Glioblastomas are characterized by an aggressive local growth pattern, a marked degree of invasiveness and poor prognosis. Tumor invasiveness is facilitated by the increased activity of proteolytic enzymes which are involved in destruction of the extracellular matrix of the surrounding healthy brain tissue. Elevated levels of matrix metalloproteinases (MMPs) were found in glioblastoma (GBM) cell-lines, as well as in GBM biopsies as compared with low-grade astrocytoma (LGA) and normal brain samples, indicating a role in malignant progression. A careful review of the available literature revealed that both the expression and role of several of the 23 human MMP proteins is controversely discussed and for some there are no data available at all. We therefore screened a panel of 15 LGA and 15 GBM biopsy samples for those MMPs for which there is either no, very limited or even contradictory data available. Hence, this is the first complete compilation of the expression pattern of all 23 human MMPs in astrocytic tumors. This study will support a better understanding of the specific expression patterns and interaction of proteolytic enzymes in malignant human glioma and may provide additional starting points for targeted patient therapy.

Gene expression studies related to cancer diagnosis and treatment are important. In order to conduct such experiment accurately, absolutely reliable housekeeping genes are essential to normalize cancer related gene expression. The most important characteristics of such genes are their presence in all cells and their expression levels remain relatively constant under different experimental conditions. However, no single gene of this group of genes manifests always stable expression levels under all experimental conditions. Incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression. Here, we examined (a) the degree of GAPDH expression regulation in Hep-1-6 mouse hepatoma and Hep-3-B and HepG2 human hepatocellular carcinoma cell lines as well as in human lung adenocarcinoma epithelial cell line (A-549) in addition to both HT-29, and HCT-116 colon cancer cell lines, under hypoxic conditions in vitro in comparison to other housekeeping genes like β-actin, serving as experimental loading controls, (b) the potential use of GAPDH as a target for tumor therapeutic approaches was comparatively examined in vitro on both protein and mRNA level, by western blot and semi quantitative RT-PCR, respectively. No hypoxia-induced regulatory effect on GAPDH expression was observed in the cell lines studied in vitro that were; Hep-1-6 mouse hepatoma and Hep-3-B and HepG2 human hepatocellular carcinoma cell lines, Human lung adenocarcinoma epithelial cell line (A-549), both colon cancer cell lines HT-29, and HCT-116. As it is the case for human hepatocellular carcinoma, mouse hepatoma, human colon cancer, and human lung adenocarcinoma, GAPDH represents an optimal choice of a housekeeping gene and/(or) loading control to determine the expression of hypoxia induced genes in tumors of different origin. The results confirm our previous findings in human glioblastoma that this gene is not an attractive target for tumor therapeutic approaches because of the lack of GAPDH regulation under hypoxia.

Glioblastomas (GBM), the most frequent malignant brain tumors in adults, are characterized by an aggressive local growth pattern and highly invasive tumor cells. This invasion is facilitated by expression of matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases. They mediate the degradation of protein components of the extracellular matrix. Twenty-three family members are known. Elevated levels of several of them have been reported in GBM. GBM cell-lines are used for in vitro studies of cell migration and invasion. Therefore, it is essential to know their MMP expression patterns. Only limited data for some of the cell-lines are published, yet. To fill the gaps in our knowledge would help to choose suitable model systems for analysis of regulation and function of MMPs during GBM tumorigenesis, cell migration and invasion.

Patients with autosomal recessive primary microcephaly have a small but architecturally normal brain containing a reduced number of neurons. Microcephalin and ASPM are two of the genes causing this disease. Both are centrosomal proteins involved in cell cycle regulation. Whereas microcephalin is a component of the DNA damage response and a repressor of telomerase function, ASPM is required for the proper formation of a central mitotic spindle and ensures symmetric, proliferative divisions of neuro-epithelial cells. Both proteins are also involved in the regulation of tumor growth. Microcephalin expression is reduced in breast cancer cell lines and human tumors of the ovary and prostate. Reduction in microcephalin mRNA expression correlates with increased chromosomal instability. ASPM mRNA is overexpressed in transformed human cell lines and tumors, and its increased expression is positively associated with proliferation of glioblastoma cells. Glioblastomas are the most prevalent malignant brain tumors in adults, characterized by increased invasiveness, an aggressive local growth pattern and short survival periods of patients. In this study, we analysed the expression of microcephalin mRNA and ASPM mRNA and protein in a panel of 15 glioblastomas and 15 astrocytoma WHO grade II by semi-quantitative RT-PCR, Western blotting and immunohistochemistry. Whereas microcephalin expression did not seem to be altered during glioma development, there was a clear increase in ASPM mRNA and protein expression that corresponded with the WHO grade of the tumor. Our findings are significant as the expression of ASPM may be used as a marker for glioma malignancy and represents a potential therapeutic target.

RAF proteins are well known oncoproteins. The B-RAF has been shown to be activated by mutations in a multitude of human cancers. Alterations of C-RAF expression are discussed to play a role in lung cancer. Only for A-RAF no link to tumorigenesis has been published so far. Malignant gliomas are the most prevalent primary brain tumors of adults. They are highly invasive and very difficult to treat, despite of surgery, γ-irradiation and chemotherapy. Although a role of the mitogenic Ras-RAF-MEK-ERK signalling cascade in brain tumor development is well established, there are only few reports available addressing alterations in RAF sequence or protein expression and function in human gliomas. We analysed the mutational status of A-RAF and B-RAF in human glioblastomas (GBM) by sequencing. Then we checked for RAF gene amplification by dot blot hybridization and examined RAF mRNA and protein expression patterns in human astrocytic gliomas of WHO grade II (LGA) and IV (GBM) by semiquantitative RT-PCR and Western blotting, respectively. The results were correlated with patients prognosis. Finally, we performed functional assays to address a putative function of A-RAF in glioma cell proliferation and migration. We showed that RAF mutations are a rare event in glioblastoma multiforme. A-raf gene amplification was more often detected and overexpression of all three RAF proteins on mRNA and protein level was regularly found in human malignant gliomas. Whereas A-RAF and C-RAF expression was negatively correlated with the patients prognosis, B-RAF expression had a positive effect. Since neither A-RAF, nor C-RAF expression had any influence on proliferation and migration of GBM cells, putative functions of C-RAF in angiogenesis and of A-RAF in regulation of metabolism are discussed. Our data indicate that RAF proteins might be valuable targets for small molecule therapies. However, initially specific functions of RAF during tumorigenesis have to be elucidated.

Glioblastoma multiforme (GBM) is the most prevalent, highly malignant, invasive and difficult-to-treat primary brain tumor in adults. At the genetic level, it is characterized by a high degree of chromosomal instability and aneuploidy. It has been shown that defects in the mitotic spindle checkpoint could lead to the development of aneuploidy as well as tumorigenesis. Additional proteins regulating sister chromatid cohesion could also be involved in maintaining the fidelity of chromosome segregation. One such protein is the precocious dissociation of sisters 5A (Pds5A), also known as sister chromatid cohesion protein 112. It is a nuclear protein, expressed from the S right through to the mitotic phase. It is highly conserved from yeast to man and plays a role in the establishment, maintenance and dissolution of sister chromatid cohesion. The mutation of Pds5A orthologs in lower organisms results in chromosome missegregation, aneuploidy and DNA repair defects. It is considered that such defects can cause either cell death or contribute to the development of cancer cells. Indeed, altered expression levels of Pds5A have been observed in tumors of the breast, kidney, oesophagus, stomach, liver and colon. Malignant gliomas, however, have not been analysed so far. Herein, we report on the cloning of Rattus norvegicus Pds5A and on the analysis of its expression pattern in rat tissue. We also show that Pds5A is significantly overexpressed at both the mRNA and protein level and that this overexpression correlates positively with the WHO grade of human gliomas. However, functional assays show that the siRNA-mediated knockdown of Pds5A affects sister chromatid cohesion but does not influence mitotic checkpoint function or the proliferation and survival of GBM cells. Although the mechanism by which Pds5A functions in GBM cells remains unclear, its overexpression in high grade gliomas implies that it could play a pivotal role during the development and progression of astrocytic tumors.