S7821 Sigma-AldrichCpGenome Universal Methylated DNA

RRP22 (Ras-related protein on chromosome 22) has been suggested as a candidate tumor suppressor in human cancers. Investigating a panel of 70 human gliomas, we found a frequent decrease in the RRP22 mRNA expression levels (67%), preferentially in high-grade gliomas [World Health Organization (WHO) grades III and IV] as compared with low-grade gliomas (WHO grade II). Moreover, reduced RRP22 mRNA expression was associated with shorter overall survival in 180 glioblastoma patients included in the National Institutes of Health Repository for Molecular Brain Neoplasia Data (NIH REMBRANDT) database. Decreased RRP22 expression levels were in part explained by 5'-CpG island hypermethylation and increased by the treatment with the demethylating agent 5-aza-2'-deoxycytidine in glioblastoma cell lines. In addition, the in vitro treatment with the histone deacetylase inhibitor trichostatin A alone resulted in RRP22 reexpression as well as a significant increase in the levels of RRP22 promoter DNA bound to pan-acetylated histone H3 and H4. Moreover, in primary human glioblastomas, we observed an increase of H3K9me3-bound and a decrease of pan-Ac-H3-bound RRP22 in comparison with non-neoplastic brain tissue, consistent with a heterochromatinization of the RRP22 promoter. Taken together, our findings demonstrate that both 5'-CpG island hypermethylation and histone modifications contribute to the frequent and prognostically unfavorable transcriptional downregulation of RRP22 in malignant gliomas.

Closed-tube PCR methods (sometimes referred to as in-tube PCR methods) for locus-specific DNA -methylation analysis are methodologies in which the amplification and analysis of bisulphite-modified DNA take place in one tube without the need to remove the PCR products for further analysis. Closed-tube methodologies lend themselves to high-throughput applications and molecular diagnostics but are also applicable as a research tool. We review three closed-tube methodologies, methylation-sensitive high-resolution melting (MS-HRM), MethyLight, and sensitive melting after real-time analysis - methylation-specific PCR (SMART-MSP). Closed-tube detection can be performed by simultaneously amplifying both methylated and unmethylated templates and subsequent melting curve analysis (MS-HRM). Alternatively, methylation-specific primers are used in real-time quantitative PCR and monitored either by a fluorescent hydrolysis probe (MethyLight) or using a double-stranded DNA binding fluorescent dye with a subsequent quality control step by melting curve analysis (SMART-MSP).

Heterogeneous DNA methylation leads to difficulties in accurate detection and quantification of methylation. Methylation-sensitive high resolution melting (MS-HRM) is unique among regularly used methods for DNA methylation analysis in that heterogeneous methylation can be readily identified, although not quantified, by inspection of the melting curves. Bisulfite pyrosequencing has been used to estimate the level of heterogeneous methylation by quantifying methylation levels present at individual CpG dinucleotides. Sequentially combining the two methodologies using MS-HRM to screen the amplification products prior to bisulfite pyrosequencing would be advantageous. This would not only replace the quality control step using agarose gel analysis prior to the pyrosequencing step but would also provide important qualitative information in its own right. We chose to analyze DAPK1 as it is an important tumor suppressor gene frequently heterogeneously methylated in a number of malignancies, including chronic lymphocytic leukemia (CLL). A region of the DAPK1 promoter was analyzed in ten CLL samples by MS-HRM. By using a biotinylated primer, bisulfite pyrosequencing could be used to directly analyze the samples. MS-HRM revealed the presence of various extents of heterogeneous DAPK1 methylation in all CLL samples. Further analysis of the biotinylated MS-HRM products by bisulfite pyrosequencing provided quantitative information for each CpG dinucleotide analyzed, and confirmed the presence of heterogeneous DNA methylation. Whereas each method could be used individually, MS-HRM and bisulfite pyrosequencing provided complementary information for the assessment of heterogeneous methylation.

A total of 343 brain tumors were studied for IDH1 and IDH2 mutations by direct sequencing and for protein expression by immunohistochemistry with mIDH1(R132H) antibody. Of these, 287 were gliomas (17 pilocytic astrocytomas, 13 grade II and 5 grade III astrocytomas, 167 primary (pGBMs) and 19 secondary (sGBMs) glioblastomas, 36 grade II and 26 grade III oligodendrogliomas and 4 grade II-III oligoastrocytomas). In gliomas, IDH1 mutations at codon R132 were identified in 22.3%, of which 93.7% were c.395G>A (p.R132H). Mutations were more frequent in oligodendrogliomas (53.2%) than in astrocytic tumors (22.8%) and in sGBMs (84.2%) upon pGBMs (1.8%). There was a statistically significant correlation between mIDH1(R132H) antibody immunostaining and the relevant mutation c.395G>A (p.R132H) (P = 0.0001). No mutations were identified in non-glial tumors which were also negative to immunohistochemistry, with the exception of one PNET. A c.515G>T (p.R172M) mutation of the IDH2 gene was only identified in a grade II oligodendroglioma patient which was wild-type for IDH1. A direct correlation with MGMT promoter hypermethylation status and an inverse correlation with EGFR amplification was found, whereas the relationships with 1p/19q co-deletion and TP53 mutations only showed a trend toward correlation. In all gliomas, a positive correlation was found between IDH1 mutations and a young age (P = 0.0001). In contrast, a correlation with overall survival could only be obtained in low-grade gliomas. Immunohistochemistry appeared to be useful in differential diagnoses, especially toward non-tumor pathologic nervous tissue, and in recognizing infiltrating glioma cells. The mIDH1(R132H) antibody positivity was complementary with Cyclin D1 expression.

The suppressor of cytokine signaling 3 (SOCS3) gene is one of eight structurally related genes of the SOCS family and has been suggested to function as a tumor suppressor by inhibition of the JAK/STAT signaling pathway. We investigated 60 human gliomas of different histological types for SOCS3 alterations and found frequent SOCS3 promoter hypermethylation and transcriptional downregulation. However, SOCS3 promoter hypermethylation was virtually absent in primary glioblastomas, which are characterized by frequent epidermal growth factor receptor (EGFR) amplification and overexpression. Assessment of the relationship between SOCS3 and EGFR aberrations revealed that SOCS3 promoter hypermethylation was inversely related to both the EGFR gene dosage as well as the EGFR protein expression, thus suggesting SOCS3 inactivation as a mechanism substituting for EGFR activation in a subset of gliomas. In support of this hypothesis, stable shRNA-mediated SOCS3 knock-down in U251 glioblastoma cells resulted in an activation of EGFR-related signaling pathways, i.e. an increase in the activation levels of STAT3, FAK and to a lesser extent MAPK, while the AKT phosphorylation levels remained unaffected. Functionally, SOCS3-depletion caused strongly increased tumor cell invasion with no obvious effect on tumor cell proliferation. In summary, our findings suggest that SOCS3 inactivation by promoter hypermethylation is mutually exclusive to EGFR activation in gliomas and preferentially promotes glioma cell invasion through STAT3 and FAK activation.

Alterations of the p53 pathway are among the most frequent aberrations observed in human cancers. We have performed an exhaustive analysis of TP53, p14, p15, and p16 status in a large series of 143 soft tissue sarcomas, rare tumors accounting for around 1% of all adult cancers, with complex genetics. For this purpose, we performed genomic studies, combining sequencing, copy number assessment, and expression analyses. TP53 mutations and deletions are more frequent in leiomyosarcomas than in undifferentiated pleomorphic sarcomas. Moreover, 50% of leiomyosarcomas present TP53 biallelic inactivation, whereas most undifferentiated pleomorphic sarcomas retain one wild-type TP53 allele (87.2%). The spectrum of mutations between these two groups of sarcomas is different, particularly with a higher rate of complex mutations in undifferentiated pleomorphic sarcomas. Most tumors without TP53 alteration exhibit a deletion of p14 and/or lack of mRNA expression, suggesting that p14 loss could be an alternative genotype for direct TP53 inactivation. Nevertheless, the fact that even in tumors altered for TP53, we could not detect p14 protein suggests that other p14 functions, independent of p53, could be implicated in sarcoma oncogenesis. In addition, both p15 and p16 are frequently codeleted or transcriptionally co-inhibited with p14, essentially in tumors with two wild-type TP53 alleles. Conversely, in TP53-altered tumors, p15 and p16 are well expressed, a feature not incompatible with an oncogenic process.

We analyzed the methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter in World Health Organization (WHO) grade III gliomas in association with other molecular markers to evaluate their prevalence.

Polymerase-mediated single-base extension (SBE) of primers using a fluorescently labeled 2',3'-dideoxynucleotide triphosphate terminator was originally commercialized as SNaPshot for analysis of single-nucleotide polymorphisms by capillary electrophoresis (CE). Application of this general method to bisulfite-converted/PCR-amplified genomic DNA (gDNA) to analytically infer polymorphic methylation status (i.e., 5-methylcytosine [5mC] vs. cytosine [C]) in CpG-rich regions of gDNA has been noted previously by others to be limited by CE mobility-shifted peaks for SBE products derived from guanine (G)/adenine (A)-mixed-base primers used to hybridize to possible polymorphic sites (i.e., C vs. thymine [T] resulting from 5mC vs. C, respectively). This limitation is precluded in the current study by using novel SNaPshot primers modified with N(6)-methoxy-2,6-diaminopurine (K), which was originally described in 1991 by Brown and Lin as a unique adenine-guanine analog capable of participating in three H-bonds with C or T in DNA. Oligonucleotides modified by K as a bispecific complement for C/T are commercially available or can be readily synthesized, and they may have utility in other assay formats used to analyze CpG methylation status.

Ferrocenylnaphthalene diimide (FND)-based electrochemical hybridization assay was applied to the detection of methylated cytosine of DNA using the products obtained after treatment with bisulfite followed by polymerase chain reaction (PCR), where unmethylated cytosine is converted to thymine and methylated one to cytosine. Twenty-meric DNA probes for the methylated (cytosine) and unmethylated (thymine) types of the part of the promoter region of cyclin D-dependent protein kinase inhibitor, p16, gene (p16(Ink4a)) were used to be immobilized on the electrochemical array (ECA) chip. Using 1 microL of 10 ng/microL of methylated sample obtained from the methylation-specific PCR of methylated genome containing 10-times excess of unmethylated one, the methylated PCR sample could be detected by the identical electrochemical signals from the two DNA probes under the settled optimum hybridization conditions.