Publications by authors named "Barbara Kaltschmidt"

82 Publications

Nanopore Sequencing Reveals Global Transcriptome Signatures of Mitochondrial and Ribosomal Gene Expressions in Various Human Cancer Stem-like Cell Populations.

Cancers (Basel) 2021 Mar 6;13(5). Epub 2021 Mar 6.

Forschungsverbund BioMedizin Bielefeld, OWL (FBMB e.V.), Maraweg 21, 33699 Bielefeld, Germany.

Cancer stem cells (CSCs) are crucial mediators of tumor growth, metastasis, therapy resistance, and recurrence in a broad variety of human cancers. Although their biology is increasingly investigated within the distinct types of cancer, direct comparisons of CSCs from different tumor types allowing comprehensive mechanistic insights are rarely assessed. In the present study, we isolated CSCs from endometrioid carcinomas, glioblastoma multiforme as well as adenocarcinomas of lung and prostate and assessed their global transcriptomes using full-length cDNA nanopore sequencing. Despite the expression of common CSC markers, principal component analysis showed a distinct separation of the CSC populations into three clusters independent of the specific type of tumor. However, GO-term and KEGG pathway enrichment analysis revealed upregulated genes related to ribosomal biosynthesis, the mitochondrion, oxidative phosphorylation, and glycolytic pathways, as well as the proteasome, suggesting a great extent of metabolic flexibility in CSCs. Interestingly, the GO term "NF-kB binding" was likewise found to be elevated in all investigated CSC populations. In summary, we here provide evidence for high global transcriptional similarities between CSCs from various tumors, which particularly share upregulated gene expression associated with mitochondrial and ribosomal activity. Our findings may build the basis for identifying novel therapeutic strategies targeting CSCs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers13051136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962028PMC
March 2021

Hyperosmolality in CHO culture: Effects on cellular behavior and morphology.

Biotechnol Bioeng 2021 Mar 10. Epub 2021 Mar 10.

Cell Culture Technology, Faculty of Technology, Bielefeld University, Bielefeld, Germany.

Exposure of Chinese hamster ovary cells (CHO) to highly concentrated feed solution during fed-batch cultivation is known to result in an unphysiological osmolality increase (>300 mOsm/kg), affecting cell physiology and morphology. Extending previous observation on osmotic adaptation, the present study investigates for the first time potential effects of hyperosmolality on CHO cells on both population and single-cell level. We intentionally exposed CHO cells to hyperosmolality of up to 545 mOsm/kg during fed-batch cultivation. In concordance with existing research data, hyperosmolality-exposed CHO cells showed a nearly triplicated volume accompanied by ablation of proliferation. On the molecular level, we observed a strong hyperosmolality-dependent increase in mitochondrial activity in CHO cells compared to control. In contrast to mitochondrial activity, hyperosmolality-dependent proliferation arrest of CHO cells was not accompanied by DNA accumulation or caspase-3/7-mediated apoptosis. Notably, we demonstrate for the first time a formation of up to eight multiple, small nuclei in single hyperosmolality-stressed CHO cells. The here presented observations reveal previously unknown hyperosmolality-dependent morphological changes in CHO cells and support existing data on the osmotic response in mammalian cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bit.27747DOI Listing
March 2021

Chronic inflammation of middle ear cholesteatoma promotes its recurrence via a paracrine mechanism.

Cell Commun Signal 2021 Feb 24;19(1):25. Epub 2021 Feb 24.

Department of Otolaryngology, Head and Neck Surgery, Medical School OWL Campus Klinikum Bielefeld, Bielefeld University, Teutoburger Str. 50, 33604, Bielefeld, Germany.

Background: Cholesteatoma disease is an expanding lesion in the middle ear. Hearing loss and facial paralysis alongside with other intracranial complications are found. No pharmaceutical treatment is available today and recurrence after surgical extraction occurs. We investigated possible TLR4-based mechanisms promoting recurrence and explore possible treatments strategies.

Methods: We isolated fibroblasts and epidermal stem cells from cholesteatoma tissue and healthy auditory canal skin. Subsequently, their expression under standard culture conditions and after stimulation with LPS was investigated by RT-qPCR. Cell metabolism and proliferation were analysed upon LPS treatment, with and without TLR4 antagonist. An indirect co-culture of fibroblasts and epidermal stem cells isolated from cholesteatoma tissue was utilized to monitor epidermal differentiation upon LPS treatment by RT-qPCR and immunocytochemistry.

Results: Under standard culture conditions, we detected a tissue-independent higher expression of IL-1β and IL-8 in stem cells, an upregulation of KGF and IGF-2 in both cell types derived from cholesteatoma and higher expression of TLR4 in stem cells derived from cholesteatoma tissue. Upon LPS challenge, we could detect a significantly higher expression of IL-1α, IL-1β, IL-6 and IL-8 in stem cells and of TNF-a, GM-CSF and CXCL-5 in stem cells and fibroblasts derived from cholesteatoma. The expression of the growth factors KGF, EGF, EREG, IGF-2 and HGF was significantly higher in fibroblasts, particularly when derived from cholesteatoma. Upon treatment with LPS the metabolism was elevated in stem cells and fibroblasts, proliferation was only enhanced in fibroblasts derived from cholesteatoma. This could be reversed by the treatment with a TLR4 antagonist. The cholesteatoma fibroblasts could be triggered by LPS to promote the epidermal differentiation of the stem cells, while no LPS treatment or LPS treatment without the presence of fibroblasts did not result in such a differentiation.

Conclusion: We propose that cholesteatoma recurrence is based on TLR4 signalling imprinted in the cholesteatoma cells. It induces excessive inflammation of stem cells and fibroblasts, proliferation of perimatrix fibroblasts and the generation of epidermal cells from stem cells thru paracrine signalling by fibroblasts. Treatment of the operation site with a TLR4 antagonist might reduce the chance of cholesteatoma recurrence. Video Abstract.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12964-020-00690-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7903614PMC
February 2021

Hepatic Vasculopathy and Regenerative Responses of the Liver in Fatal Cases of COVID-19.

Clin Gastroenterol Hepatol 2021 Jan 29. Epub 2021 Jan 29.

Department of General and Visceral Surgery, University Hospital of Bielefeld, Campus Bielefeld-Bethel, Bielefeld, Germany. Electronic address:

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects the nasopharynx and lungs and causes coronavirus disease-2019 (COVID-19). It may impact the heart, brain, kidney, and liver. Although functional impairment of the liver has been correlated with worse clinical outcomes, little is known about the pathophysiology of hepatic injury and repair in COVID-19. Histologic evaluation has been limited to small numbers of COVID-19 cases with no control subjects and demonstrated largely heterogeneous patterns of pathology..
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cgh.2021.01.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7844358PMC
January 2021

PLEKHG5 regulates autophagy, survival and MGMT expression in U251-MG glioblastoma cells.

Sci Rep 2020 12 14;10(1):21858. Epub 2020 Dec 14.

Department of Cell Biology, University of Bielefeld, Universitätsstr. 25, 33615, Bielefeld, Germany.

A signalling pathway involving PLEKHG5 (guanine exchange factor) for the Ras superfamily member RAB26 to transcription factor NF-κB was discovered in autophagy. PLEKHG5 was reported in glioblastoma multiforme (GBM) and correlates with patient survival. Thus, the generation of a cellular model for understanding PLEKHG5 signalling is the study purpose. We generated a CRISPR/Cas9-mediated knockout of PLEKHG5 in U251-MG glioblastoma cells and analysed resulting changes. Next, we used a mRFP-GFP-LC3 reporter for visualisation of autophagic defects and rescued the phenotype of PLEKHG5 wildtype via transduction of a constitutively active RAB26QL-plasmid. Effects of overexpressing RAB26 were investigated and correlated with the O-methylguanine-DNA methyltransferase (MGMT) and cellular survival. PLEKHG5 knockout showed changes in morphology, loss of filopodia and higher population doubling times. Accumulation of autolysosomes was resulted by decreased LAMP-1 in PLEKHG5-deficient cells. Rescue of PLEKHG5 restored the downregulation of RhoA activity, showed faster response to tumour necrosis factor and better cellular fitness. MGMT expression was activated after RAB26 overexpression compared to non-transduced cells. Survival of PLEKHG5 knockout was rescued together with sensitivity to temozolomide by RAB26QL. This study provides new insights in the PLEKHG5/RAB26 signalling within U251-MG cells, which suggests potential therapeutic strategies in other glioma cells and further in primary GBM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-77958-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736842PMC
December 2020

Transcriptome Analysis Reveals High Similarities between Adult Human Cardiac Stem Cells and Neural Crest-Derived Stem Cells.

Biology (Basel) 2020 Dec 1;9(12). Epub 2020 Dec 1.

Department of Cell Biology, Bielefeld University, 33615 Bielefeld, Germany.

For the identification of a stem cell population, the comparison of transcriptome data enables the simultaneous analysis of tens of thousands of molecular markers and thus enables the precise distinction of even closely related populations. Here, we utilized global gene expression profiling to compare two adult human stem cell populations, namely neural crest-derived inferior turbinate stem cells (ITSCs) of the nasal cavity and human cardiac stem cells (hCSCs) from the heart auricle. We detected high similarities between the transcriptomes of both stem cell populations, particularly including a range of neural crest-associated genes. However, global gene expression likewise reflected differences between the stem cell populations with regard to their niches of origin. In a broader analysis, we further identified clear similarities between ITSCs, hCSCs and other adherent stem cell populations compared to non-adherent hematopoietic progenitor cells. In summary, our observations reveal high similarities between adult human cardiac stem cells and neural crest-derived stem cells from the nasal cavity, which include a shared relation to the neural crest. The analyses provided here may help to understand underlying molecular regulators determining differences between adult human stem cell populations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biology9120435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761507PMC
December 2020

Isolation and Characterization of Two Novel Colorectal Cancer Cell Lines, Containing a Subpopulation with Potential Stem-Like Properties: Treatment Options by MYC/NMYC Inhibition.

Cancers (Basel) 2020 Sep 10;12(9). Epub 2020 Sep 10.

Forschungsverbund BioMedizin Bielefeld (FBMB), 33611 Bielefeld, Germany.

Cancer stem cells (CSC) are crucial mediators of cancer relapse. Here, we isolated two primary human colorectal cancer cell lines derived from a rectal neuroendocrine carcinoma (BKZ-2) and a colorectal adenocarcinoma (BKZ-3), both containing subpopulations with potential stem-like properties. Protein expression of CSC-markers prominin-1 and CD44 antigen was significantly higher for BKZ-2 and BKZ-3 in comparison to well-established colon carcinoma cell lines. High sphere-formation capacity further confirmed the existence of a subpopulation with potential stem-like phenotype. Epithelial-mesenchymal transition markers as well as immune checkpoint ligands were expressed more pronounced in BKZ-2. Both cell populations demonstrated N-myc proto-oncogene () copy number gain. Myc proto-oncogene (MYC)/NMYC activity inhibitor all-trans retinoic acid (ATRA) significantly reduced the number of tumor spheres for both and the volume of BKZ-2 spheres. In contrast, the sphere volume of ATRA-treated BKZ-3 was increased, and only BKZ-2 cell proliferation was reduced in monolayer culture. Treatment with KJ-Pyr-9, a specific inhibitor of MYC/NMYC-myc-associated factor X interaction, decreased survival by the induction of apoptosis of both. In summary, here, we present the novel colorectal cancer cell lines BKZ-2 and BKZ-3 as promising cellular in vitro models for colorectal carcinomas and identify the MYC/NMYC molecular pathway involved in CSC-induced carcinogenesis with relevant therapeutic potential.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12092582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564713PMC
September 2020

Absence of Plekhg5 Results in Myelin Infoldings Corresponding to an Impaired Schwann Cell Autophagy, and a Reduced T-Cell Infiltration Into Peripheral Nerves.

Front Cell Neurosci 2020 7;14:185. Epub 2020 Jul 7.

Department of Cell Biology, University of Bielefeld, Bielefeld, Germany.

Inflammation and dysregulation of the immune system are hallmarks of several neurodegenerative diseases. An activated immune response is considered to be the cause of myelin breakdown in demyelinating disorders. In the peripheral nervous system (PNS), myelin can be degraded in an autophagy-dependent manner directly by Schwann cells or by macrophages, which are modulated by T-lymphocytes. Here, we show that the NF-κB activator Pleckstrin homology containing family member 5 (Plekhg5) is involved in the regulation of both Schwann cell autophagy and recruitment of T-lymphocytes in peripheral nerves during motoneuron disease. -deficient mice show defective axon/Schwann cell units characterized by myelin infoldings in peripheral nerves. Even at late stages, -deficient mice do not show any signs of demyelination and inflammation. Using RNAseq, we identified a transcriptional signature for an impaired immune response in sciatic nerves, which manifested in a reduced number of CD4 and CD8 T-cells. These findings identify Plekhg5 as a promising target to impede myelin breakdown in demyelinating PNS disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fncel.2020.00185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7358705PMC
July 2020

Blood Serum Stimulates p38-Mediated Proliferation and Changes in Global Gene Expression of Adult Human Cardiac Stem Cells.

Cells 2020 06 16;9(6). Epub 2020 Jun 16.

Institute for Laboratory- and Transfusion Medicine, Heart and Diabetes Centre NRW, Ruhr University Bochum, 32545 Bad Oeynhausen, Germany.

During aging, senescent cells accumulate in various tissues accompanied by decreased regenerative capacities of quiescent stem cells, resulting in deteriorated organ function and overall degeneration. In this regard, the adult human heart with a generally low regenerative potential is of extreme interest as a target for rejuvenating strategies with blood borne factors that might be able to activate endogenous stem cell populations. Here, we investigated for the first time the effects of human blood plasma and serum on adult human cardiac stem cells (hCSCs) and showed significantly increased proliferation capacities and metabolism accompanied by a significant decrease of senescent cells, demonstrating a beneficial serum-mediated effect that seemed to be independent of age and sex. However, RNA-seq analysis of serum-treated hCSCs revealed profound effects on gene expression depending on the age and sex of the plasma donor. We further successfully identified key pathways that are affected by serum treatment with p38-MAPK playing a regulatory role in protection from senescence and in the promotion of proliferation in a serum-dependent manner. Inhibition of p38-MAPK resulted in a decline of these serum-mediated beneficial effects on hCSCs in terms of decreased proliferation and accelerated senescence. In summary, we provide new insights in the regulatory networks behind serum-mediated protective effects on adult human cardiac stem cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9061472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349155PMC
June 2020

Norepinephrine is a negative regulator of the adult periventricular neural stem cell niche.

Stem Cells 2020 09 25;38(9):1188-1201. Epub 2020 Jun 25.

Department of Neurology, University of Rostock, Rostock, Germany.

The limited proliferative capacity of neuroprogenitor cells (NPCs) within the periventricular germinal niches (PGNs) located caudal of the subventricular zone (SVZ) of the lateral ventricles together with their high proliferation capacity after isolation strongly implicates cell-extrinsic humoral factors restricting NPC proliferation in the hypothalamic and midbrain PGNs. We comparatively examined the effects of norepinephrine (NE) as an endogenous candidate regulator of PGN neurogenesis in the SVZ as well as the periventricular hypothalamus and the periaqueductal midbrain. Histological and neurochemical analyses revealed that the pattern of NE innervation of the adult PGNs is inversely associated with their in vivo NPC proliferation capacity with low NE levels coupled to high NPC proliferation in the SVZ but high NE levels coupled to low NPC proliferation in hypothalamic and midbrain PGNs. Intraventricular infusion of NE decreased NPC proliferation and neurogenesis in the SVZ-olfactory bulb system, while pharmacological NE inhibition increased NPC proliferation and early neurogenesis events in the caudal PGNs. Neurotoxic ablation of NE neurons using the Dsp4-fluoxetine protocol confirmed its inhibitory effects on NPC proliferation. Contrarily, NE depletion largely impairs NPC proliferation within the hippocampus in the same animals. Our data indicate that norepinephrine has opposite effects on the two fundamental neurogenic niches of the adult brain with norepinephrine being a negative regulator of adult periventricular neurogenesis. This knowledge might ultimately lead to new therapeutic approaches to influence neurogenesis in hypothalamus-related metabolic diseases or to stimulate endogenous regenerative potential in neurodegenerative processes such as Parkinson's disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/stem.3232DOI Listing
September 2020

Preparation of Terpenoid-Invasomes with Selective Activity against and Characterization by Cryo Transmission Electron Microscopy.

Biomedicines 2020 May 1;8(5). Epub 2020 May 1.

Thin Films & Physics of Nanostructures, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany.

Terpenoids are natural plant-derived products that are applied to treat a broad range of human diseases, such as airway infections and inflammation. However, pharmaceutical applications of terpenoids against bacterial infection remain challenging due to their poor water solubility. Here, we produce invasomes encapsulating thymol, menthol, camphor and 1,8-cineol, characterize them via cryo transmission electron microscopy and assess their bactericidal properties. While control- and cineol-invasomes are similarly distributed between unilamellar and bilamellar vesicles, a shift towards unilamellar invasomes is observable after encapsulation of thymol, menthol or camphor. Thymol- and camphor-invasomes show a size reduction, whereas menthol-invasomes are enlarged and cineol-invasomes remain unchanged compared to control. While thymol-invasomes lead to the strongest growth inhibition of , camphor- or cineol-invasomes mediate cell death and growth is not affected by menthol-invasomes. Flow cytometric analysis validate that invasomes comprising thymol are highly bactericidal to . Notably, treatment with thymol-invasomes does not affect survival of Gram-negative In summary, we successfully produce terpenoid-invasomes and demonstrate that particularly thymol-invasomes show a strong selective activity against Gram-positive bacteria. Our findings provide a promising approach to increase the bioavailability of terpenoid-based drugs and may be directly applicable for treating severe bacterial infections such as methicillin-resistant .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biomedicines8050105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7277086PMC
May 2020

A Matter of Choice: Inhibition of c-Rel Shifts Neuronal to Oligodendroglial Fate in Human Stem Cells.

Cells 2020 04 22;9(4). Epub 2020 Apr 22.

Molecular Neurobiology, University of Bielefeld, 33615 Bielefeld, Germany.

The molecular mechanisms underlying fate decisions of human neural stem cells (hNSCs) between neurogenesis and gliogenesis are critical during neuronal development and neurodegenerative diseases. Despite its crucial role in the murine nervous system, the potential role of the transcription factor NF-κB in the neuronal development of hNSCs is poorly understood. Here, we analyzed NF-κB subunit distribution during glutamatergic differentiation of hNSCs originating from neural crest-derived stem cells. We observed several peaks of specific NF-κB subunits. The most prominent nuclear peak was shown by c-REL subunit during a period of 2-5 days after differentiation onset. Furthermore, c-REL inhibition with pentoxifylline (PTXF) resulted in a complete shift towards oligodendroglial fate, as demonstrated by the presence of OLIG2/O4-oligodendrocytes, which showed , and at the transcript level. In addition c-REL impairment further produced a significant decrease in neuronal survival. Transplantation of PTXF-treated predifferentiated hNSCs into an ex vivo oxidative-stress-mediated demyelination model of mouse organotypic cerebellar slices further led to integration in the white matter and differentiation into MBP oligodendrocytes, validating their functionality and therapeutic potential. In summary, we present a human cellular model of neuronal differentiation exhibiting a novel essential function of NF-κB-c-REL in fate choice between neurogenesis and oligodendrogenesis which will potentially be relevant for multiple sclerosis and schizophrenia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9041037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226153PMC
April 2020

Bone Regeneration: A Novel Osteoinductive Function of Spongostan by the Interplay between Its Nano- and Microtopography.

Cells 2020 03 7;9(3). Epub 2020 Mar 7.

Molecular Neurobiology, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany.

Scaffold materials for bone regeneration are crucial for supporting endogenous healing after accidents, infections, or tumor resection. Although beneficial impacts of microtopological or nanotopological cues in scaffold topography are commonly acknowledged, less consideration is given to the interplay between the microscale and nanoscale. Here, micropores with a 60.66 ± 24.48 µm diameter ordered by closely packed collagen fibers are identified in pre-wetted Spongostan, a clinically-approved collagen sponge. On a nanoscale level, a corrugated surface of the collagen sponge is observable, leading to the presence of 32.97 ± 1.41 nm pores. This distinct micro- and nanotopography is shown to be solely sufficient for guiding osteogenic differentiation of human stem cells in vitro. Transplantation of Spongostan into a critical-size calvarial rat bone defect further leads to fast regeneration of the lesion. However, masking the micro- and nanotopographical cues using SiO nanoparticles prevents bone regeneration in vivo. Therefore, we demonstrate that the identified micropores allow migration of stem cells, which are further driven towards osteogenic differentiation by scaffold nanotopography. The present findings emphasize the necessity of considering both micro- and nanotopographical cues to guide intramembranous ossification, and might provide an optimal cell- and growth-factor-free scaffold for bone regeneration in clinical settings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9030654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140719PMC
March 2020

Stem Cell-Induced Inflammation in Cholesteatoma is Inhibited by the TLR4 Antagonist LPS-RS.

Cells 2020 01 14;9(1). Epub 2020 Jan 14.

Department of Cell Biology, University of Bielefeld, 33619 Bielefeld, Germany.

Cholesteatoma is a severe non-cancerous lesion of the middle ear characterized by massive inflammation, tissue destruction, and an abnormal growth of keratinized squamous epithelium. We recently demonstrated the presence of pathogenic stem cells within cholesteatoma tissue, unfortunately their potential roles in regulating disease-specific chronic inflammation remain poorly understood. In the presented study, we utilized our established human in vitro cholesteatoma stem cell model for treatments with lipopolysaccharides (LPS), tumor necrosis factor α (TNFα), and the TLR4-antagonist LPS from LPS-RS) followed by qPCR, western blot, and immunocytochemistry. Middle ear cholesteatoma stem cells (ME-CSCs) showed a significantly increased expression of TLR4 accompanied by a significantly enhanced LPS-dependent pro-inflammatory gene expression pattern of TNFα, IL-1α, IL-1ß, IL-6, and IL-8 compared to non-pathogenic control cells. LPS-dependent pro-inflammatory gene expression in ME-CSCs was driven by an enhanced activity of NF-B p65 leading to a TNFα-mediated feed-forward-loop of pro-inflammatory NF-B target gene expression. Functional inactivation of TLR4 via the TLR4-antagonist LPS-RS blocked chronic inflammation in ME-CSCs, resulting in a nearly complete loss of IL-1ß, IL-6, and TNFα expression. In summary, we determined that ME-CSCs mediate the inflammatory environment of cholesteatoma via TLR4-mediated NF-B-signaling, suggesting a distinct role of ME-CSCs as drivers of cholesteatoma progression and TLR4 on ME-CSCs as a therapeutic target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9010199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017370PMC
January 2020

A typical carcinoid of the lung - a case report with pathological correlation and propagation of the cancer stem cell line BKZ1 with synaptophysin expression.

Medicine (Baltimore) 2019 12;98(49):e18174

Department of General Thoracic Surgery, Protestant Hospital of Bethel Foundation, Burgsteig 13.

Rationale: Neuroendocrine tumors (NETs) of the lung account for 5% of all cases of lung cancer, which itself is the leading cause of cancer-related death worldwide. In accordance to its rarity, only few cell lines of NETs exist, which even often lack key characteristics of the primary tumor, making it difficult to study underlying molecular mechanisms.

Patient Concerns: The patient reported in this case is a 71-year old woman, which never smoked but suffered under dry cough.

Diagnoses: Chest CT-scan showed a paracardiac nodule of the lingula with 2 × 1.8 cm in diameter.

Interventions: The detected paracardiac nodule of the lingula was anatomically resected using video assisted thoracic surgery.

Outcomes: Histopathological diagnostic of the removed tissue identified the tumor as a well-differentiated typical carcinoid (TC), which represents one of the four subgroups of pulmonary NETs. Next to the successful treatment of the patient, we were able to propagate cancer stem cells (CSCs) out of the resected tumor tissue. To the best of our knowledge, we firstly isolated CSCs of a typical carcinoid, which were positive for the prominent CSC markers CD44, CD133 and nestin, confirming their stem cell properties. Additionally, CSCs, further referred as BKZ1, expressed the neuroendocrine marker synaptophysin, verifying their neuroendocrine origin. However, nuclear synaptophysin protein was also present in other stem cell populations, suggesting a role as general stem cell marker.

Lesson: In line with the importance of CSCs in cancer treatment and the lack of CSC-models for neuroendocrine neoplasms, the here described BKZ1 cancer stem cell line of a typical carcinoid represents a promising new model to study pulmonary carcinoids and particular NETs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/MD.0000000000018174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919531PMC
December 2019

The Therapeutic Effect of 1,8-Cineol on Pathogenic Bacteria Species Present in Chronic Rhinosinusitis.

Front Microbiol 2019 22;10:2325. Epub 2019 Oct 22.

Department of Otolaryngology, Head and Neck Surgery, Klinikum Bielefeld, Bielefeld, Germany.

Chronic rhinosinusitis (CRS) is marked by an inflamed mucosa of sinuses and is accompanied by a significantly reduced quality of live. Since no guidelines for the treatment of CRS are available, long lasting clinical histories with health care costs adding up to dozens of billion $ annually are caused by CRS. The progression of CRS is often induced by bacterial infections and/or a shift in microbiome as well as biofilm formation. The exact microbiome alterations are still unclear and the impenetrable biofilm renders the treatment with common antibiotics ineffective. This study focuses on characterizing the microbiome changes in CRS and investigating the inhibition of biofilm growth by 1,8-Cineol, a small, non-polar and hence biofilm penetrating molecule with known antimicrobial potential. We performed MALDI-TOF MS based characterization of the microbiomes of healthy individuals and CRS patients ( = 50). The microbiome in our test group was shifted to pathogens (, , and ). In contrast to published studies, solely based on cell culture techniques, we could not verify the abundance of in CRS. The inhibition of bacterial proliferation and biofilm growth by 1,8-Cineol was measured for these three pathogens. Interestingly, , the most prominent germ in CRS, showed a biofilm inhibition not simply correlated to its inhibition of proliferation. RT-qPCR confirmed that this was due to the downregulations of major key players in biofilm generation (agrA, SarA and σ) by 1,8-Cineol. Furthermore we verified this high biofilm inhibition potential in a model host system consisting out of biofilm grown on mature respiratory epithelium. A second host model, comprising organotypic slices, was utilized to investigate the reaction of the innate immune system present in the nasal mucosa upon biofilm formation and treatment with 1,8-Cineol. Interestingly , the cause of very common catheter infections, possesses a biofilm generation pathway very similar to and might be treatable in a similar fashion. The two presented model systems might be transferred to combinations of every biofilm forming bacterial with most kind of epithelium and mucosa.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2019.02325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821979PMC
October 2019

Sexual dimorphisms in adult human neural, mesoderm-derived, and neural crest-derived stem cells.

FEBS Lett 2019 12 2;593(23):3338-3352. Epub 2019 Oct 2.

Department of Cell Biology, Bielefeld University, Germany.

Sexual dimorphisms contribute, at least in part, to the severity and occurrence of a broad range of neurodegenerative, cardiovascular, and bone disorders. In addition to hormonal factors, increasing evidence suggests that stem cell-intrinsic mechanisms account for sex-specific differences in human physiology and pathology. Here, we discuss sex-related intrinsic mechanisms in adult stem cell populations, namely mesoderm-derived stem cells, neural stem cells (NSCs), and neural crest-derived stem cells (NCSCs), and their implications for stem cell differentiation and regeneration. We particularly focus on sex-specific differences in stem cell-mediated bone regeneration, in neuronal development, and in NSC-mediated neuroprotection. Moreover, we review our own recently published observations regarding the sex-dependent role of NF-κB-p65 in neuroprotection of human NCSC-derived neurons and sex differences in NCSC-related disorders, so-called neurocristopathies. These observations are in accordance with the increasing evidence pointing toward sex-specific differences in neurocristopathies and degenerative diseases like Parkinson's disease or osteoporosis. All findings discussed here indicate that sex-specific variability in stem cell biology may become a crucial parameter for the design of future treatment strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1873-3468.13606DOI Listing
December 2019

A Role for NF-κB in Organ Specific Cancer and Cancer Stem Cells.

Cancers (Basel) 2019 May 11;11(5). Epub 2019 May 11.

Department of Cell Biology, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany.

Cancer stem cells (CSCs) account for tumor initiation, invasiveness, metastasis, and recurrence in a broad range of human cancers. Although being a key player in cancer development and progression by stimulating proliferation and metastasis and preventing apoptosis, the role of the transcription factor NF-κB in cancer stem cells is still underestimated. In the present review, we will evaluate the role of NF-κB in CSCs of glioblastoma multiforme, ovarian cancer, multiple myeloma, lung cancer, colon cancer, prostate cancer, as well as cancer of the bone. Next to summarizing current knowledge regarding the presence and contribution of CSCs to the respective types of cancer, we will emphasize NF-κB-mediated signaling pathways directly involved in maintaining characteristics of cancer stem cells associated to tumor progression. Here, we will also focus on the status of NF-κB-activity predominantly in CSC populations and the tumor mass. Genetic alterations leading to NF-κB activity in glioblastoma, ependymoma, and multiple myeloma will be discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers11050655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563002PMC
May 2019

MoNa - A Cost-Efficient, Portable System for the Nanoinjection of Living Cells.

Sci Rep 2019 04 2;9(1):5480. Epub 2019 Apr 2.

Institute of Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str 1, 30625, Hannover, Germany.

Injection techniques to deliver macromolecules to cells such as microinjection have been around for decades with applications ranging from probing whole organisms to the injection of fluorescent molecules into single cells. A similar technique that has raised recent interest is nanoinjection. The pipettes used here are much smaller and allow for the precise deposition of molecules into single cells via electrokinetics with minimal influence on the cells' health. Unfortunately, the equipment utilized for nanoinjection originates from scanning ion conductance microscopy (SICM) and is therefore expensive and not portable, but usually fixed to a specific microscope setup. The level of precision that these systems achieve is much higher than what is needed for the more robust nanoinjection process. We present Mobile Nanoinjection (MoNa), a portable, cost-efficient and easy to build system for the injection of single cells. Sacrificing unnecessary sub-nanometer accuracy and low ion current noise levels, we were able to inject single living cells with high accuracy. We determined the noise of the MoNa system and investigated the injection conditions for 16 prominent fluorescent labels and fluorophores. Further, we performed proof of concepts by injection of ATTO655-Phalloidin and MitoTracker Deep Red to living human osteosarcoma (U2OS) cells and of living adult human inferior turbinate stem cells (ITSC's) following neuronal differentiation with the MoNa system. We achieved significant cost reductions of the nanoinjection technology and gained full portability and compatibility to most optical microscopes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-41648-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445100PMC
April 2019

Natural and synthetic nanopores directing osteogenic differentiation of human stem cells.

Nanomedicine 2019 04 14;17:319-328. Epub 2019 Feb 14.

Department of Cell Biology, Bielefeld University, Bielefeld, Germany; Molecular Neurobiology, Bielefeld University, Bielefeld, Germany; Bielefeld Institute for Nanoscience (BINAS), Bielefeld University, Bielefeld, Germany. Electronic address:

Bone regeneration is a highly orchestrated process crucial for endogenous healing procedures after accidents, infections or tumor therapy. Changes in surface nanotopography are known to directly affect the formation of osteogenic cell types, although no direct linkage to the endogenous nanotopography of bone was described so far. Here we show the presence of pores of 31.93 ± 0.97 nm diameter on the surface of collagen type I fibers, the organic component of bone, and demonstrate these pores to be sufficient to induce osteogenic differentiation of adult human stem cells. We further applied SiO nanoparticles thermally cross-linked to a nanocomposite to artificially biomimic 31.93 ± 0.97 nm pores, which likewise led to in vitro production of bone mineral by adult human stem cells. Our findings show an endogenous mechanism of directing osteogenic differentiation of adult stem cells by nanotopological cues and provide a direct application using SiO nanocomposites with surface nanotopography biomimicking native bone architecture.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.nano.2019.01.018DOI Listing
April 2019

NF-κB p65 directs sex-specific neuroprotection in human neurons.

Sci Rep 2018 10 30;8(1):16012. Epub 2018 Oct 30.

Molecular Neurobiology, University of Bielefeld, Bielefeld, Germany.

Protection of neurons against oxidative stress is crucial during neuronal development, maintenance and for treating neurodegenerative diseases. However, little is known about the molecular mechanisms underlying sex-specific maturation and survival of neurons. In the present study, we demonstrate NF-κB-p65 mediated neuroprotection in human glutamatergic neurons differentiated from inferior turbinate stem cells (ITSCs) in a sex-dependent manner. We successfully differentiated ITSCs into MAP-2/NF200/Synaptophysin/vGlut2-glutamatergic neurons in vitro and ex vivo and validated their functionality. TNF-α-dependent NF-κB-p65 activation was accompanied by significant neuroprotection against oxidative stress-induced neuronal death, which was surprisingly higher in neurons from female donors. Accordingly, sex-specific neuroprotection of female neurons was followed by an increased expression of special NF-κB target genes SOD2 and IGF2. Among these, SOD2 is a well known gene protecting cells against oxidative stress resulting in longevity. In addition, IGF2 is known to promote synapse formation and spine maturation, and it has antioxidant and neuroprotective effects against oxidative damage. In conclusion, we show that NF-κB-p65 is a key player in neuroprotection of human neurons, however the protective gene expression program beneath it differs between sexes. Our findings are in accordance with the increasing evidences pointing towards sex-specific differences in risk and severity of neurodegenerative diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-34394-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6207661PMC
October 2018

Primary rat LSECs preserve their characteristic phenotype after cryopreservation.

Sci Rep 2018 10 2;8(1):14657. Epub 2018 Oct 2.

Faculty of Health Sciences, Department of Medical Biology, Vascular Biology Research Group, UiT-The Arctic University of Norway, 9037, Tromsø, Norway.

Liver disease is a leading cause of morbidity and mortality worldwide. Recently, the liver non-parenchymal cells have gained increasing attention for their potential role in the development of liver disease. Liver sinusoidal endothelial cells (LSECs), a specialized type of endothelial cells that have unique morphology and function, play a fundamental role in maintaining liver homeostasis. Current protocols for LSEC isolation and cultivation rely on freshly isolated cells which can only be maintained differentiated in culture for a few days. This creates a limitation in the use of LSECs for research and a need for a consistent and reliable source of these cells. To date, no LSEC cryopreservation protocols have been reported that enable LSECs to retain their functional and morphological characteristics upon thawing and culturing. Here, we report a protocol to cryopreserve rat LSECs that, upon thawing, maintain full LSEC-signature features: fenestrations, scavenger receptor expression and endocytic function on par with freshly isolated cells. We have confirmed these features by a combination of biochemical and functional techniques, and super-resolution microscopy. Our findings offer a means to standardize research using LSECs, opening the prospects for designing pharmacological strategies for various liver diseases, and considering LSECs as a therapeutic target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-32103-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168544PMC
October 2018

Subunit-Specific Role of NF-κB in Cancer.

Biomedicines 2018 Apr 17;6(2). Epub 2018 Apr 17.

Department of Cell Biology, University of Bielefeld, 33615 Bielefeld, Germany.

The transcription factor NF-κB is a key player in inflammation, cancer development, and progression. NF-κB stimulates cell proliferation, prevents apoptosis, and could promote tumor angiogenesis as well as metastasis. Extending the commonly accepted role of NF-κB in cancer formation and progression, different NF-κB subunits have been shown to be active and of particular importance in distinct types of cancer. Here, we summarize overexpression data of the NF-κB subunits RELA, RELB, and c-REL (referring to the v-REL, which is the oncogene of Reticuloendotheliosis virus strain T) as well as of their upstream kinase inhibitor, namely inhibitor of κB kinases (IKK), in different human cancers, assessed by database mining. These data argue against a universal mechanism of cancer-mediated activation of NF-κB, and suggest a much more elaborated mode of NF-κB regulation, indicating a tumor type-specific upregulation of the NF-κB subunits. We further discuss recent findings showing the diverse roles of NF-κB signaling in cancer development and metastasis in a subunit-specific manner, emphasizing their specific transcriptional activity and the role of autoregulation. While non-canonical NF-κB RELB signaling is described to be mostly present in hematological cancers, solid cancers reveal constitutive canonical NF-κB RELA or c-REL activity. Providing a linkage to cancer therapy, we discuss the recently described pivotal role of NF-κB c-REL in regulating cancer-targeting immune responses. In addition, current strategies and ongoing clinical trials are summarized, which utilize genome editing or drugs to inhibit the NF-κB subunits for cancer treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biomedicines6020044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6027219PMC
April 2018

Stem cells in middle ear cholesteatoma contribute to its pathogenesis.

Sci Rep 2018 04 18;8(1):6204. Epub 2018 Apr 18.

Department of Otolaryngology, Head and Neck Surgery, Klinikum Bielefeld, 33604, Bielefeld, Germany.

Cholesteatoma is a potentially life-threatening middle ear lesion due to the formation of an inflamed ectopic mass of keratinizing squamous epithelium. Surgical removal remains the only treatment option, emphasizing the need to gain a better understanding of this severe disease. We show for the first time that stem cells residing in cholesteatoma tissue contribute to disease progression. Cells expressing the "stemness" markers Nestin and S100B were detected in middle ear cholesteatoma and auditory canal skin. Isolated Nestin + /S100B + -cells showed the capability for self-renewal, neurosphere formation and differentiation into mesodermal and ectodermal cell types. Compared to auditory canal skin stem cells middle ear cholesteatoma-derived stem cells displayed an enhanced susceptibility to inflammatory stimuli, and this suggested a possible contribution to the inflammatory environment in cholesteatoma tissue. Cholesteatoma derived stem cells were able to differentiate into keratinocyte-like cells using factors mimicking the microenvironment of cholesteatoma. Our findings demonstrate a new perspective on the pathogenesis of cholesteatoma and may lead to new treatment strategies for this severe middle ear lesion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-24616-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5906547PMC
April 2018

IKK1/2 protect human cells from TNF-mediated RIPK1-dependent apoptosis in an NF-κB-independent manner.

Biochim Biophys Acta Mol Cell Res 2018 Aug 7;1865(8):1025-1033. Epub 2018 Apr 7.

Department of Cell Biology, University of Bielefeld, Universitaetsstr. 25, 33501 Bielefeld, Germany. Electronic address:

TNF signaling is directly linked to cancer development and progression. A broad range of tumor cells is able to evade cell death induced by TNF impairing the potential anti-cancer value of TNF in therapy. Although sensitizing cells to TNF-induced death therefore has great clinical implications, detailed mechanistic insights into TNF-mediated human cell death still remain unknown. Here, we analyzed human cells by applying CRISPR/Cas9n to generate cells deficient of IKK1, IKK2, IKK1/2 and RELA. Despite stimulation with TNF resulted in impaired NF-κB activation in all genotypes compared to wildtype cells, increased cell death was observable only in IKK1/2-double-deficient cells. Cell death could be detected by Caspase-3 activation and binding of Annexin V. TNF-induced programmed cell death in IKK1/2 cells was further shown to be mediated via RIPK1 in a predominantly apoptotic manner. Our findings demonstrate the IKK complex to protect from TNF-induced cell death in human cells independently to NF-κB RelA suggesting IKK1/2 to be highly promising targets for cancer therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbamcr.2018.04.003DOI Listing
August 2018

Technical feasibility study for production of tailored multielectrode arrays and patterning of arranged neuronal networks.

PLoS One 2018 23;13(2):e0192647. Epub 2018 Feb 23.

Center for Spinelectronic Materials and Devices, Physics Department, Bielefeld University, Bielefeld, Germany.

In this manuscript, we first reveal a simple ultra violet laser lithographic method to design and produce plain tailored multielectrode arrays. Secondly, we use the same lithographic setup for surface patterning to enable controlled attachment of primary neuronal cells and help neurite guidance. For multielectrode array production, we used flat borosilicate glass directly structured with the laser lithography system. The multi layered electrode system consists of a layer of titanium coated with a layer of di-titanium nitride. Finally, these electrodes are covered with silicon nitride for insulation. The quality of the custom made multielectrode arrays was investigated by light microscopy, electron microscopy and X-ray diffraction. The performance was verified by the detection of action potentials of primary neurons. The electrical noise of the custom-made MEA was equal to commercially available multielectrode arrays. Additionally, we demonstrated that structured coating with poly lysine, obtained with the aid of the same lithographic system, could be used to attach and guide neurons to designed structures. The process of neuron attachment and neurite guidance was investigated by light microscopy and charged particle microscopy. Importantly, the utilization of the same lithographic system for MEA fabrication and poly lysine structuring will make it easy to align the architecture of the neuronal network to the arrangement of the MEA electrode.. In future studies, this will lead to multielectrode arrays, which are able to specifically attach neuronal cell bodies to their chemically defined electrodes and guide their neurites, gaining a controlled connectivity in the neuronal network. This type of multielectrode array would be able to precisely assign a signal to a certain neuron resulting in an efficient way for analyzing the maturation of the neuronal connectivity in small neuronal networks.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192647PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5825013PMC
April 2018

Identification of a Novel High Yielding Source of Multipotent Adult Human Neural Crest-Derived Stem Cells.

Stem Cell Rev Rep 2018 Apr;14(2):277-285

Department of Otolaryngology, Head and Neck Surgery, Klinikum Bielefeld, Teutoburger Straße 50, 33604, Bielefeld, Germany.

Due to their extraordinarily broad differentiation potential and persistence during adulthood, adult neural crest-derived stem cells (NCSCs) are highly promising candidates for clinical applications, particularly when facing the challenging treatment of neurodegenerative diseases or complex craniofacial injuries. Successful application of human NCSCs in regenerative medicine and pharmaceutical research mainly relies on the availability of sufficient amounts of tissue for cell isolation procedures. Facing this challenge, we here describe for the first time a novel population of NCSCs within the middle turbinate of the human nasal cavity. From a surgical point of view, high amounts of tissue are routinely and easily removed during nasal biopsies. Investigating the presence of putative stem cells in obtained middle turbinate tissue by immunohistochemistry, we observed Nestin/p75/S100/α smooth muscle actin (αSMA) cells, which we successfully isolated and cultivated in vitro. Cultivated middle turbinate stem cells (MTSCs) kept their expression of neural crest and stemness markers Nestin, p75 and S100 and showed the capability of sphere formation and clonal growth, indicating their stem cell character. Application of directed in vitro differentiation assays resulted in successful differentiation of MTSCs into osteogenic and neuronal cell types. Regarding the high amount of tissue obtained during surgery as well as their broad differentiation capability, MTSCs seem to be a highly promising novel neural crest stem cell population for applications in cell replacement therapy and pharmacological research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12015-017-9797-2DOI Listing
April 2018

Plekhg5-regulated autophagy of synaptic vesicles reveals a pathogenic mechanism in motoneuron disease.

Nat Commun 2017 10 30;8(1):678. Epub 2017 Oct 30.

Institute of Clinical Neurobiology, University Hospital Würzburg, 97078, Würzburg, Germany.

Autophagy-mediated degradation of synaptic components maintains synaptic homeostasis but also constitutes a mechanism of neurodegeneration. It is unclear how autophagy of synaptic vesicles and components of presynaptic active zones is regulated. Here, we show that Pleckstrin homology containing family member 5 (Plekhg5) modulates autophagy of synaptic vesicles in axon terminals of motoneurons via its function as a guanine exchange factor for Rab26, a small GTPase that specifically directs synaptic vesicles to preautophagosomal structures. Plekhg5 gene inactivation in mice results in a late-onset motoneuron disease, characterized by degeneration of axon terminals. Plekhg5-depleted cultured motoneurons show defective axon growth and impaired autophagy of synaptic vesicles, which can be rescued by constitutively active Rab26. These findings define a mechanism for regulating autophagy in neurons that specifically targets synaptic vesicles. Disruption of this mechanism may contribute to the pathophysiology of several forms of motoneuron disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-017-00689-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662736PMC
October 2017

CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle.

PLoS One 2017 2;12(8):e0182373. Epub 2017 Aug 2.

Department of Cell Biology, University of Bielefeld, Bielefeld, Germany.

Cervical cancer is the fourth common cancer in women resulting worldwide in 266,000 deaths per year. Belonging to the carcinomas, new insights into cervical cancer biology may also have great implications for finding new treatment strategies for other kinds of epithelial cancers. Although the transcription factor NF-κB is known as a key player in tumor formation, the relevance of its particular subunits is still underestimated. Here, we applied CRISPR/Cas9n-mediated genome editing to successfully knockout the NF-κB subunit c-REL in HeLa Kyoto cells as a model system for cervical cancers. We successfully generated a homozygous deletion in the c-REL gene, which we validated using sequencing, qPCR, immunocytochemistry, western blot analysis, EMSA and analysis of off-target effects. On the functional level, we observed the deletion of c-REL to result in a significantly decreased cell proliferation in comparison to wildtype (wt) without affecting apoptosis. The impaired proliferative behavior of c-REL-/- cells was accompanied by a strongly decreased amount of the H2B protein as well as a significant delay in the prometaphase of mitosis compared to c-REL+/+ HeLa Kyoto cells. c-REL-/- cells further showed significantly decreased expression levels of c-REL target genes in comparison to wt. In accordance to our proliferation data, we observed the c-REL knockout to result in a significantly increased resistance against the chemotherapeutic agents 5-Fluoro-2'-deoxyuridine (5-FUDR) and cisplatin. In summary, our findings emphasize the importance of c-REL signaling in a cellular model of cervical cancer with direct clinical implications for the development of new treatment strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0182373PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5540532PMC
September 2017

Single-particle tracking uncovers dynamics of glutamate-induced retrograde transport of NF-κB p65 in living neurons.

Neurophotonics 2016 Oct 18;3(4):041804. Epub 2016 May 18.

University of Bielefeld , Cell Biology, Universitätsstr. 25, 33501 Bielefeld, Germany.

Retrograde transport of NF-κB from the synapse to the nucleus in neurons is mediated by the dynein/dynactin motor complex and can be triggered by synaptic activation. The caliber of axons is highly variable ranging down to 100 nm, aggravating the investigation of transport processes in neurites of living neurons using conventional light microscopy. We quantified for the first time the transport of the NF-κB subunit p65 using high-density single-particle tracking in combination with photoactivatable fluorescent proteins in living mouse hippocampal neurons. We detected an increase of the mean diffusion coefficient ([Formula: see text]) in neurites from [Formula: see text] to [Formula: see text] after stimulation with glutamate. We further observed that the relative amount of retrogradely transported p65 molecules is increased after stimulation. Glutamate treatment resulted in an increase of the mean retrograde velocity from [Formula: see text] to [Formula: see text], whereas a velocity increase from [Formula: see text] to [Formula: see text] was observed for anterogradely transported p65. This study demonstrates for the first time that glutamate stimulation leads to an increased mobility of single NF-κB p65 molecules in neurites of living hippocampal neurons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1117/1.NPh.3.4.041804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4870386PMC
October 2016