FLP recombinase mediated integration into a pre-integrated FRT site is routinely used to generate highly reproducible stable transgenic cell lines. In this study, we broaden the system of site specific integration by introducing ΦC31 integrase mediated integration into attP sites.We generated a HEK293 host cell line with a single copy FRT as well as an attP site allowing site specific integration of two distinct transgenes. To achieve conditional control, we used the tetracycline and Shld1 inducible systems. By introducing fluorescent reporters we show that integration and induction of two transgenes are completely independent. We applied this new technique to investigate the effect of HNF4α on proliferation of HEK293 cells by introducing HNF4α into each integration site. We obtained in two independent cell lines highly reproducible results that prove the usefulness of this novel HEK-attP/FRT cell line.In this study we have established and applied a HEK-attP/FRT cell line that allows site specific integration of two conditional transgenes using the FLP recombinase as well as the ΦC31 integrase. Stable integration of inducible transgenes is widely used to analyze gene function in mammalian cells. To obtain highly reproducible results from different cell lines, site specific integration of one single transgene copy is essential. Integration into a pre-determined genomic locus can be achieved by FLP recombinase mediated integration  and the For many experiments the independent conditional expression of two distinct transgenes would be most desirable. Thus, an additional and independent conditional system is required. Recently, another inducible system was developed that allows conditional protein degradation . The humAn enzyme that acts completely independent of FLP and catalyses specific integration at high efficiency is the serine integrase derived from the phage ΦC31. This ΦC31 integrase mediates site-specific recombination between two DNA sequences, the phage attachment site, attP, and the bacterial attachment site, attB . ImportaIn this study, we combine the Shld1 inducible system with ΦC31 integrase mediated specific integration in the background of the tetracycline inducible FLP mediated system to have two completely independent ways of transgene activation in one and the same cell. We apply this technique to introduce the cell specific transcription factor HNF4α into a human embryonic kidney (HEK293) cell line and investigate its effect on cell proliferation.To allow site specific integration of two different transgenes we used two independent systems. The first is based on FLP recombinase mediated recombination of FRT sites Figure . By usinThe commercially available Flp-In T-Rex™ HEK293 cells contain a stably integrated single copy FRT docking site for FLP mediated integration. Using this cell line we introduced in addition for ΦC31 mediated transgene integration the newly generated attP docking site that encodes an ECFP-Neo fusion gene. After selection with G418 we selected 24 single clones with blue fluorescence. To determine the number of integrated transgene copies we performed real-time PCR on genomic DNA and selected the three double docking cell lines 12, 16, and 19 with apparent single copy integration of the docking site for further experiments.To test whether independent integration as well as independent conditional activation of two different GOI can be achieved in the double docking cell lines we generated two different integration vectors. The FRT integration vector pcDNA5/FRT/TO-DsRed contains the red fluorescent protein DsRed as GOI Figure , whereasTo test whether the two transgenes were present and conditionally active, we treated all cell lines with doxycycline, a relatively stable tetracycline derivative, or Shld1 and monitored red or yellow fluorescence Figure . In all To verify that this double conditional system can also be used to express genes interfering with cell cycle progression we introduced the transcription factor HNF4α splice variant 2. (HNF4α2) that has been shown to inhibit HEK293 cell multiplication [To address the effect of both HNF4α transgenes on cell cycle progression we treated the cell lines 19-2-2 and 19-2-6 with doxycycline and/or Shld1 and measured the cell number over five days by the MTS assay. The cell line 19-2-2 showed an about 10-fold increase of cell mass until day 5 Figure . HoweverIn summary, the two HNF4α transgenes could be independently activated by doxycycline or Shld1 and the observed effect on cell multiplication was highly reproducible between the two distinct sites of integration in two individual cell lines. Moreover, the effect of HNF4α appears to be concentration dependent, as activation of the second transgene further delays cell multiplication.In this study we created the double conditional cell line HEK-attP/FRT to independently control the expression of two stably integrated transgenes. While stable cell lines are superior to transient transfectants to investigate long term effects, there is considerable variation between different clones as the site of integration and number of transgene copies cannot be predicted using standard transfection methods. This variation becomes even more problematic, when two potentially interacting transgenes are studied. This problem has now been overcome by using in parallel FLP and ΦC31 integrase mediated integration into the corresponding pre-integrated recognition sites.When searching for the optimal HEK-attP/FRT docking cell line, it turned out that two of three host cell lines still expressed the marker for the native attP docking site after integration of the fluorescent proteins. As the cells were resistant to puromycin and the fluorescent proteins could be properly induced, we conclude that there is least one additional un-recombined copy of the attP docking site present in these cells. However, the finally selected HEK-attP/FRT cell line appears useful for single copy integration, as it allows recombination at a unique site resulting in loss of the attP marker gene expression  was cultured in DMEM supplemented with 15 μg/ml blasticidin and 100 μg/ml zeocin (InvivoGen). The HEK-attP/FRT cell line was kept in DMEM supplemented with blasticidin, zeocin, and 500 μg/ml G418 (InvivoGen). To select stable cell lines derived from the host cell line by FLP mediated integration into the FRT docking site, zeocin was substituted with 50 μg/ml hygromycin B (Invitrogen). After ΦC31 integrase mediated integration into the attP docking site, G418 was replaced by 1 μg/ml puromycin (InvivoGen). Transfections were carried out using LipofectAMINE™ (Invitrogen) or FuGENE™ (Roche).All cell lines were grown in Dulbecco's modified Eagle's medium  supplemented with 10% heat inactivated fetal calf serum (FCS), penicillin/streptomycin (100 U/ml) and 2 mM glutamine at 37°C under 8% COFor integration into the FRT docking site, cells were co-transfected with the FLP expression vector pCSFLPe  and the DNA was extracted using the DNeasy Extraction Kit (Qiagen). Real time PCR was performed using POWR-SYBR Green (Applied Biosystems) on a 7900HT Sequence Detection System (Applied Biosystems). Templates of 10 ng DNA were measured in duplicate. The number of transgene copies was determined by external calibration using the β-actin gene for normalization. Primers used were docking1: 5'-GCAAAGACCCCAACGAGAAG-3', docking2: 5'-TCACGAACTCCAGCAGGACC-3', hACTBf: 5'-GGTATCTCCCTCTGCAGC-3', hACTBr: 5'-CTATGGGCTGAGGTCTGGAT-3'.Cell pellets were lysed in RIPA buffer  supplemented with 0.2% protease inhibitor cocktail . Insoluble debris were removed by centrifugation at 14,000 g for 10 min at 4°C. The protein concentration of the supernatant was determined using a Bradford assay (Bio-Rad). Equal amounts of proteins were separated on SDS-polyacrylamide gels and transferred to nitrocellulose. Membranes were blocked with blocking reagent . For antigen detection, monoclonal anti-myc tag antibody 9E10, anti-HNF4α , anti-GFP , anti-DsRed  antibodies were employed. Peroxidase-conjugated anti-mouse IgG (Amersham), anti-rabbit IgG (Jackson ImmunoResearch) and anti-goat IgG (Sigma) were used as secondary antibodies. Immunoreactivity was detected by ECL (Amersham Biosciences).pDOCKING-Neo (Addgene plasmid 31441) contains the CMV promoter driven ECFP-neomycin fusion protein containing the blue fluorescent protein ECFP (Clonetech). A minimal attP site  is placeThe authors declare that they have no competing interests.CW designed the project, performed all assays on the HNF4α conditional cells, and drafted the manuscript. OR performed assays on the EYFP/DSRed conditional cells. FS constructed plasmids. MY constructed the docking site. NS performed western blots and transfections. GUR designed and overviewed the project. All authors have read and approved the final version of the manuscript.


Bxv-1. We also identified a gammaretrovirus in the non-small-cell lung carcinoma cell line EKVX. Prostate cancer cell lines appear to have a propensity for infection with murine gammaretroviruses, and we propose that this may be in part due to cell line establishment by xenograft passage in immunocompromised mice. It is unclear if infection with these viruses is necessary for cell line establishment, or what confounding role they may play in experiments performed with these commonly used lines. Importantly, our results suggest a need for regular screening of cancer cell lines for retroviral “contamination”, much like routine mycoplasma testing.A newly discovered gammaretrovirus, termed XMRV, was recently reported to be present in the prostate cancer cell line CWR22Rv1. Using a combination of both immunohistochemistry with broadly-reactive murine leukemia virus (MLV) anti-sera and PCR, we determined if additional prostate cancer or other cell lines contain XMRV or MLV-related viruses. Our study included a total of 72 cell lines, which included 58 of the 60 human cancer cell lines used in anticancer drug screens and maintained at the NCI-Frederick (NCI-60). We have identified gammaretroviruses in two additional prostate cancer cell lines: LAPC4 and VCaP, and show that these viruses are replication competent. Viral genome sequencing identified the virus in LAPC4 and VCaP as nearly identical to another known xenotropic MLV,  Xenotropic Murine leukemia virus-Related Virus (XMRV) was first reported in 2006 as a novel gammaretrovirus found in prostate tissues from prostate cancer patients et. al. at the 17th Conference on Retroviruses and Opportunistic Infections (CROI) provide a compelling argument that XMRV likely originated from recombination between two separate and defective endogenous MLVs that infected the CWR22 human cancer cells at some point during the process of serial passage of the cells by xenograft . Indeed, recent data presented by Pathak raft see  and it iraft see . There aThe discovery of XMRV in the CWR22Rv1 cell line prompted us to interrogate additional prostate cancer cell lines for the presence of XMRV or other murine gammaretroviruses. If the source of retroviral infection is not from the prostate cancer patient, but introduced by passage through animals or laboratory contamination, then the presence of replication competent retroviruses in commonly used prostate cancer cell lines for research could potentially have confounding effects on experimental outcomes.Source of cell lines and description of cell line tissue microarray (TMA) construction is provided in the Slides containing sections of formalin-fixed paraffin-embedded (FFPE) control cells or TMAs were deparaffinized and steamed for 25 min. in citrate-based unmasking solution for antigen retrieval (Vector Laboratories). IHC (including positive and negative controls) with either anti-p30 (MLV30) or anti-gp70 (MLV70) antisera was performed as previously described 5′-GGGCTCTCCAGAACATCATCC-3′ and GAPgen-R 5′-GTCCACCACTGACACGTTGG-3′) to verify the quality of the DNA template. MLV-specific primer (In-For) as well as the XMRV-specific reverse primer (Deletion-Rev) were as previously described 2, 200 µM dNTPs, 400 nM F and R primer, 1U AmpliTaq Gold, Applied Biosystems) using approximately 100 ng of gDNA for template and the following cycling parameters: 95°C for 2 min.; 40 cycles of 95°C for 30 sec., 54°C for 1 min. (Pan-MLV primers) or 64°C for 30 sec. (XMRV-specific primers), 72°C for 30 sec.; final extension at 72°C for 7 min. Serial dilution tests of CWR22Rv1 gDNA indicated that the degenerate Pan-MLV PCR assay had a sensitivity of detecting virus in down to 0.1 ng of CWR22Rv1 gDNA  was extracted from cultured cell pellets from each of the cell lines used in the TMA using the DNeasy Blood and Tissue Kit (Qiagen). All DNA was first amplified with genomic GAPDH PCR primers  enzyme only after infection with a replication-competent MLV. iGLuc-DERSE cells were seeded at 3.5×10∧4 cells/well in a 12-well plate. Cells were treated with 20 µg/ml DEAE-dextran for 30 min. DEAE-dextran was then removed and cells were rinsed with media. Next, 500 µl of each supernatant was added to the iGLuc-DERSE cells in triplicate. Cells were incubated for 3 hr. and 400 µl of additional media was added. Two and three days after infection, 10 µl of the media from each well was assayed for luciferase activity. At 3 days after infection, cells were passaged. Two days after cell passage, luciferase activity was measured again. Cells were passaged for a total of 13 days. Further details of the cell line will be given in a future publication.Cell line supernatants were collected after 24 hr. culture and filtered through a 0.45 µm filter. The infectivity assay uses iGLuc-DERSE cells, a 293 mCAT based cell line that produces the AseI, BspHI, BstYI, HindIII, NcoI, and PstI. Vectorette oligos were then annealed overnight at 4°C at a concentration of 1 µM using T4 DNA ligase (New England Biolabs). Vectorette PCR was conducted using a virus-specific forward primer  and a vectorette-specific reverse primer (5′-CTCTCCCTTCTCGAATCGTAA-3′). PCR products were gel purified and cloned into the pCR®2.1-TOPO vector (Invitrogen). Transformed bacterial colonies were chosen at random for sequencing.Vectorette PCR was performed as previously described CA (MLV30) or gp70SU (MLV70) As an initial screen for murine gammaretroviruses in human cell lines, we utilized a tissue microarray (TMA) containing a total of 72 cell lines, which included 58 of the 60 human cancer cell lines used in anticancer drug screens and maintained at the NCI-Frederick (NCI-60) . As showWe next used a PCR-based assay to determine whether LAPC4, VCaP and EKVX are infected with XMRV. As shown in pol and env sequences isolated from EKVX in another recent study In order to determine the identity of the viruses present in LAPC4, VCaP and EKVX, we performed long-range PCR and sequencing as described in the Bxv-1 (or XMV43) Bxv-1  viral genomes 8,657 nucleotides in length  were sequenced from the LAPC4 and VCaP cell lines. NCBI BLAST and Ensembl database searches revealed an almost perfect match (99%) to a sequence contained on Chromosome 1 of the C57BL/6J mouse genome sequence (nucleotide position 172871652–172880308). This location maps to a known murine xenotropic gammaretroviral provirus termed 3) Bxv-1 . Bxv-1 iBxv-1 provirus, we mapped multiple viral integration sites in each of these two lines using vectorette PCR To further verify that the LAPC4 and VCaP cell lines are infected with integrated Gaussia luciferase enzyme following infection with a replication-competent MLV. Gaussia luciferase activity was detected 48 hours after infection of iGLuc-DERSE cells with supernatant from the CWR22Rv1, LAPC4 and VCaP cell lines (Gaussia luciferase activity until 13 days (and 3 passages) after infection  exposed to supernatant from the DU145, MycCaP (a mouse prostate cancer cell line obtained from the C. L. Sawyers lab) or PC3 cell lines  have become contaminated with MLVs . For exaBxv-1 is present in SCID and nude mice and tumor cells passaged through these animals can become infected with xenotropic MLVs Our study demonstrates that prostate cancer cell lines appear to have a propensity for infection by murine gammaretroviruses. We propose that this tendency may be due to the fact that most of the established prostate cancer cell lines were created by passage through immunocompromised mice. In fact, et. al., which screened 411 human tumor cell lines from the Catalogue Of Somatic Mutations In Cancer (COSMIC) collection, EKVX was found to be positive for a xenotropic MLV-related virus by direct sequencing of viral gag, pol and env genes et. al. study tested all but 14 of the cell lines examined in the current study  and likewise found them to be negative for MLV. Although the authors of the study did not attempt to obtain a full-length sequence of the virus contaminating EKVX or to demonstrate that the virus is replication competent, partial pol and env sequences from the Hue et. al. study  match at 100% similarity to the near full-length viral sequence obtained in the present study (GenBank Accession #JF908817). We have now demonstrated that the virus contained in the EKVX cell line is replication competent Click here for additional data file.Figure S1A) Pan-MLV primers (B) XMRV-specific primers. Samples were tested in duplicate: 1 ng CWR22Rv1 (lane 1–2), 0.1 ng CWR22Rv1 (lane 3–4), 0.01 ng CWR22Rv1 (lane 5–6), 0.001 ng CWR22Rv1 (lane 7–8), negative control (lane 9–10). L =  molecular weight ladder.Determining the sensitivity of the Pan-MLV and XMRV-specific PCR assays. Serial dilutions of CWR22Rv1 genomic DNA were spiked into 100 ng of LNCaP (MLV-negative) genomic DNA and used as a template for PCR. ((TIF)Click here for additional data file.Figure S2Testing MLV-positive cell lines for the presence of contaminating mouse DNA by PCR assay for IAP. Lane 1 =  C57BL/6J mouse genomic DNA (positive control), lane 2 =  CWR22Rv1, lane 3 =  LAPC4, lane 4 =  VCaP, lane 5 =  EKVX, lane 6 =  negative control. All cell lines tested were negative for contaminating mouse DNA. L =  molecular weight ladder.(TIF)Click here for additional data file.Figure S3Phylogenetic analysis of virus genomes from LAPC4, VCaP and EKVX cell lines. The phylogenetic tree represents known endogenous MLV sequences, including polytropic (Pmv), modified polytropic (Mpmv) and xenotropic (Xm) viruses. This tree represents only endogenous MLVs for which the full proviral sequence is known, which is likely only a small portion of all endogenous MLVs present in mice. Exogenous MLVs  are also included for comparison.(TIF)Click here for additional data file.Figure S4Testing early passage LAPC4 for presence of virus. The pan-MLV primers described in (TIF)Click here for additional data file.Figure S5Examples of MLV-negative prostate cancer cell lines that have become contaminated with MLV during serial passage by cell culture in the laboratory. Prostate cancer cell lines obtained directly from the NCI (DU145) or ATCC (LNCaP) are negative when stained with MLV30 and MLV70 antisera. These lines were unexpectedly found to be positive for virus after serial passage in the lab.(TIF)Click here for additional data file.Table S1Complete results of IHC and PCR on 72 human cell lines.(DOC)Click here for additional data file.Table S2Primers used to sequence viral genomes.(DOC)Click here for additional data file.


Three dimensional (3D) growths of cancer cells in vitro are more reflective of in situ cancer cell growth than growth in monolayer (2D). The present study is designed to determine changes in protein and phosphoprotein that reflect adaptation of tumor cells to 3D as compared to 2D. Since relative hypoxia is a common feature of most solid tumors, the present study also aims to look at the impact of transition from normoxia to hypoxia in these two growth conditions.Using reverse-phase protein arrays, we compared levels of 121 different phosphorylated and non-phosphorylated proteins in 5 glioma and 6 adenocarcinoma lines under conditions of 3D and monolayer culture in normoxia and hypoxia. A three-way analysis of variance showed levels of 82 antibodies differed between media (2D vs. 3D) and 49 differed between treatments (hypoxia vs. normoxia). Comparing 2D to 3D growth, 7 proteins were commonly  elevated in 3D: FAK, AKT, Src, GSK3αβ, TSC2, p38, and NFκβp65. Conversely, 7 other proteins are commonly decreased: ATRIP, ATR, β-catenin, BCL-X, cyclin B1, Egr-1, and HIF-1α. Comparing normoxia to hypoxia, only NCKIPSD was commonly elevated in hypoxia; 6 proteins were decreased: cyclin B1, 4EBP1(Ser65), c-Myc, SMAD3(Ser423), S6(Ser235), and S6(Ser240). Hypoxia affected glioma cell lines differently from adenocarcinoma cell lines: 8 proteins were increased in gliomas  and none in adenocarcinomas.We identified subsets of proteins with clearly concordant/discordant behavior between gliomas and adenocarcinomas. In general, monolayer to 3D culture differences are clearer than normoxia to hypoxia differences, with anti-apoptotic, cytoskeletal rearrangement and cell survival pathways emphasized in the former and mTOR pathway, transcription, cell-cycle arrest modulation, and increased cell motility in the latter. Cancer growth and invasion reflect many genetic and molecular events. These changes cannot be easily defined in situ, because (a) many factors are difficult to reproduce outside the host and (b) simplifications made to define variables with precision can create artifacts. In this and a prior study  we addreThe importance of hypoxia to our understanding of tumor growth is based on the premise that all tumors, at some time, exhibit reduced oxygen delivery to the respiring neoplastic and stromal cells. This can be microscopic or macroscopic but can lead to proteome changes in neoplastic and stromal cells leading to impaired neoplastic growth through molecular mechanisms, resulting in cellular quiescence, differentiation, apoptosis, and necrosis ,3 and acThe importance of studying protein changes in 3-dimensional (3D) growth is also important since a feature of malignant cells is their ability to grow in 3-dimensions (3D) as spheroids and colonies. This observation has led to greater study of tumors in 3D, as it is closer to in situ growth -11 even In this study we begin to address the following questions. What protein and phosphoprotein changes reflect adaptations of tumor cells to 3D growth compared to 2D growth? What changes reflect adaptations from normoxia to hypoxia? Do tumor cells from high-grade glioma cell lines respond differently to 3D growth than adenocarcinoma cell lines? When exposed to relative hypoxic (aka microaerophilic) conditions, are changes in protein and phosphoprotein levels more affected by growth in 3D culture than they are by hypoxia?In this study, we examine levels of 121 phosphorylated and non-phosphorylated proteins using reverse-phase protein array (RPPA) [Our qualitative findings can be inferred from the p-value plots presented in Figure 5 and chose cutoffs to target false discovery rates (FDRs) of 5% and 1%. The extent of change (the height of the peak) is much more extensive for the shift from 2D to 3D than for the shift from normoxia to hypoxia. The corresponding plot for interaction terms here shows just a few significant alterations, suggesting that assessments of changes due to oxygenation conditions made in 2D are largely preserved in 3D, answering our primary question. However, the amount of change we see associated with the 2D-to-3D transition is so large that we feel quite uneasy about generalizing measurements from 2D in general without explicit testing. To determine what changes were "robust," we trichotomized residual terms for each effect (after correcting for others) by assigning scores of 1 (top 25%), -1 (bottom 25%), and 0 , and summed these values by cell line or antibody, which is an approach we found useful in an earlier study [To account for multiple testing, we fit both distributions of p-values with beta-uniform mixture (BUM) modelsThe comparisons that follow are the product of an aggregate analysis across 11 cell lines and 4 growth conditions focusing on the protein differences between 2D and 3D culture conditions. According to the BUM plots, 82 proteins were significantly different at a 5% FDR. Figures Qualitative examination of Figures 1) Levels of 7 proteins were lower in 3D than 2D cultures for the two groups: ATRIP, ATR, β-catenin, BCL-X, cyclin B1, Egr-1, and HIF-1α;2) 18 proteins showed no grossly consistent differences: AIB1, AR, ATR(Ser428), BCL2, BCL-XL, caspase 3, EGFR(Tyr992), 4EBP1(Ser65), LKB1, MGMT, p85 PI3K, p90RSK(Thr359), PCNA, PTCH, Rab25, Stat6(Tyr694), Stat5(Tyr694) and YY1; and3) Levels of 7 proteins were higher in 3D than 2D cultures: AKT, FAK, GSK3αβ(Ser21), NFκβp65(Ser536), p38(Thr180), c-Src(Tyr418), and TSC2(Thr1462).In addition to the protein changes above, differences were seen between glioma and adenocarcinoma cell lines grown in 3D and 2D cultures. In glioma cell lines, protein or phosphoprotein levels of Stat3(Thr727) and COX2 were also higher in 3D cultures, whereas in adenocarcinoma lines, additional protein increases were seen in 14-3-3-Z, TAU, ACC(Ser79), annexin, caspase 7, FOXO3, MAPK(Thr202), p70S6K, B-RAF, PARP, and PDK1(Ser241). In glioma cell lines, lower protein level in 3D cultures was seen only for cyclin D1, MSH2, Rb, S6(Ser235), and S6(Ser240), whereas in adenocarcinoma lines, lower levels were seen in ER(Ser118), FOXO3α(Ser318), c-Jun(Ser73), c-Jun, c-Myc(Thr58), c-Myc, Rb(Ser807), SMAD3(Ser423), Src(Tyr527), Stat3, and VEGFR2.The comparisons that follow are the product of an aggregate analysis across 11 cell lines and 4 growth conditions focusing on the protein differences between normoxia and hypoxia culture conditions. On the basis of the BUM plots, 50 proteins were significantly different in conditions of hypoxic and normoxic growth at a 5% FDR. Figure Qualitative examination of Figure 1) Levels of 6 proteins were lower in hypoxic conditions than in normoxic conditions in the two groups: cyclin B1, 4EBP1(Ser65), c-Myc, SMAD3(Ser423), S6(Ser235), and S6(Ser240);2) 10 proteins showed no grossly consistent differences: caspase 3, EGFR(Tyr1173), elF4E, FAK, JNK2, MGMT, PDK1, spermine synthetase, TSC2(Thr1462), and VASP; and3) 1 protein was higher in hypoxic cultures: NCKIPSD.In addition to the protein changes reported above, differences were seen between glioma and adenocarcinoma cell lines grown in hypoxia and those grown in normoxia. In glioma cell lines, protein or phosphoprotein levels were also higher for BAX, caspase 7, HIF-1α, c-JUN, MEK1, cleaved PARP, Src(Tyr527), and VEGFR2, whereas no additional protein increases were seen in adenocarcinoma lines. In glioma cell lines, hypoxia caused declines in the expression of AR, ATR(Ser428), cyclin D1, and Rb(Ser807), whereas no additional protein decreases were seen in adenocarcinoma lines.http://www.genecards.org, Cell Signaling Technology http://www.cellsignal.com/, and TOCRIS Bioscience http://www.tocris.com/ to annotate the gene-associated proteins studied. Our interpretation of protein interactions and their implications is subject to a caveat: we only have an incomplete understanding of the non-linear interactions among signaling proteins, and, therefore, can only surmise functional significance from the protein changes we observed.In order to better understand the implications of the protein changes we observed, we used the Weizmann Institute of Science site While there are a number of ways that our data could be analyzed and interpreted, we analyzed the aggregate data for 2D to 3D culture regardless of whether cells were grown in normoxia or hypoxia. From these data we concluded that the majority of cancer cell lines share some proteins that are increased to enable 3D growth and proteins that are reduced to minimize non-vital cell functions and focus. For the sake of discussion, and using available pathway analyses, we propose some relationships for the major protein changes observed for both glioma and adenocarcinoma cell lines Figure . IncreasIn addition to the general changes, there were glioma-specific changes in protein levels. Increases in Stat 3 suggest that glioma cells, responding to cytokines and growth factors, activate transcription to help establish 3D growth. Increase in inducible COX2 is known to occur in gliomas and may, through prostanoid biosynthesis, enhance mitogenesis. There were also specific changes in adenocarcinoma lines. Increases may reflect increased signaling activities and direct effects on cell adhesion and anchorage-independent growth (14-3-3-Z), fatty acid synthesis (ACC), mediation of growth-regulated tyrosine kinases (annexin 1), regulation of MAPK/ERK signaling (B-RAF), activation of apoptosis , and transcription regulation and proliferation (MAPK). Interestingly, TAU, a microtubulin-associated protein, is differentially expressed in the nervous system and was thought to be somewhat unique to the nervous system, but we found higher levels of TAU in adenocarcinoma cell lines. What this means is not clear, but, given its effect in the nervous system, it may function to stabilize cytoskeletal proteins and be part of signaling system to organize adenocarcinoma cells in a basal-antral position for glandular functions.Transitioning from normoxia to hypoxia, only 17 proteins move commonly among the glioma and adenocarcinoma cell lines. Interestingly, only one protein was elevated, NCKIPSD, a protein implicated in signal transduction as well as cell motility and stress fiber formation -27. A reGlioma cell lines behave quite differently from adenocarcinoma cell lines when exposed to hypoxia. There are 8 increased proteins in gliomas  and none in adenocarcinomas  That hypoxic conditions were insufficient in the adenocarcinoma cell lines while sufficient in the gliomas to elicit activation of HIF-1α based on the possibility that astrocytes are constitutively more sensitive and responsive to hypoxia in keeping with their function to protect neurons and this functionality carries over to the glioma  tumors studied. 2) HIF-1α levels were constitutively up regulated (oncogenic) in the glioma lines independent of hypoxic effects. In an effort to answer this question, we compared each protein across cell lines and between glioma and adenocarcinoma groups from monolayer conditions. We found only 3 proteins that had a coefficient of variation of > 0.5 and T-test p < 0.05, and they were AKTser473), AK(Thr308) and HIF-1α; they were higher in base value in the glioma lines than adenocarcinoma lines by 4.7-, 3.0-, and 2.6-fold. Since the cell lines were compared to themselves, with respect to change in protein level, under the various "treatment" conditions of 3D culture and hypoxia, the differences in absolute level did not have an adverse effect on the data we report in Figures 73, AK, gifts from Francisco Esteva (MD Anderson); a human pancreatic carcinoma (MiaPaCa), a gift from Kapil Mehta (MD Anderson); and two human ovarian carcinomas (OVCAR5 and SKOV3), purchased from the American Type Culture Collection . Five high-grade glioma lines were comprised of U87, U251HF, and SNB19, bought from the American Type Culture Collection, and LNZ308 and LN229, gifts from Oliver Bogler (MD Anderson). Cells were maintained in Dulbecco's modified Eagle's medium: nutrient mixture F-12  supplemented with10% fetal bovine serum  and 1% penicillin-streptomycin antibiotic (Invitrogen).For normoxia experiments, 2D and 3D cultured cells were incubated in a humidified incubator with constant supply of 5% CO2 at 37°C (21% oxygen). 2D and 3D cultures were grown under conditions of relative hypoxia using the Incubator Subchamber Culture System with the ProOx 110 oxygen controller  that senses oxygen inside the chamber and maintains it at the set level that was, in our experiments, 1% oxygen for relative hypoxia (for 24 hours).Numerical Preprocessing section below, the original study was initiated with 187 proteins to cover a large part of the known proteome, but because of technical issues, we were only able to study 121 different proteins in the RRPA.The antibodies used are listed in Additional file The techniques used for the 2D studies were similar to those published previously ,36, howe6 cells/mL were plated in flasks, harvested in exponential growth at about 80% confluence, and harvested using 0.25% trypsin. Cells were counted with a Vi-Cell Counter , and 5 × 106 cells were transferred to six-well plates  that were grown for 24 h at 37°C in 5% CO2 and 20% O2 [2 hypoxic environment. Duplicate cultures were performed for each treatment. After 24 h, cells were washed in phosphate-buffered saline and lysed in 1% Triton X-100, 50 nM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1 mM Na3VO4, and 10% glycerol containing freshly added protease and phosphatase inhibitors. Cellular proteins were denatured by 1% sodium dodecyl sulfate (with ß-mercaptoethanol) and diluted in five serial 1:2 dilution steps using dilution buffer (lysis buffer containing 1% sodium dodecyl sulfate).Briefly, less than 104 cells in exponential growth were pipetted into six-well plates in 5 mL of medium (DMEM/F-12) and inoculated directly into the sterile microtiter plates preloaded with lyophilized alginate sponge that had been formulated using USP-grade raw material from brown seaweed, and each plate incubated at 37°C in an atmosphere of 5% CO2 and 20% O2  to allow the cells to form spheroids. The cell lines grew similarly except for the ovarian cancer cell lines that grew a bit slower.We used the AlgiMatrix 3D Culture System six-well kit (Invitrogen), which is an animal origin-free bioscaffold that facilitates 3D cell culture. 2 × 10On day 5, we transferred half the plates to the hypoxia chamber mentioned earlier and allowed them to grow for 24 h in relative hypoxia while the remaining half served as normoxia controls. To harvest spheroids after 24 h of hypoxia , we followed the tri-sodium method described in the AlgiMatrix protocol. Briefly, 5 mL of pre-warmed iso-osmolar tri-sodium citrate solution was added to each well and incubated for 10 min at 37°C. The solution was prepared by diluting 55 mM tri-sodium citrate solution from 1 M stock solution, adding 1 g/L glucose, adjusting the osmolarity using 100 g/L NaCl solution, and adjusting the pH with 1 M citric acid solution to a pH of 7.2-7.4. After 10 min, the sponge biodegraded into the solution and the contents of each well was pipetted into a 15-mL centrifuge tube. To the tube, 5 mL of the same tri-sodium citrate solution was added, and the mixture was centrifuged for 7 min at 400 × g. The supernatant was removed, the pellet washed in phosphate-buffered saline to remove any remaining medium, and the pellet lysed using lysis buffer. The sample was then denatured, serially diluted, and arrayed on slides as in the 2D studies.We manually isolated spheroids and determined the viability of single cells by adding them to 2 mL of trypsin-EDTA in a 15-mL tube, incubating at 37°C for a few minutes, agitating the tube for 15-20 min, and counting using the Vi-Cell cell viability analyzer (Beckman-Coulter). In all cases, the proportion of viable cells was greater than 90%.Array assembly and printing were done as previously described.  In additAntibody and array staining were done as previously described ,38. BrieWe studied 11 cell lines with two replicates under the four growth conditions resulting from combining 2D and 3D under normoxia and relative hypoxia, which would have ideally yielded 88 samples for measurement. Unfortunately, because of technical problems, there was only one replicate for LNZ308 in 3D under normoxia and hypoxia and one replicate for U87 in 3D in normoxia. Thus, we studied only 85 samples. Fortunately, the 41 pairs of exact replicates that did work are adequate to let us estimate the scale of technical variation, which is much smaller (variance 0.0053) than the variance 0.4615 for the cell line, growth condition, and treatment effects studied. Consequently, the replicate to replicate variation is sufficiently small and stable across our experiments relative to other sources of error that keeping the small number of samples without replicates will not lead to any distortion of the data.http://bioinformatics.mdanderson.org/Software/OOMPA) to summarize each five-step dilution series into one log2 scale (EC50) protein concentration value. The algorithm used fits a joint four-parameter logistic model [These samples were examined using 187 antibodies in RPPAs produced by the lead author's laboratory. Array images were produced using ImageQuant software , and individual spot values were summarized using the MicroVigene RPPA module . After preprocessing was done, we used the R package SuperCurve , growth conditions (2D or 3D), and cell line of origin. We also included a term to account for potential interaction between treatment and medium.We used three-way ANOVAs, protein by protein, to model the log5 and chose cutoffs to target false discovery rates (FDRs) of 5% and 1%.To account for multiple testing, we fit distributions of p-values for each contrast with beta-uniform mixture (BUM) modelsTo determine what changes were "robust," we trichotomized residual terms for each effect (after correcting for others) by assigning scores of 1 (top 25%), -1 (bottom 25%), and 0 , and summed these values by cell line or antibody, which is an approach we found useful in an earlier study [The authors declare that they have no competing interests.VAL conceived of the project, obtained funding for the research, and was the primary author. SP was the post-doctoral student who conducted the main aspects of the study and helped write the paper. KAB and LS did the RPPA analyses and the statistical portions of this study. KAB also helped write the paper. All authors approved the final manuscript.This file contains is from the quantile matrix of trichotomized values for 3D-2D values used in Figures 2 and 3 of the paper.Click here for fileThis file contains is from the quantile matrix of trichotomized values for hypoxia-normoxia values used in Figure 4 of the paper.Click here for fileTable S1. Descriptions of 121 Distinct and 3 Duplicate Antibodies Used in Our RPPA Studies. BCL-XL, collagen VI, and Src(Tyr416/418), were used in duplicate, bringing the total number of antibodies studied in the RPPA to 124. Phosphorylation sites are indicated in parentheses.Click here for file


DNA synthesis was analysed by quantification of the S phase using flow cytometry, [methyl-3H]thymidine incorporation and expression of proliferation markers PCNA and Ki-67 using realtime PCR. One way ANOVA was used to compare groups.Human breast cancer MCF-7, MDA-MB-231 and pII, and prostate cancer PC-3 cell lines were grown to 30, 50 and 100% confluency and pulsed with 99mTc(V)-DMSA uptake were inversely related to cell density. This was paralleled by similar trends in S phase proportions, [methyl-3H]thymidine incorporation and expression of PCNA and Ki-67.In all cell lines rates of 99mTc(V)-DMSA uptake into different types of tumour cells correlate well with cell density that is useful as a non-invasive measure of tumour cellular proliferation in vivo.Rates of  This is acceptable for in vitro model systems and in animals but as long lived beta emitters, neither radionuclide is suitable for imaging in humans. Thymidine also has the disadvantage that it is rapidly catabolised and produces large amounts of re-circulating labeled catabolites following administration The ability to non-invasively detect and image cell growth and proliferation throughout the body has long been recognised to be of significant value in the diagnosis, staging and treatment of cancer. The expansion of a tumour mass is directly related to its growth fraction, which can be assessed by measuring DNA synthesis by following incorporation of  3′-deoxy-3′-fluorothymidine (18F-FLT), have been enlisted into clinical use for lymphoma, non-small-cell lung cancer and brain tumors 18F-FLT proceeds only to the triphosphate nucleoside step in the DNA synthetic pathway and not into DNA itself 18F-FLT for identifying sites of proliferative activity or malignancy in the liver and bone marrow due to the presence of high background radioactivity, and interference with pelvic lesions due to its significant excretion into the urinary bladder Positron emission tomography (PET) has emerged as a very sensitive physiological, metabolic and molecular gamma ray detection technology that is used for imaging in many research and clinical applications but principally in oncology. For several decades the most commonly used PET tracer has been the cyclotron produced 2-[fluorine-18]Fluoro-2-deoxy-D-glucose (99mTc). This radiotracer, is produced by a generator that is readily available in most hospital nuclear medicine departments and does not need an on-site cyclotron to produce it. It's half-life of 6 h is long enough to perform nuclear medicine tests and short enough to minimize radiation exposure to the patient. Thus 99mTc is a very useful isotope that has been incorporated into many tumor imaging agents such as 99mTc-hexakis-2-methoxyisobutylisonitrile , used to detect metastatic breast cancer The short half-life of PET tracers has also limited their use to centers equipped with an on-site cyclotron. The practical need for longer-lived tracers has been resolved by the use of technetium-99m (99mTc-dimercaptosuccinic acid (99mTc(V)-DMSA) formed from labelling of meso-2,3-dimercaptosuccinic acid (DMSA) with 99mTc under alkaline conditions, has been found to be a tumor-seeking agent. It was introduced to evaluate, image and manage medullary carcinoma of the thyroid Pentavalent in vivo99mTc(V)-DMSA uptake by breast lesions is related to proliferative activity, which is either directly related to tumor grade or to the mitotic activity.Several reports from Papantoniou and colleagues 99mTc(V)-DMSA uptake could be correlated with the rate of cell proliferation using an in vitro model system. Typically, attaching monolayer cultures undergo a lag phase followed by a period of rapid growth that then slows as the cells in the culturing vessel reach confluence. Confuency is the measure of % coverage of the culturing vessel. We measured 99mTc(V)-DMSA uptake into several different cancer cell lines under these different conditions and compared this with assessment of other known proliferation-associated markers.In this study we have sought to determine whether 99mTc, was obtained from a molybdenum-99-technetium-99m (99Mo-99mTc) generator located in the Clinical Nuclear Medicine Department of Mubarak Al Kabeer Hospital, Kuwait. [Methyl-3H]thymidine (specific activity 0.93 TBq/mmol) was obtained from Amersham. 2-[fluorine-18]Fluoro-2-deoxy-D-glucose (18F-FDG) was obtained from Kuwait Cancer Center (Kuwait). Dimercaptosuccinic acid  kit was purchased from Mallinckrodt Medical BV (Holland). Sodium bicarbonate was purchased from Pharmaceutical Solutions Industry (Saudi Arabia). Imatinib was purchased from LC Laboratories (USA).All general chemical reagents were purchased from Sigma (UK). Tissue culture reagents were purchased from Life Technologies (USA). Propidium iodide (PI)-ribonuclease (RNase) staining buffer (BD staining kit) was obtained from BD Biosciences (UK). Radionuclide, 2-95% air. For sub-culturing, cell monolayers were harvested with trypsin-EDTA. Cell counting was carried out using a haemocytometer  and Cell Viability Analyzer . For most experiments cells were seeded into 25 cm flasks and grown to 30%, 50% and 100% confluency. All experiments were repeated thrice and individual conditions were performed in triplicate each time.Human breast cancer MCF-7, MDA-MB-231 and prostate cancer PC-3 cell lines were purchased from Cell Lines Service . pII is an estrogen receptor down-regulated transfected cell line derived from MCF-7 in vivo99mTc(V)-DMSA uptake by particularly early breast lesions is related to proliferative activity. The evidence for this is extensively discussed in a recent review in situ tumours. We selected a well established and extensively used endocrine sensitive line (MCF-7)  as well as one that is naturally endocrine resistant (MDA-MB-231) and one that has acquired resistance (pII) as a consequence of siRNA transfection- the latter two being more aggressive in terms of metastatic propensity. As our aim was to determine whether our hypothesis was generally applicable, we also chose another cell line derived from a different tissue (prostate- PC-3) that has been shown to exhibit significantly higher phosphocholine as well as glycerophosphocholine levels, i.e., increased phospholipid metabolism We chose breast cancer cell lines as reports from Papantoniou and colleagues 99mTc(III)-DMSA), was used here for the preparation of technetium-99m (V) complex with DMSA. 99mTc(V)-DMSA complex was prepared by addition of sodium bicarbonate solution to the kit vial followed by the addition of 99mTc-eluate . The reaction mixture reached a pH value of approximately 8 (as determined with pH indicator strips) and was then left for 15 min at room temperature. This value of pH indicates the optimum conversion to 99mTc(V)-DMSA The commercial meso-2,3-dimercaptosuccinic acid (DMSA) kit (Succimer), for the preparation of trivalent technetium-99m DMSA (2O (3∶2∶3) developing mixture. The retention factor (Rf) value for 99mTc(V)-DMSA was 0.7 while for 99mTc(III)-DMSA it was 0.1 giving a very clear separation 99mTc(V)-DMSA relative to total activity on the plate. Typically we obtained efficiencies in excess of 95% conversion of 99mTc(III)-DMSA to 99mTc(V)-DMSA. The 99mTc(V)-DMSA mixture was used within 3 h of preparation.The efficiency of radiolabeling of DMSA was assessed by ascending thin-layer chromatography using Merck silica gel 60F  and n-butanol:acetic acid:H99mTc(III)-DMSA) remaining in the labeled preparation can be changed into 99mTc(V)-DMSA by bubbling with pure oxygen. We therefore performed comparative uptake experiments using 99mTc(V)-DMSA prepared with oxygen bubbling (100% oxygen) for 10 min or with no bubbling (0% oxygen).It has previously been reported 99mTc(V)-DMSA was also used for in vivo imaging in patients showing significant uptake into breast tumors (data not presented).The 99mTc(V)-DMSA (74 MBq/ml medium) for 60 min at 37°C. The culture medium was removed and counted to determine effluxed radioactivity using a dose calibrator . The cell monolayers were rapidly washed three times with ice-cold phosphate buffered saline (PBS) and detached with 0.5 ml trypsin-EDTA followed by re-suspension in 5 ml of medium. The cells were centrifuged at 10,000 g for 5 min at 4°C and solubilized with 1% sodium dodecyl sulphate (SDS) in 10 mM sodium borate. The 99mTc(V)-DMSA incorporated into the cellular lysate was counted as above.All the cell lines were pulse labeled with 99mTc(V)-DMSA radioactivity uptake in megabecquerel (MBq) per mg of protein.Results were expressed as the total 99mTc(V)-DMSA as described above.In other experiments, MCF-7 cells were grown to about 60–70% density in a 24 well plate. These were pretreated for either 24 h or 48 h with 50 µM imatinib (prepared as a 10 mM stock solution in DMSO), or for 15 min with 5 mM and 10 mM phosphonoformic acid (PFA) prior to uptake determination of 99mTc(V)-DMSA (phospholipid synthesis) was determined by extraction of cell pellets with chloroform/methanol tris (hydroxymethyl) aminomethane buffer solvent system as described by Bligh and Dyer et al99mTc(V)-DMSA content.The intracellular fate of the 18F-FDG (13.283 MBq) for 15 min at 37°C and processed as described above for 99mTc(V)-DMSA uptake.MCF-7 cells were pulse labeled with + on the uptake of 99mTc(V)-DMSA was assessed by incubating cells for 1 h, prior to performing the assay, in two types of media in place of the standard DMEM. The high Na+ medium was composed of 137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM Mg2SO4, 14 mM Tris (pH 7.4) and 0.1 mM KH2PO4. The Na+-free medium was similarly composed except that the NaCl was substituted with 137 mM N-methyl-D glucamine.The effect of Na3H]thymidine (51.8 MBq/ml of medium per flask) for 1 h at 37°C in order to assess DNA synthesis. Cells were washed thrice with ice cold PBS, trypsinized and centrifuged at 10,000 g for 5 min at 4°C. Cell pellets were suspended with ice cold PBS and left on ice for 15 min before centrifugation. Pellets were resuspended in 1 ml of 4% trichloroacetic acid (TCA)  at 4°C and again centrifuged. Washes were repeated in 4% TCA at 4°C until the radioactivity in the supernatant was reduced to background level. To solubilise the DNA (acid-insoluble fraction), the pellet was resuspended in 0.5 ml of 4% TCA and heated to 90°C for 1 h. Then the cell debris was removed by centrifugation and washed with 0.5 ml of 4% TCA. The resulting supernatants and pellets were pooled, then suspended with Ultima Gold™ scintillation fluid  and counted in a Beckman LS 6000 TA liquid scintillation counter .MCF-7, MDA-MB-231, pII and PC-3 cell lines grown to different cell densities , were pulse labeled with  we observed active uptake into several cell lines in vitro.In agreement with many studies reporting that 188Re(V)-DMSA) is a beta-emitting analogue of 99mTc(V)-DMSA, a tracer that is taken up into a variety of tumors and bone metastases that is used on a therapeutic scale for cancer therapy 188Re(V)-DMSA in cases of advanced disease with osseous and soft tissue metastatic spread could be considered if these lesions are depicted by breast and whole-body 99mTc(V)-DMSA scintigraphy 188Re(V)-DMSA was loaded with poly(lactic-co-glycolic)acid microspheres for targeted radiotherapy and for the delivery of a radiation dose to tumors This radiotracer is not only used as a diagnostic tool but even further as a therapeutic agent. Pentavalent rhenium-188 dimercaptosuccinic acid (99mTc(V)-DMSA forms a small complex as [MO(DMSA)2]-, in which the M  is coordinated square-pyramidically by the four thiolates and by an apical oxo-ligand and consists of mixtures of three stereo isomers of the square pyramidal mononuclear complex 99mTc(V)-DMSA complex possesses four negatively charged carboxylate groups, and a central anionic technetium oxobis (dithiolato) core, i.e., [TcO (DMSA)2]−1The technetium is pentavalent and coordinated by an oxo-ligand and four thiolate sulfurs of two DMSA ligands. 99mTc(V)-DMSA for imaging 99mTc(V)-DMSA as a guide for therapeutic administration of 188Re(V)-DMSA for advanced breast cancer is underway at the Vince Institute of Nuclear Sciences in Belgrade. This agent has advantages over currently employed PET radiopharmaceuticals. It is safe, cheap and readily available in many nuclear medicine centers, and can be prepared as an in house radiopharmaceutical. Unlike for example the more commonly used imaging agent 18F-FDG, there is no need for a cyclotron to produce it so it would be useful for medical centers that have no or limited access to such facilities for generating the current range of PET tracers. PET is not currently well-established in Kuwait; this study could provide some justification for considering a future trial to examine the utility of 99mTc(V)-DMSA as a nuclear medicine marker that can be synthesized locally without the need for an expensive cyclotron.Reports as recent as 2005 and 2007 describe the use of in vitro assays 99mTc(V)-DMSA uptake and cell density. It is generally the case that the growth rate of tumors slows as they increase in size, mainly as a result of poorer vascularization and necrosis at the centre. As this is an important consideration in the design of chemotherapeutic strategies, information regarding growth rates can be clinically useful, as well indicating prognosis. Our simple in vitro model aims to simulate slow and faster growing groups of cells and the results suggest that the imaging tracer 99mTc(V)-DMSA can provide additional information in this respect.The label was predominantly found in the aqueous fraction of cell extracts indicating actual uptake into the cell rather than immediate incorporation into membrane or other phospholipid. The comparative rates of uptake differed between the three breast and one prostate line, being higher in the more aggressive estrogen receptor negative MDA-MB-231 and pII lines. Interestingly, MCF-7 do not show invasive characteristics in 99mTc(V)-DMSA utilizes the type III NaPi co-transporters 99mTc(V)-DMSA uptake. Our data is consistent with studies showing that this transport is largely Na+ dependent.It is considered that 99mTc(V)-DMSA in our study after both 24 and 48 h of exposure to the drug, an agreement with previous studies. This drug has also been shown previously to reduce expression of these transporters Activated platelet-derived growth factor receptor (PDGF-R) pathway is involved in this transport mechanism. The addition of the PDGF-R inhibitor, imatinib, at least partially inhibited 99mTc(V)-DMSA to trace the antiproliferative effects of imatinib mesylate 99mTc(V)-DMSA cellular uptake studies showed that the specificaction of imatinib on PDGF-R signal pathway, in the human glioblastoma cell line U87-MG, could be followed by radioactive tracer. Furthermore, strong correlations between cellular 99mTc(V)-DMSA uptake and the effect of imatinib therapy on U87-MG proliferation, invasion and migration were obtained, likewise for 99mTc(V)-DMSA uptake and PDGF-R expression A recent study on U87-MG glioblastoma cells investigated an issue with obvious clinical implications, the ability of 3H]thymidine into cells and its subsequent incorporation into DNA is a common measure of cell proliferation. We have shown that its uptake into all four of our cell lines parallels that of 99mTc(V)-DMSA, decreasing with increasing cell density. Similarly, flow cytometry showed that the proportion of cells in s-phase was also inversely correlated with cell density. One study has reported that 99mTc(V)-DMSA brain scintitomography is a plausible non-invasive measure of glioblastoma proliferation and therapy response in vivo99mTc(V)-DMSA uptake has been correlated with the proliferation index measured by Ki-67 expression and phosphorylated focal adhesion kinase The uptake of [methyl-18F-FDG, a commonly used imaging tracer, and found that unlike 99mTc(V)-DMSA, its transport into cells was unrelated to proliferation rate, as has previously been reported As a comparison, we also measured uptake of 99mTc(V)-DMSA into 4 independent cancer cell lines and shown that this correlates well with proliferation rate using cells under conditions of fast and slow growth. The model has been validated with measurement of several parameters commonly accepted as markers of proliferation rate.In summary, we have demonstrated carrier mediated uptake of 99mTc(V)-DMSA uptake into tumors with respect to their rates of growth so that it may have an additional use beyond detection/localization of tumours.Our data indicates that it would be worthwhile to conduct further studies to examine the extent of


Long non-coding RNAs (lncRNAs) are molecules that have been recently described to participate in tumor progression, however, they remain poorly characterized and their role in brain cancers, such as medulloblastoma (MDL) has not been studied in detail as yet. This study aimed to assess the gene expression profile of a lncRNA named EVF-2 in MDL tissue samples and cell lines.We used two human MDL tissue samples, three human MDL cell lines , three human cerebellum tissue samples and two human cerebellum primary culture. The cell lines were characterized morphologically by light microscopy and immunophenotypically by flow cytometry. MDL cell lines and tissue samples, as well as, control cerebellum primary culture and tissue were evaluated for gene expression profile of the EVF-2 lncRNA gene by Quantitative Real Time PCR.MDLcell lines are morphologically presented as polygonal and fibroblastoid cells. Immunophenotypically, MDL cell lines showed a high percentage (70-99%) for CD44, CD73, CD105, CD166 and CD29 and a low or absence (0-4.2%) for CD144, CD31, CD34 and CD45. MDL cell lines were different for CD271 , CD24 (20-64%), CD146 (34-90%), CD90 , CD140b , and were positive for the cancer stem cell marker CD133 . The EVF-2 lncRNA showed 81 ± 13 times more expressed in MDL tissue samples when compared to cerebellum tissue and 119 ± 48, 476 ± 74, 227 ± 33 times more expressed in MDL cell lines UW402, UW473 and DAOY respectively, when compared to cerebellum primary culture.These data demonstrate that MDL cell lines present a immunophenotype somewhat different for some markers and that EVF-2 lncRNA is more expressed in both human MDL cell lines and tumor tissue when compared to cerebellum primary culture and tissue, respectively.FAPESP and Hemocentro de Ribeirão Preto-HCFMRP/USP.


High-grade osteosarcoma is an aggressive tumor most often developing in the long bones of adolescents, with a second peak in the 5th decade of life. Better knowledge on cellular signaling in this tumor may identify new possibilities for targeted treatment.We performed gene set analysis on previously published genome-wide gene expression data of osteosarcoma cell lines (n=19) and pretreatment biopsies (n=84). We characterized overexpression of the insulin-like growth factor receptor (IGF1R) signaling pathways in human osteosarcoma as compared with osteoblasts and with the hypothesized progenitor cells of osteosarcoma – mesenchymal stem cells. This pathway plays a key role in the growth and development of bone. Since most profound differences in mRNA expression were found at and upstream of the receptor of this pathway, we set out to inhibit IR/IGF1R using OSI-906, a dual inhibitor for IR/IGF1R, on four osteosarcoma cell lines. Inhibitory effects of this drug were measured by Western blotting and cell proliferation assays.50s below 100 nM at 72 hrs of treatment. Phosphorylation of IRS-1, a direct downstream target of IGF1R signaling, was inhibited in the responsive osteosarcoma cell lines.OSI-906 had a strong inhibitory effect on proliferation of 3 of 4 osteosarcoma cell lines, with ICin vitro rationale for using IR/IGF1R inhibitors in preclinical studies of osteosarcoma.This study provides an  The disease occurs most frequently in children and adolescents at the site where proliferation is most active, al plate . The 5-yal plate ,3. BetteWe have previously described the roles of bone developmental pathways Wnt, TGFβ/BMP, and Hedgehog signaling in osteosarcoma, but unfortunately so far could not identify suitable targets for treatment ,5. In adet al. [The role of the IGF1R pathway in growth has been illustrated in studies of knockout mice. It was shown that IGF1 null mice are 40% smaller than littermates, while IGF1R null mice are approximately 55% smaller . In dogset al. ), but thWe have analyzed genome-wide gene expression in high-grade osteosarcoma cell lines and pretreatment biopsies, and observed significantly altered activity of genes involved in IGF1R signaling when compared to profiles of mesenchymal stem cells and osteoblasts. Specifically, upstream inhibitors of IGF1R signaling were found to be downregulated in osteosarcoma, and low expression of these genes correlated with worse event-free survival. We inhibited IR/IGF1R signaling with the dual IR/IGF1R inhibitor OSI-906. This showed inhibition of phosphorylation of IRS-1 and of strong inhibition of proliferation in 3/4 osteosarcoma cell lines. Interestingly, the cell line which could not be inhibited with OSI-906, 143B, has a k-ras oncogenic transformation, which is a component of the Ras/Raf/ERK pathway, one of downstream effectors of IGF1R signaling. These results suggest that IR/IGF1R signaling may be an effective targeted for treatment of high-grade osteosarcoma patients.et al. [et al. [The 19 high-grade osteosarcoma cell lines that were used in this study were characterized and are described in Ottaviano et al. . The 12 et al. . MSCs haet al.  characte [et al. . Osteoblet al. [LIMMA[P-values <0.05 were considered to be significant. Gene set analysis was performed on KEGG pathways [P-value <1·10-5. To visualize differential expression in the IGF1R pathway, we performed Core analyses using Ingenuity Pathways Analysis .For genome-wide gene expression analysis, we used Illumina Human-6 v2.0 BeadChips. Microarray experiments and data preprocessing are described in Kuijjer et al. . Previouet al. , but sinet al. . Ethicalet al.  package l. [LIMMA in statil. [LIMMA. Probes pathways   were obtained from Cell Signaling . Rabbit polyclonal antibody against phospho-IRS-1  was purchased from Biosource, Invitrogen . A mouse monoclonal antibody against α-tubulin from Abcam  was used as a loading control . Secondary antibodies  were horseradish peroxidase (HRP) conjugated polyclonal goat-anti-rabbit IgG for components of the IR/IGF1R pathway, and HRP conjugated polyclonal goat-anti-mouse for α-tubulin. OSI-906 was purchased from Selleck Chemicals LLC .et al. [Osteosarcoma cell lines OHS, KPD, SAOS2, and 143B were treated with 0.5% DMSO or with 1 μM OSI-906 for 3 hrs, and were subsequently lysed using Mammalian Protein Extraction Reagent (Thermo Scientific 78503), to which Halt Phosphatase and Protease Inhibitor Cocktails  were added according to the manufacturer’s protocol. Concentrations of cell lysates were determined using the BioRad DC Protein Assay Kit . Per sample, 20 μg of protein was loaded on SDS-PAGE gels. Lysate of HepG2-A16 cells transfected with IR and stimulated with insulin, containing 10 μg of protein, was taken along as a positive control. Western blotting was performed as described in Schrage et al. .http://www.graphpad.com). Relative IC50s were calculated using results from the different concentrations up to the highest dose where toxicity was not yet present.OSI-906 was diluted in DMSO and stored at −20°C. OHS, SAOS2, KPD, and 143B cells were plated in 96 wells plates, using 4,000, 2,000, 12,000, and 2,000 cells per well, respectively. After 24 hrs, OSI-906 was added in triplicate at different concentrations – 0 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM. The inhibitor was incubated for 72 hrs and 96 hrs, in different experiments. The results shown are representative results from at least three independent experiments. Cell proliferation reagent WST-1 (Roche) was incubated for 2 hrs and subsequently measured using a Wallac 1420 VICTOR2 . Data were analyzed in Graphpad Prism 5.0  genes between osteosarcoma cell lines and MSCs, and 2,222 probes encoding for DE genes between osteosarcoma cells and osteoblasts. We tested the global expression patterns of KEGG pathways using globaltest [data not shown). Notably, there is no specific IGF1R signaling pathway in the KEGG database [Genome-wide gene expression data were of good quality for all cell lines. obaltest  and deteobaltest  comparedobaltest ) also redatabase . BecauseIGFBP4 and GAS6 as the most downregulated genes in osteosarcoma . IGFBP2 was also present in the top 20 results from this four-way analysis . Patients of whom biopsies had very low expression of these genes had poor event-free survival profiles . To visualize mRNA expression of the IGF1R signaling pathway members, we used Ingenuity Pathways Analysis on LIMMA toptables from osteosarcoma cells as compared with mesenchymal stem cells and from osteosarcoma cells as compared with osteoblasts  and a previously published set of osteosarcoma pretreatment biopsies  in comparison with mesenchymal stem cells (n=12) and osteoblasts (n=3) by four-way Venn analysis of all significantly affected probes with the same direction of fold change   in a four-way analysis, in which osteosarcoma pretreatment biopsies or cell lines were compared with osteoblastic cultures (n=3) or MSCs (n=12). Insulin-like growth factor binding proteins (IGFBPs) generally inhibit IGF1R signaling by competitively binding IGFs, but can under certain circumstances also stimulate IGF1R signaling [IGFBP4 and GAS6 expression have previously been shown to be downregulated in osteosarcoma cell lines . Next tntiation . IGFBP3  sarcoma . IGFBP7 arcinoma . Interes of IGF2 . Overexper types ,41. Figuet al. in direct complementation breast cancer cells for IGF1R-IGF2 and IR(A)-IGF2 [50s. Proliferation of 143B was only inhibited most likely unspecifically at high and toxic levels of the drug. The 143B cell line is a derivative of the osteosarcoma cell line HOS, transformed by a KRAS oncogene. Constitutive activation of the Ras/Raf/ERK pathway can explain why proliferation of this cell line cannot be inhibited by OSI-906. Of the cell lines that were responsive to OSI-906, KPD and OHS showed that treatment of 96 hrs was most effective, while SAOS2 already reached maximum inhibition at 72 hrs.OSI-906 is a selective small molecule dual kinase inhibitor of both IR and IGF1R . We spec(A)-IGF2 . Intereset al. [IGF1R signaling has been previously modulated in sarcoma in preclinical and clinical models. Several phase I and II clinical trials including treatment with IGF1R monoclonal antibodies are currently being conducted in sarcoma, especially in Ewing sarcoma . Monoclet al.  and in aet al. . Resultset al. . Preclinet al. -52. Howeet al. ,53,54, aet al. . Becauseet al. , blockinPhosphorylated IRS could be used as a biomarker in order to determine whether patients would respond to IGF1R inhibition. Patients with tumors exhibiting an activating mutation in downstream pathways will most likely not respond to IGF1R inhibition. Further research needs to be performed in order to assess these candidate biomarkers for response to treatment. The IGF1R pathway acts on several biological mechanisms that promote tumor progression – mitogenesis, protection from apoptosis, malignant transformation, and metastasis . It is teg several IGF binding proteins, were downregulated. As this indicated the IGF1R receptor as a potential target for treatment of osteosarcoma, we set out to inhibit this receptor in four osteosarcoma cell lines. We used OSI-906, a selective small molecule dual kinase inhibitor of both IR and IGF1R, since the insulin receptor can activate the same downstream signaling pathways as IGF1R, thereby providing a way to circumvent single inhibition of IGF1R. Treatment with OSI-906 resulted in inhibition of phosphorylation of IRS-1 Y612, a direct downstream target of IGF1R, and in strong inhibition of proliferation in 3 of 4 osteosarcoma cell lines. The non-responsive cell line, 143B, has a k-ras oncogenic transformation, and may therefore not respond to this treatment. In conclusion, we have shown that IGF1R signaling is active in osteosarcoma, and that dual inhibition of IR/IGF1R inhibits downstream signaling and proliferation of these cells. Responsiveness to this treatment may be evaluated by Western blotting against phosphorylated IRS. This study provides an in vitro rationale for using dual IR/IGF1R inhibitors in preclinical studies of osteosarcoma.Using gene set analysis of genome-wide gene expression data of high-grade osteosarcoma biopsies and cell lines, we detected an over-representation of IGF1R signaling. Specifically, different upstream inhibitors of IGF1R signaling, DE: Differentially expressed; HRP: Horseradish peroxidase; IGF: Insulin-like growth factor; IGF1R: Insulin-like growth factor receptor; IGFBPs: Insulin-like growth factor binding proteins; IPA: Ingenuity Pathways Analysis; IR: Insulin receptor; IRS: Insulin receptor substrate; KEGG: Kyoto encyclopedia of genes and genomes; MSC: Mesenchymal stem cell; OB: Osteoblast; Shc: Src homology 2 domain containing transforming protein.The authors declare that they have no competing interests.MLK performed all bioinformatics analysis and wrote the manuscript. EFPP, IHB, BEWMA performed Western blotting experiments. EFPP and BH performed inhibition studies. MS, LAMZ, and OM and were involved collection of cell line data. AMC, PCWH, BH, and MLK designed the study. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/245/prepubResult from the four-way intersection of differentially expressed probes with same direction of fold change .Click here for fileFour-way Venn diagram depicting A the number of significantly differentially expressed probes in all four analyses B the number of significantly differentially expression probes with same direction of fold change in all four analyses . In total, we detected 495 probes that were significant in all analyses. 487/495 significant probes had the same direction of fold change in all four analyses. CellvsOB: osteosarcoma cell lines vs osteoblasts, CellvsMSC: osteosarcoma cell lines vs MSCs, BiopvsOB: osteosarcoma biopsies vs osteoblasts, BiopvsMSC: osteosarcoma biopsies vs MSCs.Click here for fileValidation of expression levels or IGF1R and IRS-1. A Normalized expression levels of IGF1R and IRS-1 in the panel of 19 osteosarcoma cell lines. For both genes, we selected cell lines with relatively low and high mRNA expression (black dots), and determined protein levels on cell lysates using Western blotting. B Western blotting results of the selected cell lines.Click here for fileDose response curves up to toxic levels of OSI-906. Osteosarcoma cell lines were inhibited with different concentrations of OSI-906, for 72 (gray line) or 96 (black line) hours.Click here for file


A major bottleneck in the manufacture of recombinant therapeutic proteins is the time and effort needed for the generation of stable, high-producing mammalian cell lines. Conventional gene transfer methods for stable cell line generation rely on random transgene integration, resulting in unpredictable and highly variable levels of expression of the transgene in individual clones ,2. As a We initially determined the efficiency of the PB system in the generation of stable lines using suspension adapted mammalian cells. CHO and HEK 293 cells were co-transfected with an eGFP-bearing donor plasmid along with the PB helper plasmid. The cells were then grown in the absence of puromycin, and the percentage of GFP-expressing cells in each culture was determined by flow cytometry on a daily basis. By 21 days post-transfection, the remaining GFP-positive cells were assumed to be recombinant. Compared to conventional transfection of plasmid DNA, PB transposition resulted in an improvement in the efficiency of stable cell line generation up to 20-fold for both CHO and HEK 293 cells Figure .To further evaluate the PB system, CHO cells expressing a tumour necrosis factor receptor:Fc fusion protein (TNFR:Fc) were generated either by PB-transposition or by conventional transfection. Clonal cell lines were recovered following selection in 50 or 10 µg/mL puromycin for two weeks. Recovered lines were grown in suspension culture for 7 days in 24-well plates after which the medium was analyzed by ELISA to determine TNFR:Fc productivity. Transposition increased the frequency of high-producing clones in the transfected population Figure . To furtIn conclusion our results demonstrate that stable cell lines derived by PB transposition are efficiently generated and are more productive than cell lines generated by conventional transfection methods. Therefore, the PB system represents a valuable and practical alternative to standard plasmid transfection to efficiently generate cell clones with stable and enhanced transgene expression.


Alternative Lengthening of Telomeres (ALT) is a non-telomerase mechanism of telomere lengthening that occurs in about 10% of cancers overall and is particularly common in astrocytic brain tumors and specific types of sarcomas. Somatic cell hybridization analyses have previously shown that normal telomerase-negative fibroblasts and telomerase-positive immortalized cell lines contain repressors of ALT activity, indicating that activation of ALT results from loss of one or more unidentified repressors. More recently, ATRX or DAXX was shown to be mutated both in tumors with telomere lengths suggestive of ALT activity and in ALT cell lines. Here, an ALT cell line was separately fused to each of four telomerase-positive cell lines, and four or five independent hybrid lines from each fusion were examined for expression of ATRX and DAXX and for telomere lengthening mechanism. The hybrid lines expressed either telomerase or ALT, with the other mechanism being repressed. DAXX was expressed normally in all parental cell lines and in all of the hybrids. ATRX was expressed normally in each of the four telomerase-positive parental cell lines and in every telomerase-positive hybrid line, and was abnormal in the ALT parental cells and in all but one of the ALT hybrids. This correlation between ALT activity and loss of ATRX expression is consistent with ATRX being a repressor of ALT. In the great majority of the hybrid clones, one or other TLM was repressed, and repression of ALT correlated with wild-type ATRX expression. These data are consistent with ATRX being an ALT repressor.2 incubator.The universal hybridizer ALT cell line, GM847DM (hereinafter referred to as GM847), derived from simian virus 40 (SV40)-immortalized Lesch-Nyhan syndrome male human fibroblasts, were provided by Dr. Olivia Pereira-Smith −6M ouabain.Generation of the MeT-5A/GM847 hybrids was described previously The TRAP assay was performed essentially as described previously HinfI and RsaI (New England Biolabs) and 1 µg of digested DNA subjected to pulsed field gel electrophoresis in 0.5x TBE buffer. The gels were dried, denatured, hybridized to a telomeric-specific oligonucleotide probe, and exposed overnight to a PhosphorScreen . The PhosphorScreen was then scanned using a Typhoon imager.TRF analysis was performed as described previously 32P-labeled-(CCCTAA)3 oligonucleotide probe as previously described The C-circle assay was performed using 30 ng of genomic DNA and the signal was detected using a γ-The following primary antibodies were used for indirect immunofluorescence and/or immunoblotting: ATRX (Sigma), DAXX (Sigma), γ-tubulin (Sigma), TRF2 (Millipore), and PML (Santa Cruz). Secondary antibodies for immunofluorescence were obtained from Life Technologies: Alexa Fluor 488 donkey anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 594 donkey anti-goat IgG, and Alexa Fluor 647 donkey anti-rabbit IgG. The secondary antibody goat anti-mouse HRP used for immunoblotting was obtained from Dako.For immunodetection of ATRX and DAXX, cells fixed with 4% paraformaldehyde were permeabilized twice with 0.1% Triton X-100. ATRX and DAXX antibodies were used at a dilution of 1∶200 in 3% BSA/PBS. Reacting antibodies were detected using Alexa Fluor 488 conjugated secondary antibodies at a dilution of 1∶1,000 and cells were counterstained with DAPI (Sigma). Images were captured on an Axio-Imager M1 microscope (Carl Zeiss) using a 40x lens and analysed using AxioVision software (Carl Zeiss).Immunoblotting was performed on whole cell extracts using standard techniques. Briefly, 30 µg of protein lysate was separated on a 3–8% NuPAGE Tris-Acetate polyacrylamide SDS gel (Life Technologies), transferred to PVDF membrane (Millipore), and probed with ATRX, DAXX, and γ-tubulin antibodies. Detection was performed using SuperSignal West Pico chemiluminescent substrate (Pierce) and analyzed on an ImageQuant LAS 4000 Biomolecular Imager (Fujifilm).GTGATCGTGGCAGGAAAAGT and reverse 5′ AGCAAAAAGTTTTCCTGTTATCCA; exon 2: forward 5′ TTTCTTTGAAGCTGCCCACT and reverse 5′ AGCACATGCAACAATTTGGA, and exon 3: forward 5′ GCATCTTGCCCAGTCATTTT and reverse 5′ TGGAAAAACTGCCAATACCTG. PCR products were purified with the QIAquick PCR Purification Kit (Qiagen) and subjected to Sanger sequencing . The parental origin of the ATRX alleles in the hybrids was identified using SNP rs3088074 located on exon 9.All ATRX and DAXX exons were amplified using primers and PCR conditions as described For immunodetection of APBs, cells fixed with 4% paraformaldehyde were permeabilized with methanol. Cells were then incubated concurrently with primary antibodies TRF2, PML, and ATRX at dilutions of 1∶300, 1∶100, and 1∶500, respectively, followed by secondary antibodies to detect the protein in parentheses: Alexa Fluor 488 (TRF2) and Alexa Fluor 594 (PML) at a dilution of 1∶1,000 and Alexa Fluor 647 (ATRX) at a dilution of 1∶500. Images were captured on an Axio-Imager M1 microscope using AxioVision 4.8.2 software (Carl Zeiss) and appropriate filter sets. Nuclei containing colocalized TRF2 and PML staining were considered positive for the presence of APBs.The ALT cell line, GM847, was fused with four different telomerase-positive cell lines, and clonally derived hybrid cell lines were obtained by marker selection. The hybridization efficiency and the frequency with which immortal hybrid lines arose for each set of hybrid lines is indicated in We previously showed that fusing MeT-5A with GM847 resulted in 18 of 18 hybrid lines ceasing proliferation after 20–45 population doublings (PD), and that 13 eventually recommenced proliferation after 50–100 days  [21]. FiTelomerase activity ; Table 1Most human ALT cell lines examined to date have a characteristic pattern of telomere lengths, ranging from very short to extremely long In all of the hybrid lines examined, therefore, there was a clear-cut correlation between telomerase status and telomere length pattern: the hybrids either had telomerase activity and a TRF pattern consistent with telomere maintenance via telomerase, or they were telomerase-negative and had the long, extremely heterogeneous TRF pattern characteristic of ALT. No evidence was found for the persistence of both TLMs in any hybrid line. The hybrid cell lines that were ALT-positive were significantly more likely to have undergone a period of growth arrest .The parental and hybrid cell lines were all analyzed for C-circles , which aATRX and DAXX can be visualized using immunofluorescence as foci that colocalize with PML bodies within the nucleus ATRX immunoblotting results  correlatAnother hallmark of ALT activity is the presence of APBs, which are large aggregates of telomeric DNA and telomere binding proteins within PML nuclear domains, in a subset of cells within the population In order to determine the parental origin of the ATRX allele in the hybrid lines, we identified an ATRX sequence polymorphism (SNP). We found a SNP for which each of the parental cell lines was homozygous, and for which the GM847 allele (“G” SNP) differed from the allele in three of the four telomerase-positive parental lines (“C” SNP), and were therefore able to deduce the parental origin of the ATRX gene in three of the four sets of hybrid lines . We founWe then sequenced all exons of ATRX in each parental cell line, as well as the cell strain (GM02063) from which GM847 cells were derived and found that GM847 cells lack exon 1 of ATRX . The ampWe used somatic cell hybridization analysis to determine whether repression of ALT is associated with normal expression of ATRX/DAXX, because it was this experimental approach that had previously shown normal cells and telomerase-positive cell lines contain repressors of ALT The evidence that normal mortal cells contain repressors of ALT and telomerase indicates that it is very unlikely that ALT and telomerase are each other’s repressors. This has been verified experimentally by expressing telomerase exogenously in ALT cells and showing that the two mechanisms can co-exist within the same cell Here we addressed the question whether ATRX/DAXX represses ALT. DAXX expression was normal in each of the 24 cell lines. We found a clear-cut correlation between the ATRX status and TLM in 21/24 cell lines studied , and the correlation appeared to hold also in two of the remaining three lines which appeared to contain mixed populations. ATRX was normal in each of the 16 telomerase-positive lines  and abnormal in the ALT-positive GM847 parent and four ALT hybrid lines. A further two hybrid A549/GM847 lines, A-A and A-K, were telomerase-negative/ALT-positive by all criteria, but had low levels of ATRX protein and a small proportion of cells had ATRX foci . These two lines thus appear to contain distinct subpopulations, with the majority being ATRX-negative/ALT-positive. Consistent with this, when we examined APBs and ATRX foci in the A-K line, we did not observe any cell that contained both. Moreover, SNP analyses of the A-A and A-K lines indicated that the ATRX allele from the telomerase-positive A549 parent line was present at very low abundance. These lines were TRAP-negative, and given that the TRAP assay is usually sensitive enough to detect minor populations of telomerase-positive cells it seems likely that the subpopulations of ALT-negative cells are also telomerase-negative. Presumably these cells have telomeres that are sufficiently long to support continued proliferation in the absence of a TLM; a previous study using a different ALT cell line demonstrated that some cells were able to continue proliferating for 60 to 80 PDs after ALT activity was suppressed The data overall are therefore consistent with the hypothesis that ATRX is a repressor of ALT Although our data are consistent with ATRX being involved in repression of ALT, the low frequency with which human cells become immortalized The only cell line that was an exception to the correlation between ALT and loss of ATRX expression was the ALT-positive A549/GM847 A-R line, which had a high level of ATRX and had ATRX foci in the great majority of cells. This hybrid line retained alleles from both parent lines, as indicated by ATRX SNP analysis and the presence of exon 1 of ATRX. We looked for a loss-of-function mutation in the ATRX allele from the telomerase-positive parent, but sequence analysis indicated that ATRX remained wild-type in this hybrid line, as did DAXX and H3.3. ALT cell lines with wild-type ATRX, DAXX and H3.3 have also been observed previously Table S1Summary of hybridization efficiency and outcomes of isolated colonies.(DOCX)Click here for additional data file.


This proof-of-concept study was designed to determine if functional, pharmacodynamic profiles relevant to targeted therapy could be derived from live human melanoma samples using a novel automated platform.A series of 13 melanoma cell lines was briefly exposed to a BRAF inhibitor (PLX-4720) on a platform employing automated fluidics for sample processing. Levels of the phosphoprotein p-ERK in the mitogen-activated protein kinase (MAPK) pathway from treated and untreated sample aliquots were determined using a bead-based immunoassay. Comparison of these levels provided a determination of the pharmacodynamic effect of the drug on the MAPK pathway. A similar ex vivo analysis was performed on fine needle aspiration (FNA) biopsy samples from four murine xenograft models of metastatic melanoma, as well as 12 FNA samples from patients with metastatic melanoma.Melanoma cell lines with known sensitivity to BRAF inhibitors displayed marked suppression of the MAPK pathway in this system, while most BRAF inhibitor-resistant cell lines showed intact MAPK pathway activity despite exposure to a BRAF inhibitor (PLX-4720). FNA samples from melanoma xenografts showed comparable ex vivo MAPK activity as their respective cell lines in this system. FNA samples from patients with metastatic melanoma successfully yielded three categories of functional profiles including: MAPK pathway suppression; MAPK pathway reactivation; MAPK pathway stimulation. These profiles correlated with the anticipated MAPK activity, based on the known BRAF mutation status, as well as observed clinical responses to BRAF inhibitor therapy.Pharmacodynamic information regarding the ex vivo effect of BRAF inhibitors on the MAPK pathway in live human melanoma samples can be reproducibly determined using a novel automated platform. Such information may be useful in preclinical and clinical drug development, as well as predicting response to targeted therapy in individual patients. Molecularly targeted agents (MTAs) that block specific, critical signal transduction pathways in malignant cells have emerged as major tools in the treatment of cancer. To be most effective, these drugs must be paired with a predictive test to match the right agent with the signaling defect harbored by the patient’s tumor. However, despite some successes, most cancer patients do not yet benefit from such personalized cancer care MTA-related signaling complexities have been highlighted by the recent challenges posed by the development of BRAF inhibitors and other MAPK pathway inhibitors for metastatic melanoma To that end, we have created an automated, robust, reproducible, and potentially widely disseminated system for processing unfixed, fresh tumor samples from individual patients. This system enables “ex vivo” (that which takes place outside an organism) modulation and subsequent analysis of dynamic signal transduction networks in tumor biopsy samples to generate what we term a functional signaling profile Melanoma cell lines  were purchased from the American Type Culture Collection  and were maintained per ATCC instructions. The establishment and maintenance of the 451Lu, 451Lu-R, Mel1617, Mel1617-R, WM983B and WM983B-R cell lines has been described previously 2EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin and 1 mM PMSF; Cell Signaling, Boston, MA). Replicates were generated by additional runs on the instrument. For cell lines the number of replicates ranged from 4 to 8. For xenografts, the number of replicates was 4. For clinical human samples, which unlike cell lines and xenografts cannot be replenished, each was processed on the instrument once. Once generated, lysates were analyzed off the instrument.Cell lines, xenografts and human samples were processed on SnapPath™ , an automated cell and tissue processing platform which evokes functional signaling profiles from live cell and biopsy samples. Briefly, fresh cell and biopsy samples, containing a mixture of live and dead cells, were loaded onto the instrument in 500 ul of supportive media. Samples were then moved through five distinct processing steps on the instrument utilizing automated fluidics. Samples were first dispersed into a more homogeneous cellular suspension using mechanical shear forces. Next, samples were enriched for tumor cells by immunodepleting the samples of CD45-positive non-tumor cells (lymphocytes and macrophages) with a short 2 minute incubation with anti-CD45 antibodies conjugated to magnetic Dynal beads  then separating out bead-bound cells by a magnet on the platform. The sample was then aliquoted into two equally-sized separate temperature-controlled test chambers and treated with either vehicle (DMSO) or PLX-4720  and incubated at 37 C for 5 min. Samples were then lysed and stabilized using a cell lysis buffer  and p-ERK2 (Thr185/Tyr187). Each sample was analyzed in triplicate and results mean averaged. Data were acquired using Bio-Plex Manager™ Instrument Control (v6), then extracted and analyzed using GraphPad Prism . Levels of p-ERK detected in the control test chamber were compared with those in the PLX-4720 chamber and a percent inhibition level was determined based on the difference. For Bio-Plex analysis, inputs were normalized by total protein concentration using the Pierce® 660 nm Protein Assay . For cell lines and xenografts, the lysate concentration was 200 ug/ml and 140 ug/ml, respectively. For clinical samples, the lysates were analyzed at the highest possible concentration with a range of 25–300 ug/ml. For a given control-PLX-4720 sample pair, the protein concentration was equalized using lysis buffer.For SK-MEL-28, SK-MEL-2, A2058 and RPMI-7951 cell lines, we measured p-ERK1 (Thr202/Tyr204) and p-ERK2 (Thr185/Tyr187) using rabbit anti-p-ERK1/2  and goat anti-rabbit IgG conjugated with horseradish peroxidase . Total ERK1/2 was measured using rabbit anti-ERK1/2  and goat anti-rabbit IgG conjugated with horseradish peroxidase. As a loading control, we measured actin using mouse anti-beta-actin  and goat anti-mouse IgG conjugated with horseradish peroxidase (Bio-Rad: 1∶3000). For, p-ERK and total ERK, 10 ug of each lysate was subjected to reducing and denaturing PAGE using precast 10% PAGE Tris-HCl gels (Bio-Rad). For beta-actin, 5 ug of each lysate was used. Proteins were then transferred to nitrocellulose for probing. The Pierce ECL Western Blotting Substrate (Thermo) was used as recommended by the manufacturer. Film was scanned and densitometry conducted using ImageJ .7 cells/ml. Each mouse received a subcutaneous inoculation at one site per flank with 100 ul of cell suspension. Tumor formation varied between 4 and 8 weeks by cell line. Upon reaching 500 mm3 FNA biopsies were performed. One FNA sample consisted of 4 passes of a 23G, 1” needle on a 10cc syringe. Samples were then processed on the SnapPath as described above. Following biopsy, all mice were euthanized by CO2 and cervical dislocation.Athymic nu/nu female mice 4–6 weeks of age were obtained from Harlan Laboratories and were housed in a pathogen-free environment under controlled conditions of light and humidity and received food and water ad libitum. The mice were inoculated with SK-MEL-28, MALME-3M, COLO829 or A2058 cell lines obtained from ATCC. The cells were grown in regular growth media as described above. Subconfluent cells were removed from culture, then resuspended in Matrigel at 2×10Subjects were enrolled who had a known history of metastatic melanoma and either subcutaneous metastases amenable to palpation-guided fine needle aspiration biopsy or who were undergoing surgical resection of a metastatic lesion for clinical purposes. The clinical status of each patient at the time of biopsy, including current therapy and current response to therapy, was determined by the oncologist based on clinical assessment and available medical records. Tumor BRAF status was obtained from the individual patients medical record based on clinical testing.For the subcutaneous lesions, samples were collected using standard fine needle aspiration biopsy techniques by either an experienced cytopathologist or an oncologist who was trained and experienced in the procedure. For the surgically-excised lesions, specimens were delivered within 30 min. of excision to the pathology department where a cytopathologist examined the specimen and performed the FNA procedure on the specimen. FNA Samples were delivered within 1 hour to the laboratory for ex vivo processing on the on SnapPath™ platform.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Sobran, Inc., Baltimore, MD (Protocol Number: BIO-001-2010). The human subjects protocol and consent form were approved by the Institutional Review Boards (IRBs) of the University of Pennsylvania and Abramson Cancer Center’s Clinical Trials Scientific Review and Monitoring Committee. The study was conducted in compliance with regulations of the Health Insurance Portability and Accountability Act and the Declaration of Helsinki. Informed written consent was obtained for the biopsy procedure prior to enrollment.Our goal was to develop a reproducible and potentially distributable system to profile dynamic signal transduction events in living tumor cells from individual patients. During the development process our priorities included: the ability to accommodate multiple sample types; the inclusion of small samples from minimally invasive biopsies like FNAs; the ability to provide relatively rapid results; and automation to provide robust and reproducible results.In the resultant system, suspensions of live tumor cells are placed onto an automated fluidics-based platform where the samples are dispersed using mechanical shear forces into a more homogeneous suspension of individual cells and small cell clusters . The samAs a foundation for subsequent studies in murine xenografts and humans, a series of 13 BRAF V600E mutant melanoma cell lines was tested for sensitivity to BRAF inhibition by determining the IC50 values of each cell line to PLX-4720. As anticipated, 8 of the cell lines showed marked sensitivity to PLX-4720 (IC50<3 uM)  [18]–[19Next, functional signaling profiles were generated from each of the cell lines using the previously described automated platform. Briefly, tumor cells were grown to ∼70% confluence on 10 cm dishes, and then suspensions of tumor cell lines (1–3 million cells) that had been removed from culture plates were processed on the automated platform, which included exposure to PLX-4720 for 5 min., then levels of p-ERK were assessed from aliquots that had been treated or untreated with PLX-4720 using an immunoassay. For each sample, the level of the p-ERK phosphoprotein in the untreated aliquot was compared to the level in the PLX-4720-treated aliquot and a percent inhibition was generated such that 100% inhibition represented complete suppression of the MAPK pathway marker, p-ERK, and 0% inhibition represented complete inactivity of PLX-4720 on the MAPK pathway.In this analysis, 9 of the 14 cell lines displayed substantial ex vivo PLX-4720 suppression of the MAPK pathway (69–95% mean p-ERK inhibition) . Eight oThe ultimate value of this system lies in its ability to generate functional information from relatively small, clinically relevant samples. In addition, it has the potential to streamline preclinical studies utilizing human tumor cell-based xenografts or murine tumorgrafts of patient derived samples. To that end, we have optimized protocols to generate functional profiles from fine needle aspiration biopsy (FNA) samples. Murine models of metastatic melanoma resemble human subcutaneous melanoma metastases, displaying similar biology, histopathology, and FNA cytopathologic features 6) and tumor cell viability (14.6–51.0%) between FNA biopsy samples, successful functional profiles were obtained from most samples. The mean percent inhibition of these samples following PLX-4720 treatment ranged from 66–96%  displayed substantial ex vivo PLX-4720 suppression of the MAPK pathway (69 and 71% inhibition), seven (#3–9) showed intact MAPK pathway activity (18–56% inhibition), and three (#10–12) showed MAPK pathway activation (21–235% stimulation) upon ex vivo PLX-4720 exposure  or from surgically-excised metastases (n = 6). Cell numbers in the samples ranged from 0.228–5.95×10exposure . Two samCorrelation of these functional profiles with the known tumor BRAF genotype and clinical response to BRAF inhibitors revealed that the two patients with substantial ex vivo PLX-4720 suppression of the MAPK pathway (#1 and 2) were the only two patients whose tumors contained the BRAF V600E genotype and who showed at least a partial response to BRAF inhibitor therapy. Of the seven samples with intact MAPK pathway activity, three were from patients that were wild type for BRAF (#7–9), which would be anticipated based on the known intrinsic resistance of BRAF wild type tumors to BRAF inhibition In this study we have described a novel, automated system for the ex vivo pharmacodynamic analysis of human melanoma samples. We have termed the results of such analyses functional signaling profiles. Our preliminary studies with melanoma cell lines confirmed that functional signaling profiles from this ex vivo system accurately predicted BRAF inhibitor sensitivity in virtually all cell lines, based on their genotype, known biology, and IC50 values. These studies were extended to a clinically-relevant model system, using fine needle aspiration biopsy samples of human melanoma xenografts, which resemble human FNAs of metastatic melanoma in their cell composition and procurement method Due to the prevalence of BRAF mutations in melanoma, the MAPK pathway has emerged as a prime target of molecularly targeted agents for this disease, including an approved inhibitor of BRAF (vemurafenib) and additional emerging inhibitors of BRAF and MEK In our cell line work, the functional signaling profiles of the MAPK pathway identified ex vivo pathway blockade in every sensitive line, identified the known paradoxical pathway activation in an NRAS mutant line, and identified MAPK pathway reactivation in the known BRAF inhibitor resistant lines, with one interesting exception. The A2058 cell line contains a V600E mutation, displays ex vivo MAPK inhibition by PLX-4720, but is intrinsically resistant to BRAF inhibitors. While the exact mechanism of this resistance, despite pathway inhibition, is still under investigation, one study has suggested that it could be due to bypass pathway activation via PTEN loss and RB loss The functional signaling profiles generated in this study stratify human samples into three groups that display the following key features in the ex vivo presence of BRAF inhibition: MAPK suppression; MAPK reactivation; and MAPK stimulation. Because of the enrollment criteria and the practice patterns at the referral center where the study was conducted, most of the patients in the study were either BRAF wild type or BRAF V600E patients who were clinically progressing on a MAPK inhibitor regimen (BRAF or BRAF+MEK inhibitors). Most of these patients displayed a MAPK reactivation profile, confirming the previous evidence that MAPK reactivation plays a major role in BRAF inhibitor resistance. Interestingly, several patients displayed a MAPK activation profile, suggesting the acquisition of a previously described MAPK activation mechanism In summary, in this proof-of-concept study we have successfully generated pharmacodynamic data from human melanoma samples using a novel, automated ex vivo platform. This study focused on the MAPK pathway in metastatic melanoma, but this system is applicable to any solid tumor and multiple other molecularly targeted agents and signaling pathways. This system has potential applications in the efficient performance of preclinical drug studies in murine xenograft or human tumorgraft models. Also, it represents a novel tool for early clinical drug development in the assessment of pharmacodynamic responses to investigational drugs. Finally, it also has the potential to serve as a powerful predictive test to stratify patients in late stage clinical trials, and ultimately to guide targeted therapy.