|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1997 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1996 : ¥1,300,000 (Direct Cost : ¥1,300,000)
1) Promoter/enhancer activity of the 5'-flanking region of GM2/GD2 synthase (beta1,4-N-acetylgalactosaminyltransferase) gene.
To analyze the regulatory mechanisms of gene expression of GM2/GD2 synthase, we determined the genomic organization of the beta1,4GalNAc-T gene and defined three transcription initiation sites and the alternative usage of three exons, exon 1a, 1b and 1c. 5'-flanking region of individual initiation sites showed promoter activity when analyzed by chloramphenicolacetyltransferase assay. The high level of CAT activity was detected when the intron 1 region was added to the 5'-flanking region of exon 1a and 1b. These CAT activities corresponded to the expression levels of GM2/GD2 synthase gene.
2) Cytotoxic effects of an expression vector constructed by the combination of a cytotoxic gene and a promoter of GM2/GD2 synthase gene.
We constructed a retroviral expression vector using a cytotoxic gene (herpes simplexthymidine kinase gene) and a promoter/enhancer region (from upsteram of exon 1b to intron 1) of GM2/GD2 synthase gene. Then, we established stable transfectants of AS (astrocytoma) cell line that was a high expressant of the gene. When suppressive effects of cell proliferation was examined by adding Gancyclovir (GCV) to the culture medium, the stable transfectants showed inhibition of proliferation (>70%) at 5*10^<-6>M of GCV,while the parent cell did not even at 5*10^<-4>M of GCV.As for MeWo transfectants (a low expressant of the gene) of the retrovival vector, no inhibition of proliferation was observed. Furthermore, AS and MeWo transfectant containing the same copy number of the transfected gene were selected based on Southern blots with TK gene as a probe, then sensitivity of them to GCV was analyzed. Consequently, AS introduced with TK gene showed a growth inhibition with GCV,though MeWo with TK gene did not.