Amplification and expression of splice variants of the gene encoding the P450 cytochrome 25-hydroxyvitamin D(3) 1,alpha-hydroxylase (CYP 27B1) in human malignant glioma.

Abstract

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. 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. 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. 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.