Taxanes such as paclitaxel and docetaxel are used successfully to treat breast cancer, usually in combination with other agents. They interfere with microtubules causing cell cycle arrest; however, the mechanisms underlying the clinical effects of taxanes are yet to be fully elucidated.in vitro. We used aCGH analysis to identify mechanisms driving taxane resistance.Isogenic paclitaxel resistant (PACR) MDA‒MB‒231, paclitaxel resistant ZR75‒1 and docetaxel resistant (DOCR) ZR75‒1 cell lines were generated by incrementally increasing taxane dose in native cell lines Taxane resistant cell lines exhibited an 18-170 fold increased resistance to taxanes, with the ZR75-1 resistant cell lines also demonstrating cross resistance to anthracyclines. Paclitaxel treatment of native cells resulted in a G2/M block and a decrease in the G1 phase of the cell cycle. However, in the resistant cell lines, minimal changes were present. Functional network analysis revealed that the mitotic prometaphase was lost in the resistant cell lines.This study established a model system for examining taxane resistance and demonstrated that both MDR and mitosis represent common mechanism of taxane resistance.The online version of this article (doi:10.1186/1471-2407-14-762) contains supplementary material, which is available to authorized users. Breast tumours exhibit a wide degree of heterogeneity and diversity at both the cellular and molecular level. The taxanes, paclitaxel and docetaxel, are used successfully to treat breast cancer, alone or in combination with other agents. Taxanesin vivo, we run the risk of continuing to seek to identify novel drugs and molecular diagnostics in a stochastic and largely unfocused manner.Progress in “targeting” conventional therapeutics such as anthracyclines and taxanes has been slow and has been hampered, in part, by a lack of focus and understanding of the key molecular events that lead to drug response or resistance in the clinical setting. Without significant progress in identifying the key molecular pathways driving drug resistance Genome wide profiling of breast tumours is a powerful tool that can be used to correlate tumour characteristics to clinical outcome in patients. Many extensive studies have proposed novel and molecular subtypes of breast cancer which may have clinical relevance–12. HoweThe overall aim of this study was to generate isogenic taxane-resistant breast cancer cell lines and elucidate the mechanisms that are driving resistance to taxanes in a pre-clinical model system. The studies summarised here characterise taxane resistant cell lines derived by the incremental increase of paclitaxel or docetaxel dose. The results presented demonstrate the ZR75-1 resistant cell line harbour cross-resistance to anthracyclines. An aCGH profile demonstrated a loss of mitotic pathways in the resistant cell lines indicating a potential theranostic pathway.The breast cancer cell lines MDA-MB-231 and ZR75-1  were cultured as monolayer in DMEM supplemented with 10% foetal calf serum, 10 mM glutamine and penicillin and streptomycin. Paclitaxel , docetaxel , epirubicin , doxorubicin  and carboplatin  were dissolved in dimethyl sulphoxide (DMSO) . Concentrated stock solutions were stored at -20°C. Drug resistant isogenic daughter cell lines were derived by incremental increases in drug concentrations over time until a stable taxane resistant phenotype was acquired. Cells were in each concentration of drug for two passages and until confluent, this ranged between 1-4weeks dependent on the dose. The following isogenic sub-lines were selected for further characterisation alongside each parent line: MDA-MB-231 25nM and 50nM paclitaxel resistant (MDA-MB-231 25PACR and MDA-MB-231 50PACR), ZR75-1 25nM and 50nM paclitaxel resistant  and 25nM and 50nM docetaxel resistant .Dose response curves were set up by treating cells with increasing doses of the appropriate taxane: 0, 0.3, 1, 3, 10, 30, 100, 300, 1000 or 3000nM of either paclitaxel or docetaxel. Cross resistance to epirubicin, doxorubicin and carboplatin was assessed in a similar manner. Cell suspensions (100μl) were seeded in triplicate at a density of 30,000 cells/ml in 96 well plates and grown for 24 hours, washed and treated with drug for 72 hours. After 72 hours 100μl of growth media containing 10μl of CCK8  was added to each well for 3 hours at 37°C. The plates were then shaken for 10 minutes and optical density (OD) recorded at 450nm. IC50s were calculated using GraphPad Prism 5 . Stability of taxane resistance in MDA-MB-231 25PACR was assessed by maintaining the cells for 6 months with or without paclitaxel added to the growth medium. MDA-MB-231 parental cells were maintained without paclitaxel for an equivalent period for comparison.For cell cycle and DNA content analyses, native and resistant cells were plated in equal numbers into 6-well plates and synchronized by serum starvation overnight. Cells were then incubated with the appropriate concentration of taxane (25nM or 50nM of either docetaxel or paclitaxel), DMSO control or media alone control. The cells were collected after 24 and 48 hours, fixed with 80% ethanol and incubated with 2mg/ml RNase A  and 0.1mg/ml propidium iodide  for 30 minutes prior to analysis by flow cytometry. Data was collected by FACS Canto II and FACS Diva , and analyzed by FlowJo .DNA was extracted from cells using the Qiagen Blood and Cell Culture Maxi kit . DNA was stored in TE buffer pH 8.0 at 4°C.Cell line DNA was analysed on the Breakthrough Breast Cancer human CGH 4.6K 1.12 arrays as previously described. BrieflySlides were scanned using an Axon 4000B scanner  and images were analysed using Genepix Pro 4.1 software (Axon Instruments). The median localized background slide signal for each clone was subtracted and each clone Cy5/Cy3 ratio subjected to print-tip loess normalization. Dye swaTo examine whether genes showing common copy number gains or copy number losses across all three cell lines belong to a specific pathway, we conducted functional analysis of the common genes using Cytoscape Reactome Functional Interaction (FI) plugin in Cytoscape 3.0.2 (2013 FI network version). Genes were loaded using the gene set format with FI annotations and linker genes. Spectral clustering was performed to identify distinct network modules and subsequent pathway enrichment was calculated. Symbols were loaded as a gene set and interactions from the FI network 2012 version, including FI annotations and linker genes. Network modules were identified using spectral clustering and Pathway Enrichment computed for each module using the Reactome FI plugin functions. Reactome pathways exhibiting FDR values < 0.01 were considered enriched.5 ZR75-1 25PACR cells were transfected with Lipofectamine RNAiMAX  and siRNAs  targeting MDR1, according to manufacturer’s instructions. As controls, transfection reagents without siRNAs were added (mock transfection) and cells were transfected with siRNA targeting GAPDH. After 48h cells were lysed for RNA analysis and 72h cells were lysed for protein analysis. The differences in IC50 were analysed and calculated as described above.A total of 2.6 × 10Total protein lysates (20 μg) were separated by SDS-PAGE according to standard protocols and immu50s were compared to those from parental cell lines, with cross resistance to both forms of taxane observed in all cell lines  culture of MDA-MB-231 25PACR cells in the absence of drug, cells were re-challenged with taxanes and the responses compared to parental and resistant cells cultured in the presence of taxanes


The soluble factors secreted by mesenchymal stem cells are thought to either support or inhibit tumor growth. Herein, we investigated whether the human lung-derived mesenchymal stem cell-conditioned medium (hlMSC-CM) exerts antitumor activity in malignant pleural mesothelioma cell lines H28, H2052 and Meso4.hlMSC-CM was collected from the human lung-derived mesenchymal stem cells. Inhibition of tumor cell growth was based on the reduction of cell viability and inhibition of cell proliferation using the XTT and BrdU assays, respectively. Elimination of tumor spheroids was assessed by the anchorage-independent sphere formation assay. The cytokine profile of hlMSC-CM was determined by a chemiluminescence-based cytokine array.Our data showed that hlMSC-CM contains a broad range of soluble factors which include: cytokines, chemokines, hormones, growth and angiogenic factors, matrix metalloproteinases, metalloproteinase inhibitors and cell–cell mediator proteins. The 48- and 72-hour hlMSC-CM treatments of H28, H2052 and Meso4 cell lines elicited significant decreases in cell viability and inhibited cell proliferation. The 72-hour hlMSC-CM incubation of H28 cells completely eliminated the drug-resistant sphere-forming cells, which is more potent than twice the half maximal inhibitory concentration of cisplatin.Our findings indicate that the cell-free hlMSC-CM confers in vitro antitumor activities via soluble factors in the tested mesothelioma cells and, hence, may serve as a therapeutic tool to augment the current treatment strategies in malignant pleural mesothelioma.The online version of this article (doi:10.1186/s13287-016-0282-7) contains supplementary material, which is available to authorized users. In contrast to the early paradigm of cell replacement and differentiation of mesenchymal stem cells (MSCs) as a therapeutic mechanism, reports are accumulating that cell secretions are responsible for their beneficial effects . MSCs arWhen MSCs arrive at a tumor niche, they produce a variety of soluble factors that either positively or negatively influence tumor growth . SeveralMPM is a highly aggressive, chemoresistant lung cancer with a median survival of <1 year after diagnosis , 9. Thus2.The H28 and H2052 mesothelioma cell lines  were cultured in RPMI 1640  containing 10 % fetal bovine serum  and 1 % penicillin/streptomycin . ACC-Meso-4 cell line was obtained from Riken Cell Bank, Resource No: RBRC-RCB2293  and cultured using the above-mentioned culture medium. Cells were maintained at 37 °C, 95 % humidity and 5 % COWe have previously identified human lung parenchyma mesenchymal stromal cells, which are referred as human lung-derived mesenchymal stem cells (hlMSCs) in this study. hlMSCs were isolated by using our established protocol as reported in our previous work , which i4 in 10 cm cell culture dish containing MCDB 201 supplemented with ITS, 1 % antibiotic/antimytotic reagent (Hyclone), 1 % FBS and 20 ng/μl endothelial growth factor at 37 °C in a humid atmosphere with 5 % CO2. Upon reaching 70–80 % confluency, cells were washed once with phosphate-buffered saline (PBS) and then re-incubated with a serum-free MCDB 201 medium for 24 hours. The CM (supernatant) was then collected and centrifuged at 1200 × g for 10 minutes to remove cell components and was sterilized by passing through a 0.2-μm cell filter . Aliquots were subjected to cytokine array analysis or frozen at −80 °C for future applications. hlMSC-CM was used as it was, without dilution or addition of serum in all of the experiments in this project.Conditioned medium (CM) was collected from hlMSCs at passage 3. Cells were cultured at a density of 5 × 10The XTT and BrdU assays were performed to assess the effect of hlMSC-CM on cell viability and cell proliferation, respectively  or twice the IC50 of cisplatin . The specified concentration corresponds to the previously determined IC50 value of the three MPM cell lines: H28 (15 μM), H2052 (13 μM), and Meso4 (12 μM) [For drug treatment, cisplatin (diamminedichloroplatinum (II); Bristol Myers Squibb, Switzerland) was used. Cells were seeded at a density of 5 × 10 (12 μM) . Followip value <0.05 was considered significant.Data are reported as means ± standard deviations. For analysis of differences in multiple groups, one-way analysis of variance and Bonferroni’s post-hoc test were performed using Prism 6.0 . A We have previously identified hlMSCs exhibiting plastic adherence, the immunophenotype and trilineage differentiation capacity consistent with the established features of MSCs , 12, 13.We studied the effect of hlMSC-CM on the proliferative activity of H28, H2052 and Meso4 using the BrdU assay. The 48- and 72-hour treatments with hlMSC-CM elicited significant reductions in cell proliferation of H28 , H2052  and Meso4  relative to the nontreated cells Fig. . We alsoIn our previous work, we found cisplatin-resistant tumor spheres in H28, H2052 and Meso4 indicating the presence of putative cancer stem cells (CSCs), which may, in part, be responsible for drug resistance . Hence, 50 and twice the IC50 of cisplatin in H28 cells. The 48-hour hlMSC-CM treatment significantly reduced the sphere-forming efficiency by 74 %, whereas the 72-hour incubation completely eliminated the tumor spheres (Fig. 50 of cisplatin, we obtained significant 50 % reductions in sphere efficiency after the 48- and 72-hour treatments (Fig. 50 of cisplatin (Fig. 50 of cisplatin. Representative images of the nontreated and hlMSC-CM-treated H28 cells are shown in Fig. We then compared the inhibitory effect of hlMSC-CM on the growth of tumor spheres with that of ICres Fig. . Using tnts Fig. , and sigtin Fig. . Our resOne beneficial effect of MSCs is their inherent antitumor capacity—a current topic of investigation because of its future role in cancer therapy. Here, we report that hlMSC-CM contains a broad range of soluble factors, which conferred inhibition of cell proliferation and a decrease in cell viability in three MPM cell lines. Importantly, hlMSC-CM completely eliminated the cisplatin-resistant, sphere-forming cells.We found a huge panel of cytokines, chemokines, growth, neurotrophic, endocrine and angiogenic factors, MMPs, TIMPs and cell–cell mediator proteins in our hlMSC-CM that elicited suppression of cell growth and reduced cell viability in the tested mesothelioma cells. These data substantiate the current paradigm that MSC-dependent beneficial effects are mediated primarily by secreted factors . EvidencMSCs have diverse effects on tumor growth. In contrast to the reported tumor-promoting capacity of MSCs, our findings showed a tumor-inhibiting effect of hlMSC-CM in the tested MPM cell lines. Tumor-promoting activity is exerted either by paracrine secretion of growth factors and antiapoptotic factors, or by differentiating into tumor-associated fibroblasts, which can enhance tumor growth, metastasis formation and therapy resistance . Adverse50 of cisplatin, it stands to reason that combining hlMSC-CM with conventional drugs may improve the current treatment modalities in MPM.Sphere formation represents one of the basic features of CSCs, a rare subpopulation within a tumor, which is supposed to be a crucial player in the initiation, invasiveness and drug tolerance in different tumors including MPM , 30–35. Our study demonstrates that hlMSC-CM exerts in vitro antitumor effects in three MPM cell lines via soluble factors, which offers a potentially useful tool to augment the standard therapy in MPM.


Cancer cell lines are essential platforms for performing cancer research on human cells. We here demonstrate that, across tumor entities, human cancer cell lines harbor minority populations of putative stem-like cells, molecularly defined by dye extrusion resulting in the side population phenotype. These findings establish a heterogeneous nature of human cancer cell lines and argue for their stem cell origin. This should be considered when interpreting research involving these model systems. Cancer has long been regarded a clonal disease. While this view still applies to certain haematological malignancies, the majority of cancers, and in particular solid tumors, follow another structure and are characterized by tremendous cellular complexity. This diIn search of a common signature for ovarian CSC, we recently analysed the expression of several CSC markers in various ovarian cancer cell lines n=13). This screen revealed that of all markers tested ), only the SP marker detecting dye-extrusion by drug transporters robustly identified minor populations in virtually all cell lines investigated  , breast (n=4), lung (n=3), colorectum (n=2) and adrenal gland n=1). These experiments revealed that the SP phenotype was again a robust marker, defining distinct populations in 4/4 cell lines of breast cancer, 2/3 cell lines of lung cancer, 2/2 cell lines of colorectal cancer, 1/1 cell line of adrenal gland cancer, and still 1/5 cell lines of prostate cancer (Table . These ede novo resistance’ might therefore often be a misinterpretation of what actually happens, that is Darwinian selection of small, pre-existing cancer clones inherently showing resistance. In this respect, cancer cell lines are therefore very similar to antibiotic-treated bacterial cultures[The high conservation of SP cells in human cancer cell lines indicates a functional role of this cell compartment. In ovarian and several other cancers for instance, we and others have shown that SP cells exhibit functional SC characteristics,4,5. Thu cultures.ex vivo tumor tissue and whether these primary cells exhibit functional SC traits as well. This could not only further our understanding of in vivo tumor evolution and biology, but could also provide rationale for developing treatment strategies against a specific SC pool conserved within the tremendous complexity of solid tumors.Altogether, we propose that phenotypic and functional heterogeneity is a hallmark of most human cancer cell lines that needs to be considered for the correct interpretation of findings obtained with these model systems. Especially for observations related to SC characteristics and drug resistance, the SP minority compartment might often be responsible. Henceforth, it will be important to elucidate whether SP cells can also be found at such high frequency in


BRAFV600 or NRASQ61 somatic mutations, leads to constitutive ERK signaling. While BRAF inhibitors are initially effective for BRAF-mutant melanoma, no FDA-approved targeted therapies exist for BRAF-inhibitor-resistant BRAFV600, NRAS mutant, or wild-type melanoma.In melanoma, dysregulation of the MAPK pathway, usually via The 50% inhibitory concentration (IC50) of SCH772984, a novel inhibitor of ERK1/2, was determined in a panel of 50 melanoma cell lines. Effects on MAPK and AKT signaling by western blotting and cell cycle by flow cytometry were determined.50) < 1 μM; intermediately sensitive, IC50 1-2 μM; and resistant, >2 μM. Fifteen of 21 (71%) BRAF mutants, including 4 with innate vemurafenib resistance, were sensitive to SCH772984. All three (100%) BRAF/NRAS double mutants, 11 of 14 (78%) NRAS mutants and 5 of 7 (71%) wild-type melanomas were sensitive. Among BRAFV600 mutants with in vitro acquired resistance to vemurafenib, those with MAPK pathway reactivation as the mechanism of resistance were sensitive to SCH772984. SCH772984 caused G1 arrest and induced apoptosis.Sensitivity fell into three groups: sensitive, 50% inhibitory concentration  contains supplementary material, which is available to authorized users. BRAF mutational status. Approximately 50% of all melanomas contain an activating BRAFV600, a serine-threonine kinase which functions as an oncogenic driver. This constitutively activates the MAPK pathway via sequential phosphorylation of MEK and then ERK. Vemurafenib and dabrafenib, two type I RAF inhibitors, selectively inhibit BRAFV600, leading to improvements in both progression free survival and overall survival for patients with this disease  polymerase (PARP). After fixation and permeabilization as described above, cells were stained with anti–PARP-Alexafluor700 antibody . A minimum of 12,000 cellular events per sample were collected by flow cytometry. All flow cytometry experiments were carried out using an LSRII (BD Biosciences), using biexponential axes. Data were analyzed using FlowJo .50). Synergistic, additive or antagonistic effects of drug combinations were determined using Calcusyn software  to determine the combination indices (C.I.) as previously described [Data analysis was performed with Microsoft Excel and GraphPad Prism to determine the 50% inhibition concentration . Ninteen BRAF mutant melanoma cell lines were exposed to 0–1 μM trametinib and cell viability determined by ATP-based bioluminescence assay . Results represent mean of duplicate assay performed in three independent experiments. (PDF 47 KB)Additional file 1: Figure S1: Effect of trametinib on 50 sensitivities to SCH772984 were evaluated by Western Blot analysis for baseline pAKT levels. (PDF 31 KB)Additional file 2: Figure S2: Levels of pAKT across a group of BRAF, NRAS and double wild-type melanoma cell lines. 8 BRAF-mutant, 2 NRAS-mutant and 2 wild type melanoma cell lines in order of increasing IC50s of SCH722984 alone or in combination with MK-2206 or MK-8669. After 120 hours treatment with 0–10 μM SCH722984, SCH722984+ MK-2206 or SCH722984+ MK-8669, cell viability was determined by bioluminescence assay. Results are representative data in duplicate from three independent experiments (n = 6). B. Percent growth inhibition for two BRAF-mutant melanoma cell lines (M233 and M411) and two NRAS-mutant cell lines (M409 and WM1366). After 120 hours treatment with 0–10 μM SCH772984 + MK2206 , SCH722984 + MK-8669 , or the SCH772984 , cell viability was determined by bioluminescence assay. Results are representative data in duplicate from three independent experiments (n = 6). C. Effect ERK- inhibition alone or the combination with AKT/mTOR inhibitors on MAPK signaling. Cell lines were treated with DMSO , 1 uM SCH722984  or the combination of SCH722984+ MK-2206 and SCH722984+ MK-8669 at 1 uM for 24 hours. Western blots analyzed for phospho- and total ERK1/2, AKT and actin as loading control. (PDF 78 KB)Additional file 3: Figure S3: Effects of SCH722984 combined with MK-2206 (AKT inhibitor) and MK-8669 (mTOR inhibitor) in BRAF mutant cell lines. A. ICAdditional file 4: Figure S4: Effect of trametinib on NRAS mutant and double wild type cell lines. IC50 (nM). 14 NRAS mutant and 7 double wild-type melanoma cell lines were exposed to 0-1 μM trametinib and cell viability was determined by ATP-based bioluminescence assay . Results represent mean of duplicate assay performed in three independent experiments. (PDF 40 KB)Additional file 5: Figure S5: Combinatorial effect of Vemurafenib with SCH772984 or Trametinib. Percent growth inhibition of BRAF mutant cell lines. After 120 hours treatment with 0–10 μM vemurafenib (squares) combined with 0-10 uM SCH722984 (circles), or 0–10 μM vemurafenib combined with 0-1 μM trametinib, cell viability was determined by bioluminescence assay. Results are representative data in duplicate from two independent experiments (n = 6). (PDF 348 KB)Additional file 6: Figure S6: Effect of SCH722984, vemurafenib or the combination on cell cycle progression and apoptosis in BRAF-mutant melanoma cell lines. A sensitive cell line (M263), intermediate sensitivity (M255) and resistant to SCH722984 (M370) were exposed to DMSO as vehicle control (ControL), 1 μM vemurafenib (Vemurafenib), SCH722984 (ERKi), 50nM trametinib (Trametinib), the combination of 1 μM vemurafenib + 1 μM SCH722984 (V + E) or the combination of 1μMvemurafenib + 50nM trametinib (V + T) for 48 hours. A. Cell cycle progression example for M255 was tested by DAPI staining solution and induced apoptosis by cleaved PARP (PARP-Ax700). B. Apoptosis in response to MAPK inhibitors. Percentage of apoptotic cells positive for cleaved PARP (PARP-Ax700) in this three melanoma cell lines. B. Quantitative analysis of the cell cycle progression by DAPI staining using flow cytometry shows the percentage of cells in sub-G0, G0/G1, S phase, or G2/M. (PDF 199 KB)


Interestingly, the cancer-cell induced changes in the fibroblasts also occurred when there was no possible direct contact between the two cell lines in the co-culture. When contact was possible, the spectral changes were similar, suggesting that soluble factors but not direct cell-cell interactions were responsible for fibroblast activation. Overall, the results indicate that IR imaging could be used in the future for analyzing the microenvironment of breast tumors.It is well known that the microenvironment plays a major role in breast cancer progression. Yet, the mechanism explaining the transition from normal fibroblasts to cancer-stimulated fibroblasts remains to be elucidated. Here we report a FTIR imaging study of the effects of three different breast cancer cell lines on normal fibroblasts in culture. Fibroblast activation process was monitored by FTIR imaging and spectra compared by multivariate statistical analyses. Principal component analysis evidenced that the fibroblasts stimulated by these cancer cell lines grouped together and remained distinctly separated from normal fibroblasts indicating a modified different chemical composition in the cancer-stimulated fibroblasts. Similar changes in fibroblasts were induced by the various breast cancer cell lines belonging to different sub-types. Most significant changes were observed in the region of 2950 and 1230 cm In western woman, breast cancer has the highest rate of incidence and is the second major cause of mortality Stimulated fibroblasts, also known as myofibroblasts, are associated with cancer cells and known as cancer associated fibroblasts (CAFs). Earlier studies Genomic level characterization is a promising tool to provide individual treatment but remains expensive and is limited by the difficulty to achieve this characterization at cell level as tumors display extended heterogeneity The fibroblast cell line (CCD-1126Sk) established from a normal human breast was obtained from the American Type Culture Collection (ATCC). Breast cancer cell lines MCF-7, MDA-MB-321, SK-BR-3 were also obtained from ATCC. The characteristics of these cell lines are presented in 2 at 37°C All cell lines were grown in the conditioned media (CM) which contains: IMDM supplemented with 10% FCS, and 1% penicillin-streptomycin in a humidified incubator supplied with 5% CO2 window (transparent to IR) and cancer cell lines on the bottom of a 12 well plate culture box. Both cell types were first grown for 6 hours in different wells to let the cells settle on the surface. In a second step, the CaF2 window with the fibroblasts attached to it were transferred into the wells where cancer cells were growing. The length of the fibroblasts was measured by the Leica Microsystems microscope imaging software. Prior to FTIR measurements the cells were briefly washed on CaF2 window by distilled water. Drying was obtained either by using a gentle dry air flow or by letting the cell dry with the CaF2 windows put vertically. Visual inspection under the microscope indicated that the cells were intact in both cases. Furthermore, the IR spectra collected in the space between the cells did not contain organic materials which could indicate cell leakage. Even though it has been shown that fixation by drying or by formaldehyde followed by inclusion into paraffin largely preserves spectral differences 2 substrate. In turn, cell shape was partially preserved. For the few experiments where contact between fibroblasts and cancer cells was allowed, the two cell types were let to settle together on the CaF2 window. In all cases, the cells dried on CaF2 window were examined immediately. Storage at room temperature for 3-5 days neither changed their visual aspect nor their infrared spectra.We designed a method for co-culture the fibroblasts with different breast cancer cell lines see  with eit2 FPA  detector, with a 15× magnification. The spectral resolution was set to 8 cm−1. The data were acquired in transmission mode from sample regions of 170*170 µm2 for each unit image. Transmission mode is less prone to spectral distortion than transflection mode 2. One FTIR unit image resulted in 4,096 spectra, each one being the average of 256 scans recorded in about 5 minutes. The experiments were performed at room temperature using the Bruker spectrometer software OPUS. Each single beam spectrum was ratioed against a background spectrum obtained in the absence of sample and converted to absorbance by OPUS. Mapping larger areas  were usually achieved. All spectral processing and classifications were carried out using the program Kinetics written by our group and running under Matlab . All the spectra were preprocessed as described previously −1 as reference peak. Spectra were normalized for equal area between 1725 and 1481 cm−1. An 11-point baseline passing by 3620 2995 2800 2395 2247 1765 1724 1480 1355 1200 cm−1 was subtracted The FTIR data were acquired using a Hyperion 3000 FTIR imaging system  equipped with a liquid nitrogen cooled 64*64 MCT (Mercury-Cadmium-Telluride) 2560×2560 µmPrincipal Component Analysis (PCA) is an unsupervised multivariate method, enabling a variable reduction by building linear combinations of spectral absorbances, called Principal Component (PC), that co-vary in the selected series of spectra. The first principal component explains most of the data variance. The second principal component, uncorrelated to the first one, accounts for most of the residual variance and so on. The projection (score) of each spectrum on the different PCs indicates the importance of the contribution of that PC to the spectrum. Student's t-test was computed at every wavenumber with a significance level of α = 0.001 and a Bonferroni correction for multiple comparisons. Each marked red star indicates a statistically significant difference between the means.2 window in the presence or absence of a cancer cell line but with no direct contact between the cell types as explained in Fibroblasts were grown for 48 hours on CaF−1 is reported in −1 for fibroblasts grown with no direct contact with MCF-7 cells. A bright field microscopy corresponding image is reported in A typical raw IR image representing the absorbance at 1654 cm−1 as well as some features including amide I and amide II ranges. The major changes observed at 2950 and 1230 cm−1 could originate from νas(CH3) and P-O stretching vibrations tentatively assigned to lipids and nucleic acids respectively. The ν(CH3) (ca 2950 cm−1) to ν(CH2) (ca 2920 cm−1) ratio is an index of lipid acyl chain length Typical spectral markers illustrating the amplitude of the changes induced by the various cancer cell lines are reported in −1 could arise from the phosphate backbone of nucleic acid. The higher phosphate absorption in stimulated fibroblast potentially indicates a less compact nucleic acid materials related to increased nucleic acid synthesis activity. It has been demonstrated that the very compact DNA has an overall decreased absorbance, not because of the presence of less DNA molecules but mainly because of the local high concentration and loss of Beer law linearity The biological significance of these results is difficult to assess. As spectra were all scaled on a protein band, the increase in lipid and phosphate band intensity is relative to a same protein amount. Modification of lipid synthesis has been widely demonstrated in cancer cells FTIR spectroscopy imaging is providing spatially resolved chemical information which could add a significant value to current cancer diagnostic assessment. We observed here that fibroblasts grown alone experience very significant modifications when placed in the presence of breast cancer cell lines. In addition to changes in their shape and size, IR imaging and statistical analyses revealed that three different cancer cell lines which have different characteristic in terms of cancer classification  and fibroblasts co-culture respectively with MCF7 (B) and MDA-MB-231 (E) cancer cell line grown on CaF2 window in conditioned media (IMDM). In co-cultures there was no direct contact with the cancer cells. Arrows in B indicate markedly elongated cells due to effects of MCF7 on fibroblasts. C and F report a statistical analysis of the length of the fibroblasts in the absence and in the presence of cancer cells respectively. Error bars indicate plus or minus one standard deviation.(TIF)Click here for additional data file.


TP53 mutations represent a node of divergence in epithelial ovarian cancer histologic subtypes and may represent a therapeutic opportunity in subtypes expressing wild type, including most low-grade ovarian serous carcinomas, ovarian clear cell carcinomas and ovarian endometrioid carcinomas, which represent approximately 25% of all epithelial ovarian cancer. We therefore sought to investigate Nutlin-3a—a therapeutic which inhibits MDM2, activates wild-type p53, and induces apoptosis—as a therapeutic compound for TP53 wild-type ovarian carcinomas. Fifteen epithelial ovarian cancer cell lines of varying histologic subtypes were treated with Nutlin-3a with determination of IC50 values. Western Blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses quantified MDM2, p53, and p21 expression after Nutlin-3a treatment. DNA from 15 cell lines was then sequenced for TP53 mutations in exons 2-11 including intron-exon boundaries. Responses to Nutlin-3a were dependent upon TP53 mutation status. By qRT-PCR and WB, levels of MDM2 and p21 were upregulated in wild-type TP53 sensitive cell lines, and p21 induction was reduced or absent in mutant cell lines. Annexin V assays demonstrated apoptosis in sensitive cell lines treated with Nutlin-3a. Thus, Nutlin-3a could be a potential therapeutic agent for ovarian carcinomas expressing wild-type TP53 and warrants further investigation.Epithelial ovarian cancer is a diverse molecular and clinical disease, yet standard treatment is the same for all subtypes.  TP53. These histologic subtypes have distinct molecular origins, with correspondingly diverse and specific clinical behaviors; they are treated with the same regimen as high-grade SOC. For example, 96% of high-grade SOCs harbor TP53 mutations [TP53 appear to be more chemo-resistant [ARIDIA and PI3K aberrations, which often originate from endometriosis [KRAS or BRAF mutations and may be derived from serous borderline ovarian tumors [While ovarian cancer is the ninth most common cancer in the United States, it is the sixth most deadly: approximately 22,000 new cases of ovarian cancer are diagnosed annually, with 14,270 attributable deaths . Althougn tumors . Clinican tumors . Those wn tumors .TP53 pathway. Nutlin-3a is a small-molecule, murine double minute (MDM2) antagonist that inhibits MDM2-p53 interactions and stabilizes the p53 protein, thereby inducing cell cycle arrest and apoptosis [TP53 wild-type ovarian carcinomas.Despite these discrepancies in molecular origins, mutational characteristics, chemo-sensitivity, and overall clinical behavior, the primary standard treatment remains the same for all histologic subtypes: platinum and taxane-based chemotherapy. The Gynecologic Oncology Group (GOG) has recently established a “Rare Tumor Committee” to develop and conduct definitive phase II trials for non-high grade serous ovarian cancer especially for low-grade serous and clear cell carcinomas. Innovative therapies are needed to improve the outcomes in these patient cohorts, and one obvious node of distinction between subtypes is the poptosis . We therTP53-mutant cell line which does not express TP53 at the protein or mRNA level and was therefore used as a negative control [KRAS mutation, cell line MPSC1 contains a BRAF mutation, and cell line TOV21G contains a PIK3CA mutation (data not shown). TOV21G, SKOV3, OVCAR-3 and TOV112D were obtained from ATCC (American Type Culture Collection). MDA2774 [2 and cultured in RPMI 1640 media supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. For Western Blot and RT-PCR analyses, cell lines were treated with Nutlin-3a at their predetermined IC50, and protein and RNA were extracted at 24, 48, and 72 hours of treatment in conjunction with untreated controls. Media was exchanged with fresh Nutlin-3a every 24h.A total of 15 ovarian carcinoma cell lines were cultured: two low-grade serous (HOC7 and MPSC1); three clear cell ; five endometrioid ; three mucinous ; and two high-grade serous . The SKOV3 cell line is an established  control . HOC-7 [ control  was a gi MDA2774  was a gi MDA2774  was obta MDA2774 . OVCA420 MDA2774  were gif3 cells per well in 96-well plates. After 24h, media was exchanged and cells were treated with incremental concentrations of Nutlin-3a . After 72h of incubation, WST-1  was added to each well, and a microplate reader  was used at an absorbance of 450 nm to measure the number of remaining viable cells. Experiments were repeated with smaller titrations of Nutlin-3a as needed to determine the exact IC50 of cell lines. The IC50 was defined as the concentration at which a 50% reduction in cell viability occurred, which was calculated using Microsoft Excel 2010. Cell lines were again plated in a manner identical to above and treated with Nutlin-3a at their respective IC50, and WST-1 was added with cell viability measurement at 24, 48, and 72h.All 15 cell lines were plated at a density of 1 × 10TP53 were then sequenced for mutation analysis in all samples via Sanger Sequencing at the MD Anderson Sequencing and Microarray Facility using the BigDye Terminator v3.1 Cycle Sequencing Kits and the 3730xl DNA Analyzer . Sequences were then analyzed using both Finch TV v1.3.1 and Lasergene SeqMan Pro. The primer sequences for TP53 sequencing are listed in the DNA was extracted from cell lines according to manufacturer’s instructions using the Invitrogen Purelink Genomic DNA Mini Kit . DNA was amplified by polymerase chain reaction (PCR), and PCR products were then purified using the Invitrogen Purelink PCR Purification Kit. Exons 5–8 and exon 10 of 50 dose via Western blot analysis. Protein lysates from cell cultures were extracted with radioimmunoprecipitation assay (RIPA) buffer and quantified by Bradford method. Electrophoresis of lysates (10 μg) was carried out on a 10% sodium dodecyl sulfate-polyacrylamide gel, followed by electroblot transfer onto a PVDF membrane. After blocking in 5% nonfat milk in phosphate-buffered saline (PBS) for 1 h, the membranes were probed with the following primary antibodies: Anti-mouse p21 , anti-mouse p53 , and β-actin  were dissolved in PBS with 5% bovine serum albumin (BSA), added to the Western blots, and incubated overnight at 4°C. The blots were then rinsed and incubated with IR-dye 680–conjugated secondary antibodies . Membranes were then imaged using the LI-COR Odyssey Infrared Image Detection System  at 700 nm and 800 nm.All 15 cell lines were examined for protein expression of p21 and p53 after treatment with Nutlin-3a at the IC50 concentrations as described above, total RNA was extracted with the Ambion Minikit  and cDNA was synthesized  according to manufacturer’s instructions. Using TaqMan primer sets for p53, p21 (cyclin-dependent kinase inhibitor 1), and MDM2, qRT-PCR was performed in triplicate with the housekeeping gene cyclophilin  as normalizer. The Bio-Rad C1000 Thermal Cycler  was used for all reactions, and fold-change was calculated with the 2-△△Ct method.Gene expression of p53, p21 and MDM2 was determined by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). After treatment at IC50 and apoptosis was quantified at 24 hours of treatment with corresponding controls. Cells were centrifuged and the supernatant was removed. Annexin V APC  was added to samples and incubated in the dark for 15 min, followed by washing with binding buffer and resuspension. The FACSAria cell sorter  was used to quantify Annexin V expression; data were analyzed with FlowJo v7.6.5.The two most sensitive cell lines  were treated with Nutlin-3a at their respective ICSPSS 15.0 for Windows  was used to perform statistical analyses. A nonparametric Mann-Whitney test was used to assess the statistical significance of the differences in messenger RNA expression between RT-PCR samples. P values <0.05 were considered to be statistically significant.TP53 and known Nutlin-3a sensitivity were downloaded from the Cancer Cell Line Encyclopedia study [Gene expression data (Cel files) from 159 cancer cell lines with wild-type ia study . Cel filia study .50 of 38 μM to Nutlin-3a. The DNA sequences of exon 2–11 of TP53 in the tested cell lines and their sensitivity to Nutlin-3a were determined (TP53 mutations. The fluorescent sequencing chromatograms for the detected mutations for these nine cell lines are provided in the TP53 mutant cell lines were quite resistant to Nutlin-3a (IC50 = 20 to >70 μM). Three cell lines  with wild-type TP53 were highly sensitive to Nutlin-3a (IC50 = 4 to 6 μM). HOC-7 is a low-grade SOC cell line with a known KRAS mutation [50 = 20 μM); correspondingly, this cell line was found to have a heterozygous TP53 in-frame deletion (p.G154_S166delGTRVRAMAIYKQS) in exon 5 in this study. The Nutlin-3a sensitive OVCA429 cell line is an ovarian clear cell cell line, which has been shown to form clear cell adenocarcinoma when injected intraperitoneally in nude mice [PIK3CA mutation, which is frequently activated in ovarian clear cell carcinomas [TP53 wild-type, were relatively more sensitive to growth inhibition with Nutlin-3a (IC50 = 14 and 25 μm respectively) than the TP53 mutant cell lines. The Nutlin-3a sensitive A2780 cell line is an endometrioid-like as should not be considered as a high grader serous ovarian cancer cell line as discussed recently. A2780 carries PTEN, PIK3CA and ARID1A mutations [TP53 exon 3 and exon 4 without affecting the exons (TP53 mutations and were Nutlin-3a resistant. The mucinous ovarian cancer cell lines appeared to be most resistant to Nutlin-3a (IC50 = 40 to > 70 μm). All these mucinous cell lines carried a TP53 mutation. While RMUG-S and RMUG-L have missense mutations, MCAS cell line has a homozygous 127 bp deletion affecting the TP53 exon 4  identified in this study were generated to further delineate up-regulated genes that might be relevant to ovarian cancer cells. The gene expression profiles of three Nutlin-3a sensitive ovarian cancer cell lines  were compared to six Nutlin-3a resistant ovarian cancer cell lines . The 208 significantly up-regulated genes in Nultin-3a resistant ovarian cancer cell lines are listed in To identify genes that might be responsible for Nutlin-3a resistance, comparison of the gene expression profiles between 23 Nutlin-3a sensitive cell lines and 136 Nutlin-3a resistant cell lines were performed. The list of cell lines with wild-type TP53 harbors a set of diverse and complex functions which protect the cell from genomic damage and ensure genomic integrity. The “protective” functions appear to occur with low levels of wild-type p53; only at higher levels of activity does p53 act to terminate cell proliferation and induce apoptosis [TP53 activity is an attractive anti-cancer strategy, as TP53 is altered in ~50% of human cancers [TP53 mutations are virtually ubiquitous in high-grade serous ovarian carcinomas; however, this is not the case for other EOCs.Deemed “the guardian of the genome,” the tumor protein 53 gene poptosis . Natural cancers . TP53 muet al [TP53 activity. Others have validated the activity of Nutlin-3a in neuroblastoma, T-cell lymphoma, gastrointestinal stromal tumors [TP53 mutant. In our study, we demonstrate that Nutlin-3a has activity in TP53 wild-type ovarian carcinomas, requires an intact p53 pathway for efficacy, increases p21, and results in apoptosis. We have previously documented the utility of Nutlin-3a in low-grade serous ovarian carcinoma, with up-regulation of cell cycle control, and apoptosis genes including CDKN1A, CDKN2A, PERP, and PUMA [TP53 wild-type epithelial ovarian carcinomas which includes a low-grade SOC cell line HOC-7, an endometrioid-like cell line A2780 and a clear cell cell line OVCA429 and demonstrate that it directly enhances apoptosis.Nutlin-3a belongs to a class of compounds initially described by Vassilev et al  and funcl tumors , sarcomal tumors , renal cl tumors , and coll tumors , among ol tumors . While tand PUMA . Here, wTP53 were also relative sensitive to Nutlin-3a. On the other hand, all the mucinous cell lines had TP53 mutation and were extremely resistant to Nutlin-3a. Advanced-stage mucinous ovarian carcinomas portend a poorer prognosis than their epithelial counterparts and are chemoresistant [in vivo model; these investigations are currently ongoing.Two clear cell cell line (OVAS and TOV21G) with wild-type esistant , and conTP53  in the DNA binding domain. It is possible that Y126C is not a dominant negative mutant and the wild-type p53 is still functional to induce p21 protein expression in the presence of Nutlin-3a. On the other hand, RMUG-S had a homozygous TP53 mutation (A347V) in the tetramerisation motif and the mutant protein might still have DNA binding activity that could activate p21 protein expression. Logically, it follows that addition of cytotoxic agents may improve chemo-sensitivity—this has been demonstrated in colon, breast, and hepatocellular carcinoma cell lines, as well as melanoma and sarcoma [While inherent resistance to Nutlin-3a exists in utations  or overeutations . Others ultin-3a . As show sarcoma .TP53  and a relative lack of success with targeted agents in this field, we assert that further clinical investigation into the utility of Nutlin-3a in TP53 wild-type epithelial ovarian carcinomas is warranted.In the clinical arena, at least six phase I trials employing Nutlin-3a have been recently completed in hematologic malignancies, solid tumors, and in combination with doxorubicin in sarcomas . Preliminary clinical data indicate that RG7112  appears to be well-tolerated in patients and indicates initial evidence of clinical activity , 48. GivS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


The transgenic process allows for obtaining genetically modified animals for divers biomedical applications. A number of transgenic animals for xenotransplantation have been generated with the somatic cell nuclear transfer (SCNT) method. Thereby, efficient nucleic acid delivery to donor cells such as fibroblasts is of particular importance. The objective of this study was to establish stable transgene expressing porcine fetal fibroblast cell lines using magnetic nanoparticle-based gene delivery vectors under a gradient magnetic field. Magnetic transfection complexes prepared by self-assembly of suitable magnetic nanoparticles, plasmid DNA, and an enhancer under an inhomogeneous magnetic field enabled the rapid and efficient delivery of a gene construct (pCD59-GFPBsd) into porcine fetal fibroblasts. The applied vector dose was magnetically sedimented on the cell surface within 30 min as visualized by fluorescence microscopy. The PCR and RT-PCR analysis confirmed not only the presence but also the expression of transgene in all magnetofected transgenic fibroblast cell lines which survived antibiotic selection. The cells were characterized by high survival rates and proliferative activities as well as correct chromosome number. The developed nanomagnetic gene delivery formulation proved to be an effective tool for the production of genetically engineered fibroblasts and may be used in future in SCNT techniques for breeding new transgenic animals for the purpose of xenotransplantation.The online version of this article (doi:10.1007/s12033-016-9934-1) contains supplementary material, which is available to authorized users. Since the term “transgenic” was used for the first time , there hIn non-viral gene delivery, inorganic nanoparticles such as carbon nanotubes, gold nanoparticles, quantum dots, and magnetic nanoparticles have received increasing attention due to their unique properties including small particle size, large surface area, surface modification possibility, stability, and biocompatibility . A greatIn this study, magnetofection technology was used to deliver a pCD59-GFPBsd gene construct containing a gene encoding a protein involved in the regulation of the human immune response system, into porcine fetal fibroblasts (PFFs) Fig. . These c4 cells/cm2. Cells were cultivated on flasks coated with gelatin (30 min incubation with 0.1 % gelatin (Sigma-Aldrich) before cell seeding) at 37 °C in a humidified atmosphere containing 5 % CO2.Porcine fetal fibroblasts isolated from conceptuses, hereafter referred to as PFF cells, were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10 % fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (Sigma-Aldrich). Every 3–4 days, the cells were split 1:2 at a seeding density of 1 × 10The pCD59-GFPBsd plasmid, which expresses the human CD59 gene under the control of the EF-1α promoter in the pTracer-EF/Bsd A  vector, was obtained, amplified, and purified as described elsewhere . LuciferBr) (Sigma-Aldrich) combined with the fluorinated surfactant ZONYL FSA ethylthio]propionate) (Sigma-Aldrich), as described elsewhere propionate) and 25-kDa branched polyethylenimine (PEI-25Br) were used to associate magnetic transfection complexes. The size, morphology, ζ-potential, as well as magnetic properties of these particles were assessed. The particle sizes and morphology were evaluated by electron microscopy. The TEM micrographs show the presence of apparently spherical iron oxide cores  from 0.0625 to 4 were tested. To sediment magnetic lipoplexes, the complexes were placed on the magnetic plate and after 20 min of incubation the radioactivity was measured in the supernatant. At all tested DF-Gold-to-pDNA (v/w) ratios from 2 to 4 and PEI-Mag2-to-pDNA iron w/w ratios higher than 1, the pDNA was to a significant extent associated and magnetically sedimented  and an iron-to-DNA ratio of 2:1 (w/w) were prepared. The average size of the complexes according to the hydrodynamic diameter was approximately 1800 nm. The complexes had a positive net charge with zeta-potential values of 8 mV when measured in DMEM medium. In order to evaluate the intracellular localization of magnetic lipoplexes containing fluorescently labeled pDNA, magnetofection was conducted and fluorescent images were taken at 30 min, 4 h, and 24 h after magnetofection. The confocal microscopy confirmed rapid cellular uptake of the complexes during the magnetofection process. As shown in Fig. CD59 gene under the control of an hEF-1α promoter. After the selection process, fibroblast vitality and visual eGFP expression was evaluated. A screening procedure involving 333 and 477 bp PCR products was performed to identify transgenic fibroblast cell lines. After magnetofection, five individual cell lines (5/5) were found to be transgenic with the transgene incorporated into the nuclear genome. The results of screening for the pCD59-GFPBsd transgene are presented in Fig. n = 38).The transgenic fibroblast cell lines were produced by magnetofection of porcine fetal fibroblasts with pCD59-GFPBsd gene construct containing the human The SCNT method enables transfer of most of the manipulations from a farm to a laboratory, where the modification of somatic cells is performed and the clones characterized for transgene integration into the genome are selected . EfficieThe first essential step for efficient magnetofection is the formulation of nanomagnetic pDNA delivery vectors. For this purpose, we used PEI-Mag2 magnetic nanoparticles, which proved to be able to assemble magnetic transfection complexes and deliver different types of nucleic acid with high effectiveness , 27, 28.In the context of magnetofection, any agent that associates with MNPs and nucleic acid resulting in improvement of gene delivery can be used as an enhancer. Different parameters such as the choice of enhancer, optimal ratio of MNPs and enhancer to nucleic acid, and mixing order should be considered. The addition of cationic lipids or polymers as a pDNA condensation agent to associate complexes with MNPs led to the enhancement of MNP-mediated plasmid DNA delivery into various mammalian cells –34. We hThe size as well as electrokinetic potential of transfection complexes were found to be important factors affecting their effectiveness in cellular uptake. The DNA/(transferrin)-PEI complexes with an average size greater than 500 nm were more efficient in gene transfer compared to small complexes of 40 nm . Ross etThe sufficient magnetic properties of the complexes enable accelerated sedimentation of the magnetic lipoplexes onto the cell surface upon application of the magnetic field. The magnetic field itself does not alter the mechanism of the magnetic complex uptake . ExposureGFP gene to confirm the incorporation of the transgene with the genomic DNA. The magnetofection method which was applied to modify PFF cell lines proved to be an efficient and non-toxic strategy for producing transgenic nuclear donor cells. The magnetofected cell lines were characterized by high survival rates and proliferative activities. It was revealed that all five genotypically analyzed cell lines that had undergone the magnetofection were positive for pCD59-GFPBsd transgene screening. All of the transgenic cell lines were also eGFP-positive and expressed human CD59 gene. No morphological alterations were observed and there was a correct chromosome number and structure for magnetofected PFFs.Generation of a transgenic somatic cell line suitable for nuclear transfer requires stable transgene integration into the genome of each cell. Besides the gene of interest, the incorporated gene construct should also contain selection and visual markers. Only genetically transformed cells that survive selection can be used as a source of nuclear donor cells to reconstruct the enucleated oocytes. Various methods have been employed for the successful production of stable transformants , 10, 40.This study demonstrates that magnetofection appears to be an alternative method for viral and non-viral gene delivery into porcine fibroblasts. Magnetic complexes formulated with a gene of interest under optimized conditions in combination with an inhomogeneous magnetic field enabled effective, rapid, and non-toxic transfection of PFFs, resulting in the generation of stable transgenic cell lines. Successfully established porcine fibroblast cell lines expressing human CD59 membrane proteins can be used as a nuclei source for SCNT. Further studies are needed to determine whether the use of a magnetofection method adapted for the purpose of SCNT can result in the creation of viable transgenic cloned animals for the purpose of xenotransplantation.Supplementary material 1 (MP4 8426 kb)Supplementary material 2 (MP4 8424 kb)Below is the link to the electronic supplementary material.


BRAF-mutant melanoma, but their efficacy is limited by both intrinsic and acquired resistances. Activation of the PI3K pathway can mediate resistance to these agents, providing a strong rationale for combination therapy in melanoma. Here, a panel of nine low-passage human metastatic melanoma cell lines with BRAF mutations was tested in cell proliferation and protein expression assays for sensitivity to inhibitors of MEK (selumetinib) and BRAF (vemurafenib) as single agents and in combination with inhibitors of pan-PI3K (ZSTK474), pan-PI3K/mTOR (BEZ235), individual PI3K isoforms , or mTORC1/2 (KU-0063794). Selumetinib and vemurafenib potently inhibited cell proliferation in all cell lines, especially in those that expressed low levels of phosphorylated AKT (pAKT). ZSTK474 and BEZ235 also inhibited cell proliferation in all cell lines and enhanced the antitumor activity of selumetinib and vemurafenib in the majority of lines by either interacting synergistically or additively to increase potency or by inducing cytotoxicity by significantly increasing the magnitude of cell growth inhibition. Furthermore, ZSTK474 or BEZ235 combined with selumetinib to produce robust inhibition of pERK, pAKT, and pS6 expression and synergistic inhibition of NZM20 tumor growth. The inhibitors of individual PI3K isoforms or mTORC1/2 were less effective at inhibiting cell proliferation either as single agents or in combination with selumetinib or vemurafenib, although KU-0063794 synergistically interacted with vemurafenib and increased the magnitude of cell growth inhibition with selumetinib or vemurafenib in certain cell lines. Overall, these results suggest that the sensitivity of BRAF-mutant melanoma cells to BRAF or MEK inhibitors is at least partly mediated by activation of the PI3K pathway and can be enhanced by combined inhibition of the BRAF/MEK and PI3K/mTOR signaling pathways.BRAF and MEK inhibitors have improved outcomes for patients with  BRAF mutations leading to constitutive activation of the RAS/RAF/MEK/ERK pathway and increased cell cycle progression, differentiation, survival, migration, and angiogenesis are reported in 40–50% of melanoma cases  δ 9.49 , 8.86 , 8.52 , 8.47 , 8.39 , 8.06–8.09 , 7.95–7.99 , 7.85–7.90 , 7.73 , 7.37 , 4.85 , 3.70 , 2.39 , 1.79 ; Combustion Analysis, Calc. for C32H31N5O7S2: C, 58.1; H, 4.7; N, 10.6. Found: C, 58.0; H, 4.6; N, 10.65%.BEZ235 was prepared as a dimethanesulfonate salt by treating a suspension of the solid in methanol with 2.2 equivalents of methanesulfonic acid, to give a clear solution. Dilution with ethyl acetate gave a precipitate, which was collected by filtration and washed with further ethyl acetate. Recrystallization from methanol–ethyl acetate gave the dimethanesulfonate salt as a pale yellow solid: mp 283–286°C; 2, 5% CO2) at 37°C.A panel of nine melanoma cell lines was chosen from a series of lines generated from surgical samples of metastatic melanoma obtained with appropriate consent from patients throughout New Zealand as previously described . The celTERT was determined in the melanoma cell lines by Sequenom analysis. DNA was extracted using PureLinkTM Genomic DNA kit (Life Technologies), according to manufacturer’s protocol. To remove the EDTA-based elution buffer, DNA was re-precipitated into milliQ water. This was achieved by addition of ethanol and 5M ammonium acetate at −80°C for 2 h and centrifugation at 18,000 × g for 30 min at 4°C. The pellet was resuspended in ethanol and re-centrifuged at 18,000 × g for 10 min at 4°C, prior to resuspension in milliQ water. Extracted DNA was evaluated for gene mutations on the Sequenom MassARRAY® using the MassARRAY OncoCartaTM Panel v 1.0 and the MelaCartaTM Panel v1.0 plus hTERT, according to manufacturer’s protocol. Analysis was carried out using Sequenom MassARRAY Typer Analyzer 4.0 genotyping software. PTEN mutation status was determined by PCR sequencing as described previously  in 1% acetic acid for 30 min in the dark at room temperature. Plates were washed in 1% acetic acid, dried, and incubated with unbuffered Tris base  for 30 min on a plate shaker in the dark to solubilize the stain. The plates were read on a BioTek EL808 microplate reader at an absorbance of 490 nm with a reference wavelength of 450 nm. Absorbances of treated cells were compared to untreated cells at 0 h (100% growth inhibition) and 72 h (0% growth inhibition) after treatment. Growth inhibition above 100% indicated that fewer cells were present than when the compounds were first administered. EC50 values were calculated by fitting the inhibition data to a four-parameter logistic sigmoidal dose–response curve using GraphPad Prism 6.01. Combination indices (CI) were calculated at EC50 using the method of Chou and Talalay  at 25V for 12 min on a BioRad Trans-Blot® TurboTM semi-dry transfer machine. Following protein transfer, membranes were incubated in blocking buffer [Tris-buffered saline with 0.5% Tween®-20 (Serva) and 3% BSA] for at least 30 min then cut and incubated overnight at 4°C with antibodies at 1:1000 dilution (unless indicated) against either pAKT , pERK1/2 (Thr202/Tyr204), pS6 , AKT, ERK1/2, S6 (1:2000), PTEN (1:100), IGF1Rβ, EGFR, Insulin Receptor β, c-MET, ERBB3, MERTK , and β-actin . Membranes were washed then incubated with anti-mouse  or anti-rabbit  goat IgG HRP-conjugated secondary antibody in blocking buffer for 1 h at room temperature. After further washes, the membranes were incubated with BioRad ClarityTM ECL or Perkin Elmer Western Lighting Ultra (pAKT membranes) for 4 min prior to imaging on a LAS-4000 luminescent image analyzer (Fujifilm). After visualization of phosphorylated proteins, membranes were stripped and reprobed with total proteins and β-actin. β-actin was used to confirm equal protein loading in each blot. Each cell lysate was tested in duplicate in two to three independent experiments.Untreated cells for basal protein expression or cells treated with 500 nM of compound for 1 or 24 h were lysed in lysis buffer containing 1% protease inhibitor cocktail (Sigma-Aldrich) on ice for 15–30 min. Cells were centrifuged at 13,000 rpm for 10 min at 4°C to remove insoluble material. Protein concentration of cell lysates was determined by bicinchoninic acid assay (Thermo Scientific) against bovine serum albumin  standards at an absorbance of 562 nm on a BioTek Synergy HT plate reader using KC4 v3.4 software. Forty micrograms of each lysate was loaded onto polyacrylamide gels (10% acrylamide) and separated by SDS-PAGE at 120V for 90 min. Each gel was transferred onto an Immobilin6 NZM20 cells. Treatment was initiated when tumors reached 150 mm3 in volume as measured by electronic calipers. The dosing vehicles used were 0.5% hydroxypropyl methylcellulose with 0.2% Tween 80 (selumetinib and control vehicle), 2% carboxymethylcellulose with 1% Tween 80 (ZSTK474), and 40% PEG-400 in 20% hydroxypropyl-β-cyclodextrin (BEZ235). All vehicle constituents were supplied by Sigma-Aldrich. Treatments were administered daily for 14 days at half the single agent maximum tolerated dose as the free base equivalent by oral gavage at a volume of 10 ml/kg. Animals were monitored daily for bodyweight and any observational signs of toxicity. Tumor growth inhibition (TGI) was calculated by determining the tumor size of drug-treated mice relative to starting size as a percentage of the average relative tumor size of control mice. Synergy was inferred if the TGI of the combination was greater than the product of the TGI values of each single agent. All animal experiments followed protocols approved by the University of Auckland Animal Ethics Committee.Age-matched specific pathogen-free female NIH-III mice were subcutaneously inoculated with 5 × 10BRAF-mutant melanoma cell lines that were both developed and maintained at low-oxygen tension (5%). The mutation status of common oncogenic driver mutations in these lines was determined by Sequenom or PCR profiling. Seven of the cell lines contained V600E mutations in BRAF, while two contained V600K mutations . All PTEN-mutant lines, along with NZM30, lacked PTEN expression. pERK was expressed in all cell lines at variable levels, with the lowest expression detected in NZM7 and NZM43, while pS6 was expressed in all cell lines at similar levels relative to total protein.The basal protein expression of phosphorylated and/or total forms of AKT, PTEN, ERK1/2, and S6 were determined in the cell line panel by western blotting Figure  and 6, bBRAF-mutant and PTEN-null cell lines.Several RTKs that can activate PI3K/mTOR or RAS/RAF/MEK/ERK signaling pathways were expressed in the NZM lines Figure . High-EG50’s ranging from 12.0 ± 3.7 to 131 ± 56 nM . There was no clear relationship between basal pERK protein expression and selumetinib or vemurafenib sensitivity in the cell line panel.Each cell line in the panel was exposed to multiple concentrations of selumetinib or vemurafenib to determine the effects of these drugs on cell proliferation. Selumetinib was highly effective at inhibiting cell proliferation in all cell lines, with ECBRAF-mutant melanoma  except NZM3, NZM7, and NZM11, where selumetinib or ZSTK474 alone inhibited growth by >105% .Since the antiproliferative activity of selumetinib and vemurafenib was less pronounced in 50’s ranging from 10.2 ± 2.8 to 163 ± 100 nM , as shown for NZM34   to either vemurafenib or BEZ235 as single agents   was observed with ZSTK474 in the six cell lines that were least sensitive to vemurafenib as a single agent Figure , but sigTo determine if selumetinib in combination with ZSTK474 or BEZ235 can prevent both ERK and AKT signaling, pERK and pAKT expression levels were determined by western blotting 1 and 24 h after 500 nM drug treatment  and NZM12 (EC50 = 5.9 ± 2.7 μM)   and in NZM7, NZM20, and NZM34 cells with selumetinib and KU-0063794 (P < 0.05)  and NZM34 (P < 0.05) cells  relative to control at day 14  and 59.0 ± 7.4% (P < 0.0001) relative to control at day 14 for selumetinib with BEZ235 and ZSTK474, respectively. Treatment with selumetinib and BEZ235 prevented tumors from growing above baseline levels during the treatment period and at day 14 tumor size was significantly reduced from selumetinib (P < 0.05) or BEZ235 (P < 0.0001) treatment alone. All treatments were well tolerated over the dosing period, with minimal weight change seen in all groups  is required for cytotoxic inhibition of growth.Inhibition of PI3K alone with ZSTK474 or PI3K and mTOR with BEZ235 was effective at inhibiting cell proliferation and AKT signaling in all n status , 48, in  deleted . However therapy –31. SyneBRAF-mutant melanoma cell lines, inhibition of all or most isoforms is required due to the ability of isoforms to readily compensate for one another (Isoform-selective inhibitors of PI3K lacked potency at inhibiting cell proliferation on their own, but A66, idelalisib, and AS-252424 were able to significantly increase maximal inhibition when combined with selumetinib, but not vemurafenib, in NZM7 cells. However, since the isoform-selective PI3K inhibitors only enhanced selumetinib activity at concentrations approaching 10 μM, it is possible that these effects could be due to loss of isoform selectivity at these high concentrations. Nevertheless, the activity was less than that observed for ZSTK474, suggesting that although one or more of the PI3K isoforms may play a preferential role in promoting survival in  another , 51. Thi another . By cont another , 33, 48.in vivo, these properties will need to be evaluated in future alongside an assessment of optimal dose levels and scheduling of these agents in combination.Substantial inhibition of pS6 was observed here with selumetinib alone in the cell lines that were most sensitive to selumetinib or vemurafenib and expressed low pAKT, while only minor inhibition was detected in less sensitive lines that expressed moderate to high levels of pAKT. Further inhibition of pS6 was achieved in combination with ZSTK474 and near complete inhibition in combination with BEZ235. These results corroborate with previous findings that mTORC1 activity is primarily regulated by ERK signaling in BRAF or MEK inhibitor sensitive melanomas . HoweverBRAF-mutant melanomas, particularly those that express low pAKT and functional PTEN, can be effectively treated with single agent BRAF or MEK inhibition, for the majority of lines, enhancement of antitumor activity can be achieved by combining BRAF or MEK inhibition with an inhibitor of PI3K/mTOR signaling to block ERK, AKT, and S6 signaling. Despite either inhibition of PI3K or mTOR synergizing with BRAF/MEK inhibition, dual PI3K/mTOR inhibition appears the most promising strategy to enhance sensitivity to BRAF and MEK inhibition and possibly overcome ERK-independent intrinsic or acquired resistance.Overall, these results suggest that while some BRAF/MEK inhibitor sensitive MS, PS, and SJ conceived and designed the experiments. MS, ER, and SK performed the experimental studies. GR performed compound synthesis. BB established the melanoma cell lines. Data were analyzed by MS, SK, and SJ. SJ drafted the figures, tables, and manuscript. BB and PS critically revised the paper and all authors approved the final version.The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Availability of expanded SCLC CTC cells allowed for a screening of receptor tyrosine kinases (RTKs) expressed. The metastatic CHI3L1-negative SCLC cell line SCLC26A, established from a pleural effusion was used for comparison. The CTC cell line BHGc10 was found to exhibit increased expression of RYK, AXL, Tie-1, Dtk, ROR1/2, several ephrins (Eph) and FGF/EGF receptors compared to SCLC26A. All of these RTKs have been associated with cell motility, invasion and poor prognosis in diverse cancer entities without knowledge of their association with CTCs. The identification of RYK, AXL and ROR1/2 as pseudokinases, lacking activity, seems to be related to the observed failure of RTK inhibitors in SCLC. These kinases are involved in the noncanonical WNT pathway and their expression in SCLC CTCs represents a cancer stem cell-like phenotype.Small cell lung cancer (SCLC) has a poor prognosis and is found disseminated at first presentation in the majority of cases. The cell biological mechanisms underlying metastasis and drug resistance are not clear. SCLC is characterized by high numbers of circulating tumor cells (CTCs) and we were able to expand several CTC lines  Small cll lines . So far,ll lines , 10. Scrll lines . In the Expression of RTKs was detected on the Western blot arrays for SCLC26A and BHGc10 SCLC CTC cells, respectively. Figure Figure in vitro expanded CTCs renders analysis of the cell biologic characteristics possible [In general, CTCs are relatively rare cells but access to possible . AdditioThe RTK family regulates a wide spectrum of cell activities including proliferation, differentiation, metabolism and migration . AberranRYK (for related to tyrosine kinase) has been isolated from a DNA library of SKOV-3, an epithelial ovarian cancer cell line . Localizin vitro and in vivo [RYK overexpression has been observed in human ovarian cancer and was correlated with decreased overall survival of patients , 24. Ove in vivo . The RYK in vivo . TargeteROR1/2 are a single-span transmembrane RTKs which function in mediating the non-canonical Wnt signaling and has no essential roles known in adult tissues . ROR2 waROR2 has clearly been linked to aggressive disease patterns and has been implicated in important tumor promoting signaling pathways constituting an promising target. As a protein ROR2 is not normally expressed in adult tissues. There are several drugs targeting activated RTKs for treatment of cancers, with particular efficacy for the small-molecule inhibitors that target the ATP-binding site of the intracellular tyrosine kinase domain but these will not work for pseudokinases . ROR2 isThe TYRO3, AXL and MERTK (TAM) family of RTKs are aberrantly expressed in multiple hematological and epithelial malignancies. Their induction in tumor cells predominately promotes survival, chemoresistance and motility . AlthougEph receptors are the largest family of RTKs . ReemergCancer stem cells (CSCs) have been increasingly described for many malignancies. CSCs display many features of embryonic or tissue stem cells, and feature persistent activation of one or more conserved signal transduction pathways involved in development and tissue homeostasis, such as the Notch, Hedgehog and Wnt pathways . Wnt sigGeneration of CSCs is linked to EMT and are capable of tumor initiation and self-renewal, contributing to treatment resistance and metastases . AXL is In conclusion, RTK expression of a SCLC CTC line was here delineated for the first time. The overexpression of specific RTKs in a blood-derived line versus a metastase-derived SCLC line is in line with use of Wnt signaling, identifying this CTC line as CSC-related. The RTK pseudokinases found seem to escape targeting by RTK inhibitors but may be tackled by other means. Especially AXL may be related to the high degree of chemoresistance, observed in relapsing SCLC patients. Since the respective kinases expressed in the SCLC CTC cell line were broadly found in other tumors, this model of dissemination may hold true for other malignancies as well.SCLC26A cell line was established in our lab as well as the BHGc10 SCLC CTC line, derived from a peripheral blood sample of a relapsed SCLC patient , 12. ForRTKs were detected using a Western Blot array  according to the manufacturer's instructions. The experiments were done in duplicate. In brief, 1×107 cells/ml were washed in PBS and extracted using the appropriate buffer of the kit supplemented with protease inhibitor cocktail (Sigma-Aldrich). Extracts were centrifuged, supernantants diluted with sample buffer and applied to the nitrocellulose membranes having 49 anti-kinase catcher antibodies spotted. RTKs were finally detected using pan-anti-tyrosine-HRP conjugate and detection of binding by chemiluminescence. Spots were analyzed using GelAnalyzer  and ORIG


These findings were observed in stage I, II and III MM patients compared with healthy individuals  in regulating MM cell survival and trafficking isoforms . Thus, Mtive PCR . Specifitive PCR . Then, wtive PCR . Althougin vitro , cell cyin vitro . We nextin vivo, SCID-Bg mice were injected with MM cells silenced for p110α, β, γ and δ, and tumor development was monitored by bioluminescence imaging. Scramble-infected cells were used as control. In consistent with in vitro data demonstrating that the most significant changes were observed for adhesion of MM cells to BM-MSCs in p110β and p110δ knockdown cells, tumor progression was significantly lower in p110β- and p110δ-knockdown cell-injected mice compared with scramble cell-injected mice (P<0.05); whereas tumor growth observed in p110α- and p110γ-knockdown cell-injected mice was similar to control mice  mutation is the leading cause of activation of this pathway and is the target of many therapeutic agents in development,3 there have been no reports of this specific mutations in MM.6 Moreover, it was shown that unlike wild-type p110α, overexpression of the wild-type p110β, p110γ and p110δ is sufficient to induce an oncogenic transformation of fibroblasts in cell culture.14Interestingly, our data indicate that p110α is not critical for the survival of MM cells 9 showing expression of p110β in 38 MM cell lines in comparison to the detectable expression of p110δ in only 4 cell lines. In addition, another study8 reported similar findings in cell lines showing lack of p110δ expression in most MM cell lines. Of note, we found discrepancies in p110δ expression in cell lines between our study and prior published studies but our data was confirmed in the Cancer Cell Line Encyclopedia data at the mRNA level (data not shown).15 Importantly, Ikeda et al.8 evaluated p110δ levels in patient samples and detected its expression in all 24 MM patients. This may provide a clinical rationale for targeting p110δ despite the lack of expression of p110δ in MM cell lines.In this study, p110β was highly expressed in all MM cell lines, whereas only a minor subset expressed p110δ at the protein level , which iOverall, our data suggest that, in contrast with solid tumors, MM may be more dependent on PI3K p110β and p110δ and less dependent on PI3Kα, and these may be the focus of drug development in this hematological malignancy.


Contaminations of cell cultures with microbiological organisms are well documented and can be managed in cell culture laboratories applying reliable detection, elimination and prevention strategies. However, the presence of viral contaminations in cell cultures is still a matter of debate and cannot be determined with general detection methods. In the present study we screened 577 human cell lines for the presence of murine leukemia viruses (MLV). Nineteen cell lines were found to be contaminated with MLV, including 22RV1 which is contaminated with the xenotropic murine leukemia virus-related virus variant of MLV. Of these, 17 cell lines were shown to produce active retroviruses determined by product enhanced reverse transcriptase PCR assay for reverse transcriptase activity. The contaminated cell lines derive from various solid tumor types as well as from leukemia and lymphoma types. A contamination of primary human cells from healthy volunteers could not be substantiated. Sequence analyses of 17 MLV PCR products and five complete MLV genomes of different infected cell lines revealed at least three groups of related MLV genotypes. The viruses harvested from the supernatants of infected cell cultures were infectious to uninfected cell cultures. In the course of the study we found that contamination of human genomic DNA preparations with murine DNA can lead to false-positive results. Presumably, xenotransplantations of the human tumor cells into immune-deficient mice to determine the tumorigenicity of the cells are mainly responsible for the MLV contaminations. Furthermore, the use of murine feeder layer cells during the establishment of human cell lines and a cross-contamination with MLV from infected cultures might be sources of infection. A screening of cell cultures for MLV contamination is recommended given a contamination rate of 3.3%. Human and animal cell cultures are highly susceptible to a multitude of contaminations. These comprise cross-contamination with human or animal cells from other cell lines, leading to mixed cell populations or a complete replacement of the original cells by the contaminating cells . Other cAlthough cell lines are commonly used for the production of viruses and for the investigation of virus infections, only sporadic reports address the possible problem of unintended viral contamination of cell cultures. One reason for this dearth of attention may be the generally assumed species- and tissue-specificity of the viruses. A viral contamination is usually predicted to originate from the donor of the cells of a cell culture. Thus, either the donor organism or the cells are screened for potential virus infections, such as Epstein-Barr virus (EBV), hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human T-cell lymphotropic virus (HTLV) or other suspected human pathogenic viruses. The screening for viruses is usually motivated by safety reasons . Animal Indeed, it had been shown that the risk of unknown infection of human continuous cell cultures with the aforementioned viruses is extremely low. Only EBV is significantly prevalent in human B cell lines because this virus is widely distributed among the human population and is also used to immortalize B cells in vitro to generate B lymphoblastoid cell lines. Other human pathogenic viruses can only be found sporadically  and are not further disseminated among other cell cultures .Nevertheless, at least one group of viruses was identified to be able to infect cells from numerous species and various tissues in vitro and to occur in cell cultures: the xeno- and polytropic murine leukemia viruses (X/P-MLV) . The mouIn the past decades, several studies were published describing the contamination of cell lines with MLV. Mouse cells usually contain several full-length copies of the different host-range MLVs endogenously in their genome and produce active viruses . RegardiIn the course of screening the cell lines of our biological resource center (BRC) for XMRV we detected MLV contamination in a number of human cancer cell lines. More than 3% of the investigated cell lines were found to harbor MLV sequences and most of them are also positive for reverse transcriptase activity, indicating the production of active viruses. Most of the cell lines were presumably contaminated by xenotransplantation into mice to study the tumorigenicity of the cells. We sequenced a number of the viruses and found a high variability in specific genome regions. We also performed stimulation and cross-contamination tests to study a possible inducibility of expression and the infectiousness of any viruses released during routine cell culture.2. The basic growth media  were supplemented with 10–20% fetal bovine serum . For growth factor-dependent cell lines, specific growth factors or conditioned media containing growth factors were added. No antibiotics were added to the cultures. All cell lines were free of mycoplasma and other bacterial, yeast and fungi contaminations as tested by PCR and microbiological growth assays [–7 M 12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma Aldrich) and 3 mM Na-butyrate (Sigma Aldrich) or for 3–5 days with 1.25 μg/μl 5´-azacytidine (Sigma Aldrich).The continuous cell lines were provided for accessioning to the BRC by the original or secondary investigators . Cell lih assays . The aut6 viable cells was eluted from the columns with 200 μl dH2O. The DNA concentration and purity was determined using a Nanodrop 1000 fluorometer. Approximately 200 to 500 ng of the isolated DNA were used for PCR amplification. The integrity of the DNA was demonstrated by performing short tandem repeat cell line authenticity assays as described elsewhere [PCR was applied for the detection of proviral MLV sequences in the genomes of the human cell lines. Genomic DNA was isolated from PBS-washed cell pellets applying the High Pure PCR Template Preparation kit from Roche  according to the recommendations of the manufacturer. The DNA of 1 to 5 x 10lsewhere   were imported into Vector NTI sequence analysis program (Life Technologies) and aligned. Conserved regions were chosen to design oligonucleotides for the amplification of DNA fragments longer than 4 kbp. Further oligonucleotides were designed to produce type-specific DNA fragments for sequencing. All primers used for amplifying long PCR fragments and for sequencing are listed in Full length viral genomes from the Entrez Genome Browser online database  to detect mouse genomic DNA was performed essentially as described for the diagnostic MLV PCR, except for the exchange of MLV primer by IAP primer and the use of 20% betaine instead of 5% formamide.Total cellular RNA of the cell lines VERO-B4, NCI-H82, and MEL-JUSO was prepared employing Trizol reagent (Life Technologies) according to the manufacturer’s recommendations. Five micrograms of total RNA were used as a template for first strand cDNA synthesis applying a reverse transcriptase preamplification system kit . The reverse transcription was carried out with oligo dT primers according to the recommendations of the manufacturer. Two microliters of the first strand cDNA reaction were then used in a PCR for the detection of XPR1 transcripts which are the cellular receptors for the binding of MLV. The PCR was performed as described above for the diagnostic MLV PCR, but with specific XPR1 primers , TaKaRa The PERT assay was used to sensitively demonstrate the activity of retroviral reverse transcriptase (RT) in cell culture supernatants. The potentially present viral RT is used to produce a cDNA strand initiated by an artificial primer template hybrid consisting of MS2 bacteriophage RNA and an MS2-specific primer. The cDNA is then amplified with a second MS2-specific primer in a PCR reaction.Five ml of conditioned cell culture supernatant were harvested and centrifuged for 30 min at 2000 x g and 4°C to remove cells and cell debris. One hundred μl of the supernatant were mixed with 100 μl of PERT lysis buffer A  and 1 μl 10% Triton X-100 . The mixOne μl of PERT-RT1R primer at 10 mM was mixed with 0.4 μl bacteriophage MS2 RNA at 0.8 μg/μl  per reaction, denatured at 95°C for 5 min and subsequently incubated for 30 min at 37°C for annealing of the primer to the template. The primer template mixture was chilled on ice and processed immediately or stored at -20°C.2+-RT-buffer and 1.4 μl of primer-template mixture or with 15 μl 2 x Mn2+-RT buffer, 1.4 μl of primer-template mixture and 8.6 μl dH2O. The 1 x Mg2+-RT-buffer consisted of 56 mM KCl, 56 mM Tris-Cl pH 8.3, 9.2 mM MgCl2, 11.2 mM DTT, 0.13 μg/μl BSA, and 1 mM each of dNTPs [2+-RT buffer consisted of 60 mM KCl, 20 mM HEPES pH 7.2, 6 mM MnCl2, 4 mM spermine tetrahydrochloride, 1% triton X-100, 0.4 mM EDTA, 6 mM β-mercaptoethanol, 1 mg/ml BSA and 1 mM each of dNTPs. Then 0.1 U/μl Ribolock RNase inhibitor was added to each of the RT buffers immediately before setting up the RT reaction. The samples were incubated at 37° for 2 h. Two μl of each reaction were either directly subjected to a PCR amplification or after ethanol precipitation and redilution in 5 μl dH2O. The PCR was carried out with 100 μM of each dNTP, 0.4 μM each of PERT-RT1R and PERT-RT2F primer, 1 x PCR buffer as recommended by the manufacturer of the Taq polymerase, and 1 U TaKaRa HS ExTaq polymerase. The DNA was added at last. The Taq polymerase was activated by heating the mixture for 2 min at 96°C. The DNA amplification was carried out for 35 cycles with 10 s at 94°C denaturation, 20 s at 55°C annealing, 30 s plus 1 s per cycle at 72°C extension, and 7 min at 72°C as final extension at the end of the PCR run. The PCR products were then analyzed on a 1.3% agarose gel.Two RT reactions were carried out per sample. Five μl of the lysis reaction were combined with 23.6 μl of 1 x Mgof dNTPs . The 2 x2O and subsequently ethanol precipitated. The DNA was then resuspended in 15 μl dH2O. The DNA concentration was determined with a Nanodrop 1000 instrument. Approximately 200 ng of the purified DNA were combined with 15 pmol of the respective sequencing primer in a volume of 15 μl. The sequencing was performed by Eurofins Genomics . Sequence analysis was performed with the free software applications Vector NTI (Life Technologies), MEGA6 [http://emboss.bioinformatics.nl).Fifty microliter PCR reactions were prepared using genomic DNA and the appropriate primer combination  to ampli), MEGA6 , and "Thgag start codon as published previously [2, and 5% formamid as PCR additive provides highly sensitive reaction conditions. Furthermore, a BLAST search comparing the primer sequences with sequences from the public databases showed for the outer primer for more than 220 entries and for the inner primer for more than 130 entries complete concordance with murine leukemia viruses, particularly with XMLV and XMRV as well as with mouse chromosomal sequences attributed to endogenous MLV.In the course of screening prostate cancer and other suspected cell lines for contaminations with XMRV, we established a nested PCR assay with primers amplifying a region upstream of the eviously . DNA of Although none of the other prostate carcinoma cell lines tested (n = 5) were found to be positive for XMRV/MLV, the first PCR assays surprisingly uncovered a few cell lines that were positive for MLV sequences . This finding encouraged us to screen all human cell lines of the collection with the nested PCR assay for MLV sequences. Finally, 23 out of 577 human cell lines were positive for MLV-sequences applying nested PCR . HoweverThe MLV-positive cell lines were not restricted to specific types of cancer, but included cell lines of different leukemia and lymphoma as well as several kinds of carcinoma types. Furthermore, we found closely related cell lines  of which one or more were MLV-positive, whereas others were MLV-negative. For example, BT-B, a subclone of the HELA cell line was MLV-positive, whereas HELA itself and other HELA subclones were all MLV-negative. Similarly, only two out of five NAMALWA subclones were MLV-positive.We also investigated four samples of the cell line A2780 obtained from four different laboratories . Seventeen of the sequences revealed unequivocal and complete reads as shown in part in the first two panels of Interestingly, sequence analysis of the PCR products of mouse cell lines  invariably produced the same sequence mixture as seen with the ambiguous sequences of the human cell lines , which iAn alignment of the unambiguous sequences displayed a number of polymorphisms among the sequences. Striking are some deletions or insertions of some nucleotides found singly or repeatedly in different cell lines: (a) insertions of three, six or seven nucleotide-long stretches can be detected approximately 190 bp downstream from the forward primer in nine cell lines; (b) a six nucleotide-long insertion at approximately position 220 is seen in four cell lines, including 22RV1; (c) at the alignment position of ca. 400 three groups of sequence types can be identified: beside the complete sequence found in 11 cell lines, a 9 bp deletion displayed in five cell lines, and a 24 bp deletion only in the XMRV-positive cell line 22RV1 as already described elsewhere   PCR assay as described previously . The metn et al. . In contspecific . As show2+-buffer, a restriction to this RT reaction buffer might be possible without decreasing the sensitivity and specificity of the assay. The intensities of the PERT-PCR signals are diverse and sometimes weak, indicating variable expression of active viruses by the cell cultures.The enzymes of the MLV retroviruses are initially packaged during the replication process of the retrovirus as inactive proteins linked together in the Gag-Pol polyprotein consisting of Gag, the structural protein of the virus capsid, and Pol consisting of the protease, RT, and the integrase. During the following maturation process of the already released complete viruses the proenzyme is proteolytically cleaved to produce the various active enzymes that are necessary for the infection of cells . Thus, tThe weakly PERT-positive cell line SK-GT-2 was treated with the chemical induction reagents 5-azacytidine, and a combination of 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate. Although the reagents induced a differentiation of the cells as determined by morphological changes and cessation of cell proliferation, no stimulation of MLV expression was detected employing the PERT assay. According to these results, the activation of infectious viruses during conventional cell cultivation is unlikely.To investigate a possible cross-contamination of MLV from one cell culture to another, we performed infection experiments of MLV-negative cell cultures with cell culture supernatants of MLV-positive cell lines. The cell lines 22RV1, COLO-677, DEL, EVSA-T, KELLY, KYSE-70, LCL-HO, NAMALWA.CSN/70, NCEB-1, and SCLC-21H were all shown by PERT PCR assay to release active MLV into the cell culture supernatant. Cell free culture supernatants were used as MLV source to infect the MLV-negative cell lines MEL-JUSO, NCI-H82, and VERO-B4, of which the latter two were previously shown to be sensitive for MLV infections: the VERO-B4 cell line was used for infection studies , whereasAs shown in 2+ and Mn2+-reaction buffer (NCI-H82/SCLC-21H MLV).On the other hand, we observed proviruses in two inoculated cell cultures, but without any virus production  and a third culture with an extremely low virus expression which was hardly detectable with the MgMEL-JUSO was infected with cell culture supernatant of the cell lines 22RV1 and COLO-677. Although the cell line does not express XPR-1 surface protein, XMRV of the cell line 22RV1 was able to infect the cells and PERT-PCR demonstrated the presence of active viruses in the virus cross-contaminated cell culture. This result was unexpected, because XMRV was assigned to the xenotropic MLV and should therefore use the XPR-1 membrane protein of the eukaryotic cells for internalization of the viruses. Hence, the virus seems to be able to use an alternative receptor or it does not use XPR-1 at all and has to be assigned to another MLV type. Infection studies of the MEL-JUSO cell line with MLV of COLO-677 revealed no sign of infection, neither integration into the genome of the host cells nor release of active viruses into the cell culture supernatant.The results indicate that MLV infections can be spread by cross-contamination from an infected culture to a non-infected culture.Although all human genomic DNAs were previously analyzed for authenticity and cell line cross-contamination by short tandem repeat (STR) analysis , we addiFive DNA preparations of human cell lines were found to be positive for both, IAP and MLV. The NCEB-1 cells represent human-mouse hybrid cells and were described to stably carry 5–8 mouse chromosomes in their nuclei. The origin of these chromosomes is still a matter of debate, but it is suspected that they were introduced into the cells by co-culture with mouse feeder layer cells , 22. In Controlling the DNA preparations, it turned out that the double-positive DNAs had been prepared in parallel with mouse DNAs. Presumably, they had been contaminated with small amounts of the mouse genomic DNAs. Newly prepared DNA was then negative in both PCR assays. In conclusion, identifying MLV sequences in the human genome does not necessarily indicate a virus contamination of the cells. It is also possible that the DNA is contaminated with traces of mouse cell line DNA. Mouse IAP PCR or sequencing of the PCR products can verify the purity of the DNAs.The contamination of human DNA with traces of murine DNA is concordant with results of a study performed by Zhang et al.  who founOver the last decades several reports have been published on the detection of retroviruses from different mammalian species in human cell lines, indicating contaminations with feline retroviruses , murine The most comprehensive study for XMRV was performed by Hué et al.  who screIn the current study, much emphasis was placed on the prevention of false-positive and false-negative results due to PCR inhibition or contamination of DNA or PCR reagents. Hence, false results are unlikely because the PCR products were sequenced and revealed diverse sequences. Additionally, almost all PCR-positive cell lines were also positive in the PERT assay.The published history of a cell line clone may be a first indication to pinpoint the source of a contamination. As already reported by others  and as dOn the other hand, a number of the tested human cell lines are reportedly tumorigenic in mice as determined by xenotransplantation into mice (see datasheets of the DSMZ cell lines), but are negative for MLV. Thus, the handling of mice or murine cells adjacently to the human cell cultures does not necessarily lead to a contamination with murine retroviruses. This situation resembles very much mycoplasma contaminations: the extent of infections seem to depend on the quality of cell culture technique . In manyWe recognized not only a contamination problem regarding XMRV occurrence in the cell lines, but also regarding contamination of the human genomic DNA with mouse-derived DNA detected by IAP-PCR. This problem became also evident in the context of false-positive XMRV contaminations in human prostate carcinoma and chronic fatigue syndrom patients , 23. PerWe were able to show by PERT-PCR that most of the contaminated cell cultures also produce MLV. As the viruses use cellular enzymes for the reproduction, it is likely that the cells are affected in many ways. First of all, the viral genome is integrated into the cellular chromosomes. As the integration site is random, the initial infection occurs in many cells at different locations. This increases the chance that an important gene is affected by the integration, resulting in activation or inactivation. Furthermore, the production of new viruses may significantly alter the physiology of the cells because the retroviruses are shed continuously from the cells without lysing and killing the cells. This suggests that the producing cells are constantly altered in their physiology.It is currently unknown, whether all cells of an MLV positive cell line are infected with the virus or not. If only a subpopulation would carry MLV, the non-infected cells might be separated from the infected cells by single cell cloning. In an effort to move forward in this field, we are currently establishing a fluorescence in-situ hybridization assay to visualize the viral sequences in the cells.2+- or Mn2+-buffers did not clearly differentiate between magnesium and manganesium dependent retroviruses. Thus, the RT activity might also result from other retrovirus contaminations. However, until now only very few reports were published describing the occurrence of retroviruses in human cell lines. For example, the NAMALWA and LCL-HO cell lines are known to be infected with squirrel monkey retroviruses (SMRV) [The PERT-PCR assay does not detect MLV reverse transcriptase specifically. Even the use of Mgs (SMRV) . In caseSequencing of the PCR products and of whole genomes of MLV revealed various genotypes of MLV. All different genotypes could be found in the NCBI databases either as discrete MLV sequence entries or as part of murine genomic DNA sequences. The latter are supposedly proviruses integrated into the murine genome. However, highly related sequences were already found to be contaminants of cell lines: EKVX, LAPC4, VCaP , DG-75 , RKO 1919, the pThe contamination of human cell cultures with MLV raises the question whether the contaminated cells carry a higher risk regarding the handling of the cell cultures. In contrast to most other viruses, MLV is completely inactivated in the human body by antibody-independent serum components –42. The In conclusion, we could demonstrate that a) 19 of 577 (3.3%) different human cell lines were contaminated with MLV; b) 17 of the MLV-positive cell lines produce active viruses; c) the infecting MLV constitute at least three evolutionary related groups, d) the retroviruses are transmissible from one infected cell culture to another. Other sources of infection could be heterotransplantation of the human cells and the use of murine feeder layer.S1 Table(XLSX)Click here for additional data file.