Glioblastoma (GBM) is one of the most aggressive human cancers with a median survival of less than two years. A distinguishing pathological feature of GBM is a high degree of inter- and intratumoral heterogeneity. Intertumoral heterogeneity of GBM has been extensively investigated on genomic, methylomic, transcriptomic, proteomic and metabolomics levels, however only a few studies describe intratumoral heterogeneity because of the lack of methods allowing to analyze GBM samples with high spatial resolution. Here, we applied TOF-SIMS (Time-of-flight secondary ion mass spectrometry) for the analysis of single cells and clinical samples such as paraffin and frozen tumor sections obtained from 57 patients. We developed a technique that allows us to simultaneously detect the distribution of proteins and metabolites in glioma tissue with 800 nm spatial resolution. Our results demonstrate that according to TOF-SIMS data glioma samples can be subdivided into clinically relevant groups and distinguished from the normal brain tissue. In addition, TOF-SIMS was able to elucidate differences between morphologically distinct regions of GBM within the same tumor. By staining GBM sections with gold-conjugated antibodies against Caveolin-1 we could visualize border between zones of necrotic and cellular tumor and subdivide glioma samples into groups characterized by different survival of the patients. Finally, we demonstrated that GBM contains cells that are characterized by high levels of Caveolin-1 protein and cholesterol. This population may partly represent a glioma stem cells. Collectively, our results show that the technique described here allows to analyze glioma tissues with a spatial resolution beyond reach of most of other omics approaches and the obtained data may be used to predict clinical behavior of the tumor.
Most of the mammalian genome consists of nucleotide sequences not coding for proteins. Exons of genes make up only 3% of the human genome, while the significance of most other sequences remains unknown. Recent genome studies with high-throughput methods demonstrate that the so-called noncoding part of the genome may perform important functions. This hypothesis is supported by three groups of experimental data: 1) approximately 10% of the sequences, most of which are located in noncoding parts of the genome, is evolutionarily conserved and thus can be of functional importance; 2) up to 99% of the mammalian genome is being transcribed forming short and long noncoding RNAs in addition to common mRNA; and 3) mutations in noncoding parts of the genome can be accompanied by progression of pathological states of the organism. In the light of these data, in the review we consider the functional role of numerous known sequences of noncoding parts of the genome including introns, DNA methylation regions, enhancers and locus control regions, insulators, S/MAR sequences, pseudogenes, and genes of noncoding RNAs, as well as transposons and simple repeats of centromeric and telomeric regions of chromosomes. The assumption is made that the intergenic noncoding sequences without definite/clear functions can be involved in spatial organization of genetic loci in interphase nuclei.
The tumor suppressor PTEN controls multiple cellular functions, including cell cycle, apoptosis, senescence, transcription, and mRNA translation of numerous genes. In tumor cells, PTEN is frequently inactivated by genetic mutations and epimutations. The aim of this study was to investigate the methylation patterns of the PTEN gene and its pseudogene PTENP1 as potential genetic markers of endometrial hyperplasia (EH) and endometrial carcinoma (EC). Methylation of the 5-terminal regions of the PTEN and PTENP1 sequences was studied using methyl-sensitive PCR of genomic DNA isolated from 57 cancer, 43 endometrial hyperplasia, and normal tissue samples of 24 females aged 17-34 years and 19 females aged 45-65 years, as well as 20 peripheral venous blood samples of EC patients. None of the analyzed DNA samples carried a methylated PTEN gene. On the contrary, the PTENP1 pseudogene was methylated in all analyzed tissues, except for the peripheral blood. Comparison of PTENP1 methylation rates revealed no differences between the EC and EH groups (0.80 p 0.50). In all these groups, the methylation level was high (71-77% in patients vs. 58% in controls). Differences in PTENP1 methylation rates between normal endometrium in young (4%) and middle-aged and elderly (58%) females were significant (p 0.001). These findings suggest that PTENP1 pseudogene methylation may reflect age-related changes in the body and is not directly related to the endometrium pathology under study. It is assumed that, depending on the influence of a methylated PTENP1 pseudogene on PTEN gene expression, the pseudogene methylation may protect against the development of EC and/or serve as a marker of a precancerous condition of endometrial cells.
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