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

Purpose: Recently, we reported the isolation of six novel genes termed glio ma-amplified sequences (GASs) from the glioblastoma cell line TX3868 using microdissected mediated cDNA capture (U. Fischer et at, Rum. Mol. Genet., 5 : 595-600, 1996), The aim of this study was to further characterize the gen e GAS89. Experimental Design: To determine the amplification frequency, we performed comparative PCR studies and Southern blot hybridization experiments. To id entify 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 libra ry, we isolated an incomplete gene sequence showing identity with the gene for 25-hydroxyvitamin D-3 1,alpha -hydroxylase. Different full-length clone s were then isolated using PCR primers chosen from the 3'- and 5 ' -untrans lated regions, As determined by sequencing, the clones represent various sp lice variants of the 25-hydroxyvitamin D-3 I,alpha -hydroxylase gene. The c lones encode truncated proteins but also one potentially functional enzyme variant. Reverse transcription-PCR studies revealed overexpression of sever al 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-hydroxyv itamin D-3 1,alpha -hydroxylase and the appearance of mRNA splice variants in glioblastoma multiforme, The endogenous expression of the 25-hydroxyvita min 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.