11. Nutlgnalling ,12-14, ao Nutlin -17.MDM2 is frequently amplified in these tumours [WtTP53/Ampl MDM2liposarcomas [Wt TP53but do not have amplified MDM2 (are WtMDM2), it would be of interest to investigate if Nutlin-3a could potentiate the response of WtMDM2/Wt TP53tumours to conventional chemotherapy. Since p53 mutations are very heterogeneous, different mutations sites could also imply different response to combined therapy. Nutlin has been shown to be synergistic with genotoxic drugs  in haematological malignancies, lymphoma, neuroblastoma and hepatocellular carcinoma, and with radiation in lung cancer [TP53 tumours, suggesting that clinical combination studies in sarcoma are warranted.Early studies suggest that MDM2 antagonists may be particularly effective in sarcomas because  tumours ,18,19. Wsarcomas . Howeverg cancer ,21-26, bg cancer . Paclitag cancer . Here, wg cancer -31. The Wt TP53and two with MutTP53. Two cell lines had Ampl MDM2and three had WtMDM2 was amplified in five distinct fragments by using flanking intronic primers with M13 tails [Genomic DNA was extracted from cell lines using the standard phenol chloroform method. The total protein coding region of 13 tails , generatCellular integrity was measured as total cellular protein by the Sulforhodamine B (SRB) assay, which was performed essentially as described previously . BrieflyNutlin-3a: IC50x, ratio 1:2 = IC50Nutlin-3a: 2 times IC50x and ratio 2:1 = 2 times IC50Nutlin-3a: IC50x , where x is the chemotherapy. For Methotrexate we used the 1:1 ratio only. When the ratio was set, a mixture of the two drugs was made and serially diluted to obtain a good dosage range.All cell lines were exposed to the drugs alone or in combination. The IC50 of the individual drug was determined where after combinations were made based on the ratio of the IC50's. Drugs were added in a constant ratio design. For Doxorubicin and Cisplatin three fixed ratios were set: Ratio 1:1 = IC50After exposure, cells were fixed with 50% Trichloroacetic acid , washed and stained with 0.4% Sulforhodamine B  dissolved in 1% acetic acid , subsequently washed five times with 1% acetic acid to remove unbound stain. Protein bound stain was dissolved in 150 μl 10 mM Tris base (Merck). Optical density was measured at 540 nm in a Victor Wallac 1420 multi-label counter . Relative Growth was calculated as follows:treated - ODzero > 0; Cell death occurred if ODtreated - ODcontrol < 0, where ODtreated represents the optical density of treated cells at the day of the assay, ODcontrol the optical density of the untreated control cells and ODzero the optical density at the moment of drug addition. From the relative growth we obtain the fraction affected (Fa) as follows:when ODTo evaluate the pharmacological interaction of the different combinations of drug treatments, we followed the method proposed by Chou et al.  Briefly,x)1 and (Dx)2 are the doses of drug 1 and drug 2 alone inhibiting x % whereas (D1) is the dose of drug 1 in combination with drug 2 and (D2) the dose of drug 2 in combination with drug 1 that gives the experimentally observed x inhibition. Since our aim was to achieve maximal effect of the drugs tested on cancer cells, a mean CI was calculated from data points with fraction affected (Fa) > 0.5. (Fa) < 0.5 would imply lower growth inhibition and a large fraction of the cell population would still grow. Fa < 0.5 was therefore considered not relevant. Furthermore we evaluated how much each drug dose in a synergistic combination could be reduced at a given effect level compared with the doses for each drug alone, the so-called dose-reduction index (DRI):where  as illustrated by the example: if drug A and drug B each inhibit 50% and if (0.5A+ 0.5B) also inhibits 50% and both drugs have no overlapping toxicity toward the host, DRI ≥ 1 may still be beneficial .In the drug sensitivity assay we chose a constant ratio design with three fixed ratios as described above. By only including the 1:1 ratio in the western analysis, our intent was to eliminate the potential disturbing effect of different drug concentrations on the molecular mechanisms. Cells were treated for 24 hours with Nutlin-3a, Doxorubicin, Cisplatin, Methotrexate or described drug combinations. They were lysed in lysis buffer (supplemented with phosphatase and protease inhibitor cocktails (Sigma-Aldrich)) and sonicated. 15-30 μg of protein lysate was separated on 4-12% NuPage BIS-Tris gradient gels  and transferred to 0.45 μm PVDF membranes  in blotting buffer with 20% (v/v) methanol using wet blot equipment from BIO-RAD, CA USA. Antibodies used : anti-p53 (1:3000 Santa Cruz sc-6243), anti-MDM2 , anti-MDM4 (1:20000 Bethyl Labs), anti-PUMA , anti-β-actin , and polyclonal anti-p21 . All HRP-conjugated secondary antibodies were from Dako, Denmark. Bands were visualized using SuperSignal West Dura ECL  and Kodak X-VIS film and software . Pictures were merged and presented using Photoshop .P < 0.05.The Statistical package SPSS 13.0 was used. 2-tailed unpaired Student's T-test on transformed results was used for statistical analysis. Changes were considered to be significant when Ampl/TP53WtMDM2) as a control cell line. As representative of Wt/TP53WtMDM2, we chose the osteosarcoma cell line U2OS. Finally, SaOS-2 and RMS13 were chosen as examples of two different cell lines with mutated p53. SaOS-2 is known to be NullTP53[WtTP53, did not respond to Nutlin-3a, and was found to have a transversion in codon 280, exon 8, changing the amino acid from arginine to serine in the resulting protein. Protein analysis uncovered high levels of probably dysfunctional p53 protein and constitutively high p21 and PUMA (see below). This is consistent with a report that the Rh30 cell line, established independently from a xenograft from the same patient, also had this mutation [Wt TP53were validated by sequencing.We investigated the effect of combining Nutlin-3a with the current standard of sarcoma therapy  on the growth of five sarcoma cell lines. The characteristics and sources of the cell lines used are described in Table  TP53Null. RMS13, mutation  and illuAmpl MDM2cell lines (OSA and T778) were sensitive to Nutlin-3a and showed clear dose dependence, whereas the Wt/TP53Wt MDM2U2OS was less sensitive, with an IC50 about twice that for the Ampl MDM2cell lines. Consistent with previous results, we were not able to reach 50% growth inhibition for the Mut TP53cell lines (SaOS-2 and RMS13) when using Nutlin-3a alone. Cisplatin, Doxorubicin and Methotrexate also inhibited cell growth in a concentration-dependent manner. Figure AmplMDM2/Wt TP53OSA and WtMDM2/Mut TP53RMS13 exposed to single drugs. In all cell lines tested, Doxorubicin and Methotrexate were more cytotoxic than Cisplatin or Nutlin-3a when used alone  values (indicating the fraction of cells inhibited after drug exposure) were obtained after exposure of the cells to a series of drug concentrations. To indicate the effects at different Fa values, combination index (CI) values were calculated for each Fa. For the 75, IC90 and IC95 are reported in Table Ampl MDM2cell lines, Doxorubicin could be reduced 10-fold and in T778 the Cisplatin dose could also be significantly reduced when combined with Nutlin at a 2:1 ratio. In addition, in cell lines being Ampl MDM2or WtMDM2, T778 and U2OS respectively, we found that reduced dose of Methotrexate would suffice when combined with Nutlin-3a. Some combinations that were not synergistic also maintained efficacy with reduced level of standard cytotoxic drugs (DRI > 1) when combined with Nutlin-3a. This holds for all combinations with Doxorubicin or Methotrexate as well as some with Cisplatin. Moreover, as depicted in Table As expected, synergism, corresponding to CI < 1, always yielded a favourable dose reduction index (DRI > 1) for both drugs. The DRI values at ICAmpl MDM2cell lines  was more sensitive to all three genotoxic agents tested than the TP53 null cell line (SaOS-2). Methotrexate was more effective in the lines with MDM2 amplification, suggesting some relation between MDM2 and the Methotrexate mechanism of action that warrants further investigation.The genotoxic agents tested in this study are commonly used in sarcoma treatment. Doxorubicin and Methotrexate were quite effective in these cell lines, whereas higher doses of Cisplatin and Nutlin-3a were required in the MDM2Wt/Wt TP53U2OS cell line. This is consistent with previous results in colon (HCT116) and ovarian (A2780) cancer cells, where Nutlin was cytoprotective in combination with Cisplatin [Mut TP53cell lines, RMS13 and SaOS-2, showed a significant sensitization towards Cisplatin and Doxorubicin at IC50 when combined with Nutlin-3a. This apparently p53-independent effect of Nutlin-3a is in line with previous publications [TP53-null or-mutant cells. It was reported that when cells with dysfunctional TP53 were exposed to high concentrations of Nutlin (e.g. 20-30 μM), p73 was displaced from its binding to MDM2, resulting in cell cycle arrest and apoptosis [TP53, most likely due to E2F1-mediated up-regulation of p73 activity [The DRI values clearly illustrated the benefit of the synergistic combinations, with 10-fold reductions in Doxorubicin and Cisplatin when combined with Nutlin-3a, but also combinations with less than additive effects showed promising DRI values. Nutlin-3a and Doxorubicin or Cisplatin were less than additive in the isplatin . Compellications -48, and poptosis ,46,49. NWtTP53/MDM2Ampl and WtTP53/MDM2Wt cell lines and DRI values that implied that Methotrexate could be reduced ten-fold when combined with Nutlin-3a. In the MDM2Wt cell line U2OS, treatment with Methotrexate decreased MDM4 to undetectable levels both alone and in combination with Nutlin-3a. According to the current hypothesis, reduction of MDM4 could lead to more unbound p53 [Mut TP53cell line (RMS13), could not be explained by this and clearly needs further investigation.The third chemotherapy tested here, Methotrexate, acts through nucleotide metabolism, for which E2F1 is a central transcription factor ,51. Addiound p53 , and recound p53 . It is tOur in vitro data suggest that MDM2 antagonists could offer effective therapy for sarcoma either as single agent or in combination with prevailing chemotherapy, and may significantly reduce the genotoxic burden with the same or better antitumour effect. Further work with animal models of sarcoma and ultimately clinical studies needs to be done to evaluate and optimize combinations of Nutlin and current chemotherapies. Since clear biomarkers are still lacking, clinical trials should be followed up with detailed tumour profiling.Lyubomir Vassilev is employed by Roche, the company which developed Nutlins, and as such could be perceived to have a vested interest in these results. This has however had no consequences for how the studies were performed and the results interpreted.HOO carried out the drug sensitivity assays and the combination analysis, performed the statistical analysis and drafted the manuscript. EBP carried out the immunoassays. PN participated in the design of the study and drug sensitivity assays. MB and RAL carried out the sequence analysis. LTV participated in the study design and helped to draft the manuscript. OM conceived the study, participated in its design and coordinated and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/211/prepub


In vitro functional analysis demonstrated that the metastatic cell line had higher invasion and migration ability when compared with the primary liver tumor cell line. These cell lines were subjected to microRNA (miRNAs) microarray analysis to identify differentially expressed miRNAs which were associated with the developement of metastasis in vivo. Fifteen human miRNAs, including miR-106b, were differentially expressed in 2 metastatic cell lines compared with the primary tumor cell lines. The clinical significance of miR-106b in 99 HCC clinical samples was studied. The results demonstrated that miR-106b was over-expressed in HCC tumor tissue compared with adjacent non-tumor tissue (p = 0.0005), and overexpression of miR-106b was signficantly correlated with higher tumor grade (p = 0.018). Further functional studies demonstrated that miR-106b could promote cell migration and stress fiber formation by over-expressing RhoGTPases, RhoA and RhoC. In vivo functional studies also showed that over-expression of miR-106b promoted HCC metastasis. These effects were related to the activation of the epithelial-mesenchymal transition (EMT) process. Our results suggested that miR-106b expression contributed to HCC metastasis by activating the EMT process promoting cell migration in vitro and metastasis in vivo.Hepatocellular carcinoma (HCC) is one the the most fatal cancers worldwide. The poor prognosis of HCC is mainly due to the developement of distance metastasis. To investigate the mechanism of metastasis in HCC, an orthotopic HCC metastasis animal model was established. Two sets of primary liver tumor cell lines and corresponding lung metastasis cell lines were generated.  Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. It is the third leading cause of cancer deaths worldwide MicroRNAs (miRNAs) are small non-coding RNA molecules which possess regulatory functions for gene expression. Development of metastasis is associated with alteration of gene expression, hence, miRNAs are suspected to be associated with the development of cancer metastasis. Previous studies have demonstrated that miRNAs play a critical role in development of cancer and metastasis. miR-21 and miR-122a can regulate important tumor suppressors, PTEN and CyclinG1 in HCC Animal models are widely used in cancer research for studying the development of cancer and metastasis. Microenvironment affects the development of cancer and metastasis HCC cell lines PLC8024, HepG2, Hep3B, Huh7 , MHCC97H  HCC clinical samples were collected with informed written consent and approval by the Institutional Review Board of the University of Hong Kong from HCC patients who underwent hepatectomy in Department of Surgery, Queen Mary Hospital, University of Hong Kong Medical Center . Clinicopathological data were collected.5 cells were orthotopically implanted into the liver of 4–6 weeks old CB17 SCID mice under anesthesia. Tumor formation was monitored by IVIS100 in vivo imaging system  with intra-peritoneal injection of 3 mg luciferin. After 10–12 weeks of inoculation, mice were sacrificed. Primary liver tumor and metastatic lung nodules were isolated and subjected to primary culture. Stable cell lines were checked by luciferase activity to confirm the cells originated from the parental HCC cell lines.Animal studies were approved by Committee on the Use of Live Animals in Teaching and Research at the University of Hong Kong. HCC cell lines, PLC8024 and MHCC97H cells were labeled with luciferase by lentivirus infection. 5×10Cell migration ability was measured by wound-healing assay. Full confluent cells were seeded into 24-wells plate. Acellular area was created by scraping using a pipette tip. Wound closure was measured at 24 and 48 hours interval.5 cells were seeded into the upper chamber with serum-free DMEM. DMEM with 10% FBS were put into lower compartment as chemo-attractant. Cells were allowed to invade for 48 hours. Remaining cells in the upper chamber were scraped out by cotton swap. Matrigel membranes were fixed with ice-cold methanol and stain with 0.1% crystal violet solution. Membranes were then destained and visualized under microscope. Each experiment were performed in triplicate and repeated twice.Trans-well invasion assay was performed using matrigel invasion chamber . 2×10Cells were cultured on chamber slide for 24 hours and serum-starved for another 24 hours. After 48 hours, cells were fixed with 4% paraformaldehyde and cell membranes were permeabilized with 0.1% TritonX100. Slides were then blocked by 1% BSA, and FITC-conjugated or TRITC-conjugated phalloidin  was hybridized onto the slide at 37°C for 1 hour. Images were then visualized by fluorescent microscopy.Total RNAs from cell lines and HCC clinical samples were extracted by Trizol reagent  following manufacture's protocol.miRNA microarray analysis was carried out by NCode miRNA expression profiling service . Total RNAs (10 µg) were enriched by PureLink miRNA isolation kit . Enriched miRNAs were polyadenylated and subsequently tagged with specific sequences to enable the detection of fluorescents. Tagged miRNAs were then purified and hybridized onto NCode Multi-Species miRNA Microarray V2.0 , containing probes for Sanger mirBASE 9.0, overnight at 52°C. Slides were then subjected to stringency wash and hybridized to AlexaFluor3 or AlexaFluor5 at 62°C for 4 hours. Slides were then washed and scan using GenePix4000B microarray scanner . Data were captured and analyzed by GenePix Pro software .First-strand cDNA synthesis for miRNA QPCR analysis was performed by TaqMan miRNA Reverse Transcription Kit and MegaPlex Primer Pool . Total RNA (350 ng) was subjected to reverse transcription with MegaPlex Primer Pool as RT-primer.−ddCt methods.TaqMan MicroRNA Assays were used for QPCR analysis. Reaction mixture containing 1XTaqMan Universal PCR Master Mix , 1XTaqMan MicroRNA Assay and 1∶15 diluted cDNA was subjected to thermal cycling on 7900HT Fast Real-Time PCR System . U6 snRNA was used as reference for the expression of the mature miRNAs. The cycling conditions were 95°C 10 min, followed by 40 cycles at 95°C 15 sec and 60°C 1 min. Relative miRNAs expression was calculated by 2miRNA-106b LNA knock-down probe and the scramble control  were transfected into the miR-106b over-expressed cell line, PLC-LM, using Lipofectamine 2000  according to manufacturer's protocol.Pre-miR-106b was cloned into pCDH-CMV-MCS-EF1-copGFP vector . Pseudoviral particles were prepared by the LentiStarter Kit  following the manufacturer's protocol. Pseudoviral particles were used for transduction in PLC-PT, Huh7, and Hep3B cell lines. One week after transduction, GFP+ cells were sorted using MoFlow cell sorting system . GFP+ cells were harvested and QPCR analysis was employed to confirm the over-expression of miR-106b.Protein lysate were obtained from cell lines using RIPA buffer . RhoGTPases, RhoA and RhoC , and EMT markers, E-cadherin, N-cadherin , Vimentin , and TWIST1 , were immune-blotted as previously described Statistical analysis was performed by SPSS16.0. Continuous data and categorical data were analyzed by Student's t-test and chi-square test respectively. P-value<0.05 was consider as statistically significant.HCC cell lines PLC8024 and MHCC97H, labeled with luciferase, were orthotopically implanted into liver of SCID mice . After 1Cell proliferation rates of both primary and metastatic cell lines were analyzed by MTT assay. No obvious differences between primary tumor and lung metastasis cell lines for both PLC8024 and MHCC97H derived cell lines were observed (Data not shown).Migration ability was measured by wound-healing assay. Cell invasion ability was measured by trans-well invasion assay . The invMetastatic ability of the PLC-PT and PLC-LM cell lines were compared by using the orthotopic animal model (Data not shown). The PLC-PT cell shows a lower metastatic ability compared to the parental PLC8024 and PLC-LM after 10 weeks of inoculation. For the PLC-LM, it has the highest metastatic ability amount three cell lines and most of the mice develop lung metastasis at week 10.Cytoskeleton structure was visualized by phalloidin staining . PLC-LM The primary tumor and lung metastasis cell lines of the two sets of phenotypically similar cell lines (PLC-PT vs PLC-LM and MHCC97H-PT vs MHCC97H-LM) were compared by NCode miRNA microarray profiling service. Array data were deposited at the Gene Expression Omnibus (GEO accession no.: GSE43445). MicroRNA candidates were selected by >2-fold difference in expression with at least 4 out of 6 spots were statistically significant . With criteria mentioned above, 67 miRNAs candidates were identified from PLC8024 derived cell lines, whereas 30 candidates were selected from MHCC97H derived cell lines. Fifteen common candidate human miRNAs were identified from the microarray analysis from these two sets of cell line .To validate microarray results, PLC8024 and MHCC97H derived cell lines were subjected to QPCR analysis. miR-106b expression in other HCC cell lines was also compared. All HCC cell lines show higher expression of miR-106b than normal hepatocyte cell line, MIHA . Furtherin vitro and metastatic ability in vivo, were identified by microarray analysis. The clinical relevance of top 8 over-expressing miRNAs from microarray analysis was first analyzed in 20 pairs of matched HCC tumor and non-tumor sample. Amount these miRNAs, miR-21 and miR-106b show clinical significance in this 20 HCC clinical sample cohort. This two miRNA targets were further analyzed by QPCR in another 79 pairs HCC sample . miR-21 and miR-106b showed significant up-regulation in tumors compared with the non-tumor tissues (p = 0.0059 and 0.0005 respectively)  . We corrctively) . The onlQPCR analysis showed successful miR-106b knock-down in PLC-LM cells by Locked Nucleic Acid (LNA) with >50% efficiency from Day 1 to Day 4 after transfection , while tTo confirm the functional role of miR-106b in other HCC cell lines, wound healing assay and phalloidin staining were performed in other 2 HCC cell lines, Huh7 and Hep3B. The wound healing assay showed that miR-106b over-expression can enhance the cell migration ability in vitro in both cell lines . In addiPLC-PT cells stably over-expressing miR-106b were implanted into liver of SCID mice. Mice were scarified at week 10, lungs were analyzed by H&E staining . Lung meExpression of RhoGTPases, RhoA and RhoC, were analyzed by western blot . Over-exin vitro and metastasis in vivo. Meanwhile, cells transduced with lentivirus over-expressing miR-106b  also show activation of EMT compare to their corresponding vector control   .HCC is a cancer prone to vascular invasion and metastasis, and most patients die from metastatic disease. Understanding the biology of metastasis in HCC is critical to improve patient prognosis. As primary treatment for metastatic HCC is usually not by surgery, it is difficult to collect metastatic tumor specimens from patients. In this study, orthotopic metastasis HCC animal model is used to study the role of microRNAs in development of metastasis in HCC.in vitro. The PLC-LM cell line was highly invasive and had higher migration ability. However, the proliferation rates are similar in PLC-PT and PLC-LM cell line. This suggested that metastasis development in vivo is related to enhanced cell migration and invasion ability rather than cell proliferation. In contrast, we did not observe higher migration capacity in MHCC97H-LM when compared to the MHCC97H-PT cell line. This may be due to the facts that MHCC97H is already a highly metastatic cell line, some of the molecular pathways involved in promoting the aggressive phenotype are deregulated, so the difference between MHCC97H-LM and MHCC97H-PT were not obvious. Even we can observed miR-106b expression is higher in MHCC97H-LM, this does not make any differences in the in vitro functional characteristics of this two cell lines since some of the down-stream molecule are deregulated due to the aggressive nature in the parental cell line, MHCC97H.The primary tumor and lung metastatic cell lines derived from PLC8024 showed different invasiveness in vitro between the primary tumor and lung metastasis cell lines. The metastatic cell line showed higher migration and invasion abilities than the primary tumor cell line in PLC8024 derived cell lines, but not in the MHCC97H derived cell lines. However, metastatic ability in vivo was similar between PLC8024 and MHCC97H cell lines and those commonly differential expressed miRNAs maybe related to the development of metastasis. By comparing miRNA microarray profile of the PLC8024 and MHCC97H derived cell lines, 15 human miRNAs were differentially expressed in primary tumor cell lines versus the metastatic cell lines. Although our focus in this study was miR-106b, the highest differentially expressed miRNA was miR-21, which is a well-known miRNA involved in various kinds of cancer including HCC in vitro and in vivo studies.To explore the role of miRNAs in development of metastasis, two metastatic cell lines derived from PLC8024 and MHCC97H were compared with the corresponding primary tumor cell lines by microarray analysis. More miRNAs were differentially expressed in the PLC8024 derived cell lines. These genotypic differences may contribute to the phenotypic differences QPCR analysis for miR-21 and miR-106b expression in HCC and nontumorous liver was performed. U6 was chosen as the reference in our studies as it is stably expressed in all cell lines and clinical sample cohort. Several studies have demonstrated the critical role of miR-21 in HCC carcinogenesis in vitro were transient and >50% knock-down could be attained within 4 days post-transfection. We performed a series of experiments including MTT assay, migration and invasion assay to study the functions of miR-106b. Our results suggested that migration ability decreased upon miR-106b knock-down without affecting cell proliferation rate and invasion ability, suggesting the main functional role of miR-106b in enhancing cell motility. To confirm this finding, we also over-expressed miR-106b in PLC-PT cell line. Migration ability was increased when we over-expressed miR-106b in the cells without affecting cell proliferation rate and invasion ability. Since miR-106b could increase cell migration ability in PLC-PT cell lines, we visualized the stress fiber formation, which can promote cell migration, by phalloidin staining. In order to confirm our finding in other HCC cell lines, we perform both wound healing assay and phalloidin staining in Huh7 and Hep3B cell lines. We observed higher cell migration ability with increase in stress fiber formation upon miR-106b over-expression. We demonstrated that over-expression of miR-106b can increase stress fiber formation, hence, enhancing migration ability in vitro.To investigate the functional role of miR-106b, we knocked down miR-106b in the lung metastasis cell line with high miR-106b expression and over-expressed miR-106b in the primary tumor cell lines with low miR-106b expression. LNA knock-down in vivo metastatic potential. We observed lung metastatic nodules in 80% of mice inoculated with miR-106b over-expressing cells, whereas only 20% mice developed lung metastasis in the empty vector control group. This showed that miR-106b expression could promote formation of lung metastatic nodules in mice, further corroborating a critical role of miR-106b in development of HCC metastasis.PLC-PT cell line with stable over-expression of miR-106b was orthotopically implanted into the mice to examine the in vitro studies demonstrated miR-106b over-expression can increase cancer cell motility by inducing stress fiber formation. Since RhoA and RhoC are essential RhoGTPases involved in stress fiber formation and cell migration, expression level of RhoA and RhoC were analyzed. Our results demonstrated that miR-106b over-expressing cells, PLC-LM, have higher expression level for RhoA and RhoC than the PLC-PT. Knock-down of miR-106b significantly down-regulate RhoA and RhoC expression, but not in the scramble control. This indicated that expression of miR-106b can indirectly regulate the expression of RhoGTPases. This observation can be confirmed by up-regulation of RhoGTPases in miR-106b over-expressing transfectant, PLC-PT/106b+, Huh7/106b+, and Hep3b/106b+. Up-regulation of RhoA and RhoC are essential for cell migration and cancer metastasis in various cancers in vitro and metastasis development in vivo were affected by the indirect regulation of RhoA and RhoC through miR-106b regulation.RhoGTPases, RhoA and RhoC, are reported to be involved in regulation of actin polymerization and organization of actin cytoskeleton resulting in stress fiber formation in vitro and in vivo data supported this hypothesis. With higher miR-106b expression, the cells exhibited higher metastatic potential. When miR-106b expression was down-regulated, expression of epithelial marker, E-cadherin, increased while mesenchymal markers, N-cadherin, Vimentin, and TWIST1, decreased. Taken together, our data suggest a role of miR-106b in metastasis of HCC via activation of EMT process and enhancement of cell motility. This is the first study reporting the role of miR-106b in HCC metastasis and implies a potential strategy to inhibit metastasis in HCC by targeting miR-106b.Previous studies have already demonstrated that miR-106b was involved in various important signaling pathways such as PTEN tumor-suppressive pathway Figure S1QPCR analysis in cell lines and clinical sample. (A) miR-106b expression in HCC cell lines is higher than immortalized normal hepatocyte cell line MIHA. (B) Higher miR-106b expression was observed in metastatic cell lines than the primary tumor cell lines. (C) miR-21 and (D) miR-106b expression were analyzed in HCC clinical sample by QPCR. Expression of miR-21 and miR-106b was significantly higher in HCC tumor tissue. (E) Successful knock-down of miR-106b expression was attained within 4 days after transfection. (F) Over-expression of miR-106b in PLC-PT, Huh7 and Hep3B cell lines can be attained by lentiviral transduction by comparing with the empty vector control.(TIF)Click here for additional data file.


In vitro and in vivo assays uncovered the involvement of SOCS2 in the regulation of cell growth and apoptosis. Functionally, SOCS2 knockdown inhibited PCa cell proliferation and xenograft growth in a CAM assay. Decreased cell growth after SOCS2 downregulation was associated with cell-cycle arrest and apoptosis. In addition, we proved that SOCS2 expression is significantly elevated upon androgenic stimulation in androgen receptor (AR)-positive cell lines, providing a possible mechanistic explanation for high SOCS2 levels in PCa tissue. Consequently, SOCS2 expression correlated with AR expression in the malignant tissue of patients. On the whole, our study linked increased SOCS2 expression in PCa with a pro-proliferative role in vitro and in vivo.Deregulation of cytokine and growth factor signaling due to an altered expression of endogenous regulators is well recognized in prostate cancer (PCa) and other cancers. Suppressor of cytokine signaling 2 (SOCS2) is a key regulator of the GH, IGF, and prolactin signaling pathways that have been implicated in carcinogenesis. In this study, we evaluated the expression patterns and functional significance of SOCS2 in PCa. Protein expression analysis employing tissue microarrays from two independent patient cohorts revealed a significantly enhanced expression in tumor tissue compared with benign tissue as well as association with Gleason score and disease progression.  Prostate cancer (PCa) represents the most common malignancy in men in the Western world . FurtherSOCS proteins have originally been described as the feedback inhibitors of cytokine-induced Janus kinase (JAK/STAT) signaling  and are Socs2 knockout as well as transgenic mice have been reported to display gigantism  (Innsbruck-TMA) containing tissue cores obtained from 90 PCa patients who underwent radical prostatectomy at the University Hospital Innsbruck was constructed and immunohistochemically stained as described elsewhere  assay were assessed using a quantitative duplex PCR. PCR composition and thermal cycling were used as described previously  and cultured as indicated. The DUCaP-R subline was established by cultivation in the presence of 1 nM R1881  following the manufacturer's instructions. Western blotting was carried out as described previously  or luciferase (shLuc) . The generation and transfection of constructs have been described in pCMV6-AC-GFP and pCMV-AC-GFP-SOCS2 plasmids were obtained from Origene . The cells were transfected with DNA (0.5 μg/six wells) using 3 μl of Fugene HD (Roche) for 48 h. For stable overexpression, the cells were selected with 0.5 mg/ml of G418 (neomycin) for 7 days.3H]thymidine incorporation assay as described in Proliferation was assessed using thymidine incorporation and clonogenic assays revealed a significant decrease in both proliferation .In the present study, we investigated SOCS2 expression patterns in benign and low- and high-Gleason score tumor samples as well as in prostate cell lines. Differences between SOCS2 mRNA and protein levels observed in the cell lines are not surprising, because SOCS2 protein is highly unstable as a consequence of active degradation (in vivo. Secondly, SOCS2 knockdown results in a substantial decrease in cell proliferation and clonogenicity in AR-negative and -positive cell lines. We could link this growth inhibition to S-phase and G2/M-phase cell-cycle arrests with a slight increase in apoptosis in AR-negative PC3 and DU145 cells. Interestingly, SOCS2-depleted LNCaP cells exhibit a dramatic increase in apoptosis, suggesting an additional role for SOCS2 in AR-positive cells, which renders them more sensitive to SOCS2 knockdown.In concordance with the tissue expression profile, our functional assays point to a potential growth-promoting activity of SOCS2 in PCa. First, SOCS2 downregulation leads to a significantly diminished tumor growth Socs2 knockout and transgenic mice display gigantism (SOCS2 knockdown in PCa cells resulted in growth inhibition.The concept of SOCS2 as a growth promoter rather than as an inhibitor is supported by data obtained from other studies. Both igantism . We thervice versa (In addition, we proved that SOCS2 is upregulated by androgens in AR-positive cell lines. SOCS2 is induced upon androgenic stimulation in a dose- and time-dependent manner at both mRNA and protein levels. Androgen responsiveness of SOCS2 was further confirmed in an experimental setting, where androgens were depleted. Although we cannot exclude alternative mechanisms, a regulation via STAT5 might be a possible explanation. AR increases the transcriptional activity of STAT5 and ce versa .in vitro and in vivo. Furthermore, we demonstrated androgen responsiveness of SOCS2 in AR-positive cell lines, providing a mechanistic explanation for the high SOCS2 expression in PCa via androgenic stimulation. These findings render SOCS2 an interesting candidate for further investigations to clarify specific pathways involved in SOCS2-mediated effects on PCa cell growth.While this manuscript was in preparation, http://dx.doi.org/10.1530/ERC-13-0446.This is linked to the online version of the paper at in vitro experiments except the Affymetrix analysis and wrote the manuscript. J Kern and E Gunsilius carried out and analyzed the CAM assay. P Ofer established shSOCS2 and shLuc LNCaP cell lines. I E Eder and J Rainer conducted the Affymetrix experiment. G Schäfer scored IHC of the Innsbruck-TMA in cooperation with MP. D Dietrich and G Kristiansen planned and coordinated the methylation analysis and IHC staining of the Bonn-TMA. S Geley cloned the plasmids for shRNA transfection. H Klocker was responsible for the generation of the Innsbruck-TMA. Z Culig supervised J Hoefer and helped writing the manuscript. M Puhr supervised J Hoefer, established shSOCS2 and shLuc PC3 and DU145 cell lines, organized the in vivo experiments, analyzed the Innsbruck-TMA and helped writing the manuscript. In addition, all the co-authors improved the manuscript and approved its final version. M Puhr and Z Culig are joint corresponding authors.J Hoefer carried out all the