PURPOSE: Recently, we reported the isolation of six novel genes termed glioma-amplified sequences (GASs) from the glioblastoma cell line TX3868 using microdissected mediated cDNA capture (U. Fischer et al., HUM: MOL: GENET:, 5: 595-600, 1996). The aim of this study was to further characterize the gene GAS89. EXPERIMENTAL DESIGN: To determine the amplification frequency, we performed comparative PCR studies and Southern blot hybridization experiments. To identify full-length clones of GAS89 we screened a HybriZAP library. Reverse transcription-PCR was performed to isolate splice variants and to determine expression levels. RESULTS: We identified for the gene GAS89 an amplification frequency of 25% in 28 examined glioblastoma multiforme samples. Screening a HybriZAP library, we isolated an incomplete gene sequence showing identity with the gene for 25-hydroxyvitamin D(3) 1,alpha-hydroxylase. Different full-length clones were then isolated using PCR primers chosen from the 3'- and 5'-untranslated regions. As determined by sequencing, the clones represent various splice variants of the 25-hydroxyvitamin D(3) 1,alpha-hydroxylase gene. The clones encode truncated proteins but also one potentially functional enzyme variant. Reverse transcription-PCR studies revealed overexpression of several variants in glioblastoma samples with GAS89 amplification in comparison with normal brain RNA and glioblastoma without GAS89 amplification. CONCLUSIONS: This is the first report of gene amplification for 25-hydroxyvitamin D(3) 1,alpha-hydroxylase and the appearance of mRNA splice variants in glioblastoma multiforme. The endogenous expression of the 25-hydroxyvitamin D(3) 1,alpha-hydroxylase gene and the appearance of alternative splice variants reveal a new feature of the molecular pathogenesis of glioblastoma and may represent a new target for glioma therapy.
PURPOSE: Recently, we reported the isolation of six novel genes termed glioma-amplified sequences (GASs) from the glioblastoma cell line TX3868 using microdissected mediated cDNA capture (U. Fischer et al., HUM: MOL: GENET:, 5: 595-600, 1996). The aim of this study was to further characterize the gene GAS89. EXPERIMENTAL DESIGN: To determine the amplification frequency, we performed comparative PCR studies and Southern blot hybridization experiments. To identify full-length clones of GAS89 we screened a HybriZAP library. Reverse transcription-PCR was performed to isolate splice variants and to determine expression levels. RESULTS: We identified for the gene GAS89 an amplification frequency of 25% in 28 examined glioblastoma multiforme samples. Screening a HybriZAP library, we isolated an incomplete gene sequence showing identity with the gene for 25-hydroxyvitamin D(3) 1,alpha-hydroxylase. Different full-length clones were then isolated using PCR primers chosen from the 3'- and 5'-untranslated regions. As determined by sequencing, the clones represent various splice variants of the 25-hydroxyvitamin D(3) 1,alpha-hydroxylase gene. The clones encode truncated proteins but also one potentially functional enzyme variant. Reverse transcription-PCR studies revealed overexpression of several variants in glioblastoma samples with GAS89 amplification in comparison with normal brain RNA and glioblastoma without GAS89 amplification. CONCLUSIONS: This is the first report of gene amplification for 25-hydroxyvitamin D(3) 1,alpha-hydroxylase and the appearance of mRNA splice variants in glioblastoma multiforme. The endogenous expression of the 25-hydroxyvitamin D(3) 1,alpha-hydroxylase gene and the appearance of alternative splice variants reveal a new feature of the molecular pathogenesis of glioblastoma and may represent a new target for glioma therapy.
Authors: Long N Truong; Shashikant Patil; Sherry S Martin; Jay F LeBlanc; Anil Nanda; Mary L Nordberg; Marie E Beckner Journal: Diagn Pathol Date: 2012-06-12 Impact factor: 2.644
Authors: Inês Crespo; Hermínio Tão; Ana Belen Nieto; Olinda Rebelo; Patrícia Domingues; Ana Luísa Vital; Maria del Carmen Patino; Marcos Barbosa; Maria Celeste Lopes; Catarina Resende Oliveira; Alberto Orfao; María Dolores Tabernero Journal: PLoS One Date: 2012-09-28 Impact factor: 3.240