Mdr Hamster-Cells Exhibiting Multiple Altered Gene-Expression - Effects of Dexniguldipine-Hcl (B859-35), Cyclosporine-a and Buthionine Sulfoximine

An actinomycin D selected, multidrug-resistant (MDR) hamster CHO subline showed strong expression of the P-glycoprotein and sorcin genes together with several other alterations such as a (i) reduced growth rate, (ii) lowered topoisomerase II, (iii) lowered glutathione-S-transferase-P gene expression, and (iv) the emergence of a 15.5 kDa protein. Besides high resistances to adriamycin, actinomycin D, and vincristine, we observed a lowered sensitivity towards bleomycin, a rather hydrophilic drug usually not involved in P-glycoprotein associated MDR. Moreover, the MDR subline showed a pronounced collateral (enhanced) sensitivity towards the sterically pure dihydropyridine anticancer drug dexniguldipine-HCl (B859-35) preventing its characterization for MDR modulation here. At a non-cytotoxic dose (10 muM) the immunosuppressive cyclic peptide cyclosporin A completely abolished the resistance to vincristine, partially reversed the resistance to teniposide and strongly enhanced the sensitivity towards bleomycin, while not influencing the drug sensitivities of the parental cell line. Buthionine sulfoximine (BSO), an agent depleting cellular glutathione levels, distinctly increased the sensitivity towards teniposide at nontoxic doses (50 muM) exclusively in the MDR subline, while it did not alter vincristine or bleomycin cytotoxicity

Abundance of Flt3 and its ligand in astrocytic tumors

C Eßbach,1 N Andrae,1 D Pachow,1 J-P Warnke,2 A Wilisch-Neumann,1 E Kirches,1 C Mawrin11Department of Neuropathology, Otto-von-Guericke University, Magdeburg, 2Department of Neurosurgery, Paracelsus Hospital, Zwickau, GermanyBackground: Molecular targeted therapies for astrocytic tumors are the subject of growing research interest, due to the limited response of these tumors, especially glioblastoma multiforme, to conventional chemotherapeutic regimens. Several of these approaches exploit the inhibition of receptor tyrosine kinases. To date, it has not been elucidated if fms-like tyrosine kinase-3 (Flt3) and its natural ligand (Flt3L) are expressed in astrocytic tumors, although some of the clinically intended small-molecule receptor tyrosine kinase inhibitors affect Flt3, while others do not. More importantly, the recent proof of principle for successful stimulation of the immune system against gliomas in preclinical models via local Flt3L application requires elucidation of this receptor tyrosine kinase pathway in these tumors in more detail. This therapy is based on recruitment of Flt3-positive dendritic cells, but may be corroborated by activity of this signaling pathway in glioma cells.Methods: Receptor and ligand expression was analyzed by real-time polymerase chain reaction in 31 astrocytic tumors (six diffuse and 11 anaplastic astrocytomas, 14 glioblastomas) derived from patients of both genders and in glioblastoma cell lines. The two most common activating mutations of the Flt3 gene, ie, internal tandem duplication and D835 point mutation, were assessed by specific polymerase chain reaction.Results: A relatively high abundance of Flt3L mRNA (4%–6% of the reference, β2 microglobulin) could be demonstrated in all tumor samples. Flt3 expression could generally be demonstrated by 40 specific polymerase chain reaction cycles and gel electrophoresis in 87% of the tumors, including all grades, although the small quantities of the receptor did not allow reliable quantification. Expression of both mRNAs was verified in the cell lines, excluding a derivation solely from contaminating lymphocytes or macrophages. No activating mutations were found.Conclusion: Our results warrant further analysis of endogenous Flt3 signaling in these tumors prior to application of immunotherapy in human patients.Keywords: fms-like tyrosine kinase-3, fms-like tyrosine kinase-3 ligand, glioblastoma multiforme, receptor tyrosine kinase

Resveratrol decreases B-cell lymphoma-2 expression and viability in GH3 pituitary adenoma cells of the rat

Benjamin Voellger,1 Elmar Kirches,2 Annette Wilisch-Neumann,2 Andreas Weise,1 Jorge Humberto Tapia-Perez,1 Rosita Rupa,1 Christian Mawrin,2 Raimund Firsching11Department of Neurosurgery, 2Department of Neuropathology, Otto von Guericke University, Magdeburg, GermanyObjective: Resveratrol is a phytoestrogen with various antiproliferative and proapoptotic effects. This in vitro study aimed to analyze the effect of resveratrol on the viability and expression of modulators of apoptosis in GH3 pituitary adenoma cells of the rat.Methods: GH3 cells were incubated with resveratrol concentrations from 20 to 100 µM for 48–72 hours. Cell viability was quantified using a hemocytometer. We assessed the ability of resveratrol to kill GH3 cells by an enzyme-linked immunosorbent assay (ELISA) of nucleosome liberation and by DNA degradation (unidimensional gel electrophoresis). Relative messenger RNA (mRNA) expression of survivin, B-cell lymphoma-2 protein (BCL-2) and BCL-2-associated X protein (BAX) normalized to β2 microglobulin was measured using quantitative real-time polymerase chain reaction (qRT-PCR).Results: GH3 cell survival significantly decreased with increasing concentrations of resveratrol. In GH3 cells treated with 100 µM resveratrol, ELISA demonstrated a significant rise of nucleosome liberation, which typically occurs during apoptosis. In parallel, gel electrophoresis showed degradation of DNA into random fragments, pointing to a necrotic mode of cell death in most GH3 cells. In GH3 cells treated with 100 µM resveratrol, qRT-PCR detected a significant decrease of BCL-2 mRNA expression and a decrease of survivin mRNA expression, whereas a change of BAX mRNA expression could not be found. The BAX/BCL-2 ratio was significantly increased in GH3 cells after resveratrol treatment.Conclusions: Resveratrol reduces GH3 cell viability in a dose-dependent manner by inducing nonapoptotic cell death and apoptosis. Apoptosis in GH3 cells is probably mediated by resveratrol-dependent downregulation of apoptosis inhibitors, namely BCL-2 and possibly survivin. Further investigation of the potential effects of resveratrol on pituitary adenoma cells is warranted.Keywords: resveratrol, phytoestrogens, viability, GH3 cell

The spectrum of age-related small vessel diseases: potential overlap and interactions of amyloid and non-amyloid vasculopathies

Deep perforator arteriopathy (DPA) and cerebral amyloid angiopathy (CAA) are the commonest known cerebral small vessel diseases (CSVD), which cause ischaemic stroke, intracebral haemorrhage (ICH) and vascular cognitive impairment (VCI). While thus far mainly considered as separate entities, we here propose that DPA and CAA share similarities, overlap and interact, so that "pure" DPA or CAA are extremes along a continuum of age-related small vessel pathologies. We suggest blood-brain barrier (BBB) breakdown, endothelial damage and impaired perivascular β-amyloid (Aβ) drainage are hallmark common mechanisms connecting DPA and CAA. We also suggest a need for new biomarkers (e.g. high-resolution imaging) to deepen understanding of the complex relationships between DPA and CAA. This article is protected by copyright. All rights reserved

Invited Review: The spectrum of age-related small vessel diseases: potential overlap and interactions of amyloid and nonamyloid vasculopathies

Deep perforator arteriopathy (DPA) and cerebral amyloid angiopathy (CAA) are the commonest known cerebral small vessel diseases (CSVD), which cause ischaemic stroke, intracebral haemorrhage (ICH) and vascular cognitive impairment (VCI). While thus far mainly considered as separate entities, we here propose that DPA and CAA share similarities, overlap and interact, so that ‘pure’ DPA or CAA are extremes along a continuum of age-related small vessel pathologies. We suggest blood-brain barrier (BBB) breakdown, endothelial damage and impaired perivascular β-amyloid (Aβ) drainage are hallmark common mechanisms connecting DPA and CAA. We also suggest a need for new biomarkers (e.g. high-resolution imaging) to deepen understanding of the complex relationships between DPA and CAA.</p

miRNA-145 is downregulated in atypical and anaplastic meningiomas and negatively regulates motility and proliferation of meningioma cells

Meningiomas are frequent, mostly benign intracranial or spinal tumors. A small subset of meningiomas is characterized by histological features of atypia or anaplasia that are associated with more aggressive biological behavior resulting in increased morbidity and mortality. Infiltration into the adjacent brain tissue is a major factor linked to higher recurrence rates. The molecular mechanisms of progression, including brain invasion are still poorly understood. We have studied the role of micro-RNA 145 (miR-145) in meningiomas and detected significantly reduced miR-145 expression in atypical and anaplastic tumors as compared with benign meningiomas. Overexpression of miR-145 in IOMM-Lee meningioma cells resulted in reduced proliferation, increased sensitivity to apoptosis, reduced anchorage-independent growth and reduction of orthotopic tumor growth in nude mice as compared with control cells. Moreover, meningioma cells with high miR-145 levels had impaired migratory and invasive potential in vitro and in vivo. PCR-array studies of miR145-overexpressing cells suggested that collagen type V alpha (COL5A1) expression is downregulated by miR-145 overexpression. Accordingly, COL5A1 expression was significantly upregulated in atypical and anaplastic meningiomas. Collectively, our data indicate an important anti-migratory and anti-proliferative function of miR-145 in meningiomas

A Novel 2.5D Culture Platform to Investigate the Role of Stiffness Gradients on Adhesion-Independent Cell Migration

Current studies investigating the role of biophysical cues on cell migration focus on the use of culture platforms with static material parameters. However, migrating cells in vivo often encounter spatial variations in extracellular matrix stiffness. To better understand the effects of stiffness gradients on cell migration, we developed a 2.5D cell culture platform where cells are sandwiched between stiff tissue culture plastic and soft alginate hydrogel. Under these conditions, we observed migration of cells from the underlying stiff substrate into the alginate matrix. Observation of migration into alginate in the presence of integrin inhibition as well as qualitative microscopic analyses suggested an adhesion-independent cell migration mode. Observed migration was dependent on alginate matrix stiffness and the RhoA-ROCK-myosin-II pathway; inhibitors specifically targeting ROCK and myosin-II arrested cell migration. Collectively, these results demonstrate the utility of the 2.5D culture platform to advance our understanding of the effects of stiffness gradients and mechanotransductive signaling on adhesion-independent cell migration