Abundance of Flt3 and its ligand in astrocytic tumors.

BACKGROUND: 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, b2 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.

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.

Dysregulation of iron protein expression in the G93A model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by selective loss of motor neurons which leads to progressive paralysis and death by respiratory failure. Although the cause of sporadic ALS is still unknown, oxidative stress is suggested to play a major role in the pathogenesis of this disease and of the rare familial form, which often exhibits mutations of the superoxide dismutase 1 (SOD1) gene. Since enhanced iron levels are discussed to participate in oxidative stress and neuronal death, we analyzed the expression levels of Fe-related mRNAs in a cell culture ALS model with the G93A mutation of SOD1. We observed an increased total iron content in G93A-SOD1 SH-SY5Y neuroblastoma cells compared to wild-type (WT)-SOD1 cells. mRNA expression for transferrin receptor 1 (TfR1) and divalent metal transporter 1 was increased in G93A-SOD1 cells, which was in accordance with higher iron uptake. Experiments with the iron chelator deferoxamine revealed a normal reaction of WT and mutant cells to cytoplasmic iron depletion, i.e. TfR1 upregulation, suggesting a basically conserved function of the iron-responsive element/iron regulatory protein (IRE/IRP) pathway, designed to adapt gene expression to iron levels. Expression levels of mitoferrin 1 and 2, frataxin, and iron-sulfur cluster scaffold protein were also significantly increased in G93A-SOD1 cells, suggesting higher mitochondrial iron import and utilization in biosynthetic pathways within the mitochondria. Moreover, expression of these transcripts was further enhanced, if G93A-SOD1 cells were differentiated by retinoic acid (RA). Since RA treatment increased cytoplasmic reactive oxygen species (ROS) levels in these cells, an IRE/IRP independent, ROS-mediated mechanism may account for dysregulation of iron-related genes.