PARP-1 protein expression in glioblastoma multiforme.
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SUBMITTER: Galia A
PROVIDER: S-EPMC3352138 | biostudies-literature | 2012 Feb
REPOSITORIES: biostudies-literature
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PARP-1 protein expression in glioblastoma multiforme.
Galia A A Calogero A E AE Condorelli R R Fraggetta F F La Corte A A Ridolfo F F Bosco P P Castiglione R R Salemi M M
European journal of histochemistry : EJH 20120227 1
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 nuc ...[more]
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Instead, VAV1 is found in non-tumoral astrocyte-like cells that are located either peritumoraly or perivascularly, suggesting that its expression is linked to synergistic signalling cross-talk between cancer and infiltrating cells. Conclusions/Significance: Interestingly, we show that the pattern of expression of VAV1 is a good prognostic factor to unveil populations of high-grade glioma patients with different survival and progression free survival rates. 1. Oligonucleotide microarray analyses Total RNAs were extracted using the Triazol reagent (Life Technologies, Gaithersburg, MD, USA) and purified with the RNeasy Mini kit (Qiagen, Valencia, CA, USA). The integrity of RNA samples obtained was assessed using the 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA). Double-stranded cDNAs and biotinylated cRNAs were synthesized using a T7-polyT primer and the BioArray RNA labelling Kit (Enzo Farningdale, NY, USA), respectively. Labelled RNAs were then fragmented and hybridised to HU-133A oligonucleotide arrays (Affymetrix, Santa Clara, CA, USA) according to standard Affymetrix protocols. After hybridization and washes, arrays were scanned using the Gene Array Scanner (Affymetrix), and the expression value for each probe set calculated using the MAS 5.0 software (Affymetrix). All examples had a scaling factor lower than threefold and 3’/5’ of GAPDH probe set <2.5. Gene levels were transformed to base two logarithms. A median normalization approach was applied. Only genes with al least three “present” calls across all samples were selected. All these steps were done at the Genomics and Proteomics Unit of the “Centro de Investigación del Cáncer, Salamanca”. 2. Microarray data analyses To visualize clusters of genes with similar expression patterns, we used a hierarchical clustering method (Cluster and TreeView software) based on the average-linkage method with the centred correlation metric [26]. A multidimensional scaling method (BRB Arrays Tools version 3.0) was also utilized by using Euclidean distance criteria [27]. Supervised learning was used to identify genes with statistically significant changes in expression among different classes by using the Significant Analysis of Microarrays (SAM) algorithm [28]. All data were permuted over 100 cycles by using the two-class (unpaired) and multi-class response format. Significant genes were selected based on the lowest false discovery ratio (between 0.6 and 0.9). In addition, nonparametric tests such as Wilcoxon rank sum test and Kruskal-Wallis test to compare more than two unpaired group were also used (SPSS 18, SPSS Inc). 3. Functional annotation of microarray data Probe sets showing significant expression change were functionally annotated and grouped according to biological function criteria using GeneOntology biological process descriptions. The functional analysis to identify the most relevant biological mechanism, pathways and functional categories in gene dada sets was generated using the Ingenuity Pathway software (Ingenuity Systems, Mountain View, CA, USA) available in the web (www.ingenuity.com) [29]. A functional network was considered significant when it fulfilled the following criteria: i) to have a minimal score of 15; ii) to have a minimum of 20 direct functional interactions among the network members. 4. Quantitative reverse transcription-PCR Total RNA was quantified in a RNA 6000 Nano Chip (Agilent Technologies) and quantitative PCR performed using the QuantiTect SYBR Green RT–PCR kit (Qiagen). To quantify VAV1 mRNA levels, we used two different sets of probes: PAIR A (5’-AAC AAC GGG AGG TTC ACC CT-3’ and 5’-GGT CCC TCA TGG CAT CCA-3’) and PAIR B (5’-AGC CAT TGG ACC CTT TCT ACG-3’ and 5’-GCC ATG GAC ATA GGG CTT CA-3’). Amplifications were performed using the iCycler apparatus (Bio-Rad Laboratories, California, USA). Analyses of data were done using the iCycler iQ Optical System Software, version 3.0a (Bio-Rad Laboratories). Primers to GAPDH were used as intersample normalizing controls. Variations in expression of VAV1 mRNA were represented as the mean value of the fold change respect the VAV1 expression levels detected in sample #19209 with both pairs of oligonucleotide primers. 5. Immunohistochemical analyses The VAV1 antibody was generated in rabbits using a synthetic peptide and purified by affinity chromatography in Bustelo’s laboratory. This antibody recognizes VAV1 proteins from humans and mice but it does not recognize other VAV family members (unpublished data). For immunostaining, tissue sections were washed thrice with Xylene and once with 100% ethanol, rehydrated by sequential changes in 80%, 70%, and 50% ethanol and a final incubation in phosphate-buffered saline (PBS). Each rehydrating step involved 3 min incubations with the indicated solutions. Endogenous peroxidases were quenched by the addition of a 3% H2O2 solution in methanol for 30 min at room temperature (RT). Tissue sections were subsequently washed twice with PBS. Antigen retrieval was performed by incubation in 1 mM EDTA for 30 min at 37°C. The slides were washed twice in PBS and blocked in blocking buffer (Zymed, CA, USA) for 30 min at RT. Specimens were then incubated with the primary antibody (1:250 dilution) in blocking buffer. After an 1 hr incubation at 37°C, slides were washed three times in PBS, incubated with a biotinylated secondary antibody for 30 min at 37°C, washed thrice in PBS, incubated with horseradish peroxidase-streptavidin for 30 min at 37°C, washed three times in PBS, and developed using the AEC substrate (Zymed). Slides were then washed twice in water, counterstained with hematoxilin (Zymed), washed again in water, and mounted with GVA (Zymed). Samples were analyzed by light microscopy and images acquired suing an Axiophot imaging system (Zeiss, Munich, Germany). 6. Fluorescence in situ hybridization analyses FISH experiments were carried out in 40 cases of glioblastoma multiforme (grade IV) positive for VAV1 expression. For this purpose, we performed dual-colour FISH analyses with locus-specific probes for centromere 7 (Abbott Molecualr, Des Plaines) exactly as previously described.[30] Polysomies were defined when more than 10% of the nuclei surveyed contained three or more CEP signals (chromosome-specific FISH probes that hybridize to highly repetitive human satellite DNA sequences, usually located near centromeres). 7. Immunohistochemistry and fluorescence in situ hybridization (FISH) in paraffin-embedded tumours Four um sections were cut from routinely processed paraffin blocks and mounted onto glass slides with a charged coating. Sections were dewaxed in Xylene and then rehydrated using increasing concentrations of alcohol before being rinsed briefly in water. Slides were heated 2 min in 1 mM EDTA (pH 9.0) in a microwavable pressure cooker. After antigen retrieval, slides were incubated 1 h at RT in a moist chamber with a primary antibody diluted in PBS supplemented with 10% foetal calf serum. Slides were incubated for 1 h with fluorochrome-conjugated antibodies to the appropriate IgG isotypes in a moist chamber in the dark. Finally, slides were washed thrice in PBS containing 0.5% Tween 20 three before FISH analysis. 8. Degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) analyses After the staining of tissue sections with VAV1 antibodies (see above), the regions of the tumour were identified, microdissected, and collected using the PALM® microscope system (P.A.L.M. Microlaser Technologies, Munich, Germany). The genomic DNA was extracted as indicated by Isola et al [31] with modifications to small DNA amounts. Those included the resuspension of the microdissected sections in extraction buffer followed by a digestion with proteinase K (0.6 mg/ml). All samples were resuspended in 10 ul of 10 mM Tris-HCl (pH 7.4) and 0.1 mM EDTA. DOP-PCR amplification was performed in two steps. For the first, low-stringency step, 1 ul of sample was added to 4 ul of buffer A (2.5 ul of 600 uM dNTPs (Roche, Pleasanton, CA), 0.5 ul of 10 uM DOP primer 5’-CCGACTCGAGNNNNNNNATGTGG-3’, where N= A, C, G, or T) [32] and 1 ul of 5x Sequenase Reaction Buffer (Amersham, Cleveland, OH). Reactions were performed using 5 cycles of 30ºC for 5 min, 37ºC for 2 min, and 96ºC for 2 min, adding 0.65 units of Sequenase in each 30ºC step. The first phase product was then subjected to the second step usin
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Project description:The Polycomb group (PcG) proteins play a critical role in histone mediated epigenetics which has been implicated in the malignant evolution of glioblastoma multiforme (GBM). By systematically interrogating The Cancer Genome Atlas (TCGA), we discovered widespread aberrant expression of the PcG members in GBM samples compared to normal brain. The most striking differences were upregulation of EZH2, PHF19, CBX8 and PHC2 and downregulation of CBX7, CBX6, EZH1 and RYBP. Interestingly, changes in EZH2, PHF19, CBX7, CBX6 and EZH1 occurred progressively as astrocytoma grade increased. We validated the aberrant expression of CBX6, CBX7, CBX8 and EZH2 in GBM cell lines by Western blotting and qRT-PCR, and further the aberrant expression of CBX6 in GBM tissue samples by immunohistochemical staining. To determine if there was functional significance to the diminished CBX6 levels in GBM, CBX6 was overexpressed in GBM cells resulting in decreased proliferative capacity. In conclusion, aberrant expression of PcG proteins in GBMs may play a role in the development or maintenance of the malignancy.
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Project description:Chromosomal abnormalities and genomic instability are common features of, and possible driving forces in, tumorigenesis. Recently, several mitotic proteins that are critical to proper chromosome segregation have been identified. Members of the Aurora kinase family have been identified as having important roles in mitosis; overexpression induces multicellularity and fosters polyploidy. As aneuploidy is a common feature of malignant gliomas, particularly glioblastomas (GBMs), we examined 25 prospectively collected GBMs to assess the role that overexpression of one member of this family, Aurora B, might have in the clinical behaviour of GBMs. Aurora B expression levels were markedly correlated with a shortened survival. Aurora B expression was not directly related to age, tumour proliferation status or to several common molecular changes found in GBMs. These results suggest that Aurora B may be a prognostic feature of impaired survival and a novel therapeutic target in some patients.
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Project description:Glioblastoma Multiforme (GBM) is the most frequent and lethal primary brain cancer. Due to its therapeutic resistance and aggressiveness, its clinical management is challenging. Platelet-derived Growth Factor (PDGF) genes have been enrolled as drivers of this tumour progression as well as potential therapeutic targets. As detailed understanding of the expression pattern of PDGF system in the context of GBM intra- and intertumoral heterogeneity is lacking in the literature, this study aims at characterising PDGF expression in different histologically-defined GBM regions as well as investigating correlation of these genes expression with parameters related to poor prognosis. Z-score normalised expression values of PDGF subunits from multiple slices of 36 GBMs, alongside with clinical and genomic data on those GBMs patients, were compiled from Ivy Glioblastoma Atlas Project - Allen Institute for Brain Science data sets. PDGF subunits show differential expression over distinct regions of GBM and PDGF family is heterogeneously expressed among different brain lobes affected by GBM. Further, PDGF family expression correlates with bad prognosis factors: age at GBM diagnosis, Phosphatase and Tensin Homolog deletion and Isocitrate Dehydrogenase 1 mutation. These findings may aid on clinical management of GBM and development of targeted curative therapies against this devastating tumour.
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Project description:BackgroundReprogramming of cancers into normal-like tissues is an innovative strategy for cancer treatment. Recent reports demonstrate that defined factors can reprogram cancer cells into pluripotent stem cells. Glioblastoma multiforme (GBM) is the most common and aggressive malignant brain tumor in humans. Despite multimodal therapy, the outcome for patients with GBM is still poor. Therefore, developing novel therapeutic strategy is a critical requirement.MethodsWe have developed a novel reprogramming method that uses a conceptually unique strategy for GBM treatment. We screened a kinase inhibitor library to find which candidate inhibitors under reprogramming condition can reprogram GBM cells into neurons. The induced neurons are identified whether functional and loss of tumorigenicity.ResultsWe have found that mTOR and ROCK kinase inhibitors are sufficient to reprogram GBM cells into neural-like cells and "normal" neurons. The induced neurons expressed neuron-specific proteins, generated action potentials and neurotransmitter receptor-mediated currents. Genome-wide transcriptional analysis showed that the induced neurons had a profile different from GBM cells and were similar to that of control neurons induced by established methods. In vitro and in vivo tumorigenesis assays showed that induced neurons lost their proliferation ability and tumorigenicity. Moreover, reprogramming treatment with ROCK-mTOR inhibitors prevented GBM local recurrence in mice.ConclusionThis study indicates that ROCK and mTOR inhibitors-based reprogramming treatment prevents GBM local recurrence. Currently ROCK-mTOR inhibitors are used as anti-tumor drugs in patients, so this reprogramming strategy has significant potential to move rapidly toward clinical trials.
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