Gene expression profile of glioblastoma multiforme invasive phenotype points to new therapeutic targets.
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ABSTRACT:
SUBMITTER: Hoelzinger DB
PROVIDER: S-EPMC1490313 | biostudies-literature | 2005 Jan
REPOSITORIES: biostudies-literature
Publications
Gene expression profile of glioblastoma multiforme invasive phenotype points to new therapeutic targets.
Hoelzinger Dominique B DB Mariani Luigi L Weis Joachim J Woyke Tanja T Berens Theresa J TJ McDonough Wendy S WS Sloan Andrew A Coons Stephen W SW Berens Michael E ME
Neoplasia (New York, N.Y.) 20050101 1
The invasive phenotype of glioblastoma multiforme (GBM) is a hallmark of malignant process, yet molecular mechanisms that dictate this locally invasive behavior remain poorly understood. Gene expression profiles of human glioma cells were assessed from laser capture-microdissected GBM cells collected from paired patient tumor cores and white matter-invading cell populations. Changes in gene expression in invading GBM cells were validated by quantitative reverse transcription polymerase chain rea ...[more]
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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). 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