Today's biopharmaceutical industry is under increasing pressure considering cost efficient development. Short timeframes rule the progress starting from the generation of producer cell lines to the establishment of a final production process. Hence, the timescale for optimization of cell culture media is small, but on the other hand it contains high potential for global process improvement. In this scope, our specially tailored media development platform, which allows a fast and reliable introduction of high-performance basis media and feeds, establishes new perspectives for an efficient process development.For the design and development of TeutoCell's new media platform various cell lines and expression systems were comprehensively analyzed and incorporated. The results gained from cultivations and extensive analysis of culture supernatant and e.g. product glycosylation were integrated in a cyclic development strategy, utilizing theoretical and empirical formulation optimizations. Special applications like single clone selection were integrated into our platform as well.The cell lines used for the development of our media platform include CHO-DG44, CHO-GS and CHO-K1 clones. Cultivations were carried out in shaking flasks as well as closed-loop controlled 0.5 - 2.0 L bioreactor systems in batch und fed-batch mode using standard conditions. An industrially relevant, protein-free and chemically defined medium was used as a reference.Media development for single clone selection by limited dilution was performed with different CHO suspension cells in microtiter plates (from 96- to 6-wells) up to shaking flaks. Analysis of single clone colonies was done with a Cellscreen System.Samples of a model antibody produced in commercially available CHO reference medium and TeutoCell's platform medium using two different producer clones were desalted, denaturated and treated with PNGaseF. Glycans were concentrated via solid-phase extraction and analyzed by MALDI-TOF mass spectrometry. Signal-to-noise ratios of specific masses were used for calculations of relative amounts.6 cells/mL and product titer of 1015 mg/L were reached. An overview of these results is illustrated in Figure The performance of the platform medium was evaluated using a set of eleven different CHO cell lines in comparison to an industrially relevant, protein-free and chemically defined medium. For all tested cell lines, the maximum viable cell density (vcd) as well as the integrated viable cell density (ivcd) and the product titer were higher compared to the reference. In numbers, the improvement in vcd ranged between a factor of 1.7 and 2.5, in ivcd between a factor of 1.2 and 4.2 and in product titer between a factor of 1.4 and 2.7 in batch cultures. By this improvement viable cell densities of up to 17.53·10Furthermore, the potential influence of the utilized medium on product glycosylation was examined. For this, antibody harvest from two different clones cultivated in reference and platform medium was analyzed. The results of relative quantification of glycan structures by mass spectrometry showed highly comparable profiles for the reference and platform medium. An overview of the glycoanalysis is given in Table As an additional application, the platform medium was successfully utilized as a basis for a chemically defined cloning medium in limited dilution experiments. For different cell lines single cell growth was achieved and cells were effectively expanded from 96-well plate format up to shaking flask cultures.Within this work a chemically defined and animal-component free media platform was successfully implemented, which supports high performance growth and productivity without supplementation of proteins or growth hormones. In addition, its streamlined formulation of less than 50 components increases the design space for the efficient development of custom formulations. The suitability as a platform medium was verified by the successful cultivation of a wide range of cell lines including CHO-DG44, CHO-GS and CHO-K1 clones and the feature of easy adaption from serum containing and commercially available formulations. For all tested cell lines stable high performance cultivations with high product yields were achieved, with consistent glycosylation profiles. As a further field of application, the platform medium provides the basis for single cell growth following limited dilution.


XMRV is a gammaretrovirus closely related to xenotropic murine leukemia viruses (MuLVs). XMRV was first identified in familial cases of prostate cancer tissue using a virus gene array. Although, initial reports have identified XMRV predominantly in the prostate, recent reports of detection of XMRV in blood cells of patients with Chronic Fatigue Syndrome suggests that blood cells could act as a primary target and reservoir for XMRV to help disseminate infection throughout the body. The aim of this study is to elucidate possible routes of transmission and to determine the host range and cellular tropism of XMRV.Culture supernatants containing infectious virus from 22RV-1 or DU145-clone-7 cells were used to infect human cell lines Jurkat, H9, HL60, U937, primary PBMC and monocyte-derived macrophages representing the hematopoietic system. In addition, a variety of epithelial cells, lung epithelial cell line A549 and cervical epithelial cell lines CaSki, HeLa and SiHa were evaluated for infectivity with prostate cancer lines DU145 and LNCaP serving as positive controls. Infected cells were monitored for XMRV replication over a period of 5-7 days. XMRV replication was quantitated by RT-PCR, DNA PCR and real-time PCR. The ability of these cell lines to produce infectious viral particles was also determined.Replication of XMRV could be observed in cervical and lung epithelial cells, T-cell lines Jurkat and H9, B-cell line HL60, U937 cells and in primary PBMC and monocyte-derived macrophages. The levels of XMRV transcripts were lower in primary monocytes compared to T-cell lines suggesting less efficient replication in these cells.Viral replication could be identified in primary hematopoietic cells and a variety of epithelial cells in addition to the previously described prostate cancer derived cell lines investigated. Viral replication was considerably lower in primary monocytes, suggesting less efficient replication in these cells. These observations will help to further our understanding of XMRV pathogenesis and provide insights into the modes of transmission involved in XMRV infection.


Tumours are commonly hypoxic and this can be associated with aggressive tumour type, metastasis and resistance to therapy. Heat shock proteins (hsps) are induced in response to hypoxia, provide cancer cells with protection against tumour-associated stressors and chaperone oncoproteins that drive tumour proliferation. This study examined the effect of different oxygen concentrations on the expression of hsps in melanoma cell lines.2. Expression of Hsp90, Hsp70, Hsp60, Hsp40 and Hsp32 proteins were determined by flow cytometry.Melanoma cell lines were cultured in 2% and 20% O2. Hsp expression was different in 2% compared to 20% O2 and changes in Hsp90 expression correlated with cell line generation time (P<0.005) and viability (P<0.01). Greater total hsp expression correlated with improved viability in 2% but not 20% O2 (P<0.05). Relative expression of the different hsps was consistent across cell lines and each correlated with the others (P = 0.0001) but not with Hsp32. Hsp expression was inversely correlated with cell line adhesion to laminin as well as collagen type IV and Breslow depth of the original primary tumour tissue (P<0.05), but not with Clark level or patient survival. All five hsps were identified on the cell surface.Growth rates and viability were reduced in the majority of cell lines by culture in 2% O2 variably altered hsp expression in a panel of melanoma cell lines. Hsp expression was associated with certain cell line characteristics and clinical parameters of the originating tumour.Culture in 2% O It is well established that hypoxia is a feature of human tumours The major mechanism by which cells respond to hypoxic stress is by rapid modulation of the expression of the HIF transcription factor in vivo. Hsps have been investigated in melanoma tumour tissue and play multiple roles important for cancer growth, but have not been extensively studied in melanoma cell lines. Here, we present a preliminary study that sought to assess hsp expression in relation to melanoma cell line characteristics, patient clinical parameters and low and high oxygen culture conditions in a relatively large panel of melanoma cell lines.The relevance of hypoxia and hsps to cancer is well documented, but has rarely been studied in the context of human cancer cell cultures. Laboratory cell culture is routinely performed under hyperoxic conditions (i.e. in air) and this may be a limitation of this model for the study of human cancer cells which frequently experience hypoxia http://www.medizin.uni-tuebingen.de/estdab/) which is the largest existing collection of melanoma cell lines. The ESTDAB cell lines have been thoroughly characterised and controlled with mycoplasma testing and DNA finger printing Cell lines: Melanoma cell lines were selected from the European Searchable Tumour Line Database  for five days in an incubator  in air (20% O2) and in 2% O2 using the Concept 1000 Invivo2 . Seeding cell number was adjusted according to the generation time (time required for one population doubling) of each cell line in order to avoid confluence and to obtain similar cell numbers at the end of the culture period. Following the culture period cells were washed with Hank's Buffered Salt Solution (HBSS)  and detached from culture flasks by incubation with Trypsin-EDTA   for approximately three minutes. Trypsin was inactivated by adding of an equal volume of RPMI medium containing 10% FCS.Cell culture: Cell lines were cultured with 35 ml RPMI 1640 medium  supplemented with 10% heat inactivated Foetal Calf Serum (FCS)  in 75 cmDetermination of cell number and viability: The trypan blue exclusion method was used to determine the number of living and dead cells. Melanoma cells were suspended in 10 ml of HBSS and mixed thoroughly. Equal volumes of a 0.4% trypan blue solution (Sigma) and the cell sample were mixed and applied to a Neubauer haemocytometer. Phase contrast microscopy was used to distinguish between stained (dead) and unstained (living) cells.5 cells were washed with PFEA  and Fc receptors blocked with GAMUNEX  on ice for ten minutes. Cells were washed with PFEA before being permeablised and fixed with Cytofix/Cytoperm and Perm/Wash  according to the manufacturer’s instructions. Single fluorochrome staining was performed where antibody was diluted in Perm/Wash and incubated for 30 minutes on ice. PE-conjugated antibodies were used at the following concentrations: Hsp90 1:300 (product #SPA-830PE), Hsp70 1:100 (product #SPA-820PE), Hsp60 1:100 (product #SPA-807PE), Hsp40 1:100 (product #SPA-400PEF), Hsp32 1:100 (product #OSA-111PE) . Following incubation cells were washed twice with Perm/Wash before being suspended in 120 µL of 1% formaldehyde solution  in PFEA and stored on ice in a darkened container. For cell surface staining, PFEA was used in place of Cytofix/Cytoperm and Perm/Wash. Cells were immediately analysed on an LSR II flow cytometer with FACSDiva software (BD Biosciences). Data was analysed using FlowJo software . To perform data analysis, the main population of cells was gated on a forward scatter versus side scatter dot plot according to size and granularity. For each sample a fluorescence index (FI) was calculated in order to allow the comparison of fluorescence values for each cell line. The FI is the fold increase of the mean fluorescence intensity of the main population of cells in the stained cell sample by comparison with the corresponding unstained sample. FI values of less than two were considered negative.Flow cytometry: Following trypsinisation, 3.0×10Statistical analysis: Statistical analysis was performed using Prism software version 4.02 . Cell line ligand adhesion data was obtained from ESTDAB. Within matching data sets, changes of less than 5.0% were not considered to be different.Paired two-tailed non-parametric t tests (Wilcoxon matched pairs tests) were used to assess significance between matching data points between two conditions.Significance between two groups was assessed with two-tailed non-parametric (Mann-Whitney U) tests.Correlations were assessed with non-parametric two-tailed (Spearman) correlation tests.Trends across four grouping variables were assessed with two-tailed chi-squared contingency tests.2. Twelve of 18 cell lines showed a mean increase of 70% in generation time when cultured in 2% O2 compared with the 20% O2 condition (P = 0.054). Five of the remaining six cell lines showed reduced generation times (mean decrease 26%), while the remaining cell line did not change , while one cell line showed no change for both generation time and viability (EST 143).Eighteen melanoma cell lines were selected for culture in 2% Ot change  A. Cell t change  B , but the remaining cell lines up- and down-regulated these four hsps in response to low oxygen tension. Of note is that the four cell lines that showed faster generation times and improved viability in the 2% O2 condition showed a corresponding increase in Hsp90 expression in the 2% O2 culture condition.Comparing hsp expression between the 2% and 20% Opression . For eac2 culture conditions. With the exception of Hsp60 in 20% O2 (P<0.03), total hsp expression was not associated with generation time in either condition (data not shown). When examining the increase or decrease in hsp expression in response to low oxygen tension, associations between the change in Hsp90 expression with cell line generation time  but not in the 20% O2 culture condition ) (2).Given that the relative expression of hsps was observed to be consistent, relationships between the expression of these hsps were investigated. The expression of Hsp90, Hsp70, Hsp60 and Hsp40 was found to correlate, but not Hsp32 (in 2% (P = 0.015) and 20% OHsp90 is involved in cell adhesion The expression of hsps in melanoma tissue has been shown to correlate with clinical parameters, but few, if any, studies have been performed on hsp expression in melanoma cell lines in this context. Patient survival time, Clark level and Breslow depth of the primary tumour tissue was known for a limited number of the metastasis-derived cell lines in the biobank used here, and were tested for a correlation with the expression of hsps. The expression of Hsp90 and Hsp40 was found to inversely correlate with Breslow depth, but no relationships were found with patient survival or Clark level . Since hypoxia is a common and important feature of cancer, culture of these cells under high oxygen conditions may contribute to the generation of misleading results. For this reason we investigated viability, generation time and hsp expression in order to assess melanoma cell line response to low oxygen tension. Although hypoxia is a feature of human melanoma, the majority of the cell lines tested here displayed retarded growth and reduced viability under low oxygen conditions. This may be due to the fact that they have been generated and cultured under hyperoxic conditions which have prevented the selection of hypoxia-resistant cells that would normally occur Relative hsp expression was observed to be consistent across the cohort of cell lines and levels of Hsp 90, 70, 60 and 40 expression were shown to correlate with one another but not with Hsp32. Hsp90 is essential for eukaryotic cell viability and it chaperones a large number of overexpressed client proteins in cancer. This may explain why Hsp90 was expressed at a level many fold greater than the other hsps examined in vitro conditions. Similar reports exist in other contexts et al. demonstrated MMP-2 mRNA levels in renal cancer cell lines to inversely correlate with patient survival in vivo processes is that it is not known how reflective the parameters measured under these conditions are of in vivo conditions. The data presented here coupled with at least one previous report may suggest that cancer cell lines maintain a degree of relevance to patient clinical parameters and thus may be of use as models for cancer. This relevance may be maintained indirectly, since results from previous studies using melanoma tissue suggest that in contrast to the results of this study using cell lines, that Breslow depth is positively associated with hsp expression. Since in these cell lines hsp expression was relevant to viability in 2% but not 20% O2, culture under low oxygen tension may be more relevant to in vivo growth conditions, but further studies are needed to confirm these preliminary findings. Of note is that the inverse relationship between hsp expression and Breslow depth was observed in 20% O2, and it is not known if hsp expression in 2% O2 also correlates with Breslow depth. No remarkable differences were observed between the primary-derived and metastasis-derived cell lines in this study.Hsp expression was observed to correlate with Breslow depth but not Clark level or patient survival. Studies using primary melanoma tissue have shown increased Hsp90 expression to be associated with greater Breslow depth and Clark level in vivo, as the level of hsp expression observed in these cell lines may not be reflective of the level in vivo. This could be due to the influence of the artificial cell culture environment. Follow-up studies in tumours and cell lines will confirm or refute the preliminary findings presented here. If this observation is confirmed, further studies could examine other clinical and biological features such as the expression of inflammatory cytokines, tumour ulceration, regional differences in the distribution of hsp expression , histological sub-type or other factors that may interact with or separately explain this observation. If the level of hsp expression observed in this study is reflective of in vivo expression, then studies using melanoma tissue that have shown higher hsp expression to be associated with greater Breslow depth It should be noted that the observation of an inverse correlation between hsp expression and Breslow depth does not imply that this relationship is present in tumours in vivo importance of hypoxia coupled with the finding that hsp expression is relevant to melanoma cell viability in low but not high oxygen tension suggests cancer cell cultures should more frequently be subjected to oxygen tension that more closely resembles in vivo levels in order to cast more light on the largely unknown influence that this condition has on the myriad of facets investigated in modern cancer research.Although this investigation is not an extensive study into the effects of low oxygen tension on cancer cell cultures, it does present novel findings with respect to the potential role of hsps and demonstrates that further investigation into the effects of oxygen tension on cancer cell cultures is warranted. These data suggest that hsps associate closely in melanoma cells and that their expression is associated with tolerance to low oxygen tension and patient clinical parameters. The


There is an error in paragraph two, sentence two of the "Identification of cell lines with 'claudin-low' features" section of Results. The sentence should read: "All lines exhibit low levels of E-cadherin and Claudin-7 in comparison to normal mammary epithelial cells ."


A fructose containing cell culture medium has the advantage of low lactate production and a small pH change, leading to cell and product stability. But, not all cell lines grow well in the medium, and the fructose transporter, GLUT5, is related to it . Thus, wAs a model, an IgG and GLUT5 co-expression vector was constructed and transfected into cells. When the transfected CHO-K1 and SC-01MFP cells were cultured in the fructose-based medium, both IgG productions were increased up to about two-fold of that cultured in the glucose-based medium (Table


Ovarian cancer is the leading cause of death among gynaecological malignancies. Multiple drug resistance makes cancer cells insensitive to chemotherapy. In this study, we developed sixprimary ovarian cancer cell lines  resistant to drugs such as methotrexate, cisplatin, doxorubicin, vincristine, topotecan, and paclitaxel. A chemosensitivity assay MTT test was performed to assess drug cross-resistance. Quantitative real-time polymerase chain reaction and Western blot were also performed to determine mRNA and protein expression of genes involved in chemoresistance. We observed high cross-resistance to doxorubicin, vincristine, and paclitaxel in the cell lines resistant to these agents. We also found a significant correlation between resistance to these drugs and increased expression of P-gp. Two different mechanisms of topotecan resistance were observed in the W1TR and W1PR cell lines. We did not observe any correlation between MRP2 transcriptand protein levels. Cell lines resistant to agents used in ovarian cancer treatment remained sensitive to methotrexate. The main mechanisms of drug resistance were due to P-gp expression in the doxorubicin, vincristine, and paclitaxel resistant cell lines and BCRP expression in the topotecan resistant cell line. Ovarian cancer represents the most common cause of death among gynaecological malignancies in Europe and North America. The average 5-year survival rate is approximately 40%; however, patients with advanced disease (stages III and IV) have a significantly lower survival rate of only 10%–20%  at 37°C in a 5% CO2 atmosphere.The primary human ovarian cancer cell line W1 was established in our department (December 2009) from tissue obtained from an untreated patient diagnosed with ovarian cancer. Sublines resistant to methotrexate , cisplatin , doxorubicin , vincristine , topotecan , and paclitaxel  were generated by exposure of the W1 line to incremental increasing concentrations of each drug. The cells were seeding in the concentration of 10 thousand cells/cm. (1993) . These c. (1993) . All celμL of the MTT labelling reagent was added to the medium , and the cells were incubated for an additional 4 h. Following this process, 100 μL of solubilisation solution was added to each well. The absorbance of each sample was measured in a microplate reader at 570 nm with a reference wavelength of 720 nm, according to the manufacturer's protocol. The negative control was conducted using cell-free culture medium containing both the MTT reagent and solubilisation solution. The experiments were repeated three times, and each concentration in a given experiment was tested in duplicates. Cell viability was expressed as a percentage of the untreated control (means ± SEM).The drug sensitivity of the W1 cell line and the drug resistant cell lines were confirmed by the MTT cell survival assay. Briefly, all cell lines were seeded at a density of 5,000 cells/well in 96-well plates. The cells were allowed to grow for 48 hours and subsequently treated with fresh medium supplemented with or without increasing concentrations of drugs and incubated for 72 h at 37°C. After 72 h of exposure, 10 μg of RNA was used to cDNA synthesis. Real-time PCR was performed using the Eppendorf PCR System , Maxima SYBR Green/ROX qPCR Master Mix (Fermentas) and sequence-specific primers, as indicated in β-actin, hypoxanthine-guanine phosphoribosyltransferase 1 (HRPT1) and beta-2-microglobulin (β2M) served as the normalising genes (geometric mean) against which changes in the examined genes expression were compared. Gene expression was analysed using the relative quantification (RQ) method. RQ estimates the difference at the level of gene expression against a calibrator (W1 drug sensitive line) . The W1 cell line was used as the calibrator. The analysis was conducted employing the standard formula: RQ = 2−ΔΔCt (where ΔΔCt = ΔCt for the sample (drug-resistant line) −ΔCt for the calibrator (drug sensitive line)). The graphs were made using Sigma Plot.Changes in ABCB1, ABCC1, ABCC2, ABCG2 and LRP gene expression in the W1 and drug-resistant cell lines were examined. RNA was isolated using the Gene Matrix Universal RNA purification Kit (EURx Ltd.), as described by the manufacturer's protocol. Reverse transcription was performed using the M-MLV reverse transcriptase (Invitrogen) as described in the manufacturer's protocol using a thermal cycler . 2 μL of Maxima SYBR Green/ROX qPCR Master Mix (Fermentas), 1 μL of each primer   followed by 50 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. After amplification, Melt Curve analysis was performed to analyze product melting temperature. The amplification products were also resolved by 3% agarose gel electrophoresis and visualized by ethidium bromide staining.For amplification, 12,5  Poland) , 9,5 μL 6 cells/50 μL lysis buffer) were lysed in buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM EDTA, Protein Inhibitor Cocktail (ROCHE) and 1% Triton X-100 for 60 min at 4°C. The lysates were centrifuged at 12000 ×g for 15 min at 4°C, and protein concentration was determined using the Bio-Rad  protein assay system. Thirty micrograms of the protein was resuspended in a solution of 40 μL of 200 mM Tris-HCl (pH 6.8), 5% SDS, 10% glycerol, 0.25% 2-mercaptoethanol, and 0.1% bromophenol blue. The resuspended protein was loaded into each well and separated on a 7% Tris-glycine gel using the SDS-PAGE technique. The proteins were transferred to a PVDF membrane and blocked with 5% milk in TBS/Tween , followed by immunodetection with either mouse anti-P-gp Ab (C219) at 1 : 500 dilution, mouse anti-MRP1 Ab (IU2H10) at 1 : 500 dilution, goat anti-MRP2 Ab (H-17) at 1 : 500 dilution, rabbit anti-ABCG2 Ab (H-70) at 1 : 500 dilution, or mouse anti-MVP/LRP (MVP37) Ab at 1 : 500 dilution with the appropriate HRP-conjugated secondary Ab. Chemiluminescence detection of the bands was performed using the enhanced chemical luminescence (ECL) kit and Hyperfilm ECL from Amersham . The Western blot was quantified by densitometry analysis of the band intensity in the autoradiogram using the GelDoc-It Imaging System and the Vision WorkLS software. To normalise protein loading of the lanes, the membranes were stripped and reblotted with rabbit anti-GADPH Ab (FL-335) at 1 : 500 dilution, donkey anti-goat HRP-conjugated Ab, and goat anti-rabbit HRP-conjugated Ab.Cells . In contrast, the MDR1 transcript level significantly decreased in the methotrexate-resistant cell line (P < 0.01).The transcript level of MDR1 significantly increased in the doxorubicin, vincristine, and paclitaxel  resistant cell lines (ell line  (P < 0.0P < 0.001) and significantly lower in the paclitaxel-resistant cell line (P < 0.01) (The MRP2 transcript level was significantly higher in the methotrexate-resistant cell line ( < 0.01) . P < 0.01) and topotecan (P < 0.001) resistant cell lines. However, expression of BCRP was variable in these two cell lines. We observed approximately sixfold higher transcript levels in the W1VR cells, and expression in the W1TR cells increased by a factor of more than 1,000. In contrast, the expression of BCRP in the W1PR cells significantly decreased (P < 0.01) (BCRP expression increased in the vincristine ( < 0.01) . Western blot analysis  of the PTo assess whether expression of MDR genes was correlated with resistance to a specific drug treatment, correlation analyses of MDR1, MRP2, and BCRP with IC50 levels for methotrexate, cisplatin, doxorubicin, vincristine, topotecan, and paclitaxel were performed. We observed a high degree of correlation between the MDR1 transcript and protein and resistance to doxorubicin, vincristine and paclitaxel Tables  and 4. SIn our study, we compared development of multiple drug resistance to the parental W1 ovarian cancer cell line in response to cytostatic agents used in ovarian cancer chemotherapy, all of which have different mechanisms of action. The drug cross-reactivity study showed that the parental W1 cell line was sensitive to all investigated drugs with IC50 below their therapeutic concentration . CompariThe drug most commonly prescribed in ovarian cancer treatment is cisplatin . In our A similar pattern of cross-resistance to doxorubicin, vincristine, and paclitaxel has been observed in the investigated cell lines. The cell lines studied here can be divided in two groups: sensitive to the tested drugs  and those resistant to the drugs . These results may have been expected because cross-resistance between cell lines resistant to these drugs has frequently been documented in the literature –33. FurtThe W1PR line has shown a similar pattern of response to the topotecan like topotecan-resistant cell line, W1TR; however, the W1TR line showed no resistance to paclitaxel. Here, we observed only a one-sided cross-resistance, suggesting that topotecan may not serve as a good cytostatic for ovarian cancer treatment because resistance to paclitaxel has already developed. Therefore, topotecan might be better applied as a drug for first-line chemotherapy. The best recognised protein responsible for MDR is P-gp, encoded by the ABCB1 (MDR1) gene. We observed high levels of the transcript and the protein in cell lines resistant to doxorubicin, vincristine and paclitaxel. This observation is consistent with published data showing that P-gp has broad substrate specificity, including natural products such as anthracyclines, vinca alkaloids, and taxanes –16, 34. The lack of correlation between the transcript and protein levels of MRP2 may result from increased transcription or increased transcript stability caused by methotrexate treatment. It is also possible that there exists another so far not described MRP2 isoform, and the difference between the transcript and protein levels may reflect different MRP2 isoforms. Regardless, we observed a pronounced correlation between the MRP2 transcript level and methotrexate resistance in our cell lines. This correlation suggests that MRP2 plays a critical role in methotrexate resistance in the investigated cell lines, which is also consistent with several previously published data showing a correlation between MRP2 expression and methotrexate resistance , 36. HigHigh BCRP transcript and protein levels in the topotecan resistant cell line have been well established , 41, 42 Contrary to other published results, we observed methotrexate sensitivity in our cell lines expressing high levels of P-gp. According to previously published reports, methotrexate is a substrate for P-gp , but ourIn summary, some of mechanisms of drug resistance are well known, and our results suggest that it is possible to predict cross-resistance to other drugs when the classical MDR, which is correlated with P-gp expression, is involved. Cases of other resistances such as topotecan, methotrexate, and cisplatin resistance appear to be more complex, and further analyses of MDR development must be explored. Our results confirm that cisplatin is an effective drug for first-line chemotherapy in ovarian cancer treatment. The efficacy of topotecan and doxorubicin as the second lines of chemotherapy may be limited because of their cross-resistance with paclitaxel, which is used as a first line of chemotherapeutic treatment. Therefore, methotrexate may be considered to be an alternative therapy for ovarian cancer treatment because no cross-resistance was observed in our cell lines.


TAK733 is a novel allosteric, non-ATP-binding, inhibitor of the BRAF substrates MEK-1/2.The growth inhibitory effects of TAK733 were assessed in a panel of 27 cutaneous and five uveal melanoma cell lines genotyped for driver oncogenic mutations. Flow cytometry, Western blots and metabolic tracer uptake assays were used to characterize the changes induced by exposure to TAK733.BRAFV600E mutant cell lines being highly sensitive with IC50s below 1 nM. The five uveal melanoma cell lines had GNAQ or GNA11 mutations and were either moderately or highly sensitive to TAK733. The tested cell lines wild type for NRAS, BRAF, GNAQ and GNA11 driver mutations were moderately to highly resistant to TAK733. TAK733 led to a decrease in pERK and G1 arrest in most of these melanoma cell lines regardless of their origin, driver oncogenic mutations and in vitro sensitivity to TAK733. MEK inhibition resulted in increase in pMEK more prominently in NRASQ61L mutant and GNAQ mutant cell lines than in BRAFV600E mutant cell lines. Uptake of the metabolic tracers FDG and FLT was inhibited by TAK733 in a manner that closely paralleled the in vitro sensitivity assays.Fourteen cutaneous melanoma cell lines with different driver mutations were sensitive to the antiproliferative effects of TAK733, with a higher proportion of The MEK inhibitor TAK733 has antitumor properties in melanoma cell lines with different oncogenic mutations and these effects could be detectable by differential metabolic tracer uptake. NRAS or BRAF genes in melanomas of skin primary, c-Kit in acral and mucosal melanomas, and GNAQ and GNA11 in uveal melanomas FDG. The lowest degree of inhibition was in the two most resistant cell lines, the BRAFV600E mutant M233 and the NRASQ61K mutant M244  did not clearly differentiate naturally sensitive and resistant BRAFV600E mutant cutaneous melanomas to the BRAF inhibitor vemurafenib, but downstream signaling studies did suggest that the PI3K/AKT pathway may be involved [NRAS or BRAF mutant melanomas, which could be due to hyperactive receptor tyrosine kinase signaling leading to resistance [Initial data testing MEK inhibitors in melanoma cell lines suggested a high level and selective sensitivity in ncogenes . Furtherhibitors ,14. The urafenib ,22. The involved ,22. In tsistance .BRAFV600E mutation frequently have high sensitivity to MEK inhibitors in vitro[BRAFV600E mutant cutaneous metastatic melanoma enrolled in clinical trials testing MEK inhibitors [in vitro and in vivo results with MEK inhibitors is not clearly understood at this time, but it may be related to a lower therapeutic window of MEK inhibitors in the clinic compared to type I BRAF inhibitors. This could be explained by the paradoxical activation of the MAPK pathway in BRAF wild type cutaneous cells, where type I BRAF inhibitors increase (or do not change) MAPK signaling in normal cells, while they efficiently block the MAPK pathway downstream of oncogenic BRAFV600. On the contrary, MEK inhibitors can equally block the MAPK pathway downstream of both oncogenic and wild type BRAF. This lack of differentiation most likely causes the dose limiting toxicities (DLT) at exposures in vivo that do not adequately block the MAPK pathway in BRAFV600 mutant melanoma. Despite this, MEK inhibitors are likely to have a role in the treatment of cancers with constitutive MAPK signaling from oncogenic mutations upstream of MEK. In particular the combination of MEK and RAF inhibitors may be beneficial by inducing higher MAPK inhibition in mutant cells and therefore lowering the cancer escape mechanisms and also decreasing toxicities from paradoxical MAPK activation [BRAF has only MEK as a substrate for activation , and as  in vitro. Howeverhibitors ,26 have hibitors ,13,27. Ttivation , such astivation .GNAQ or GNA11, resulting in overlapping functions in melanoma cells with the constitutive upregulation of the MAPK pathway [GNAQ mutation resulted in sensitivity to downstream blocking of the MAPK pathway with a MEK inhibitor [in vitro and clinical results when blocking oncogenic MAPK signaling using MEK inhibitors may apply to uveal melanomas.The majority of uveal melanomas bear a mutually exclusive activating mutation in either  pathway . In precnhibitor . Our dat3H-radiolabeled compounds that are trapped intracellularly upon metabolic processing allows testing their potential future use as PET probes in the clinical development of a new agent. It is anticipated that these radiolabeled metabolic probes can provide non-invasive pharmacodynamic information with the use of clinical PET scanners. In our studies, the highly sensitive cell lines had a decrease in the uptake of radiolabeled thymidine and deoxy-glucose that seemingly correlated with the cell viability and cell cycle results. However, there were variable changes in the highly resistant cell lines that did not directly correlate with the cell viability assay results (ex. M263 with marked decrease in the uptake of both tracers despite its resistance to TAK733). The metabolic tracer uptake studies were performed at a slightly earlier time point than the proliferation/viability assays to capture earlier events, and may be the reason of the discrepancy in results. These results raise the point that earlier PET scans with these tracers to detect early pharmacodynamic changes may not fully predict the later restaging imaging CT scan results.The differential uptake of in vitro against a large subset of melanoma cell lines. We confirmed the previously reported cytotoxic effect of a MEK inhibitor against cell lines with BRAFV600E mutations, but in addition the cytotoxic activity was evident in a high proportion of melanoma cell lines with NRAS, GNAQ or GNA11 driver mutations. The antiproliferative and cell metabolism effects of this MEK inhibitor against melanoma cell lines can be detected with metabolic probes that could be tested with caution in the clinical development of this agent using PET imaging.In conclusion, inhibition of oncogenic MAPK signaling through MEK1 and MEK2 by TAK733 results in antitumor activity TAK-733 was obtained under a materials transfer agreement (MTA) from Millennium Pharmaceuticals, Inc.  and dissolved in dimethyl sulfoxide  to a stock concentration of 10 mM. The cutaneous melanoma cell lines of the M series were established from biopsies of metastatic melanoma of cutaneous origin as previously described  under thCell lines were analyzed for known oncogenic activating mutations and deletions using multiplex PCR as well as by MALDI-TOF mass spectrometry  . Point mMelanoma cell lines were treated with TAK-733 or parallel DMSO vehicle control at the given concentrations for 72 hours. Cell viability was measured using a tetrazolium compound [3--5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS)-based colorimetric cell proliferation assay  as previously described .Cells were treated with different concentrations of TAK-733 (50 and 500 nM) or parallel vehicle control for 48 hours, fixed by Cytofix/Cytoperm solution and washed by Perm/Wash buffer according to fixation and pereabilization method recommended by BD bioscience, and then stained in sterile PBS containing 1.0% albumin bovine serum, 0.1% Nonidet P-40 (Sigma-Aldrich) and 3 μM DAPI . Flow cytometry was analyzed using FlowJo .Western blotting was performed as previously described . Primary4 cells/well were plated on 0.001% poly-L-lysine (Sigma-Aldrich) pre-incubated filter bottom 96-well plates  and rested for 24 hours. 0.1 and 1 μM of TAK733 or parallel DMSO vehicle control were added in triplicate for 20 hours. Cells were incubated for 1 hour with 2.0 μCi with metabolic tracers chosen as analogues of PET tracers: 3H-DDG  in glucose-free RPMI 1640 (Invitrogen), or methyl-3H-thymidine  in RPMI 1640. Extracellular metabolic tracer was washed off using a multiscreen HTS vacuum manifold system (Millipore). 100 μL scintillation fluid  was added to each well and tritium count was measured on a 1450 microbeta trilux microplate (Perkin Elmer).3 x 10Erika von Euw, Mohammad Atefi, Narsis Attar, Connie Chu, Sybil Zachariah, Barry L. Burgess, Stephen Mok, Charles Ng, Deborah J.L. Wong, Bartosz Chmielowski, Richard C. Koya, Tara A. McCannel: Have no competing interests. David I. Lichter, Elena Izmailova: Are employees of Millennium Pharmaceuticals, Inc., the manufacturer of TAK733. Antoni Ribas: Is a compensated ad hoc advisor to Millennium Pharmaceuticals, Inc., the manufacturer of TAK733.EE, MA, NA, CC, SZ, BLB, SM, CN, DJLW, DIL, RCK: Performed experiments. EE, MA, TAM, EI, AR: Designed the studies. BLB, BC, EI, AR: Provided key reagents. EE, MA, AR: Wrote the manuscript. EE, MA, NA, CC, SZ, BLB, SM, CN, DJLW, BC, DIL, RCK, TAM, EI, AR: All authors approved the final manuscript.Figure S1TAK733 MTS-based colorimetric cell proliferation assay curves in melanoma cell lines of cutaneous origin according to their BRAF (A) or NRAS (B) mutational status, WT (C) and of uveal origin (D). Modulation of the melanoma cell line viability at a range of different concentrations of TAK733. The effects of TAK733 on cell growth and viability were analyzed after 72 hours of treatment using an MTS assay.Click here for fileFigure S2Time-course analyses of the effects of TAK733 on the signaling of the MAPK and PI3K/AKT pathways by Western blot. Two BRAFV600E melanoma cell lines were exposed for varying time points to TAK733. A) The sensitive BRAFV600E mutated cutaneous melanoma cell line M229; B) The resistant BRAFV600E mutated cutaneous melanoma cell line M233.Click here for file


Fatty acid binding proteins (FABP) play an important role in carcinogenesis. Modified FABP expression patterns were described for prostate, bladder and for renal cell carcinoma. Studies on metabolic relationships and interactions in permanent cell lines allow a deeper insight into molecular processes. The aim of this study is therefore a systematic overview on mRNA and protein expressions of seven FABPs in frequently used urological cell lines.Nine cell lines of renal carcinomas, seven of urinary bladder carcinomas, and five of prostate carcinomas were investigated. Quantitative RT-qPCR and western blotting were used to determine different FABPs. In addition, 46 paired cancerous and noncancerous tissue samples from nephrectomy specimen with renal cell carcinomas were investigated regarding the ileum FABP mRNA expression level and associated with survival outcome.General characteristics of all urological carcinoma cell lines were the expression of E-and IL-FABP on mRNA and protein level, while the expressions differed between the cell lines. The protein expression was not always congruent with the mRNA expression. Renal cell carcinoma cell lines showed expressions of L-, H- and B-FABP mRNA in addition to the general FABP expression in five out of the eight investigated cell lines. In bladder cancer cell lines, we additionally found the expression of A-FABP mRNA in six cell lines, while H-FABP was present only in three cell lines. In prostate cancer cell lines, a strong reduction of A- and E- FABP mRNA was observed. The expression of B-FABP mRNA and protein was observed only in the 22 RV-1 cells. IL-FABP mRNA was over-expressed in renal tumour tissue. The IL-FABP ratio was identified as an independent indicator of survival outcome.Distinctly different FABP expression patterns were observed not only between the cell lines derived from the three cancer types, but also between the cell lines from the same cancer. The FABP patterns in the cell lines do not always reflect the real situation in the tumours. These facts have to be considered in functional studies concerning the different FABPs. Fatty acid binding proteins (FABPs) represent a family of cytosolic proteins containing 9 different types. The designations for the FABPs were derived from the tissue from which they had originally been isolated and include: 1) liver (L-FABP); 2) intestine (I-FABP); 3) heart (H-FABP); 4) adipocyte (A-FABP); 5) epidermis (E-FABP); (6) ileal (IL-FABP); (7) brain (B-FABP); (8) myelin (M-FABP) and (9) testis (T-FABP). The FABPs bind different fatty acids and take on the transport to cellular compartments. Thus, FABPs are involved in lipid metabolism and regulation of gene expression [ liver  kidney specimens from primary tumours were used for total RNA isolation. The samples were derived from patients with renal cell carcinomas (RCC) undergoing radical tumour nephrectomy at the Department of Urology, Charité - Universitätsmedizin Berlin, Germany between 2003 and 2005. Staging met the UICC 2002 criteria. Histological classification was performed according to the WHO criteria; tumour grading was accomplished according to Fuhrman. The clinico-pathological parameters in all RCC were summarised in table 6 cells. All preparation steps were performed according to the supplier's protocol. The sample pairs from the same kidney were collected immediately after surgery in RNAlater® Stabilization Reagent , stored overnight at 4°C and at -80°C afterwards. The RNA isolation was performed as formerly described by our group [The total RNA from cells and tissue material were isolated using the RNeasy mini Kit from Qiagen . The cell number was adjusted between 2 and 6 × 10One μg RNA of the various urological cell lines and of the kidney tissues respectively, was reverse transcribed using the Transcriptor First Strand cDNA Synthesis Kit  by random hexamer priming method according to the manufacturer's recommendations. The detailed procedure was previously described . All use-2 .The previously characterised conditions  were use7 cells were washed two times in PBS and then sedimented by 300 × g centrifugation for 10 min. Cell lysates were prepared according to the method of Zigrino et al. [Suspensions of 2 - 3 × 10o et al. . Proteino et al. . All proStatistical analysis was performed with SPSS, version 19.0 . P values < 0.05 were considered significant.The general characteristic of all urological carcinoma cell lines is the expression of E-and IL-FABP both on mRNA and protein level Figure , while tThe cell line HK-2 derived from normal renal tissue only expressed mRNAs of E-FABP and IL-FABP. Its mRNA expression pattern differed in comparison to the five carcinoma cell lines 786-0, A 704, ACHN, Caki-1 and Caki-2, which showed additional FABPs. The 786-0 and Caki-1 cells expressed L-FABP, ACHN cells expressed H-FABP and A 704 and Caki-2 cells expressed B-FABP.The protein expression was not always congruent with the mRNA expression and the levels of E- and IL-FABP in these cells were different in the cell lines. In 786-0, A 498 and Caki-2 cells, the E-FABP protein expression was very high Figure . Caki-2 E- and IL-FABP were ubiquitously detectable as mRNA and protein in all bladder cell lines Figure . The proE-FABP was found to be the main FABP in prostate carcinoma cell lines Figure , while tThe benign prostate hyperplasia cell line (BPH-1) was characterised by the two additional mRNAs of H-FABP and A-FABP. Two cell lines derived from metastasis, DU-145 and LNCaP, lost of these two mRNAs (H-FABP and A-FABP). The 22 RV-1 cells, derived from a primary prostate carcinoma, were marked by the loss of A-FABP mRNA and appearance of B-FABP mRNA and the corresponding protein. Protein detection of H- and A-FABP was not possible in all cell lines, so that differences between mRNA and protein expression appeared there, too.s = -0.143, p = 0.343; rs = -0.181, p = 0.228). Samples from patients without metastasis at the time of radical nephrectomy (27 cases) displayed a 139-fold (median) over-expression  in RCC than in the normal tissue  reached significance for survival in Kaplan-Meier analysis Figure . PatientApproximately one quarter of all newly diagnosed cancer patients have urological tumours . Thus, tOur data show three essential features: (a) the expression of E- and IL-FABP and the lack of expression of I-FABP on mRNA and protein levels in all urological cell lines Figure , (b) theDiscrepancies between RNA and protein could be explained by different analytical sensitivities of the methods, in case of RT-PCR and Western blot technique, and more probably, by biological reasons. As extensive cellular machinery is involved in the process of translation, possible differences between RNA and protein expression can be caused by posttranscriptional regulatory mechanisms. These processes are still poorly understood. One possibility is that small non-coding RNAs might influence the translation.In addition to the general expression of E- and IL-FABP in all cell lines, five out of the eight investigated renal cell carcinoma cell lines showed expressions of L-, H- and B-FABP mRNA. Only the Caki-2 cells represented the situation in renal cell carcinoma tissue, particularly the decreased or absent expression of L- and H-FABP and the up-regulation of B-FABP mRNA. Protein of B-FABP was exclusively detectable in the Caki-2 cells. It was known from earlier studies ,5 that rThe results of IL-FABP expression and our previous observations regarding the clinical significance of B- and L-FABPs in renal cell carcinoma  promptedIn summary, this and the former study showed that the renal cell carcinoma tissue was characterised by decreased L- and H-FABPs and increased B- and IL-FABPs. The changes of these four FABP types indicate fundamental alterations in the fatty acid metabolism in the RCC carcinogenesis. Most of the investigated renal carcinoma cell lines only partly reflected these changes in tumour tissue.In bladder cancer cell lines, we found in addition to the general expression of E- and IL-FABP the expression of A-FABP in six examined cell lines, while H-FABP was present only in three cell lines . A high expression of A-FABP mRNA and protein was observed in the well-differentiated RT-4 and RT-112 cells, while the other cell lines, which had been derived from less-differentiated, high-grade bladder tumours such as HT-1376 cells showed lower A-FABP mRNA expression. This result corresponds to data of Ohlsson et al.  reportedThe expression pattern of various FABP types in prostate cancer cell lines was previously described by Das et al. . The autOur study is the first report about the mRNA and protein expression of various FABP types in frequently used cell lines from prostate, bladder, and kidney cancers. Regarding to carcinoma cell lines, distinct differences of FABP expression patterns were observed between the cells derived from the three cancer types but also between the organ-specific cell lines. In addition, the FABP patterns in the cell lines do not reflect the real situation in the tumours. It was suggested that in normal tissues a balance exists between different FABP types and that this balance is destroyed in cancerogenesis causing a new composition of FABP types . To studThe authors declare that they have no competing interests.AT coordinated the study, performed statistical analyses and wrote the paper. SS performed the RT-PCR and western blots. MJ realised the primer design and controlled the quality of PCR reactions. KJ supported statistical analyses and revised the paper. CS provided samples and clinico-pathological data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/302/prepub


To report three low-passage cell lines from primary choroidal melanoma with metastatic outcome, which were stable for cytogenetic patterns and expression profiles of the primary melanoma.125iodine brachytherapy or immediately after enucleation. Cells were examined for cytopathology, evaluated by fluorescence in-situ hybridization (FISH) for the centromere of chromosome 3, analyzed by 250K whole genome Mapping Array and U133 plus 2.0 Expression Array, and placed in cell culture. At passage 3, the cell lines were analyzed by Mapping Array and Expression Array.In patients with choroidal melanoma, transscleral fine needle aspiration biopsy (FNAB) was performed immediately before plaque placement for Three cell lines were propagated from primary choroidal melanomas in three patients who subsequently developed metastasis. Two cell lines were stable for the entire chromosomal aberration pattern of the respective primary tumor. In the third, necrotic material from the biopsy prevented further analysis, yet resulted in a stable cell line. Each cell line had chromosome 3 loss, 6q loss, 8p loss, multiple 8q gain, and 16q loss. Additionally, two cell lines had chromosome 6p gain. Two cell lines had RNA expression profiles similar to the respective primary tumors; the third cell line had a similar RNA expression profile relative to the other two cell lines.FNAB of primary choroidal melanomas resulted in highly characterized, low-passage cell lines, which were stable for the cytogenetic patterns and expression profiles found in the primary tumor. These cell lines represent novel tools for the study of metastatic choroidal melanoma biology. Choroidal melanoma (melanoma arising primarily from the ciliary body or choroid) is the most common primary intraocular malignancy in adults. Despite treatment with brachytherapy, external beam radiation or enucleation, long-term follow-up is associated with melanoma-related death in approximately 50% of patients .Most strongly correlated with increased risk of metastasis are cytogenetic aberrations and gene expression abnormalities in melanoma cells -5. Loss Melanoma cytogenetic abnormalities provide information regarding the fundamental molecular biology of choroidal melanoma and may uncover potential genes for targeted therapy ,12. RecoAll studies were performed in accordance with the United States Health Insurance Portability and Accountability Act (HIPAA) of 1996. All participants gave informed consent and all studies were approved by the Office of the Human Research Protection Program  of the University of California, Los Angeles, Los Angeles, CA.125iodine brachytherapy or immediately after enucleation. As described elsewhere, cells obtained from the biopsy were examined for cytopathology, evaluated by fluorescence in-situ hybridization (FISH) for the centromere of chromosome 3, analyzed by 250K Mapping Array  for chromosomal copy number variation, U133 plus 2.0 Arrays (Affymetrix) for gene expression, and placed in cell culture [In patients with primary choroidal melanoma and no clinical evidence of metastasis, transscleral fine needle aspiration biopsy (FNAB) was performed immediately before plaque placement for  culture -13.2. Primary cultures were seeded in 12.5 cm2 vented flasks and grown to confluency (about 12 weeks) replacing half of the medium every 3 days.Triturated biopsy cells were cultured in a growth medium of DMEM, 10% human AB serum, 5 µg/ml bovine insulin and glutamine-pen-strep at 37 °C, 7% CO2 flasks and evaluated at passage 3. Briefly, trypsinized cells were stabilized in RNAprotect Cell Reagent  and analyzed by 250K Mapping Array and U133 plus 2.0 Expression Array (Affymetrix) as described hereafter. Beginning at passage 3, portions of the cell lines were cryogenically preserved for use in future studies and passaged in cell culture to document the characteristics of continued proliferation.These spontaneous cultures were expanded into 75 cmGenomic DNA and total RNA were sequentially isolated from trypsinized cell cultures using an AllPrep DNA/RNA Mini Kit (Qiagen). Isolated DNA was quantified using a NanoDrop ND-1000 Spectrophotometer . No DNA sample was subjected to whole genome amplification techniques. DNA copy number was assessed using GeneChip Human 250K NSPI Mapping Arrays (Affymetrix). Probe preparation, hybridization, and reading were performed by the UCLA DNA Microarray Core according to the standard 96-well protocol published by Affymetrix. Copy number variation was computed using Genome Console software from Affymetrix .RNA was quantified on a NanoDrop Spectrophotometer and analyzed on a 2100 Bioanalyzer  for integrity. RNA had an A260/280 ratio of >1.90 and RNA integrity number (RIN) of 8.5 or higher as determined by the 2100 Bioanalyzer. Prepared RNA was hybridized to GeneChip Human Genome U133 Plus 2.0 Arrays (Affymetrix) at the UCLA DNA Microarray Core Facility using the standard Affymetrix protocol.GNAQ) was determined for both cell line genomic DNA and available germ line DNA from blood samples via PCR and sequencing on an ABI 3130 Genetic Analyzer using BigDye 2.0 chemistry , and intronic sequence primers 5′-TTC CCT AAG TTT GTA AGT AGT GC-3′ and 5′-AGA AGT AAG TTC ACT CCA TTC C-3′ encompassing exon 5 of GNAQ.Additionally, the nucleotide sequence of exon 5 of the guanine nucleotide binding protein (G protein) q polypeptide  showed variable propagation . Among cThe three characterized cell lines with monosomy 3 were from patients with choroidal melanoma who developed clinical evidence of melanoma metastasis <1.5 years after primary melanoma diagnosis and treatment; all three patients subsequently died from liver failure due to metastatic disease. All three cell lines demonstrated stable propagation through at least passage 6 over a period of 2.5 years. Two of the three cell lines persisted in melanin production beyond passage 6 and none of the cultures exhibited contact inhibition in over-growth conditions .Cytogenetically, two of the three cell lines (MEL20–06–039 and MEL20–07–070) demonstrated the chromosomal aberration pattern present in the respective primary melanoma, including monosomy 3, 6q loss, 8p loss, multiple 8q gain and 16q loss . The thiGNAQ demonstrated heterozygous mutations in codon 209 in two of the three cultures (resulting in either Q209L or Q209P) and wild-type sequence in the third culture  origin from primary choroidal melanomas that developed clinical evidence of metastasis <1.5 years after treatment of the primary tumor, (2) demonstration of cellular propagation through at least 6 passages, (3) documentation of equivalent cytogenetic aberrations and similar gene expression patterns in the primary melanomas and in the cell lines at passage 3, and (4) preservation of cell lines by freezing at passage 3 to provide highly characterized cell lines for future studies.Limitations of the three cell lines as surrogates for study of melanoma biology, metastasis and therapy relate to (1) the limited but potentially significant differences in chromosomal aberration and gene expression between primary melanomas and corresponding cell lines, (2) the likelihood that, with long-term propagation and multiple passages, cell lines will undergo culture-related evolution in both chromosomal structure and gene expression, and (3) recognition that cancer biology is influenced by the in vivo micro-environment that is not replicated with in vitro models. However, the fact that the cell lines differ slightly between themselves may not be as important as having a diverse genetic background under which therapeutic drugs may be tested to determine biologic pathways in tumor development.In summary, FNAB of primary choroidal melanomas that developed clinical metastases resulted in the development of three low-passage, highly characterized cell lines that exhibited the chromosomal aberrations and gene expression profiles present in the primary choroidal melanomas. These cell lines, with portions frozen beginning at passage 3 and portions continuing to propagate, provide in vitro models for study of choroidal melanoma biology, metastasis and therapy.


G12D;Pdx1-Cre (KC) mouse at 50 weeks of age, whereas UN-KPC-960 and UN-KPC-961 cell lines were derived from pancreatic tumors of KrasG12D;Trp53R172H;Pdx1-Cre (KPC) mice at 17 weeks of age. The cancer mutations of these parent mice carried over to the daughter cell lines . The cell lines showed typical cobblestone epithelial morphology in culture, and unlike the previously established mouse PDAC cell line Panc02, expressed the ductal marker CK19. Furthermore, these cell lines expressed the epithelial-mesenchymal markers E-cadherin and N-cadherin, and also, Muc1 and Muc4 mucins. In addition, these cell lines were resistant to the chemotherapeutic drug Gemcitabine. Their implantation in vivo produced subcutaneous as well as tumors in the pancreas (orthotopic). The genetic mutations in these cell lines mimic the genetic compendium of human PDAC, which make them valuable models with a high potential of translational relevance for examining diagnostic markers and therapeutic drugs.Pancreatic cancer (PC) remains one of the most lethal human malignancies with poor prognosis. Despite all advances in preclinical research, there have not been significant translation of novel therapies into the clinics. The development of genetically engineered mouse (GEM) models that produce spontaneous pancreatic adenocarcinoma (PDAC) have increased our understanding of the pathogenesis of the disease. Although these PDAC mouse models are ideal for studying potential therapies and specific genetic mutations, there is a need for developing syngeneic cell lines from these models. In this study, we describe the successful establishment and characterization of three cell lines derived from two (PDAC) mouse models. The cell line UN-KC-6141 was derived from a pancreatic tumor of a Kras The analysis of functions and characteristics of specific genetic mutations and PC biomarkers present in these cell lines can shed light on the design of promising diagnostic and therapeutic strategies.Despite many advances in the understanding of molecular mechanisms involved in pancreatic cancer (PC) pathogenesis over the last four decades, the disease remains one of the top malignancies with worst prognosis KRAS, CDKN2A, TP53, and SMAD4/DPC4 genes are commonly observed in PDAC tumors from PC patients Kras and Trp53 mutations. The role of oncogenic Ras in PC was examined by directing endogenous expression of G12DKras in the progenitor cells of the pancreas in KrasG12D;Pdx1-Cre (KC) mice R172HTrp53 and G12DKras was examined in the pancreas of KrasG12D;Trp53R172H;Pdx1-Cre (KPC) mice Mutations in in vitro and in vivo characterization of these cell lines are comprehensively described, including markers currently known for pancreatic tumors.Mouse PDAC cell lines with greater clinical relevance to PC are highly needed. The currently available Panc02 cell line has been used over the past three decades The complete medium consisted of DMEM containing heat inactivated FBS, L-Glutamine (200 mM), 100x non-essential amino acids (100 mM), sodium bicarbonate, HEPES buffer, Gentamicin (50 mg/ml), and Penicillin/Streptomycin (100 µg/ml). Immediately after resecting the tumor, 200 mg of pancreatic tumor tissue was transferred into a petri dish containing sterile PBS and Gentamicin (20 µg/ml).The collected tissues were washed 3 times with PBS-gentamicin and transferred into a petri dish containing the complete medium. The tumor was finely-minced with a sterile scalpel and transferred into a sterile centrifuge tube with the complete medium containing Collagenase P  (10 mg/ml).The mixture was incubated at 37°C for 30 min. The tubes were inverted every three min to ensure proper mixing. Following two washing cycles in complete medium, the pellet obtained after centrifugation was suspended in the complete medium. After letting the tube stand for 2 min, cell suspension without tissue debris was transferred into a new sterile 10 cm Petri dish.2, the medium was replaced with fresh medium. Cells were trypsinized once confluent. To prevent the growth of fibroblasts, differential trypsinization was carried out, where trypsin was added to the cells and after 10 sec, the cells were washed with PBS and fresh medium was added. Additionally, medium was supplemented with cholera toxin (400 ng/ml) for the first 7 passages as it has been previously reported that it has an enhanced mitogenic effect on epithelial cells, but not on fibroblast cells 2 in a humidified incubator. Three cell lines were established successfully. The cell line derived from a KrasG12D;Pdx1-Cre (KC) mouse was named UN-KC-6141 and the two cell lines derived from KrasG12D;Trp53R172H;Pdx1-Cre (KPC) mice were named UN-KPC-960 and UN-KPC-961 . In vitro characterization and tumorigenic studies were done after 35 passages in cell culture. The murine PDAC cell line Panc02 was included in the in vitro functional assays.After incubating overnight at 37°C in 5% COKras codon 12 (exon 1) and p53 codons 172 (Exon 5) was carried out by using set of primers to each gene , which results in a 168 bp (Kras) and 229 bp (p53) PCR products. The PCR products were purified with PCR product purification kit  and the purified PCR products were sequenced using Kras-seqR:5'-TGACCTGCTGTGTCGAGAAT-3' for Kras and p53-seqF: 5'-CACGTACTCTCCTCCCCTCA-3' for p53 gene.The sub-confluent cultures of UN-KC-6141, UN-KPC-960, and UN-KPC-961 were lysed with cell lysis solution containing beta-mercaptoethanol and total RNA was isolated with RNeasy Minikit . cDNA was synthesized from total RNA using oligo(dT)18 primer and SuperScript II reverse transcriptase . PCR amplification of murine D) for each cell line was calculated during the exponential growth phase using the formula TD = 0.693t/ln(Nt/N0), where t = time difference in h, Nt = absorbance value at time t, and N0 = absorbance value at initial time For growth kinetics studies, each cell line was seeded in quadruplicate in a 96-well plate  in complete medium. After overnight incubation, the medium was replaced with medium supplemented with 1% FBS and Penicillin/Streptomycin (100 µg/ml). Every day, 10 µl of the Cell Proliferation Reagent WST-1  was added to each well and after 3 h of incubation, absorbance values were measured at 450 nm. Absorbance values were subtracted from values recorded at the reference wavelength (600 nm), as indicated by the manufacturer. The procedure was repeated for 7 days. The doubling time  cells using the RNeasy Minikit . cDNA was synthesized from total RNA using oligo(dT)18 primer and SuperScript II reverse transcriptase  as described by us in a previous publication The anti-CK19 hybridoma (TROMA-III) developed by Dr. Rolf Kemler was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained at The University of Iowa . Antibody against murine E-cadherin was obtained from Cell Signaling . N-cadherin antibody was a kind gift from Dr. Keith R. Johnson from UNMC . β-actin and Amylase antibodies were purchased from Sigma Aldrich . For confocal studies the secondary antibodies conjugated to Alexa Fluor®  were obtained from Life Technologies™ . The secondary antibodies conjugated to horseradish peroxidase used for western blot analysis were obtained from GE Healthcare Life Sciences . Anti-mouse Muc1 antibody  was purchased from Abcam® . The anti-Muc4  antibody was designed in this lab and developed by GenScript  as has been described previously 5 cells suspended in complete medium were seeded on glass cover slips placed in a 12-well plate. The next day, cells were fixed in ice cold methanol. After washing in PBST and blocking with 10% goat serum , the cells were incubated with antibodies for Amylase (1:500), CK19 , E-cadherin (1:50), N-cadherin (1:20), and Muc1 (1:100) overnight. They were washed four times with PBST, 5 min per wash. The cells were incubated with the respective secondary antibodies (mouse/rabbit) conjugated to Alexa Fluor   and after repeating the washing steps, the glass coverslips were mounted on glass slides with vectashield mounting medium . The cells were imaged on a laser confocal microscope LSM 510  in the respective wavelengths at a magnification of ×63.Protein expression was analyzed by confocal microscopy. 2×10For western blot analysis, cells were seeded on each well of a 6-well plate in complete medium. At ∼80% confluency, cells were lysed with radioimmunoprecipitation assay buffer (RIPA) containing protease and phosphatase inhibitors. The lysates were subjected to several freeze-thaw cycles to ensure complete lysis. The concentration of lysates was determined with the micro–bicinchoninic acid protein estimation kit . The protein concentrations were adjusted and solutions were prepared under reducing conditions (i.e. β-mercaptoethanol). 2% SDS-agarose gels were used for analysis of Muc4 expression. 40 µg of protein lysates were loaded and the gel was run for 4 h at 100 V. E-cadherin and N-cadherin expression levels were analyzed by SDS-PAGE. 40 µg of protein lysates were loaded and separation was done at 80 mA for 1 h. Resolved proteins were transferred onto polyvinylidene difluoride membranes, blocked in 5% milk in PBS, and incubated with the primary antibodies overnight. After washing with PBST, the corresponding secondary antibodies were added and after repeating the washing steps, the proteins were detected by luminol  after exposure to X-ray films.4 cells suspended in complete medium were seeded in each well of a 96-well plate. The next day, the cells were treated with different concentrations of Gemcitabine solution (100 nM-100 µM) in quadruplicate wells. After incubating the cells with Gemcitabine for 48 h, media containing thiazolyl blue tetrazolium bromide reagent  was added to the cells. After 4 h of incubation, the formazan crystals produced by metabolically active cells were dissolved with 100 µl of DMSO. Absorbance values at 540 nm were used to calculate cytotoxicity percentages. The half maximal inhibitory concentration (IC-50) of Gemcitabine was determined in each cell line from interpolating values in the graph (% Cytotoxicity vs. Gemcitabine Concentration).The cytotoxic effect of the chemotherapeutic drug Gemcitabine on KC and KPC cell lines was compared to the cytotoxic effect on Panc02. The injectable solution of Gemcitabine, GEMZAR®  was kindly provided by the pharmacy at the Lied Transplant Center at UNMC. For the cytotoxic assay, 1×106) were injected in C57BL/6 mice, whereas UN-KPC-960 and UN-KPC-961 cells (1×106) were injected in mixed background (i.e. B6.129) mice (N = 7). Mouse PC cells suspended in 50 µl sterile PBS were injected orthotopically using the same procedure described by us 6 cells were injected (N = 12) in mixed B6.129 background mice on the lateral chest. Tumor growth was monitored by palpation/Vernier caliper measurements in case of subcutaneous tumors. Throughout the experiment, animals were provided with food and water ad libitum and subjected to a 12-h dark/light cycle. Animal studies were performed in accordance with the U.S. Public Health Service "Guidelines for the Care and Use of Laboratory Animals" under an approved protocol by the University of Nebraska Medical Center Institutional Animal Care and Use Committee (IACUC). After euthanization, pancreatic tumors were dissected out, weighed and fixed in 10% formalin  for H&E staining. Gross metastatic lesions were examined in distant organs and processed for histological analysis.The tumorigenicity of the cell lines was determined after orthotopic implantation of the mouse PC cell lines (UN-KC-6141/UN-KPC-960/UN-KPC-961) into the head of the pancreas after 35 passages. Based on the mice background from where the cell lines were generated, UN-KC-6141 cells  were cut. The sections were deparaffinized using EZ-DeWaxTM  and rehydrated progressively. Afterwards, the sections were stained with hematoxylin and eosin (H&E) stains and examined by a certified pathologist.p-value .Significant differences in the experimental values were determined by calculating p-values using the JMP® Statistical Discovery Software . A Student’s t-test was used to calculate the corresponding G12D;Pdx1-Cre (KC) and the KrasG12D;Trp53 R172H;Pdx1-Cre (KPC) mice at 50 weeks and 17 weeks of age, respectively. One cell line derived from the KC mice was named UN-KC-6141, and two other cell lines derived from the KPC mice were named UN-KPC-960 and UN-KPC-961. All three cell lines grew for over 50 passages in normal media with no sign of senescence. Monolayer cultures of all three cell lines showed typical cobblestone morphology with closed contact island formation  of 33 h (p < 0.0001 compared to Panc02). UN-KPC-960 and Panc02 grew at similar rates (TD ≈ 60 h), while UN-KC-6141 showed the slowest growth rate (TD of 70 h)  compared to Panc02  and Muc4 (p < 0.05) were significantly higher in UN-KC-6141, UN-KPC-960 and UN-KPC-961 cells  instead of Gemcitabine The establishment of the KC and KPC cell lines in culture provided the opportunity to examine their tumorigenicity in appropriate mouse background from which they were derived. The UN-KC-6141 cells were tested in the C57BL/6 mice, while UN-KPC-960 and UN-KPC-961 cells were examined in mice of mixed B6.129 background. One month after orthotopic implantation of UN-KC-6141 cells in C57BL/6 mice, tumors formed in their pancreas Fig. 7A,For PDAC, histological grading is assigned by the extent of glandular differentiation Genetically engineered mouse models are presently a promising approach for understanding the pathogenesis and progression of PDAC and for evaluating novel therapeutic agents  that should translate to high clinical success G12D;Pdx1-Cre (KC) and KrasG12D;Trp53R172H;Pdx1-Cre (KPC) mouse models. The three cell lines derived in these studies, named UN-KC-6141, UN-KPC-960, and UN-KPC-961, have been maintained in cell culture for over 50 passages without any sign of senescence. These cell lines show typical epithelial cobblestone morphology, and expressed high levels of epithelial and mesenchymal markers such as CK19, E-cadherin, N-cadherin, Muc1, and Muc4  in all the KC and KPC-derived cell lines, and the Trp53 mutation (R172H) was only present only in the KPC cell lines. Two recent publications have reported the well-established Panc02 cell line has a Smad4 mutation, but both Kras and Trp53 mutations are absent KRAS and TP53 mutations and ductal characteristics, the newly derived murine PDAC cell lines UN-KC-6141, UN-KPC-960, and UN-KPC-961 should be very valuable preclinical studies and have excellent translational significance.The most commonly occurring mutations in PDAC include those in the Gemcitabine is the most used chemotherapeutic treatment for PC patients, but tumors may acquire resistance, as evidenced by the poor survival statistics In conclusion, the UN-KC-6141, UN-KPC-960, and UN-KPC-961 cells described here represent a valuable tool for yielding a better understanding of PC pathogenesis and to study novel treatments for this lethal disease. The genotypic and phenotypic characteristics of these novel cell lines mimic human PDAC, which make them valuable models with significant translational relevance.


Platinum derivatives are used widely for the treatment of many cancers. However, the toxicity that is observed makes imperative the need for new drugs, or new combinations. Anvirzel™ is an extract which has been demonstrated with experimental data that displays anticancer activity. The aim of the present study is to determine whether the combination of Cisplatin and Anvirzel™ has a synergistic effect against different types of cancer.To measure the efficacy of treatment with Cisplatin and Anvirzel™, methyl-tetrazolium dye (MTT) chemosensitivity assays were used incorporating established human cancer cell lines. Measurements were performed in triplicates, three times, using different incubation times and different concentrations of the two formulations in combination or on their own. t-test was used for statistical analysis.In the majority of the cell lines tested, lower concentrations of Anvirzel™ induced a synergistic effect when combined with low concentrations of Cisplatin after an incubation period of 48 to 72 h. The combination of Anvirzel™/Cisplatin showed anti-proliferative effects against a wide range of tumours.The results showed that the combination of Anvirzel™ and Cisplatin is more effective than monotherapy, even when administered at low concentrations; thus, undesirable toxic effects can be avoided. Nerium oleander comprised primarily of Oleandrin and Oleandrigenin [Many studies demonstrate the anti-proliferative activity of Oleandrin. These properties make it attractive for use as a treatment for cancer -5; howevdrigenin . Recent drigenin -12. Cispdrigenin ,14. Becadrigenin , it is cdrigenin ,17. A redrigenin . Therefodrigenin -23.The human carcinoma cell lines used were obtained from the ECACC-HPA . PC3, LNCaP and 22Rv1 are human prostate cancer cell lines, MDA-MB 231, T47D, and MCF-7 are human breast cancer lines, CALU-1, COLO699N and COR-L 105 are non-small cell lung carcinoma lines (NSCLC), HCT-116, HT55 and HCT-15 are colorectal cancer lines, and A375 and PANC-1 are melanoma and pancreatic cancer cells lines, respectively. MTT chemosensitivity assays were used to determine the efficacy of combined treatment compared with that each drug alone. The incubation times used in the study were 24, 48 and 72 h at concentrations ranging from 0.01 ng/ml to 10 ng/ml for Anvirzel™ and from 0.1 μg/ml to 100 μg/ml for Cisplatin.2 flasks  in the recommended media supplemented with the appropriate amount of heat inactivated Fetal Bovine Serum  and 2 mM L-Glutamine . The cells were maintained at 37°C in a 5% CO2 atmosphere.Cells were cultured in 75 cmCells were detached by trypsinisation  during the logarithmic phase of growth and plated in 96-well plates  at a density of 18,000 cells/well in a final volume of 200 μl medium per well. When the cells reached 70–80% confluence, the medium was removed and Anvirzel™  and Cisplatin  were added to the cells at different concentrations. Absorbance was measured after 24, 48 and 72 h of incubation.For the MTT assay, methyl-tetrazolium dye  was added to each well at a concentration of 5 mg/ml (diluted in PBS) and the plates incubated for 3 h at 37°C. The medium was then discarded and the cells rinsed with PBS. Finally, the formazan crystals were dissolved in dimethylsulphoxide .To calculate the fold-decrease in staining the absorbance was calculated using the Beer-Lambert law: A = εcl, where “A” is the absorbance, “ε” is an extinction coefficient, “l” is the distance the light travels through the material, and “c” is the concentration of the absorbing species .The optical density of the plate was measured using a μQuant spectrophotometer (μQuant Biomolecular Spectrophotometer MQX200) and the data analysed with Gen5 software . Absorbance was measured at 570 nm and a second wavelength at 630 nm was measured to subtract background “noise”.All treatments for each cell line were performed in triplicate, three times. The statistical significance of all effects was evaluated using the “difference of the means” test. A p value < 0.05 was considered significant.Different results were observed not only between each type of cancer but also between each cell line within the same carcinoma group. In all cell lines has been studied LC50 , which is the concentration that kill half of the sample population, and GI50 (growth inhibition 50), which is the concentration required to inhibit growth by 50%. In all cell lines tested, administration of the combined formulation at lower concentrations was more effective than monotherapy. The formulation that elicited the most effective results in most of the cell lines was a combination of 0.01 ng/ml Anvirzel™ and 0.1 μg/ml Cisplatin, with incubation period of 48 h or 72 h. The combination of 0.01 ng/ml Anvirzel™ and 1 μg/ml Cisplatin was more effective in pancreatic cancer cell line, as well in PC3 and MDA-MB231 cell lines. The Figures Nerium oleander (family Apocynaceae) that contains two toxic cardiac glycosides, Oleandrin and Oleandrigenin [Nerium oleander is often used for its healing properties [+ and Ca2+[+/K+-ATPase and thus inhibits the export of FGF-2 [Anvirzel™ is an extract of drigenin , which hoperties . Oleandr and Ca2+. Other s and Ca2+,28. Olea and Ca2+. Recent of FGF-2 ,28. Cispof FGF-2 ,14. In hof FGF-2 . Many stof FGF-2 ,20.It has been confirmed that Anvirzel™ has better activity at lower concentrations in many cancer cell lines, after 48 or 72 h of incubation. In contrast, low Cisplatin concentrations (<1 μg/ml), do not indicate activity in the same cells. The effect of platinum is evident at concentrations greater than 10 μg/ml. However, these concentrations have high cytotoxicity effects. The combination of the two formulations at very low concentrations is able to inhibit cell growth by more than 50% in an exposure time of 72 hours. The same concentration may also reduce the number of live cells at rates up to 85% in the same time of exposure. Lowered effect observed in cell lines that are hormone-dependent, cases such as breast and prostate cancer.The MTT viability assays were used to test the hypothesis that combined treatment with Cisplatin and Anvirzel™ would be more effective than either drug alone. The results of all three assays were both concentration and cell line-dependent. It is noteworthy that increased efficacy was observed at lower concentrations of both substances, 0.01 ng/ml for Anvirzel™ and 0.1 μg/ml for Cisplatin, than of either drug alone. The results were neither reliable nor reproducible at higher concentrations of Anvirzel™ and Cisplatin. The results were also time-dependent: treatment was effective after 48 h and 72 h of incubation, but not after 24 h.The present study contributes to demonstrate an effective interaction between Anvirzel™ and another widely used drug with cytostatic effects. Based on these data, it is crucial to perform further studies to identify and characterize the interaction between Anvirzel™ and other drugs currently used to treat cancer.CDDP: Cisplatin; MTT: 3--2,3-diphenyltetrazolium bromide; NSCLC: Non-small cell lung cancerThe authors declare that they have no competing interests.PA carried out the chemosensitivity assays, drafted the manuscript and performed the statistical analysis. MT participated in the chemosensitivity assays. MC participated in the chemosensitivity assays. EI carried out the cell lines culture. DK participated in the design of the study and coordination. JN participated in the design of the study and coordination. DK participated in the design of the study. IP supervised the assays and the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/2050-6511/14/18/prepub


Melanoma is an often fatal form of skin cancer which is remarkably resistant against radio- and chemotherapy. Even new strategies that target RAS/RAF signaling and display unprecedented efficacy are characterized by resistance mechanisms. The targeting of survival pathways would be an attractive alternative strategy, if tumor-specific cell death can be achieved. Bcl-2 proteins play a central role in regulating survival of tumor cells. In this study, we systematically investigated the relevance of antiapoptotic Bcl-2 proteins, i.e., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1, in melanoma cell lines and non-malignant cells using RNAi. We found that melanoma cells required the presence of specific antiapoptotic Bcl-2 proteins: Inhibition of Mcl-1 and A1 strongly induced cell death in some melanoma cell lines, whereas non-malignant cells, i.e., primary human fibroblasts or keratinocytes were not affected. This specific sensitivity of melanoma cells was further enhanced by the combined inhibition of Mcl-1 and A1 and resulted in 60% to 80% cell death in all melanoma cell lines tested. This treatment was successfully combined with chemotherapy, which killed a substantial proportion of cells that survived Mcl-1 and A1 inhibition. Together, these results identify antiapoptotic proteins on which specifically melanoma cells rely on and, thus, provide a basis for the development of new Bcl-2 protein-targeting therapies. Melanoma is one of the deadliest types of skin cancer with strongly rising incidence. Due to its therapy resistance in advanced stages, melanoma is the skin cancer with the highest mortality. Fewer than 20% of melanoma patients respond to chemotherapy, which does not prolong the survival of late stage melanoma patients The targeted activation of apoptotic pathways may be an alternative antitumor strategy and may be valuable to overcome de novo or acquired resistance to conventional chemotherapy or MAP kinase inhibition. Apoptosis can be initiated via two pathways, the mitochondrial and the death receptor-mediated pathway The group of antiapoptotic Bcl-2 proteins consists of five members, i.e., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1. The expression level and relevance for survival of each antiapoptotic member varies between different cell lineages. Antiapoptotic proteins can promote survival and, accordingly, the expression or the activity of antiapoptotic proteins can be increased in cancer. In addition, cellular stress in tumors, e.g., generated by genomic alterations, exaggerated proliferation, or insufficient nutrition can result in the requirement of antiapoptotic proteins for tumor cell survival. This situation, termed synthetic lethality, can make tumor cells vulnerable and offers the opportunity for therapeutic intervention In this study, we systematically investigated the relevance of antiapoptotic Bcl-2 proteins in melanoma cell lines utilizing RNA interference. In addition, primary human fibroblasts from skin were studied in order to identify those antiapoptotic Bcl-2 proteins whose loss specifically affects melanoma cells while sparing non-malignant cells. It was found that melanoma cell lines – in contrast to non-malignant fibroblasts - required specific antiapoptotic Bcl-2 proteins for survival. Inhibition of Mcl-1 and A1 resulted in cell death of melanoma cell lines and targeting both antiapoptotic Bcl-2 proteins simultaneously enhanced cell death to 80% whereas non-malignant cells remained unaffected. In addition, cell death induction could be further increased by exposure to chemotherapeutic drugs.Expression levels of antiapoptotic Bcl-2 proteins were analyzed by immunoblotting. Because sensitivity of commercial A1 antibodies was found to be not sufficient for detection, expression levels of A1 were analyzed on RNA level. Compared to primary human keratinocytes and fibroblasts, primary human melanocytes expressed lower levels of Bcl-xL and Mcl-1 whereas levels of Bcl-2 and A1 were higher . This meThese results suggest that melanocytes express a characteristic expression pattern of antiapoptotic Bcl-2 proteins, which is clearly altered in melanoma cells. In contrast to melanocytes, the expression profile in melanoma cells was inconsistent among different cell lines. Overall, no general increase in the expression of antiapoptotic Bcl-2 proteins was observed in melanoma cell lines, which would point towards a pro-survival role.Next, the relevance of antiapoptotic Bcl-2 proteins for melanoma cell survival was assessed by silencing them utilizing RNA interference. Effects caused by silencing of Bcl-2, Bcl-xL, Bcl-w, Mcl-1, or A1 were analyzed in the metastatic melanoma cell line 1205Lu. In addition, non-malignant cells were analyzed in order to dissect Bcl-2 proteins that are specifically important for melanoma survival. For this, primary human fibroblasts instead of melanocytes were used as a non-malignant reference. Fibroblasts represent a more prevalent cell type and, in a therapeutic perspective, unspecific cell death is expected to be more serious in ubiquitous cell types than in melanocytes. Furthermore, transfection efficacy of siRNAs is insufficient in melanocytes resulting in inefficient inhibition. ShRNA expression is applicable in melanocytes, e.g., by utilizing lentiviral shRNA-encoding constructs, however, in general, we observe lower silencing efficacies of shRNAs compared to siRNAs. Therefore, it is unlikely to generate the same inhibition efficacies in melanocytes as in melanoma cells. In fibroblasts and melanoma cells, the applied siRNAs efficiently abrogated expression of the respective Bcl-2 molecule . The effTogether, these data show that silencing of individual Bcl-2 proteins does not induce cell death in non-malignant fibroblasts. In contrast, melanoma cells were found to be dependent on the presence of specific antiapoptotic proteins: Inhibition of Mcl-1 or A1 caused significant cell death without further apoptotic stimulus.The results suggested that targeting of the appropriate Bcl-2 proteins, i.e., Mcl-1 or A1, represents a strategy to specifically induce apoptosis in melanoma. A consistent, but not significant increase was also observed when Bcl-2, Bcl-xL, and Bcl-w were targeted in 1205Lu melanoma cells . We, theNext, cell death was analyzed. Non-malignant fibroblasts survived the simultaneous inhibition of A1 and Bcl-2, Bcl-xL, or Bcl-w, respectively. Similarly, these cells were not affected by inhibition of Mcl-1 together with Bcl-2 or Bcl-w, respectively, while co-inhibition of Mcl-1 and Bcl-xL strongly induced cell death . In 1205Among the tumor-specific combinations, simultaneous inhibition of Mcl-1 and A1 most efficiently induced cell death in 1205Lu melanoma cells. Without further stimulus, around 60% of melanoma cells died whereas fibroblasts remained healthy . The comNext, these analyses were extended to other melanoma cell lines derived from different tumor stages and to primary human keratinocytes, representative for another non-malignant skin cell type. Similar to fibroblasts, keratinocytes were not sensitive to the inhibition of Mcl-1 or A1, nor to the inhibition of both proteins simultaneously . HoweverTogether, the results show that the combined inhibition of Mcl-1 and A1 efficiently induces apoptosis in melanoma cell lines, whereas primary cells remain unaffected.We next examined whether cell death induced by inhibition of A1, Mcl-1, or both, can further be enhanced by exposure to chemotherapy, i.e., 5-fluorouracil (5-FU), while maintaining the tumor specificity. Cells were treated with siRNAs with or without 5-FU. Three and 6 days after treatment cell viability was measured by a fluorimetric assay and cell death induction was measured by FACS analysis. On day 3, melanoma cell viability was decreased by inhibition of A1 and Mcl-1, respectively, and – most prominently – by inhibition of both . On day Together, the data suggest that chemotherapy cooperates with Mcl-1 and A1 by killing cells that survive A1 or Mcl-1 inhibition. This results in increased cell death while tumor specificity was maintained.Several studies have analyzed the levels of antiapoptotic Bcl-2 proteins in melanoma, however the results were conflicting and the relevance of Bcl-2 molecules is still unclear In this systematic screen, Mcl-1, but also A1, have been identified as potential targets whose loss led to strong cell death induction in melanoma cell lines, whereas non-malignant cells remained unaffected. Inhibition of Mcl-1 effectively induced apoptosis in most melanoma cell lines. Newly, A1 was identified as a molecule that is of relevance for melanoma cell survival. Despite less efficient apoptosis induction compared to Mcl-1, combined targeting of Mcl-1 and A1 caused a substantial increase in cell death in the majority of melanoma cell lines tested, without inducing apoptosis in fibroblasts or keratinocytes. Nevertheless, melanoma cell lines were identified that were primarily dependent on Mcl-1 (WM1232) or A1 (WM239A) and the combined treatment did not show an additional benefit. However, all melanoma cell lines tested in our study were sensitive to at least one of both Bcl-2 members. This demonstrates that - even in the presence of varying expression levels and sensitivities among melanoma cell lines - targeting of A1 at least broadens the apoptotic sensitivity of melanoma cells to Mcl-1 inhibition. In a therapeutic perspective, this may be important for the development of multi-specific BH3 mimetics. The intrinsic sensitivity of melanoma cells to Mcl-1 and A1 loss is also supported by several studies that observed melanoma cell death that was associated with induction of the BH3-only protein Noxa In contrast to melanoma, inhibition of single antiapoptotic proteins did not lead to cell death in non-malignant fibroblasts. However, when targeting two antiapoptotic Bcl-2 proteins simultaneously, the approach to identify tumor-specific combinations turned out to be very important: Inhibition of Mcl-1 in combination with the inhibitor ABT-737 strongly induced cell death, which was also observed by others Other studies reported that melanoma cells require Mcl-1 for survival under certain stress conditions, like endoplasmatic reticulum stress, lack of cell adhesion, or proapoptotic stimuli that target the death receptor-mediated apoptotic pathway Chemotherapy represents an apoptotic stimulus that activates apoptosis via multiple pathways and, therefore, may function independently of the more focused effects of Mcl-1 and A1 inhibition. Despite high efficacy in cell death induction by Mcl-1 and A1 inhibition, we observed a small subpopulation of surviving cells (around 20%). The chemotherapeutic agent 5-fluorouracil (5-FU) resulted in significant cell death in this surviving subpopulation. 5-FU reduces DNA synthesis by inhibiting thymidylate synthase, causes DNA damage, and promotes apoptosis via the DNA damage pathway Together, this study demonstrates that it is possible to efficiently induce cell death specifically in melanoma cells by targeting certain antiapoptotic Bcl-2 proteins. Targeting of more than one Bcl-2 molecule clearly increased efficacy and broadened sensitivity among cell lines without loosing tumor specificity. Further increase can be achieved by the combination with chemotherapy, a treatment that kills 90% of melanoma cell lines without affecting non-malignant fibroblasts. These data provide a basis for future therapeutic approaches based on targeting of Bcl-2 proteins, e.g, by BH3 mimetics. They further underscore the importance to address each Bcl-2 molecule specifically to ascertain that cell death or drug sensitization is restricted to the tumor.Anti–Bcl-2 (Ab-1) antibody was purchased from Merck Biosciences . Anti–Bcl-xL, anti–Bcl-w, anti-caspase 3, anti-caspase 9, and HRP-conjugated secondary antibodies were obtained from New England Biolabs . Anti-Mcl-1 (22) antibody was purchased from BD Bioscience . Anti–β-actin (AC-15) and Anti-α-tubulin (DM1A) antibodies were obtained from Sigma-Aldrich . PCR primers and siRNAs were purchased from Eurofins MWG Operon . ABT-737 was purchased from Abbott Laboratories . zVAD-FMK was from Bachem .GGAAGAAUUGUAACCAUAU, A1b: CGGAUGUGGAUACCUAUAA, Bcl-2: GGGAGAUAGUGAUGAAGUA, Bcl-w: GGCAGACUUUGUAGGUUAU; Bcl-xL: GGAACUCUAUGGGAACAAU; Mcl-1: GGCAGUCGCUGGAGAUUAU; and Mcl-1b: CCAUGUAGAGGACCUAGAA. Depicted is the 19nt portion corresponding to the sense strand of the targeted mRNA. The sequence of the non-silencing control siRNA (ctrl) was GCGCAUUCCAGCUUACGUA.siRNAs were designed as described previously Human melanoma cell lines were a gift of M. Herlyn . All were isolated from clinically and histologically defined lesions. Keratinocytes, melanocytes and fibroblasts were isolated from neonatal human foreskins. Cultivation of cells was described previously Transfections were performed in 6-well dishes in a volume of 1 ml. To enable direct comparison of single and co-inhibition experiments, equal amounts of siRNAs (40 nM) were transfected. In single inhibition experiments, 20 nM of specific siRNA was transfected along with 20 nM control siRNA utilizing 1.6 µl Lipofectamine RNAiMAX  according to the manufacturer's protocol. In co-inhibition experiments, 20 nM of each specific siRNA was used.Viable cells were quantified in 6-well dishes by using a fluorometric assay . Viable cells with intact metabolism were identified by their ability to reduce cell-permeable resazurin to fluorescent resorufin. Medium was replaced with 750 µl of culture medium and 150 µl of CellTiter-Blue reagent. After 1 hour incubation at 37°C, fluorescence was measured.Adherent and supernatant cells were analyzed by staining with FITC-labeled Annexin V  and propidium iodide (Sigma-Aldrich) as described previously Total RNA was extracted from cells using QIAzol Lysis Reagent  as described by the manufacturer and analyzed by quantitative RT-PCR. Reverse transcription and quantification procedures using the LightCycler TaqMan Master Kit (Roche) together with the Universal Probe Library system (Roche) were described previously SureLock Mini-Cell apparatus (Invitrogen) as described previously Adherent and supernatant cells were lysed in buffer containing 50 mM Tris, pH 7.4, 0.25 M NaCl, 1 mM EDTA, 0.1% Triton X-100, phosphatase inhibitors , and protease inhibitors . Gel electrophoresis, blotting and protein detection was carried out using the Xcell For statistical analysis, 2-tailed Student's t test was used to assess the significance of mean differences. Differences were considered significant at a P value of 0.05 or less.