Independent Cell Lines Research Articles

Reduction of organelle motility by removal of potassium and other solutes.

There are surprisingly few studies that describe how the composition of cell culture medium may affect the trafficking of organelles. Here we utilize time lapse multi-channel fluorescent imaging to show that short term exposure of Huh-7 cells to medium lacking potassium, sodium, or chloride strongly reduces but does not eliminate the characteristic back and forth and cell-traversing movement of fluorescent EGF (FL-EGF) containing organelles. We focused on potassium because of its relatively low abundance in media and serum and its energy requiring accumulation into cells. Upon exposure to potassium free medium, organelle motility declined steadily through 90 min and then persisted at a low level. Reduced motility was confirmed in 5 independent cell lines and for organelles of the endocytic pathway (FL-EGF and Lysotracker), autophagosomes (LC3-GFP), and mitochondria (TMRE). As has been previously established, potassium free medium also inhibited endocytosis. We expected that diminished cellular metabolism would precede loss of organelle motility. However, extracellular flux analysis showed near normal mitochondrial oxygen consumption and only a small decrease in extracellular acidification, the latter suggesting decreased glycolysis or proton efflux. Other energy dependent activities such as the accumulation of Lysotracker, TMRE, DiBAC4(3), and the exclusion of propidium iodide remained intact, as did the microtubule cytoskeleton. We took advantage of cell free in vitro motility assays and found that removal of potassium or sodium from the reconstituted cytosolic medium decreased the movement of endosomes on purified microtubules. The results indicate that although changes in proton homeostasis and cell energetics under solute depletion are not fully understood, potassium as well as sodium appear to be directly required by the motile machinery of organelles for optimal trafficking.

Characterization of the microDNA through the response to chemotherapeutics in lymphoblastoid cell lines.

Recently, a new class of extrachromosomal circular DNA, called microDNA, was identified. They are on average 100 to 400 bp long and are derived from unique non-repetitive genomic regions with high gene density. MicroDNAs are thought to arise from DNA breaks associated with RNA metabolism or replication slippage. Given the paucity of information on this entirely novel phenomenon, we aimed to get an additional insight into microDNA features by performing the microDNA analysis in 20 independent human lymphoblastoid cell lines (LCLs) prior and after treatment with chemotherapeutic drugs. The results showed non-random genesis of microDNA clusters from the active regions of the genome. The size periodicity of 190 bp was observed, which matches DNA fragmentation typical for apoptotic cells. The chemotherapeutic drug-induced apoptosis of LCLs increased both number and size of clusters further suggesting that part of microDNAs could result from the programmed cell death. Interestingly, proportion of identified microDNA sequences has common loci of origin when compared between cell line experiments. While compatible with the original observation that microDNAs originate from a normal physiological process, obtained results imply complementary source of its production. Furthermore, non-random genesis of microDNAs depicted by redundancy between samples makes these entities possible candidates for new biomarker generation.

222 Dexamethasone Induces Mesenchymal Trans-differentiation and Promotes Hallmarks of Cancer in Glioblastoma

Abstract INTRODUCTION Dexamethasone is the most commonly used drug in oncologic patients. In brain cancer and specifically glioblastoma, the drug is used in high doses to treat brain tumor associated vasogenic edema. Besides the rather immediate anti-inflammatory properties of dexamethasone, the longer term molecular effects have not been sufficiently studied. This work uncovers possibly deleterious dexamethasone-mediated effects through mesenchymal trans-differentiation and promotion of cellular hallmarks of cancer in glioblastoma patients. METHODS Three independent glioblastoma stem cell (GSC) lines with tumorigenic potential were used for the experiments. RT-PCR, Western Blot, Immunohistochemistry, invasion assays, whole-genome expression profiling, and pathway analyses were done with dexamethasone-exposed versus control cells both in vitro and in vivo orthotopic mouse xenograft models. GSCs were also co-exposed to dexamethasone, temozolomide, and camptothecin; the latter as a potential neutralizer of dexamethasone-induced deleterious effects. A dexamethasone induced gene signature was interrogated for survival prognostication in two large independent clinico-genomic databases comprising more than 500 patients. RESULTS >Dexamethasone induced the mesenchymal activator CCAAT/enhancer binding protein beta (CEBPB) in a dose dependent manner in vitro and in dexamethasone-treated orthotopic tumors. Significantly altered cellular functions included cell movement, cell survival, apoptosis, and angiogenesis. Dexamethasone-treated GSCs were protected against temozolomide-induced apoptosis and had significant increase of invasion both in vitro and in vivo. Furthermore, dexamethasone exposure resulted in significant elevation of cell proliferation and angiogenesis in vivo. A prognostic dexamethasone induced 33 gene signature was derived from orthotopic tumor expression profile analysis and validated in large clinico-genomic databases (p-value: 0.0007). Camptothecin was validated as potential partial neutralizer of dexamethasone-induced oncogenic effects. CONCLUSION Dexamethasone exposure induces a genetic program and CEBPB expression in GSCs that adversely affects key cellular functions and response to therapeutics. High risk scores associated with these genes have negative prognostic value in patients. Our findings further suggest well tolerated low-dose camptothecin as a potential neutralizer of adverse dexamethasone-mediated effects.

Regeneration of cell suspension derived Apium graveolens L. protoplasts

Cytoplasmatic male sterility (CMS), which can be achieved by protoplast fusion and regeneration, has potential to greatly facilitate hybrid breeding of celery (Apium graveolens L.). Therefore as a first step we developed a simple and efficient protoplast isolation and regeneration protocol for three commercial A. graveolens varieties (green and white celery and celeriac). To this end, cell suspensions from independent cell lines of open pollinated cultivars and inbred lines were initiated as a source for protoplast isolation. Comparative analyses showed that culturing was most successful in modified Kao and Michayluk liquid medium supplemented with 0.3 mg l−1 2,4-D. The cytokinin type (TDZ or zeatin) and concentration had no significant effect on regeneration efficiency. Microcalli were obtained within 15 days to 5 weeks after protoplast isolation. Supplementing the culture medium with 25% conditioned medium increased microcolony formation for some of the cultured lines. Plants were obtained within 2 months of microcallus culturing and these were all diploid, suggesting genetic inheritance consistency. The efficiency of regeneration mainly depended on the specific genotype, with outcrossing genotypes displaying high heterogeneity in regeneration responses whereas inbred lines did not regenerate. The protocol presented here enables to implement protoplast fusion in celery breeding.

Abstract 5835: Oncogenic activation of the serine synthesis pathway by the scaffolding protein menin

Abstract Developmental transcription programs are epigenetically regulated by multi-protein complexes including the MLL-containing trithorax (TrxG) complexes, which methylate H3K4 to promote gene transcription. We recently reported that the TrxG protein MLL1 and its binding partner menin are overexpressed by and function as oncogenes in Ewing sarcoma. Moreover, small molecule inhibition of the menin-MLL1 protein-protein interaction leads to loss of menin and MLL1 protein expression and to inhibition of growth and tumorigenicity. For the current study we investigated the mechanistic basis of menin-MLL1 mediated oncogenic activity in Ewing sarcoma. Bru-seq analysis was performed to identify changes in nascent gene transcription in Ewing sarcoma cells following exposure to the menin-MLL interaction inhibitor MI-503. Identified targets were validated by qRT-PCR, western blot, immunocytochemistry and by interrogation of published databases. Loss of function was achieved by lentiviral shRNAs and by inhibitor treatment. Exposure of three independent Ewing sarcoma cell lines to MI-503 resulted in significant and reproducible modulation of nearly 100 genes. Bioinformatics analysis of MI-503-regulated genes revealed that the serine synthesis pathway (SSP) was the most significantly impacted pathway downstream of MI-503 treatment. The SSP has recently emerged as a major mediator of tumorigenicity in multiple human cancers. qRT-PCR confirmed reduced expression of SSP enzyme-encoding genes PHGDH, PSAT1 and PSPH in MI-503 treated cells. To determine if the SSP contributes to Ewing sarcoma pathogenesis we evaluated expression of SSP genes and proteins in tumors and cell lines. Our findings showed that Ewing sarcomas express high levels of PHGDH, PSAT1, and PSPH and interrogation of the Cancer Cell Line Encyclopedia revealed that PHGDH expression is higher in Ewing sarcoma than in any other cancer in the database. Studies of Ewing sarcoma cells in serine and glycine free media showed that cells retain viability but that exposure to the PHGDH inhibitor, CBR-5884, inhibits survival in both standard and serine/glycine-free conditions, confirming the importance of the pathway to tumor pathogenesis. To determine how menin-MLL1 promote SSP activation we performed loss of function studies and discovered that knockdown of menin, but not of MLL1, leads to a significant reduction in SSP gene expression, implicating menin as the primary mediator of SSP gene activation. Transcriptional up regulation the SSP pathway has been linked to ATF4 in non-small cell lung cancer. Significantly, ATF4 protein expression is high in Ewing sarcoma cells and ATF4 transcript and protein expression are down regulated following MI-503 treatment and menin knockdown. Together these data demonstrate that the SSP is highly active in Ewing sarcoma and that its activation is induced, at least in part, by a novel menin-ATF4 transcriptional axis. Citation Format: Laurie Kathleen Svoboda, Selina Shiqing Teh, Sudha Sud, Armand Bankhead, Brian Magnuson, Mats Ljungman, Costas Lyssiotis, Tomasz Cierpicki, Jolanta Grembecka, Elizabeth R. Lawlor. Oncogenic activation of the serine synthesis pathway by the scaffolding protein menin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5835. doi:10.1158/1538-7445.AM2017-5835

Abstract 1390: Deregulation of RNA N6-adenosine methylation contribute to liver carcinogenesis

Abstract Primary liver cancer, particularly the most common type hepatocellular carcinoma (HCC), is the third leading cause of cancer death worldwide. Case is even worse in Asia due to extremely high prevalence of HBV/HCV infection, which enhances the risk of HCC. However, the exact mechanism of how HCC develops is still not clearly defined. Previous studies have shown strong evidence for epigenetic alterations in human carcinogenesis, which mainly focus on abnormal DNA methylation, histone modifications, and chromatin remodeling. Recent studies suggest that diverse chemical modifications on RNAs, also known as “epi-transcriptome”, constitute another layer of gene expression regulation. N-6-methyladenosine (m6A) is the most abundant modification on eukaryotic mRNA, which has been found to functionally influence mRNA stability, alternative splicing, and translation efficiency. However, the roles of m6A deregulation in human carcinogenesis remain unclear. Through whole-transcriptome sequencing, we identified that METTL3, the major component of m6A methyltransferase, was significantly up-regulated in human HCC. Up-regulation of METTL3 negatively associated with poor patient overall and disease free survival. Functionally knockdown/knockout of METTL3 dramatically inhibited HCC proliferation, migration and colony formation abilities in vitro and significantly suppressed in vivo tumorigenicity. In contrast, overexpression of METTL3 using CRISPR/dCas9 SAM system modestly but significantly enhanced HCC proliferation, migration, anchorage-independent growth in vitro and tumor formation in vivo. In order to find the downstream mechanisms which might attributed to METTL3 oncogenic function we performed RNA-Seq on two independent METTL3 knockdown cell lines to interrogated their transcriptome changes. Based on RNA-Seq data, we identified a prominent tumor suppressor SOCS2 as a potential down-stream target of METTL3. With m6A-RNA immunoprecipitation (RIP) assay, we confirmed that SOCS2 mRNA was subject to m6A modification in cancer cells. Inactivation of METTL3 by RNAi or treatment of methylation inhibitor (DAA) impaired m6A mediated mRNA degradation and thereby stabilized SOSC2 mRNA. We further confirm SOCS2 tumor suppressor function in HCC. Downregulation of SOCS2 was associated with poor patient overall survival and disease free survival of HCC patients. Knockdown of SOCS2 significantly promoted HCC proliferation, migration in HCC cells. In conclusion, our findings suggested that deregulation of METTL3 and its associated m6A modification could contribute to human carcinogenesis by imposing an epigenetic control on the stability and expressions of critical tumor suppressor genes. Citation Format: Mengnuo Chen, Larry L. Wei, Cheuk Ting Law, Felice HC Tsang, Iris MJ Xu, Joyce Lee MF Lee, Carmen Wong CL Wong, Irene OL Ng, Chun-Ming Wong. Deregulation of RNA N6-adenosine methylation contribute to liver carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1390. doi:10.1158/1538-7445.AM2017-1390

Abstract 1672: Workflow automation and parallelization improves the isolation and analysis of tumor-infiltrating immune subpopulations

Abstract Immunotherapy against cancer has proven clinical efficacy and tremendous potential in multiple tumor entities. Syngeneic mouse tumor models represent the gold standard to analyze effects of immunotherapy due to their fully competent immune-repertoire. However, the amount and composition of tumor infiltrating leukocytes (TIL) is highly variable, complicating the targeted analysis of subpopulations. In particular, small subpopulations cannot be analyzed properly but may be lost in the background noise. When working with large cohort sizes, even the immune-phenotyping of TIL by flow cytometry is time consuming and data processing highly work intensive making pre-enrichment methods for sample debulking attractive. We have established an automated workflow combining tissue dissociation with TIL specific isolation to improve and accelerate downstream analysis. Tumor dissociation was automated using the gentleMACS™Octo Dissociator and optimized for epitope conservation to overcome bias in immune-phenotyping caused by dissociation with aggressive of impure enzymes. Next, isolation of TIL was improved by developing a new CD45-specific enrichment reagent for the magnetic cell sorting (MACS) based isolation directly from dissociated tumor tissue. The whole workflow takes only about 90 min. To validate this method on starting material showing variable frequencies of TIL infiltration, we used syngeneic mouse tumors derived by injection three independent tumor cell lines. Tumors derived from injection of B16.F10 melanoma showed TIL frequencies of 2-4%, CT26.WT colon carcinoma tumors 15-21%, and 4T1 breast carcinoma tumors 32-37% among total viable cells after dissociation. Using a manual separation system, TIL were enriched to purities above 90% at yields above 70% for CT26.WT and 4T1 tumors, and purities above 80% at high yields above 95% for B16.F10 tumors. To address the need of parallelization and automation for sample processing in large cohort sizes used in in vivo studies, a fully automated version of the MultiMACS™ Cell24 Separator was developed by integrating the instrument in a liquid handling robotic platform. This new system, the MultiMACS™ X, can process 24 cell separations in parallel with minimal hands-on time. When compared to the manual system, equal purities were achieved whereas the overall yield of target cells was increased from 70% to 90% as shown in the case of CT26.WT tumors. Importantly, while the TIL enrichment significantly reduced the time and reagent costs in immune subset analysis, the composition of infiltrating immune cells was not affected, excluding the risk of introducing a bias by this method. Taken together, we have developed an automated workflow for the isolation of TIL from mouse tumors reducing time and costs of downstream analysis while standardizing and enhancing the detection and quantification of immune cell subpopulations. Citation Format: Janina Brauner, David Agorku, Anne Langhammer, Lukas Pohlig, Jan Drewes, Thomas Dino Rockel, Oliver Schilling, Stephan Mehlhose, Wa'el Al Rawashdeh, Andreas Bosio, Olaf Hardt. Workflow automation and parallelization improves the isolation and analysis of tumor-infiltrating immune subpopulations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1672. doi:10.1158/1538-7445.AM2017-1672

Abstract 2555: ZC3H8 associates with PML bodies and influences aggressive tumor cell behavior

Abstract The ZC3H8 gene encodes a protein with three zinc finger motifs in the C-terminal region, suggesting a role as an RNA binding protein, but whose specific function remains unclear. ZC3H8 protein localizes to both PML bodies and Cajal bodies within the nucleus, as confocal microscopy demonstrates colocalization of ZC3H8 with both PML and coilin proteins. ZC3H8 has a casein kinase 2 phosphorylation site in the N-terminal region, and treatment with a casein kinase 2 inhibitor causes the numerous PML bodies and their associated ZC3H8 to coalesce to a few larger bodies. Removal of the inhibitor restores PML bodies to their original state. We constructed a mutant of ZC3H8 that removed the predicted CK2 phosphorylation site by replacing the serine with an alanine residue. Cells transfected with this mutant had reduced numbers of PML bodies, similar to cells treated with the CK2 inhibitor. In contrast, a mutant constructed with a glutamic acid in place of the phosphorylatable serine was predicted to behave as a constitutive mutant. This mutant did not exhibit differences from wild type localization. These experiments suggest that ZC3H8 integrity is key to maintenance of PML bodies. Other studies have identified ZC3H8 as a component of the LEC (little elongation complex), and we found that another identified component, ICE2 (Narg) also localizes to PML bodies. The ZC3H8 gene is overexpressed in a number of human and mouse breast cancer cell lines, and elevated mRNA levels are associated with a poorer prognosis for women with breast cancer. We have used RNA silencing to decrease levels of expression in two independent mouse mammary tumor cell lines. Cells with lower ZC3H8 expression have dramatic phenotypic changes, showing decreased rates of proliferation, slower migration in wound healing assays, formation of fewer and smaller colonies in soft agar assays, and decreased ability to invade through a basement membrane in transwell assays. Further, ZC3H8-depleted cells have decreased rates of growth as spheroids and decreased growth in vivo in syngeneic mice. We developed an inducible vector driving expression of ZC3H8. Upon treatment with the inducer, doxycycline, cells assume a more aggressive phenotype. We introduced a synonymous mutant of ZC3H8 unable to be silenced by the RNAi into the knockdown cells, and rescued the aggressive phenotype of the original tumor cells. Overexpression of ZC3H8 in non-tumorigenic COMMA-D cells leads to a less pronounced, but opposite, increase in proliferation, motility, and invasion. We have engineered the synonymous ZC3H8 with disrupted zinc finger motifs, and preliminary results suggest that disruption of the first zinc finger is not sufficient to cause loss of the aggressive phenotype. We suggest that ZC3H8 is an oncogene with substantial effects on the cancer cell phenotype. These effects are hypothesized to be associated with ZC3H8 function in PML bodies. Citation Format: John A. Schmidt, Emily Duffner, Gerard Walker, Keith G. Danielson, Janice E. Knepper. ZC3H8 associates with PML bodies and influences aggressive tumor cell behavior [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2555. doi:10.1158/1538-7445.AM2017-2555

Granulin-epithelin precursor interacts with 78-kDa glucose-regulated protein in hepatocellular carcinoma

BackgroundGranulin-epithelin precursor (GEP) is a secretory growth factor, which has been demonstrated to control cancer growth, invasion, drug resistance and immune escape. Our previous studies and others also demonstrated its potential in targeted therapy. Comprehensive characterization of GEP partner on cancer cells are warranted. We have previously shown that GEP interacted with heparan sulfate on the surface of liver cancer cells and the interaction is crucial for GEP-mediated signaling transduction. This study aims to characterize GEP protein partner at the cell membrane with the co-immunoprecipitation and mass spectrometry approach.MethodsThe membrane fraction from liver cancer model Hep3B was used for capturing binding partner with the specific monoclonal antibody against GEP. The precipitated proteins were analyzed by mass spectrometry. After identifying the GEP binding partner, this specific interaction was validated in additional liver cancer cell line HepG2 by co-immunoprecipitation using GRP78 and GEP antibodies, respectively, as the bait. GRP78 transcript levels in hepatocellular carcinoma (HCC) clinical samples (n = 77 pairs) were examined by real-time quantitative RT-PCR. GEP and GRP78 protein expressions were investigated by immunohistochemistry on paraffin sections.ResultsWe identified the GEP-binding protein as 78-kDa glucose-regulated protein (GRP78, also named heat shock 70-kDa protein 5, HSPA5). This interaction was validated in independent HCC cell lines. Increased GRP78 mRNA levels were demonstrated in liver cancer tissues compared with the paralleled liver tissues (t-test, P = 0.002). GRP78 and GEP transcript levels were significantly correlated (Spearman’s correlation, P = 0.001), and the proteins were also detectable in the cytoplasm of liver cancer cells by immunohistochemical staining.ConclusionsGRP78 and GEP are interacting protein partners in liver cancer cells and may play a role in GEP-mediated cancer progression in HCC.

A Transcriptional Program for Detecting TGFβ-Induced EMT in Cancer.

Most cancer deaths are due to metastasis, and epithelial-to-mesenchymal transition (EMT) plays a central role in driving cancer cell metastasis. EMT is induced by different stimuli, leading to different signaling patterns and therapeutic responses. TGFβ is one of the best-studied drivers of EMT, and many drugs are available to target this signaling pathway. A comprehensive bioinformatics approach was employed to derive a signature for TGFβ-induced EMT which can be used to score TGFβ-driven EMT in cells and clinical specimens. Considering this signature in pan-cancer cell and tumor datasets, a number of cell lines (including basal B breast cancer and cancers of the central nervous system) show evidence for TGFβ-driven EMT and carry a low mutational burden across the TGFβ signaling pathway. Furthermore, significant variation is observed in the response of high scoring cell lines to some common cancer drugs. Finally, this signature was applied to pan-cancer data from The Cancer Genome Atlas to identify tumor types with evidence of TGFβ-induced EMT. Tumor types with high scores showed significantly lower survival rates than those with low scores and also carry a lower mutational burden in the TGFβ pathway. The current transcriptomic signature demonstrates reproducible results across independent cell line and cancer datasets and identifies samples with strong mesenchymal phenotypes likely to be driven by TGFβ.Implications: The TGFβ-induced EMT signature may be useful to identify patients with mesenchymal-like tumors who could benefit from targeted therapeutics to inhibit promesenchymal TGFβ signaling and disrupt the metastatic cascade. Mol Cancer Res; 15(5); 619-31. ©2017 AACR.

Resistance to Taxanes in Triple-Negative Breast Cancer Associates with the Dynamics of a CD49f+ Tumor-Initiating Population.

SummaryTaxanes are a mainstay of treatment for breast cancer, but resistance often develops followed by metastatic disease and mortality. Aiming to reveal the mechanisms underlying taxane resistance, we used breast cancer patient-derived orthoxenografts (PDX). Mimicking clinical behavior, triple-negative breast tumors (TNBCs) from PDX models were more sensitive to docetaxel than luminal tumors, but they progressively acquired resistance upon continuous drug administration. Mechanistically, we found that a CD49f+ chemoresistant population with tumor-initiating ability is present in sensitive tumors and expands during the acquisition of drug resistance. In the absence of the drug, the resistant CD49f+ population shrinks and taxane sensitivity is restored. We describe a transcriptional signature of resistance, predictive of recurrent disease after chemotherapy in TNBC. Together, these findings identify a CD49f+ population enriched in tumor-initiating ability and chemoresistance properties and evidence a drug holiday effect on the acquired resistance to docetaxel in triple-negative breast cancer.

AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines.

Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

Gene-modified NK-92MI cells expressing a chimeric CD16-BB-ζ or CD64-BB-ζ receptor exhibit enhanced cancer-killing ability in combination with therapeutic antibody.

Natural killer (NK) cells play a pivotal role in monoclonal antibody-mediated immunotherapy through the antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. NK-92MI is an interleukin-2 (IL-2)-independent cell line, which was derived from NK-92 cells with superior cytotoxicity toward a wide range of tumor cells in vitro and in vivo. Nonetheless, the Fc-receptor (CD16) that usually mediates ADCC is absent in NK-92 and NK-92MI cells. To apply NK-92MI cell-based immunotherapy to cancer treatment, we designed and generated two chimeric receptors in NK-92MI cells that can bind the Fc portion of human immunoglobulins. The construct includes the low-affinity Fc receptor CD16 (158F) or the high-affinity Fc receptor CD64, with the addition of the CD8a extracellular domain, CD28 transmembrane domains, two costimulatory domains (CD28 and 4-1BB), and the signaling domain from CD3ζ. The resulting chimeric receptors, termed CD16-BB-ζ and CD64-BB-ζ, were used to generate modified NK-92MI cells expressing the chimeric receptor, which were named NK-92MIhCD16 and NK-92MIhCD64 cells, respectively. We found that NK-92MIhCD16 and NK-92MIhCD64 cells significantly improved cytotoxicity against CD20-positive non-Hodgkin's lymphoma cells in the presence of rituximab. These results suggest that the chimeric receptor-expressing NK-92MI cells may enhance the clinical responses to currently available anticancer monoclonal antibodies.

The G-protein alpha-subunit gene CGA1 is involved in regulation of resistance to heat and osmotic stress in Chlamydomonas reinhardtii.

In eukaryotic cells, many important functions of specific G-proteins have been identified, but microalgal G-proteins are poorly studied. In this work, we characterized a gene (CGA1) encoding the G-protein α-subunit in Chlamydomonas reinhardtii. Independent knockdown mutants of CGA1 were generated via RNA interference (RNAi). CGA1 expression levels were consistently and significantly reduced in both independent CGA1 mutant cell lines (cga1). Both cga1 mutants had a higher survival rate at 35°C in comparison with the wild type. This stronger resistance of the cga1 mutants became more evident during simultaneous exposure to heat and osmotic stress. The stronger resistance of the CGA1 knockdown mutants to the two stressors was accompanied with significant morphological alterations-both cell size and cell wall thickness were different from those of the wild type. This finding supports the roles of CGA1 in C. reinhardtii morphology in response to stressors. To further understand biochemical mechanisms of the CGA1-mediated resistance, we thoroughly analyzed the level of reactive oxygen species (ROS) and the expression of several heat shock proteins or MAP kinase genes as possible downstream effectors of CGA1. Our data clearly indicated that CGA1 is implicated in the regulation of resistance to heat or osmotic stress in C. reinhardtii via HSP70A and MAPK6. Because the G-protein α-subunit is highly conserved across microalgal species, our results should facilitate future biotechnological applications of microalgae under extreme environmental conditions.

Characterization of Human Cancer Cell Lines by Reverse-phase Protein Arrays

Cancer cell lines are major model systems for mechanistic investigation and drug development. However, protein expression data linked to high-quality DNA, RNA, and drug-screening data have not been available across a large number of cancer cell lines. Using reverse-phase protein arrays, we measured expression levels of ∼230 key cancer-related proteins in >650 independent cell lines, many of which have publically available genomic, transcriptomic, and drug-screening data. Our dataset recapitulates the effects of mutated pathways on protein expression observed in patient samples, and demonstrates that proteins and particularly phosphoproteins provide information for predicting drug sensitivity that is not available from the corresponding mRNAs. We also developed a user-friendly bioinformatic resource, MCLP, to help serve the biomedical research community.

Probing the prostate tumour microenvironment I: impact of glucose deprivation on a cell model of prostate cancer progression.

In the developed world, prostate cancer is the most common cancer diagnosis in men. Although prostate cancer initially presents as a non life-threatening disease, 90% of patients will develop castration resistant prostate cancer (CRPC), which preludes distant metastasis and is largely accountable for prostate cancer associated deaths. This is because as yet, there are no viable molecular therapeutic targets for effective treatment of CRPC. It is now widely accepted that cancer cells can alter their metabolic profile during the course of tumourgenesis and metastasis such that they are able to survive in oxygen and nutrient-poor environments. This work was aimed towards gaining greater mechanistic understanding of how such stresses in the tumour microenvironment impact on both androgen sensitive (LNCaP) and androgen independent (LNCaP-abl and LNCaP-abl-Hof) prostate cancer cell lines. Here we have applied technically robust and reproducible label-free liquid chromatography mass spectrometry analysis for comprehensive proteomic profiling of prostate cancer cell lines under nutrient deficient (low glucose) conditions. This led to the identification of approximately 4,000 proteins - one of the largest protein datasets for prostate cancer cell lines established to date. The biological and clinical significance of proteins showing a significant change in expression as result of low glucose conditions was established. Novel, intuitive workflows were subsequently implemented to ensure the verification of selected proteins of interest in a robust, reproducible and high throughput manner. Overall, these data suggest that this strategy supports identification of protein biomarkers of prostate cancer progression and potential therapeutic targets for CRPC.

Probing the prostate tumour microenvironment II: Impact of hypoxia on a cell model of prostate cancer progression

Approximately one in six men are diagnosed with Prostate Cancer every year in the Western world. Although it can be well managed and non-life threatening in the early stages, over time many patients cease to respond to treatment and develop castrate resistant prostate cancer (CRPC). CRPC represents a clinically challenging and lethal form of prostate cancer. Progression of CRPC is, in part, driven by the ability of cancer cells to alter their metabolic profile during the course of tumourgenesis and metastasis so that they can survive in oxygen and nutrient-poor environments and even withstand treatment. This work was carried out as a continuation of a study aimed towards gaining greater mechanistic understanding of how conditions within the tumour microenvironment impact on both androgen sensitive (LNCaP) and androgen independent (LNCaP-abl and LNCaP-abl-Hof) prostate cancer cell lines. Here we have applied technically robust and reproducible label-free liquid chromatography mass spectrometry analysis for comprehensive proteomic profiling of prostate cancer cell lines under hypoxic conditions. This led to the identification of over 4,000 proteins – one of the largest protein datasets for prostate cancer cell lines established to date. The biological and clinical significance of proteins showing a significant change in expression as result of hypoxic conditions was established. Novel, intuitive workflows were subsequently implemented to enable robust, reproducible and high throughput verification of selected proteins of interest. Overall, these data suggest that this strategy supports identification of protein biomarkers of prostate cancer progression and potential therapeutic targets for CRPC.

Investigating the role of caveolin-2 in prostate cancer cell line

Prostate cancer is a worldwide problem. While the role of caveolin-1 has been extensively studied, little is known about the role of caveolin-2 (CAV2) in prostate cancer. Up-regulation of CAV2 in androgen independent PC3 cells compared to normal prostate cell line and androgen dependent prostate cancer cell lines has been observed. Recent studies suggest that up-regulation of CAV2 plays an important role in androgen independent prostate cancer. This study investigates whether CAV2 is important in mediating the aggressive phenotypes seen in androgen independent prostate cancer cells. The androgen independent prostate cancer cell line, PC3 was used that has been shown to express CAV2, and CAV2 knock down was performed using siRNA system. Changes to cell number, migration and invasion were assessed after knocking down CAV2. Our results showed that down-regulating CAV2 resulted in reduced cell numbers, migration and invasion in PC3 cells. This preliminary study suggests that CAV2 may act to promote malignant behavior in an androgen independent prostate cancer cell line. Further studies are required to fully elucidate the role of CAV2 in androgen independent prostate cancer.

Bifunctional conjugates with potent inhibitory activity towards cyclooxygenase and histone deacetylase

We herein disclose a series of compounds with potent inhibitory activities towards histone deacetylases (HDAC) and cyclooxygenases (COX). These compounds potently inhibited the growth of cancer cell lines consistent with their anti-COX and anti-HDAC activities. While compound 2b showed comparable level of COX-2 selectivity as celecoxib, compound 11b outperformed indomethacin in terms of selectivity towards COX-2 relative to COX-1. An important observation with our lead compounds (2b, 8, 11b, and 17b) is their enhanced cytotoxicity towards androgen dependent prostate cancer cell line (LNCaP) relative to androgen independent prostate cancer cell line (DU-145). Interestingly, compounds 2b and 17b arrested the cell cycle progression of LNCaP in the S-phase, while compound 8 showed a G0/G1 arrest, similar to SAHA. Relative to SAHA, these compounds displayed tumor-selective cytotoxicity as they have low anti-proliferative activity towards healthy cells (VERO); an attribute that makes them attractive candidates for drug development.

Cell cycle and apoptosis pathway modulation by Tualang honey in ER-dependent and -independent breast cancer cell lines

We previously demonstrated that Tualang honey (TH) induced apoptosis in breast cancer cell lines, via activation of caspase-3/7, -8, and -9 and mitochondrial membrane depolarization. Here we investigated the effect of TH on cell cycle progression and its regulatory molecules as well as apoptosis-related signaling molecules in estrogen receptor-dependent MCF-7 and -independent breast MDA-MB-231 cancer cells. Flow cytometry analysis shows that 24 h treatment with 1% TH caused G2/M phase arrest in MCF-7 but S phase arrest in MDA-MB-231 cells. Significant changes in the expression of apoptosis-related genes were observed in MCF-7 cells but not in MDA-MB-231 cells. We further demonstrated by Western blotting that TH increased the expression of p53, p21, and FADD proteins in MCF-7 cells and TRADD, FADD, and p21 proteins in MDA-MB-231 cells. This study shows that TH differentially affects the cell cycle progression of MCF-7 and MDA-MB-231 cells via p53-dependent and -independent p21 signaling, respectively, and apoptosis via the death receptor pathway is indicated. Taken together, the ability of TH to modulate cell signaling pathways suggest the potential value of a readily consumed honey as a chemopreventive agent for breast cancer.