- Open Access
Attenuated Expression of DFFB is a Hallmark of Oligodendrogliomas with 1p-Allelic Loss
© McDonald et al; licensee BioMed Central Ltd. 2005
Received: 18 July 2005
Accepted: 12 September 2005
Published: 12 September 2005
Allelic loss of chromosome 1p is frequently observed in oligodendroglioma. We screened 177 oligodendroglial tumors for 1p deletions and found 6 tumors with localized 1p36 deletions. Several apoptosis regulation genes have been mapped to this region, including Tumor Protein 73 (p73), DNA Fragmentation Factor subunits alpha (DFFA) and beta (DFFB), and Tumor Necrosis Factor Receptor Superfamily Members 9 and 25 (TNFRSF9, TNFRSF25). We compared expression levels of these 5 genes in pairs of 1p-loss and 1p-intact tumors using quantitative reverse-transcriptase PCR (QRTPCR) to test if 1p deletions had an effect on expression. Only the DFFB gene demonstrated decreased expression in all tumor pairs tested. Mutational analysis did not reveal DFFB mutations in 12 tested samples. However, it is possible that DFFB haploinsufficiency from 1p allelic loss is a contributing factor in oligodendroglioma development.
Oligodendroglial tumors with allelic losses on 1p usually display loss of relatively long regions, a phenomenon that has made the identification of putative 1p tumor suppressor genes difficult [1–6]. However, the vast majority of reported oligodendroglioma cases with 1p-loss have involved the 1p36 region, with several breakpoints within the region observed [5–9]. It is important to note that several apoptotic genes have been mapped to 1p36. Diminished apoptosis has been recognized as one of the hallmarks of most types of cancer, representing one of the major ways known for a tumor-cell population to expand . Therefore, we tested TP73, TNFRSF9, TNFRSF25, DFFA, and DFFB, all of which are 1p genes involved in apoptosis, for differential expression in 1p-status subsets of oligodendroglioma. QRTPCR analysis of match-paired samples demonstrated that levels of DFFB were decreased in all 1p-allelic loss cases. In contrast, the other tested genes showed heterogeneous patterns of expression. This result suggests DFFB to be a key molecule affected by 1p-deletion in oligodendroglioma.
Materials and methods
The records of 177 patients who underwent treatment for oligodendroglial tumors at the University of Texas M.D. Anderson Cancer Center (UTMDACC) between 1981 and 2002 were collected and reviewed. These patients were initially diagnosed as having low-grade oligodendroglioma or mixed oligoastrocytoma, anaplastic oligodendroglioma or mixed oligoastrocytoma, or glioblastoma multiforme with significant oligodendroglial component by neuropathologists from UTMDACC and later confirmed by two of the authors (KA and GF). Mixed tumors were included in this study since clear pathologic discrimination between glioma subtypes is sometimes difficult, and as a group, oligoastrocytomas often have 1p deletions . In fact, both the oligodendroglial and astrocytic components of mixed tumors have been observed to have this genetic signature .
Tissue for DNA isolation was obtained from paraffin-embedded samples. Each tissue block was histologically assessed for tumor by a neuropathologist (KA). Sections were directly cut from the block for DNA isolation if at least 90% of the tissue was determined to be tumor. If the proportion of tumor was <90%, 10 to 20 unstained slides were prepared from the block and tumor tissue was dissected from normal tissue. DNA was isolated by digesting deparaffinized tumor sections for 3 to 5 days with proteinase K at 55°C (0.5 mg/ml in 100 mmol/L NaCl, 10 mmol/L Tris-HCl, pH 8.0, 25 mmol/L ethylenediaminetetraacetic acid, 0.5% sodium dodecyl sulfate), followed by a phenol:chloroform:isoamyl alcohol extraction and isopropanol precipitation.
Tissue for RNA isolation was obtained from fresh/frozen samples. Each frozen section was histologically assessed for tumor by a neuropathologist (GF or KA) and used only if at least 90% of the tissue was determined to be tumor. For RNA isolation, up to 50 mg of tissue was frozen in liquid nitrogen, crushed into powder using a mortar and pestle, and dissolved in 1 ml of Trizol® Reagent. 200 μl chloroform was added to the sample, vortexed at high speed for 15 seconds, and centrifuged at 12,000 × g for 15 minutes at 4°C. After transfer of the aqueous phase to fresh 1.5 ml Eppendorf tube, an equal volume of 70% ethanol was added and mixed by tube inversion. The sample was then loaded onto a QIAGEN RNeasy® mini column and centrifuged at 16,000 × g for 20 seconds (QIAGEN Sciences, Germantown, MD). The column was washed twice with 500 μl of QIAGEN's RPE buffer. RNA was eluated off the column in 50 μl of nuclease-free water. RNA was quantified using a spectrophotometer and qualitated with an Agilent BioAnalyzer 2100 (Agilent Technologies, Palo Alto, CA).
Detection of 1p allelic loss
Quantitative Reverse Transcription-Polymerase Chain Reaction (QRTPCR) Analysis
Initial experiments were performed to determine the valid range of RNA concentrations and to demonstrate the similarity of PCR efficiencies for each gene of interest compared to the endogenous control gene cyclophilin. To determine fold-changes in each gene, QRTPCR was performed on the ABI Prism 7700 using the commercially available gene expression assay for p73, DFFA, and DFFB (Hs00232088_m1, Hs00189336_m1, Hs00237077_m1, respectively) and the cylophilin Vic-labeled Pre-Developed Assay Reagent (Applied Biosystems, Foster City, CA) without multiplexing. In triplicate, we amplified 50 ng cDNA for each sample for each assay in a reaction containing 1× TaqMan® Universal PCR Master Mix without AmpErase UNG and 1× gene expression assay with the following cycling conditions: 10 minutes at 95°C, then 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. Calculations were performed using the δδCt method to determine fold-difference in 1p-loss cases relative to the matched 1p-intact cases. Fold changes for TNFSF5, TNFRSF9, TNFRSF11a, and TNFRSF25 were determined in a similar fashion, using commercially available gene expression assays (Hs00374176_m1, Hs00155512_m1, Hs00187189_m1, and Hs00237054_m1, respectively) and the 18S rRNA TaqMan® Endogenous Control (Hs99999901_s1).
Primer sets used for amplifying and sequencing the coding regions of DFFB.
F primer seq
R primer seq
Results and Discussions
The second part of our strategy included the identification of abrogated cellular pathways in oligodendrogliomas with 1p/19q allelic loss. The transcriptsomes of eight pairs of gender- and age-matched tumors were measured using a Pathway microarray consisting of 1,500 functionally characterized genes constructed in our Cancer Genomics Core Laboratory. We used paired sample tests (the Sign Rank test and the paired t-test) to identify differentially expressed genes. While the sign rank test uses the null hypothesis that the medians for the two classes are same, the paired t-test uses the null hypothesis that the means of the two classes are same. We recognize a gene as significant when the sample data for the gene gives a p-value less than 0.01 (99% confidence). This analysis revealed a number of genes that demonstrated robust differential expression (McDonald and Zhang, unpublished results).
We addressed the candidacy of DFFB as an oligodendroglioma tumor suppressor gene by mutation analysis. Twelve tumors with 1p-allelic loss were screened for mutations by sequencing the 1.2 kb coding region of DFFB. No coding region mutations were detected in any of the samples, which may indicate that haploinsufficiency of DFFB is enough of a genetic insult to contribute to tumorigenesis. In order to thoroughly test this hypothesis, it will be necessary to further investigate DFFB, perhaps by determining if the DFFB promoter has been hypermethylaed and/or if intronic sequence has been mutated in tumor samples.
DFFB-null mouse lines have been established via gene targeting . Resultant mice developed normally but their lymphocytes were more susceptible to DNA damage. These animal model experiments suggest that DFFB is a weak tumor suppressor, which may only manifest its function in the presence of stress and DNA damage. Brain tissue samples from three six-month-old specimens revealed neuropil spongiosis, but no tumor development was observed (data not shown). We are not clear at present the implication of the neuropil spongiosis phenotype. Consistent with our data, a sequencing effort in a neuroblastoma study did not reveal a tumor-specific mutation in DFFB . The gene expression level of DFFB was not analyzed in that study.
Thus, this study revealed that attenuated expression of the DFFB gene is a signature of oligodendrogliomas with 1p-allelic loss. Since DFFB contributes to both chromosomal condensation and DNA degradation during apoptosis, decreased expression of DFFB may subject cells to DNA damage stresses, which in turn may contribute to both tumorigenesis and better response to DNA damaging chemotherapy. Further studies are needed to investigate the role of the DFFB gene in the etiology of oligodendroglioma.
This work was partially supported by a gift from the "Anthony Bullock III Research fund".
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