Georg-Speyer-Haus
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UPF1 regulates myeloid cell functions and S100A9 expression by the hnRNP E2/miRNA-328 balance
(2016)
UPF1 is a key player in nonsense mediated mRNA decay (NMD) but also involved in posttranscriptional gene regulation. In this study we found that UPF1 regulates the expression of genes with functions in inflammation and myeloid cell differentiation via hnRNP E2. The majority of the UPF1-regulated genes identified in monocytic cells contain a binding site for hnRNP E2 within 5′ UTR located introns with hnRNP E2 acting here as splicing regulator. We found that miRNA-328 which is significantly induced during monocytic cell differentiation acts independently from its gene silencing function as RNA decoy for hnRNP E2. One representative gene controlled by the hnRNP E2/miRNA-328 balance is S100A9 which plays an important role in cell differentiation and oxidative stress response of monocytes. Induction of miRNA-328 expression during cell differentiation antagonizes the blockade by hnRNP E2 which results in the upregulation of CD11b expression and ROS production in monocytic cells. Taken together, our data indicate that upregulation of miR-328 is responsible for the induction of hnRNP E2 target genes during myeloid cell differentiation.
Although effective antibody-based vaccines have been developed against multiple viruses, such approaches have so far failed for the human immunodeficiency virus type 1 (HIV-1). Despite the success of anti-retroviral therapy (ART) that has turned HIV-1 infection into a chronic disease and has reduced the number of new infections worldwide, a vaccine against HIV-1 is still urgently needed. We discuss here the major reasons for the failure of “classical” vaccine approaches, which are mostly due to the biological properties of the virus itself. HIV-1 has developed multiple mechanisms of immune escape, which also account for vaccine failure. So far, no vaccine candidate has been able to induce broadly neutralizing antibodies (bnAbs) against primary patient viruses from different clades. However, such antibodies were identified in a subset of patients during chronic infection and were shown to protect from infection in animal models and to reduce viremia in first clinical trials. Their detailed characterization has guided structure-based reverse vaccinology approaches to design better HIV-1 envelope (Env) immunogens. Furthermore, conserved Env epitopes have been identified, which are promising candidates in view of clinical applications. Together with new vector-based technologies, considerable progress has been achieved in recent years towards the development of an effective antibody-based HIV-1 vaccine.
Hypoxia-induced long non-coding RNA Malat1 is dispensable for renal ischemia/reperfusion-injury
(2018)
Renal ischemia-reperfusion (I/R) injury is a major cause of acute kidney injury (AKI). Non-coding RNAs are crucially involved in its pathophysiology. We identified hypoxia-induced long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) to be upregulated in renal I/R injury. We here elucidated the functional role of Malat1 in vitro and its potential contribution to kidney injury in vivo. Malat1 was upregulated in kidney biopsies and plasma of patients with AKI, in murine hypoxic kidney tissue as well as in cultured and ex vivo sorted hypoxic endothelial cells and tubular epithelial cells. Malat1 was transcriptionally activated by hypoxia-inducible factor 1-α. In vitro, Malat1 inhibition reduced proliferation and the number of endothelial cells in the S-phase of the cell cycle. In vivo, Malat1 knockout and wildtype mice showed similar degrees of outer medullary tubular epithelial injury, proliferation, capillary rarefaction, inflammation and fibrosis, survival and kidney function. Small-RNA sequencing and whole genome expression analysis revealed only minor changes between ischemic Malat1 knockout and wildtype mice. Contrary to previous studies, which suggested a prominent role of Malat1 in the induction of disease, we did not confirm an in vivo role of Malat1 concerning renal I/R-injury.
The signal transducer and activator of transcription 5 (STAT5) regulates differentiation, survival, proliferation and transformation of hematopoietic cells. Upon cytokine stimulation, STAT5 tyrosine phosphorylation (pYSTAT5) is transient, while in diverse neoplastic cells persistent overexpression and enhanced pYSTAT5 are frequently found. Post-translational modifications might contribute to enhanced STAT5 activation in the context of transformation, but the strength and duration of pYSTAT5 are incompletely understood. We found that O-GlcNAcylation and tyrosine phosphorylation act together to trigger pYSTAT5 levels and oncogenic transcription in neoplastic cells. The expression of a mutated hyperactive gain-of-function (GOF) STAT5 without O-GlcNAcylation resulted in decreased tyrosine phosphorylation, oligomerization and transactivation potential and complete loss of oncogenic transformation capacity. The lack of O-GlcNAcylation diminished phospho-ERK and phospho-AKT levels. Our data show that O-GlcNAcylation of STAT5 is an important process that contributes to oncogenic transcription through enhanced STAT5 tyrosine phosphorylation and oligomerization driving myeloid transformation. O-GlcNAcylation of STAT5 could be required for nutrient sensing and metabolism of cancer cells.
Mammalian oocytes are arrested in the dictyate stage of meiotic prophase I for long periods of time, during which the high concentration of the p53 family member TAp63α sensitizes them to DNA damage-induced apoptosis. TAp63α is kept in an inactive and exclusively dimeric state but undergoes rapid phosphorylation-induced tetramerization and concomitant activation upon detection of DNA damage. Here we show that the TAp63α dimer is a kinetically trapped state. Activation follows a spring-loaded mechanism not requiring further translation of other cellular factors in oocytes and is associated with unfolding of the inhibitory structure that blocks the tetramerization interface. Using a combination of biophysical methods as well as cell and ovary culture experiments we explain how TAp63α is kept inactive in the absence of DNA damage but causes rapid oocyte elimination in response to a few DNA double strand breaks thereby acting as the key quality control factor in maternal reproduction.
Inhibition of the IκB kinase complex (IKK) has been implicated in the therapy of several chronic inflammatory diseases including inflammatory bowel diseases. In this study, using mice with an inactivatable IKKα kinase (IkkαAA/AA), we show that loss of IKKα function markedly impairs epithelial regeneration in a model of acute colitis. Mechanistically, this is caused by compromised secretion of cytoprotective IL-18 from IKKα-mutant intestinal epithelial cells because of elevated caspase 12 activation during an enhanced unfolded protein response (UPR). Induction of the UPR is linked to decreased ATG16L1 stabilization in IkkαAA/AA mice. We demonstrate that both TNF-R and nucleotide-binding oligomerization domain stimulation promote ATG16L1 stabilization via IKKα-dependent phosphorylation of ATG16L1 at Ser278. Thus, we propose IKKα as a central mediator sensing both cytokine and microbial stimulation to suppress endoplasmic reticulum stress, thereby assuring antiinflammatory function during acute intestinal inflammation.
Yeast large ribosomal subunit (LSU) precursors are subject to substantial changes in protein composition during their maturation due to coordinated transient interactions with a large number of ribosome biogenesis factors and due to the assembly of ribosomal proteins. These compositional changes go along with stepwise processing of LSU rRNA precursors and with specific rRNA folding events, as revealed by recent cryo-electron microscopy analyses of late nuclear and cytoplasmic LSU precursors. Here we aimed to analyze changes in the spatial rRNA surrounding of selected ribosomal proteins during yeast LSU maturation. For this we combined a recently developed tethered tertiary structure probing approach with both targeted and high throughput readout strategies. Several structural features of late LSU precursors were faithfully detected by this procedure. In addition, the obtained data let us suggest that early rRNA precursor processing events are accompanied by a global transition from a flexible to a spatially restricted rRNA conformation. For intermediate LSU precursors a number of structural hallmarks could be addressed which include the fold of the internal transcribed spacer between 5.8S rRNA and 25S rRNA, the orientation of the central protuberance and the spatial organization of the interface between LSU rRNA domains I and III.
Hematopoietic differentiation is controlled by key transcription factors, which regulate stem cell functions and differentiation. TAL1 is a central transcription factor for hematopoietic stem cell development in the embryo and for gene regulation during erythroid/megakaryocytic differentiation. Knowledge of the target genes controlled by a given transcription factor is important to understand its contribution to normal development and disease. To uncover direct target genes of TAL1 we used high affinity streptavidin/biotin-based chromatin precipitation (Strep-CP) followed by Strep-CP on ChIP analysis using ChIP promoter arrays. We identified 451 TAL1 target genes in K562 cells. Furthermore, we analysed the regulation of one of these genes, the catalytic subunit beta of protein kinase A (PRKACB), during megakaryopoiesis of K562 and primary human CD34+ stem cell/progenitor cells. We found that TAL1 together with hematopoietic transcription factors RUNX1 and GATA1 binds to the promoter of the isoform 3 of PRKACB (Cβ3). During megakaryocytic differentiation a coactivator complex on the Cβ3 promoter, which includes WDR5 and p300, is replaced with a corepressor complex. In this manner, activating chromatin modifications are removed and expression of the PRKACB-Cβ3 isoform during megakaryocytic differentiation is reduced. Our data uncover a role of the TAL1 complex in controlling differential isoform expression of PRKACB. These results reveal a novel function of TAL1, RUNX1 and GATA1 in the transcriptional control of protein kinase A activity, with implications for cellular signalling control during differentiation and disease.
Background: Legionella pneumophila (L. pneumophila) is a causative agent of severe pneumonia. It is highly adapted to intracellular replication and manipulates host cell functions like vesicle trafficking and mRNA translation to its own advantage. However, it is still unknown to what extent microRNAs (miRNAs) are involved in the Legionella-host cell interaction.
Methods: WT and MyD88-/- murine bone marrow-derived macrophages (BMM) were infected with L. pneumophila, the transcriptome was analyzed by high throughput qPCR array (microRNAs) and conventional qPCR (mRNAs), and mRNA-miRNA interaction was validated by luciferase assays with 3´-UTR mutations and western blot.
Results: L. pneumophila infection caused a pro-inflammatory reaction and significant miRNA changes in murine macrophages. In MyD88-/- cells, induction of inflammatory markers, such as Ccxl1/Kc, Il6 and miR-146a-5p was reduced. Induction of miR-125a-3p was completely abrogated in MyD88-/- cells. Target prediction analyses revealed N-terminal asparagine amidase 1 (NTAN1), a factor from the n-end rule pathway, to be a putative target of miR-125a-3p. This interaction could be confirmed by luciferase assay and western blot.
Conclusion: Taken together, we characterized the miRNA regulation in L. pneumophila infection with regard to MyD88 signaling and identified NTAN1 as a target of miR-125a-3p. This finding unravels a yet unknown feature of Legionella-host cell interaction, potentially relevant for new treatment options.
Gallbladder cancer (GBC) is a lethal cancer with poor prognosis associated with high invasiveness and poor response to chemotherapy and radiotherapy. New therapeutic approaches are urgently needed in order to improve survival and response rates of GBC patients. We screened 130 small molecule inhibitors on a panel of seven GBC cell lines and identified the HSP90 inhibitor 17-AAG as one of the most potent inhibitory drugs across the different lines. We tested the antitumor efficacy of 17-AAG and geldanamycin (GA) in vitro and in a subcutaneous preclinical tumor model NOD-SCID mice. We also evaluated the expression of HSP90 by immunohistochemistry in human GBC tumors.
In vitro assays showed that 17-AAG and GA significantly reduced the expression of HSP90 target proteins, including EGFR, AKT, phospho-AKT, Cyclin B1, phospho-ERK and Cyclin D1. These molecular changes were consistent with reduced cell viability and cell migration and promotion of G2/M cell cycle arrest and apoptosis observed in our in vitro studies.
In vivo, 17-AAG showed efficacy in reducing subcutaneous tumors size, exhibiting a 69.6% reduction in tumor size in the treatment group compared to control mice (p < 0.05).
The HSP90 immunohistochemical staining was seen in 182/209 cases of GBC (87%) and it was strongly expressed in 70 cases (33%), moderately in 58 cases (28%), and weakly in 54 cases (26%).
Our pre-clinical observations strongly suggest that the inhibition of HSP90 function by HSP90 inhibitors is a promising therapeutic strategy for gallbladder cancer that may benefit from new HSP90 inhibitors currently in development.
FUSE Binding Protein 1 (FUBP1) is a transcriptional regulator, which is overexpressed in various cancer entities, including hepatocellular carcinoma (HCC) and colorectal cancer (CRC). It fulfills pro-proliferative and anti-apoptotic functions in cancer cells, resulting in increased proliferation and reduced sensitivity towards apoptotic stimuli.
Previously, camptothecin (CPT) and its clinically used analog 7-ethyl 10hydroxycamptothecin (SN-38) were shown to inhibit FUBP1 in biophysical interaction displacement assays (AlphaScreen; surface plasmon resonance, SPR), and first insights into the cellular effects of FUBP1 inhibition were obtained. CPT and SN-38 are known to potently inhibit topoisomerase 1 (TOP 1), and until today, these inhibitors were thought to be specific for this target. This could be disproved by our FUBP1 binding studies. An open issue, which is addressed in this thesis, was the contribution of FUBP1 inhibition to SN-38-mediated apoptosis apoptosis.
During this thesis, a low micromolar efficacy of CPT/SN-38-induced inhibition of FUBP1 binding to the Far Upstream Sequence Element (FUSE) oligonucleotide of p21 was determined. Furthermore, FUBP1 was for the first time shown to directly interact with a potential FUSE sequence upstream of the transcription start in pro-apoptotic gene BIK. In proof of-principle experiments, an effective inhibition of the binding of FUBP1 to the FUSE BIK DNA by CPT/SN-38 was verified.
One of the main goals of this thesis was to further elucidate the contribution of cellular FUBP1-inhibition by CPT/SN-38 to the anti-cancer potential of these substances. For this purpose, the TOP 1 mutant and TOP 1 wild type colorectal cancer sub-cell lines HCT116 G7 and HCT116 S were used. CPT/SN-38 was shown to induce apoptosis in single and combinatorial treatments with mitomycin c (MMC), independently of the TOP 1 mutation status of the cells. Furthermore, a prominent induction of a FUBP1 target gene signature was observed upon treatment of both cell lines with CPT/SN-38. Consequently, CPT/SN-38 was able to fulfill its anticancer effects in these cells, although TOP 1 could not be the main target in the mutant cell line.
In a second approach to gain indirect evidence for FUBP1 dependent effects of CPT/SN-38, the TOP 1-specific inhibitors topotecan (TTN) and β lapachone (BL) were used for the treatment of HCC and CRC cell lines. Interestingly, the TOP 1 inhibitors TTN and BL exhibited a reduced potency in apoptosis induction compared to the dual (FUBP1 and TOP 1) inhibitor SN-38.
Finally, two independent screens for a specific FUBP1 inhibitor were performed. In the first approach, a small number of structural and functional CPT-derivatives that exhibited a reduced inhibitory potential against TOP 1, were tested for their ability to interfere with the FUBP1/FUSE binding. Two particular indenoisoquinoline derivatives revealed potent in vitro inhibition of FUBP1 with low micromolar IC50 values.
In a second approach, previously identified candidate FUBP1 inhibitors that had been isolated from the Maybridge Hit Finder library served as lead structures for a structure activity relationship (SAR) study of the inhibition of FUBP1 binding to the FUSE oligonucleotide. After two cycles of optimization, a medium-potent FUBP1 inhibitor was obtained that induced effective deregulation of FUBP1 target genes in cell culture experiments.
Es war kein Aprilscherz, als das »Time Magazine« am 1. April 2013 auf der Titelseite ankündigte, wie man Krebs heilen kann. Anlass war die Gründung einer Initiative zur besseren Vernetzung von klinischen Forschern und Grundlagenwissenschaftlern, um so neue Therapieansätze wie »Checkpoint-Inhibitoren« bei malignem Melanom (schwarzem Hautkrebs), auch auf andere Krebserkrankungen übertragen zu können. Checkpoint-Inhibitoren sind der erste echte Durchbruch in der Therapie von fortgeschrittenen Krebserkrankungen.
Background: Due to the steadily increasing number of cancer patients worldwide the early diagnosis and treatment of cancer is a major field of research. The diagnosis of cancer is mostly performed by an experienced pathologist via the visual inspection of histo-pathological stained tissue sections. To save valuable time, low quality cryosections are frequently analyzed with diagnostic accuracies that are below those of high quality embedded tissue sections. Thus, alternative means have to be found that enable for fast and accurate diagnosis as the basis of following clinical decision making.
Methods: In this contribution we will show that the combination of the three label-free non-linear imaging modalities CARS (coherent anti-Stokes Raman-scattering), TPEF (two-photon excited autofluorescence) and SHG (second harmonic generation) yields information that can be translated into computational hematoxylin and eosin (HE) images by multivariate statistics. Thereby, a computational HE stain is generated resulting in pseudo-HE overview images that allow for identification of suspicious regions. The latter are analyzed further by Raman-spectroscopy retrieving the tissue’s molecular fingerprint.
Results: The results suggest that the combination of non-linear multimodal imaging and Raman-spectroscopy possesses the potential as a precise and fast tool in routine histopathology.
Conclusions: As the key advantage, both optical methods are non-invasive enabling for further pathological investigations of the same tissue section, e.g. a direct comparison with the current pathological gold-standard.
Cancer research has become a global enterprise, and the number of researchers, as well as the cost for their activities, has skyrocketed. The budget for the National Cancer Institute of the United States National Institutes of Health alone was US$5.2 billion in 2015. Since most of the research is funded by public money, it is perfectly legitimate to ask if these large expenses are worth it. In this brief commentary, we recapitulate some of the breakthroughs that mark the history of breast cancer research over the past decades and emphasize the resulting benefits for afflicted women. In 1971, only 40% of women diagnosed with breast cancer would live another 10 years. Today, nearly 80% of women reach that significant milestone in most developed countries. This dramatic change has afforded breast cancer patients many productive years and a better quality of life. Progress resulted largely from advances in the understanding of the molecular details of the disease and their translation into innovative, rationally designed therapies. These developments are founded on the revolution in molecular and cellular biology, an entirely new array of methods and technologies, the enthusiasm, optimism, and diligence of scientists and clinicians, and the considerable funding efforts from public and private sources. We were lucky to be able to spend our productive years in a period of scientific upheaval in which methods and concepts were revolutionized and that allowed us to contribute, within the global scientific community, to the progress in basic science and clinical practice.
Gallbladder cancer (GBC) is a highly malignant tumor characterized by a poor response to chemotherapy and radiotherapy. We evaluated the in vitro and in vivo antitumor efficacy of mTOR inhibitors, rapamycin and WYE-354. In vitro assays showed WYE-354 significantly reduced cell viability, migration and invasion and phospho-P70S6K expression in GBC cells. Mice harboring subcutaneous gallbladder tumors, treated with WYE-354 or rapamycin, exhibited a significant reduction in tumor mass. A short-term treatment with a higher dose of WYE-354 decreased the tumor size by 68.6% and 52.4%, in mice harboring G-415 or TGBC-2TKB tumors, respectively, compared to the control group. By contrast, treatment with a prolonged-low-dose regime of rapamycin almost abrogated tumor growth, exhibiting 92.7% and 97.1% reduction in tumor size, respectively, compared to control mice. These results were accompanied by a greater decrease in the phosphorylation status of P70S6K and a lower cell proliferation Ki67 index, compared to WYE-354 treated mice, suggesting a more effective mTOR pathway inhibition. These findings provide a proof of concept for the use of rapamycin or WYE-354 as potentially good candidates to be studied in clinical trials in GBC patients.
MiR144/451 expression is repressed by RUNX1 during megakaryopoiesis and disturbed by RUNX1/ETO
(2016)
Abstract: A network of lineage-specific transcription factors and microRNAs tightly regulates differentiation of hematopoietic stem cells along the distinct lineages. Deregulation of this regulatory network contributes to impaired lineage fidelity and leukemogenesis. We found that the hematopoietic master regulator RUNX1 controls the expression of certain microRNAs, of importance during erythroid/megakaryocytic differentiation. In particular, we show that the erythorid miR144/451 cluster is epigenetically repressed by RUNX1 during megakaryopoiesis. Furthermore, the leukemogenic RUNX1/ETO fusion protein transcriptionally represses the miR144/451 pre-microRNA. Thus RUNX1/ETO contributes to increased expression of miR451 target genes and interferes with normal gene expression during differentiation. Furthermore, we observed that inhibition of RUNX1/ETO in Kasumi1 cells and in RUNX1/ETO positive primary acute myeloid leukemia patient samples leads to up-regulation of miR144/451. RUNX1 thus emerges as a key regulator of a microRNA network, driving differentiation at the megakaryocytic/erythroid branching point. The network is disturbed by the leukemogenic RUNX1/ETO fusion product.
Author Summary: The regulatory network between transcription factors, epigenetic cofactors and microRNAs is decisive for normal hematopoiesis. The transcription factor RUNX1 is important for the establishment of a megakaryocytic gene expression program and the concomitant repression of erythroid genes during megakaryocytic differentiation. Gene regulation by RUNX1 is frequently disturbed by mutations and chromosomal translocations, such as the t(8;21) translocation, which gives rise to the leukemogenic RUNX1/ETO fusion protein. We found that RUNX1 regulates microRNAs, which are of importance at the megakaryocytic/erythroid branching point. Specifically, RUNX1 down-regulates expression of the microRNA cluster miR144/451 during megakaryocytic differentiation by changing the epigenetic histone modification pattern at the locus. We could show that miR451, one of the micorRNAs of the miR144/451 cluster, supports erythroid differentiation. We found that expression of miR451 is repressed by the RUNX1/ETO fusion protein, resulting in up regulation of miR451 target genes. Our data support the notion that RUNX1 suppresses the erythroid gene expression program including the erythroid microRNA miR451 and that the RUNX1/ETO fusion protein interferes with normal gene regulation by RUNX1.
The transcription factor Tal1 is a critical activator or repressor of gene expression in hematopoiesis and leukaemia. The mechanism by which Tal1 differentially influences transcription of distinct genes is not fully understood. Here we show that Tal1 interacts with the peptidylarginine deiminase IV (PADI4). We demonstrate that PADI4 can act as an epigenetic coactivator through influencing H3R2me2a. At the Tal1/PADI4 target gene IL6ST the repressive H3R2me2a mark triggered by PRMT6 is counteracted by PADI4, which augments the active H3K4me3 mark and thus increases IL6ST expression. In contrast, at the CTCF promoter PADI4 acts as a repressor. We propose that the influence of PADI4 on IL6ST transcription plays a role in the control of IL6ST expression during lineage differentiation of hematopoietic stem/progenitor cells. These results open the possibility to pharmacologically influence Tal1 in leukaemia.
Immunohistochemical assessment of phosphorylated mTORC1-pathway proteins in human brain tumors
(2015)
Background: Current pathological diagnostics include the analysis of (epi-)genetic alterations as well as oncogenic pathways. Deregulated mammalian target of rapamycin complex 1 (mTORC1) signaling has been implicated in a variety of cancers including malignant gliomas and is considered a promising target in cancer treatment. Monitoring of mTORC1 activity before and during inhibitor therapy is essential. The aim of our study is to provide a recommendation and report on pitfalls in the use of phospho-specific antibodies against mTORC1-targets phospho-RPS6 (Ser235/236; Ser240/244) and phospho-4EBP1 (Thr37/46) in formalin fixed, paraffin embedded material.
Methods and findings: Primary, established cell lines and brain tumor tissue from routine diagnostics were assessed by immunocyto-, immunohistochemistry, immunofluorescent stainings and immunoblotting. For validation of results, immunoblotting experiments were performed. mTORC-pathway activation was pharmacologically inhibited by torin2 and rapamycin. Torin2 treatment led to a strong reduction of signal intensity and frequency of all tested antibodies. In contrast phospho-4EBP1 did not show considerable reduction in staining intensity after rapamycin treatment, while immunocytochemistry with both phospho-RPS6-specific antibodies showed a reduced signal compared to controls. Staining intensity of both phospho-RPS6-specific antibodies did not show considerable decrease in stability in a timeline from 0–230 minutes without tissue fixation, however we observed a strong decrease of staining intensity in phospho-4EBP1 after 30 minutes. Detection of phospho-signals was strongly dependent on tissue size and fixation gradient. mTORC1-signaling was significantly induced in glioblastomas although not restricted to cancer cells but also detectable in non-neoplastic cells.
Conclusion: Here we provide a recommendation for phospho-specific immunohistochemistry for patient-orientated therapy decisions and monitoring treatment response.
Background: The complex cellular networks within tumors, the cytokine milieu, and tumor immune escape mechanisms affecting infiltration and anti-tumor activity of immune cells are of great interest to understand tumor formation and to decipher novel access points for cancer therapy. However, cellular in vitro assays, which rely on monolayer cultures of mammalian cell lines, neglect the three-dimensional architecture of a tumor, thus limiting their validity for the in vivo situation.
Methods: Three-dimensional in vivo-like tumor spheroid were established from human cervical carcinoma cell lines as proof of concept to investigate infiltration and cytotoxicity of NK cells in a 96-well plate format, which is applicable for high-throughput screening. Tumor spheroids were monitored for NK cell infiltration and cytotoxicity by flow cytometry. Infiltrated NK cells, could be recovered by magnetic cell separation.
Results: The tumor spheroids were stable over several days with minor alterations in phenotypic appearance. The tumor spheroids expressed high levels of cellular ligands for the natural killer (NK) group 2D receptor (NKG2D), mediating spheroid destruction by primary human NK cells. Interestingly, destruction of a three-dimensional tumor spheroid took much longer when compared to the parental monolayer cultures. Moreover, destruction of tumor spheroids was accompanied by infiltration of a fraction of NK cells, which could be recovered at high purity.
Conclusion: Tumor spheroids represent a versatile in vivo-like model system to study cytotoxicity and infiltration of immune cells in high-throughput screening. This system might proof useful for the investigation of the modulatory potential of soluble factors and cells of the tumor microenvironment on immune cell activity as well as profiling of patient-/donor-derived immune cells to personalize cellular immunotherapy.
Epigenetic silencing of transgene expression represents a major obstacle for the efficient genetic modification of multipotent and pluripotent stem cells. We and others have demonstrated that a 1.5 kb methylation-free CpG island from the human HNRPA2B1-CBX3 housekeeping genes (A2UCOE) effectively prevents transgene silencing and variegation in cell lines, multipotent and pluripotent stem cells, and their differentiated progeny. However, the bidirectional promoter activity of this element may disturb expression of neighboring genes. Furthermore, the epigenetic basis underlying the anti-silencing effect of the UCOE on juxtaposed promoters has been only partially explored. In this study we removed the HNRPA2B1 moiety from the A2UCOE and demonstrate efficient anti-silencing properties also for a minimal 0.7 kb element containing merely the CBX3 promoter. This DNA element largely prevents silencing of viral and tissue-specific promoters in multipotent and pluripotent stem cells. The protective activity of CBX3 was associated with reduced promoter CpG-methylation, decreased levels of repressive and increased levels of active histone marks. Moreover, the anti-silencing effect of CBX3 was locally restricted and when linked to tissue-specific promoters did not activate transcription in off target cells. Thus, CBX3 is a highly attractive element for sustained, tissue-specific and copy-number dependent transgene expression in vitro and in vivo.