Kevin Mellert

Enhancing Endosomal Escape of Transduced Proteins by Photochemical Internalisation

Induced internalisation of functional proteins into cultured cells has become an important aspect in a rising number of in vitro and in vivo assays. The endo-lysosomal entrapment of the transduced proteins remains the major problem in all transduction protocols. In this study we compared the efficiency, cytotoxicity and protein targeting of different commercially available transduction reagents by transducing a well-studied fluorescently labelled protein (Atto488-bovine serum albumin) into cultured human sarcoma cells. The amount of internalised protein and toxicity differed between the different reagents, but the percentage of transduced cells was consistently high. Furthermore, in all protocols the signals of the transduced Atto488-BSA were predominantly punctual consistent with an endosomal localisation. To overcome the endosomal entrapment, the transduction protocols were combined with a photochemical internalisation (PCI) treatment. Using this combination revealed that an endosomal disruption is highly effective in cell penetrating peptide (CPP) mediated transduction, whereas lipid-mediated transductions lead to a lower signal spreading throughout the cytosol. No change in the signal distribution could be achieved in treatments using non-lipid polymers as a transduction reagent. Therefore, the combination of protein transduction protocols based on CPPs with the endosomolytic treatment PCI can facilitate protein transduction experiments in vitro.

Preclinical Characterization of Novel Chordoma Cell Systems and Their Targeting by Pharmocological Inhibitors of the CDK4/6 Cell-Cycle Pathway

Chordomas are tumors that arise at vertebral bodies and the base of the skull. Although rare in incidence, they are deadly owing to slow growth and a lack of effective therapeutic options. In this study, we addressed the need for chordoma cell systems that can be used to identify therapeutic targets and empower testing of candidate pharmacologic drugs. Eight human chordoma cell lines that we established exhibited cytology, genomics, mRNA, and protein profiles that were characteristic of primary chordomas. Candidate responder profiles were identified through an immunohistochemical analysis of a chordoma tissue bank of 43 patients. Genomic, mRNA, and protein expression analyses confirmed that the new cell systems were highly representative of chordoma tissues. Notably, all cells exhibited a loss of CDKN2A and p16, resulting in universal activation of the CDK4/6 and Rb pathways. Therefore, we investigated the CDK4/6 pathway and responses to the CDK4/6-specific inhibitor palbociclib. In the newly validated system, palbociclib treatment efficiently inhibited tumor cell growth in vitro and a drug responder versus nonresponder molecular signature was defined on the basis of immunohistochemical expression of CDK4/6/pRb (S780). Overall, our work offers a valuable new tool for chordoma studies including the development of novel biomarkers and molecular targeting strategies.

H3F3A mutation in giant cell tumour of the bone is detected by immunohistochemistry using a monoclonal antibody against the G34W mutated site of the histone H3.3 variant

Giant cell tumour of the bone (GCTB) is a neoplasm predominantly of long bones characterized by the H3F3A mutation G34W. Conventional diagnosis is challenged by the tumours giant cell‐rich morphology, which overlaps with other giant cell‐containing lesions of the bone. Recently, a monoclonal antibody specific for the H3F3A mutation has been generated. Our aim was to test this antibody on a cohort of giant cell‐containing lesions.

Aberrant Single Exon Skipping is not Altered by Age in Exons of NF1, RABAC1, AATF or PCGF2 in Human Blood Cells and Fibroblasts

In human pre-mRNA splicing, infrequent errors occur resulting in erroneous splice products as shown in a genome-wide approach. One characteristic subgroup consists of products lacking one cassette exon. The noise in the splicing process, represented by those misspliced products, can be increased by cold shock treatment or by inhibiting the nonsense mediated decay. Here, we investigated whether the splicing noise frequency increases with age in vivo in peripheral bloods cells or in vitro in cultured and aged fibroblasts from healthy donors. Splicing noise frequency was measured for four erroneously skipped NF1 exons and one exon of RABAC1, AATF and PCGF2 by RT-qPCR. Measurements were validated in cultured fibroblasts treated with cold shock or puromycin. Intragenic but not interpersonal differences were detected in splicing noise frequencies in vivo in peripheral blood cells of 11 healthy donors (15 y–85 y) and in in vitro senescent fibroblasts from three further donors. No correlation to the age of the donors was found in the splicing noise frequencies. Our data demonstrates that splicing error frequencies are not altered by age in peripheral blood cells or in vitro aged fibroblasts in the tested exons of the four investigated genes, indicating a high importance of correct splicing in these proliferating aged cells.

Partial Blindness to Submicron Topography in NF1 Haploinsufficient Cultured Fibroblasts Indicates a New Function of Neurofibromin in Regulation of Mechanosensoric

Cells sense physical properties of their extracellular environment and translate them into biochemical signals. In this study, cell responses to surfaces with submicron topographies were investigated in cultured human NF1 haploinsufficient fibroblasts. Age-matched fibroblasts from 8 patients with neurofibromatosis type 1 (NF1+/–) and 9 controls (NF1+/+) were cultured on surfaces with grooves of 200 nm height and lateral distance of 2 µm. As cellular response indicator, the mean cell orientation along microstructured grooves was systematically examined. The tested NF1 haploinsufficient fibroblasts were significantly less affected by the topography than those from healthy donors. Incubation of the NF1+/– fibroblasts with the farnesyltransferase inhibitor FTI-277 and other inhibitors of the neurofibromin pathway ameliorates significantly the cell orientation. These data indicate that NF1 haploinsufficiency results in an altered response to specific surface topography in fibroblasts. We suggest a new function of neurofibromin in the sensoric mechanism to topographies and a partial mechanosensoric blindness by NF1 haploinsufficiency.

Cyclic stretch increases splicing noise rate in cultured human fibroblasts

BackgroundMechanical forces are known to alter the expression of genes, but it has so far not been reported whether they may influence the fidelity of nucleus-based processes. One experimental approach permitting to address this question is the application of cyclic stretch to cultured human fibroblasts. As a marker for the precision of nucleus-based processes, the number of errors that occur during co-transcriptional splicing can then be measured. This so-called splicing noise is found at low frequency in pre-mRNA splicing.FindingsThe amount of splicing noise was measured by RT-qPCR of seven exon skips from the test genes AATF, MAP3K11, NF1, PCGF2, POLR2A and RABAC1. In cells treated by altered uniaxial cyclic stretching for 18 h, a uniform and significant increase of splicing noise was found for all detectable exon skips.ConclusionOur data demonstrate that application of cyclic stretch to cultured fibroblasts correlates with a reduced transcriptional fidelity caused by increasing splicing noise.

HOXA7 , HOXA9 , and HOXA10 are differentially expressed in clival and sacral chordomas

Chordomas are rare tumours of the bone arising along the spine from clivus to sacrum. We compared three chordoma cell lines of the clivus region including the newly established clivus chordoma cell line, U-CH14, with nine chordoma cell lines originating from sacral primaries by morphology, on genomic and expression levels and with patient samples from our chordoma tissue bank. Clinically, chordomas of the clivus were generally smaller in size at presentation and patients with sacral chordomas had more metastases and more often recurrent disease. All chordoma cell lines had a typical physaliphorous morphology and expressed brachyury, S100-protein and cytokeratin. By expression analyses we detected differentially expressed genes in the clivus derived cell lines as compared to the sacral cell lines. Among these were HOXA7, HOXA9, and HOXA10 known to be important for the development of the anterior-posterior body axis. These results were confirmed by qPCR. Immunohistologically, clivus chordomas had no or very low levels of HOXA10 protein while sacral chordomas showed a strong nuclear positivity in all samples analysed. This differential expression of HOX genes in chordomas of the clivus and sacrum suggests an oncofetal mechanism in gene regulation linked to the anatomic site.

Restoring functional neurofibromin by protein transduction

In Neurofibromatosis 1 (NF1) germ line loss of function mutations result in reduction of cellular neurofibromin content (NF1+/−, NF1 haploinsufficiency). The Ras-GAP neurofibromin is a very large cytoplasmic protein (2818 AA, 319 kDa) involved in the RAS-MAPK pathway. Aside from regulation of proliferation, it is involved in mechanosensoric of cells. We investigated neurofibromin replacement in cultured human fibroblasts showing reduced amount of neurofibromin. Full length neurofibromin was produced recombinantly in insect cells and purified. Protein transduction into cultured fibroblasts was performed employing cell penetrating peptides along with photochemical internalization. This combination of transduction strategies ensures the intracellular uptake and the translocation to the cytoplasm of neurofibromin. The transduced neurofibromin is functional, indicated by functional rescue of reduced mechanosensoric blindness and reduced RasGAP activity in cultured fibroblasts of NF1 patients or normal fibroblasts treated by NF1 siRNA. Our study shows that recombinant neurofibromin is able to revert cellular effects of NF1 haploinsuffiency in vitro, indicating a use of protein transduction into cells as a potential treatment strategy for the monogenic disease NF1.

U-CH17P, -M, and -S, a new cell culture system for tumour diversity and progression in chordoma

Chordoma is a rare bone tumor with a known intrinsic heterogeneity. Here, we address this tumor heterogeneity in a new cell culture model for tumor diversity and progression in chordoma. The three cell lines U‐CH17P, U‐CH17M, and U‐CH17S were established from a primary sacral chordoma and its derived metastases, a soft tissue and a skin metastasis, respectively. The lesions had divergent differentiation patterns which are conserved in the derived cell lines making them a suitable in vitro model for the analysis of tumorigenesis in chordoma. A common feature of the three cell lines is the expression of typical chordoma markers, such as Brachyury, vimentin, cytokeratins, EMA and S100 protein. A comparison of the genomic aberrations by array comparative genomic hybridization of the cell lines and the corresponding parental tumor tissues revealed that the precursor cells of U‐CH17P, U‐CH17M and U‐CH17S were already present in the primary tumor. Therefore, we show that clonal diversity of this chordoma exists in the primary tumor and that not all of these subclones tend to metastasize. All cell lines had a CDKN2A loss. A comparison of the gene expression profiles of the cell lines revealed significant differences in the expression of several genes like MAGEC2 and SEMA6A known to be associated with the tendency to metastasize or proliferation and migration. Since the underlying mechanisms of tumor progression in chordoma are still largely unclear, the three U‐CH17 cell lines are a suitable in vitro model for elucidating chordoma oncobiology.