IDH1 mutations is prognostic marker for primary glioblastoma multiforme but MGMT hypermethylation is not prognostic for primary glioblastoma multiforme

Purpose

To establish the frequency of IDH1 mutations and MGMT methylation in primary glioblastomas.

Experimental design

We screened primary glioblastoma multiforme (GBM) in a population-based study for IDH1 mutations and MGMT methylation and correlated them with clinical data.

Results

IDH1 mutations were detected in 5 of 40 primary glioblastomas (12,5%). Primary GBM patients carrying IDH1 mutations were significantly younger, mean age of 41 ± 5.06 years, than patients with wild-type IDH1, mean age of 57 ± 2,29 years, p = 0.011. The mean survival time of all GBM patients with and without IDH1 mutations was 19 months (5 cases) and 16 months (35 cases), respectively (p > 0,05). MGMT methylation was detected in 13 of the 40 patients (32,5%). MGMT-promoter methylation did not correlate with overall survival (OS; p > 0,05).

Conclusion

In summary, our study is the first study to investigate the IDH1 mutation status and MGMT methylation in primary GBMs in Turkish population and confirmed IDH1 mutation as a genetic marker for also primary GBMs. Our data are still insufficient for definite ascertainment; and our preliminary results suggest: IDH1 status shows an association with younger age and there is a lack of association between IDH1 mutation and survival time. Furthermore MGMT promoter methylation had no prognostic value and lower frequency in primary glioblastomas.     

Element distribution is altered in a zone surrounding human glioblastoma multiforme

Recent data indicate that A₁ adenosine receptor (A₁AR) density is increased in a zone surrounding human and experimental gliomas. On the contrary, tumor tissue and adjacent brain tissue show low to intermediate A₁AR densities. In order to assess whether changes in A₁AR expression are indicating further processes of a chemical reorganization of the peritumoral zone, we investigated element concentrations and distribution patterns of copper and zinc in six human glioblastoma multiforme (GBM) specimens by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). Uranium and lead were used as external standards.

Copper and zinc levels were increased in a peritumoral zone corresponding to the region of elevated A₁AR density. They showed a lower density in the solid tumor in comparison to surrounding brain tissue, although the cellular density was higher within GBM.

Our findings suggest that the immediate vicinity of GBM is characterized by increased levels of copper and zinc supporting the view that higher A₁AR density surrounding GBM is not an isolated alteration of peritumoral tissue but an indicator of complex changes in the vicinity of infiltrative tumors. Further research is needed to explore the pathophysiological consequences of altered peritumoral element distribution.     

GAPDH is not regulated in human glioblastoma under hypoxic conditions

Background

Gene expression studies related to cancer diagnosis and treatment are becoming more important. Housekeeping genes that are absolutely reliable are essential for these studies to normalize gene expression. An 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 regulation of GAPDH expression in human glioblastoma cells 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 and (c) differences in GAPDH expression between low-grade astrocytomas and glioblastomas, for which modest and severe hypoxia, respectively, have been previously demonstrated. GAPDH and β-actin expression was comparatively examined in vivo in human low-grade astrocytoma and glioblastoma on both protein and mRNA level, by Western blot and semiquantitative RT-PCR, respectively. Furthermore, the same proteins were determined in vitro in U373, U251 and GaMG human glioblastoma cells using the same methods. HIF-1α protein regulation under hypoxia was also determined on mRNA level in vitro in GaMG and on protein level in U251, U373 and GaMG cells.

Results

We observed no hypoxia-induced regulatory effect on GAPDH expression in the three glioblastoma cell lines studied in vitro. In addition, GAPDH expression was similar in patient tumor samples of low-grade astrocytoma and glioblastoma, suggesting a lack of hypoxic regulation in vivo.

Conclusion

GAPDH represents an optimal choice of a housekeeping gene and/or loading control to determine the expression of hypoxia induced genes at least in glioblastoma. Because of the lack of GAPDH regulation under hypoxia, this gene is not an attractive target for tumor therapeutic approaches in human glioblastoma.     

PARP-1 protein expression in glioblastoma multiforme

One of the most common type of primary brain tumors in adults is the glioblastoma multiforme (GBM) (World Health Organization grade IV astrocytoma). It is the most common malignant and aggressive form of glioma and it is among the most lethal ones. Poly (ADP-ribose) polymerase 1 (PARP-1) gene, located to 1q42, plays an important role for the efficient maintenance of genome integrity. PARP-1 protein is required for the apoptosis-inducing factor (AIF) translocation from the mitochondria to the nucleus. PARP-1 is proteolytically cleaved at the onset of apoptosis by caspase-3. Microarray analysis of PARP-1 gene expression in more than 8,000 samples revealed that PARP-1 is more highly expressed in several types of cancer compared with the equivalent normal tissues. Overall, the most differences in PARP-1 gene expression have been observed in breast, ovarian, endometrial, lung, and skin cancers, and non-Hodgkin's lymphoma. We evaluated the expression of PARP-1 protein in normal brain tissues and primary GBM by immunohistochemistry. Positive nuclear PARP-1 staining was found in all samples with GBM, but not in normal neurons from controls (n=4) and GBM patients (n=27). No cytoplasmic staining was observed in any sample. In conclusion, PARP-1 gene is expressed in GBM. This finding may be envisioned as an attempt to trigger apoptosis in this tumor, as well as in many other malignancies. The presence of the protein exclusively at the nucleus further support the function played by this gene in genome integrity maintenance and apoptosis. Finally, PARP-1 staining may be used as GBM cell marker.     

Equine herpesvirus type 1-mediated oncolysis of human glioblastoma multiforme cells

The cytolytic animal virus equine herpesvirus type 1 (EHV-1) was evaluated for its oncolytic potential against five human glioblastoma cell lines. EHV-1 productively infected four of these cell lines, and the degree of infection was positively correlated with glioma cell death. No human major histocompatibility complex class 1 (MHC-I) was detected in the resistant glioma line, while infection of the susceptible glioma cell lines, which expressed human MHC-I, were blocked with antibody to MHC-I, indicating that human MHC-I acts as an EHV-1 entry receptor on glioma cells.     

AP-1 membrane-cytoplasm recycling regulated by mu1A-adaptin.

The adaptor protein complex AP-1 mediates vesicular protein sorting between the trans Golgi network and endosomes. AP-1 recycles between membranes and the cytoplasm together with clathrin during transport vesicle formation and vesicle uncoating. AP-1 recycles independent of clathrin, indicating binding to unproductive membrane domains and premature termination of vesicle budding. Membrane recruitment requires ADP ribosylation factor-1-GTP, a transmembrane protein containing an AP-1-binding motif and phosphatidyl-inositol phosphate (PI-4-P). Little is known about the regulation of AP-1 membrane-cytoplasm recycling. We identified the N-terminal domain of micro1A-adaptin as being involved in the regulation of AP-1 membrane-cytoplasm recycling by constructing chimeras of micro1A and its homologue micro2. The AP-1* complex containing this mu2-micro1A chimera had slowed down recycling kinetics, resulting in missorting of mannose 6-phosphate receptors. The N-terminal domain is only accessible from the cytoplasmic AP-1 surface. None of the proteins known to influence AP-1 membrane recycling bound to this micro1A domain, indicating the regulation of AP-1 membrane-cytoplasm recycling by an yet unidentified cytoplasmic protein.     

Investigation of serum proteome alterations in human glioblastoma multiforme

Glioblastoma multiforme (GBM) or grade IV astrocytoma is the most common and lethal adult malignant brain tumor. The present study was conducted to investigate the alterations in the serum proteome in GBM patients compared to healthy controls. Comparative proteomic analysis was performed employing classical 2DE and 2D‐DIGE combined with MALDI TOF/TOF MS and results were further validated through Western blotting and immunoturbidimetric assay. Comparison of the serum proteome of GBM and healthy subjects revealed 55 differentially expressed and statistically significant (p <0.05) protein spots. Among the identified proteins, haptoglobin, plasminogen precursor, apolipoprotein A‐1 and M, and transthyretin are very significant due to their functional consequences in glioma tumor growth and migration, and could further be studied as glioma biomarkers and grade‐specific protein signatures. Analysis of the lipoprotein pattern indicated elevated serum levels of cholesterol, triacylglycerol, and low‐density lipoproteins in GBM patients. Functional pathway analysis was performed using multiple software including ingenuity pathway analysis (IPA), protein analysis through evolutionary relationships (PANTHER), database for annotation, visualization and integrated discovery (DAVID), and GeneSpring to investigate the biological context of the identified proteins, which revealed the association of candidate proteins in a few essential physiological pathways such as intrinsic prothrombin activation pathway, plasminogen activating cascade, coagulation system, glioma invasiveness signaling, and PI3K signaling in B lymphocytes. A subset of the differentially expressed proteins was applied to build statistical sample class prediction models for discrimination of GBM patients and healthy controls employing partial least squares discriminant analysis (PLS‐DA) and other machine learning methods such as support vector machine (SVM), Decision Tree and Naïve Bayes, and excellent discrimination between GBM and control groups was accomplished.     

Loss of heterozygosity on chromosome 10 in human glioblastoma multiforme

Recessive mutations, revealed by loss of the wild-type allele, have been associated with the development of a variety of cancers in children and adults. Polymorphic chromosome 10 markers were used to screen paired tumor and lymphocyte DNA samples in 13 patients with glioblastoma multiforme. Ten patients showed loss of constitutional heterozygosity in the tumor samples. This finding suggests that a recessive gene involved in the development of glioblastoma multiforme is present on chromosome 10.     

APRIL is increased in serum of patients with brain glioblastoma multiforme

A PRoliferation-Inducing Ligand (APRIL) is a cytokine with the ability to induce tumorigenesis. The aim of the study was to measure serum APRIL levels in patients with brain glioblastoma multiforme. Twenty five patients with brain tumor and a control group of 25 subjects took part in the study. APRIL was measured by the enzyme-linked immunosorbent method. The study showed increased APRIL levels in the serum of patients with brain glioblastoma multiforme compared to the control group (p < 0.05). However, there was no significant difference in the level of this cytokine between groups of patients divided according to their clinical state and tumor size (p > 0.05). Inflammation parameters such as C-reactive protein (CRP) and polymorphonuclear leucocytes (PMN) were also increased in patients with brain tumor compared to controls (p < 0.05). There was a significant correlation between APRIL and CRP and PMN (p < 0.05). Results from the study suggest that APRIL may play a role in the pathogenesis of brain glioblastoma multiforme. It is possible that anti-APRIL therapy might be useful in this disease. However, this cytokine cannot be regarded as a marker of tumor size or of severity of the clinical condition of patients.     

Expression of adipose differentiation-related protein (ADRP) is conjointly regulated by PU.1 and AP-1 in macrophages

ADRP is associated with intracellular lipid droplets. We demonstrate the regulatory mechanism for ADRP expression in RAW264.7 macrophages. The ADRP mRNA expression was stimulated by PMA, and synergistically enhanced in association with its protein level in the presence of lipids. A proteasome inhibitor protected the protein from degradation under the lipid-free conditions. One of the possible sites of the PMA action was proved to be an Ets/AP-1 element in the promoter, since mutations of this site reduced the PMA-induced promoter activity, and ligation of this element led to a significant increase in the PMA-responsiveness of homologous or heterologous promoters. Mutations of this site diminished the synergistic effect on the promoter activity induced by PMA and oleic acid, suggesting a possible interaction between this site and the downstream PPARdelta site. EMSA revealed that PU.1 and AP-1 conjointly bound to this site. The juxtaposition of the two sequences was requisite for full activity, since spacer sequences between them decreased the PMA-induced activity. PI3 kinase inhibitor was found to reduce the PMA-induced mRNA expression and promoter activity in parallel with PU.1/AP-1 complex formation on EMSA. From these results, we concluded that the Ets/AP-1 site is an important cis-acting element that regulates the ADRP gene expression in macrophages.     

AGAP2-AS1 serves as an oncogenic lncRNA and prognostic biomarker in glioblastoma multiforme

Long noncoding RNA (lncRNA) AGAP2 antisense RNA 1 (AGAP2-AS1) has been suggested to function as an oncogenic lncRNA in lung cancer, breast cancer, and anaplastic glioma. However, the expression pattern and molecular mechanism of AGAP2-AS1 in glioblastoma multiforme (GBM) remains unknown. The purpose of this study is to present more evidence about the clinical and biological function of AGAP2-AS1 in GBM. In our results, we found AGAP2-AS1 expression was increased in GBM compared with adjacent normal brain tissues or low-grade glioma tissues, and there was no significantly different between low-grade glioma tissues and normal tissues. Kaplan-Meier survival analysis indicated patients with GBM having high-expression of AGAP2-AS1 had shorter overall survival time than those with low expression of AGAP2-AS1. The loss-of-function studies showed that downregulation of AGAP2-AS1 depressed cell proliferation, migration, and invasion, and promoted cell apoptosis in GBM. In summary, AGAP2-AS1 is a prognostic biomarker for patients with GBM, and functions as an oncogenic lncRNA to modulate GBM cell proliferation, apoptosis, migration, and invasion, which suggests that AGAP2-AS1 is potential therapeutic target for GBM.     

A developmentally regulated, putative serine/threonine protein kinase is essential for development in Dictyostelium.

Using PCR technology, we have cloned parts of three developmentally regulated putative serine/threonine kinases from Dictyostelium. All show significant homology to members of the cAMP-dependent protein kinase A/protein kinase C subfamilies. A genomic clone encoding one of these, DdPK3, has been isolated and sequenced. The open reading frame encodes a protein of 648 amino acids with the conserved kinase domain in the C-terminal half. The protein encoded by this gene is unusual in that it contains long homopolymer runs in the N-terminal half of the protein, including a long run of 88 amino acids in which 73 are glutamine residues. To examine the function of DdPK3, a gene disruption was created via homologous recombination. Ddpk3- cells do not aggregate by themselves but will co-aggregate with wild-type cells. However, after aggregation these cells are 'sloughed off' and do not proceed further through development, but are found as a discrete mass alongside the fruiting body formed by the wild-type cells. Analysis of signal transduction pathways indicates that cAMP pulse-induced expression of aggregation stage-specific genes is normal in Ddpk3- cells, as is induction of the prestalk gene Ddras in single cell assays. However, cAMP induction of the late promoters of cAMP receptor cAR1 and of two prespore-specific genes is absent under similar conditions. These cells show normal activation of adenylate cyclase and normal phosphorylation of the G alpha protein G alpha 2 in response to cAMP. The possible role of DdPK3 in Dictyostelium development is discussed.