RESUMO
During this study, we analysed the pericentric inversion that distinguishes human chromosome 12 (HSA12) from the homologous chimpanzee chromosome (PTR10). Two large chimpanzee-specific duplications of 86 and 23 kb were observed in the breakpoint regions, which most probably occurred associated with the inversion. The inversion break in PTR10p caused the disruption of the SLCO1B3 gene in exon 11. However, the 86-kb duplication includes the functional SLCO1B3 locus, which is thus retained in the chimpanzee, although inverted to PTR10q. The second duplication spans 23 kb and does not contain expressed sequences. Eleven genes map to a region of about 1 Mb around the breakpoints. Six of these eleven genes are not among the differentially expressed genes as determined previously by comparing the human and chimpanzee transcriptome of fibroblast cell lines, blood leukocytes, liver and brain samples. These findings imply that the inversion did not cause major expression differences of these genes. Comparative FISH analysis with BACs spanning the inversion breakpoints in PTR on metaphase chromosomes of gorilla (GGO) confirmed that the pericentric inversion of the chromosome 12 homologs in GGO and PTR have distinct breakpoints and that humans retain the ancestral arrangement. These findings coincide with the trend observed in hominoid karyotype evolution that humans have a karyotype close to an ancestral one, while African great apes present with more derived chromosome arrangements.
Assuntos
Centrômero/genética , Quebra Cromossômica/genética , Inversão Cromossômica/genética , Cromossomos Humanos Par 12/genética , Pan troglodytes/genética , Homologia de Sequência do Ácido Nucleico , Animais , Linhagem Celular , Cromossomos Artificiais Bacterianos/genética , Cromossomos Artificiais de Bacteriófago P1/genética , Cromossomos de Mamíferos/genética , Evolução Molecular , Duplicação Gênica , Rearranjo Gênico/genética , Genes/genética , Gorilla gorilla/genética , Humanos , Hibridização in Situ Fluorescente/métodos , Dados de Sequência MolecularRESUMO
Glioblastomas are highly aggressive brain tumours and are characterised by substantial cellular heterogeneity within a single tumour. A sub-population of glioblastoma stem-like cells (GSCs) that shares properties with neural precursor cells has been described, exhibiting resistance to therapy and therefore being considered responsible for the high recurrence rate in glioblastoma. To elucidate the underlying cellular processes we investigated the role of phosphatases in the GSC phenotype, using an in vitro phosphatome-wide RNA interference screen. We identified a set of genes, the knockdown of which induces a significant decrease in the glioma stem cell marker CD133, indicating a role in the glioblastoma stem-like phenotype. Among these genes, the ecto-nucleotidase ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) was found to be highly expressed in GSCs compared with normal brain and neural stem cells. Knockdown of ENPP1 in cultured GSCs resulted in an overall downregulation of stem cell-associated genes, induction of differentiation into astrocytic cell lineage, impairment of sphere formation, in addition to increased cell death, accumulation of cells in G1/G0 cell cycle phase and sensitisation to chemotherapeutic treatment. Genome-wide gene expression analysis and nucleoside and nucleotide profiling revealed that knockdown of ENPP1 affects purine and pyrimidine metabolism, suggesting a link between ENPP1 expression and a balanced nucleoside-nucleotide pool in GSCs. The phenotypic changes in E-NPP1-deficient GSCs are assumed to be a consequence of decreased transcriptional function of E2F1. Together, these results reveal that E-NPP1, by acting upstream of E2F1, is indispensable for the maintenance of GSCs in vitro and hence required to keep GSCs in an undifferentiated, proliferative state.
Assuntos
Neoplasias Encefálicas/genética , Glioblastoma/genética , Células-Tronco Neurais/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Antígeno AC133 , Antígenos CD/biossíntese , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Pontos de Checagem do Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula , Fator de Transcrição E2F1/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Glicoproteínas/biossíntese , Humanos , Peptídeos , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genéticaRESUMO
Deletions of chromosomal arms 1p and 19q are frequent in oligodendroglial tumours and linked to radio- and chemotherapy response as well as longer survival. The molecular mechanisms underlying this clinically important association are as yet unknown. Here, we studied the peroxiredoxin 1 (PRDX1) gene at 1p34.1 for promoter methylation and expression in primary gliomas and investigated its role in radio- and chemosensitivity of glioma cells in vitro. In total, we screened primary glioma tissues from 93 patients for methylation of the 5'-CpG island of PRDX1 by sodium bisulfite sequencing. PRDX1 mRNA and protein expression levels were determined in subsets of the tumours by quantitative PCR and western blot analysis, respectively. PRDX1 hypermethylation and reduced expression were frequently detected in oligodendroglial tumours and secondary glioblastomas, but not in primary glioblastomas. In oligodendroglial tumours, both PRDX1 hypermethylation and reduced mRNA expression were significantly associated with 1p/19q-deletion. Stable knockdown of PRDX1 by lentiviral transduction of short-hairpin (sh)RNA constructs significantly increased apoptosis and reduced cell viability of Hs683 glioma cells exposed to ionizing irradiation or temozolomide in vitro. Taken together, our findings indicate that epigenetic silencing of PRDX1 is frequent in 1p/19q-deleted oligodendroglial tumours and likely contributes to radio- and chemosensitivity of these tumours.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 1/genética , Glioma/patologia , Oligodendroglia/metabolismo , Peroxirredoxinas/genética , Regiões Promotoras Genéticas/genética , Tolerância a Radiação/genética , Adulto , Idoso , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Ilhas de CpG/genética , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/efeitos da radiação , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Regulação para Baixo/genética , Feminino , Técnicas de Silenciamento de Genes , Inativação Gênica , Glioma/tratamento farmacológico , Glioma/genética , Glioma/radioterapia , Humanos , Isocitrato Desidrogenase/genética , Masculino , Pessoa de Meia-Idade , Mutação , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/patologia , Oligodendroglia/efeitos da radiação , Peroxirredoxinas/deficiência , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos da radiação , Temozolomida , Adulto JovemRESUMO
The concept of cancer stem-like cells (CSCs) has gained considerable attention in various solid tumors including glioblastoma, the most common primary brain tumor. This sub-population of tumor cells has been intensively investigated and their role in therapy resistance as well as tumor recurrence has been demonstrated. In that respect, development of therapeutic strategies that target CSCs (and possibly also the tumor bulk) appears a promising approach in patients suffering from primary brain tumors. In the present study, we utilized RNA interference (RNAi) to screen the complete human kinome and phosphatome (682 and 180 targets, respectively) in order to identify genes and pathways relevant for the survival of brain CSCs and thereby potential therapeutical targets for glioblastoma. We report of 46 putative candidates including known survival-related kinases and phosphatases. Interestingly, a number of genes identified are involved in metabolism, especially glycolysis, such as PDK1 and PKM2 and, most prominently PFKFB4. In vitro studies confirmed an essential role of PFKFB4 in the maintenance of brain CSCs. Furthermore, high PFKFB4 expression was associated with shorter survival of primary glioblastoma patients. Our findings support the importance of the glycolytic pathway in the maintenance of malignant glioma cells and brain CSCs and imply tumor metabolism as a promising therapeutic target in glioblastoma.