Specific Inhibition of c-sis Protein Synthesis and Cell Proliferation with Antisense Oligodeoxynucleotides in Human Glioma Cells

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THE PROTEIN ENCODED by c-sis is overexpressed in human neuroglial tumors and has been hypothesized to play an important role in tumorigenesis. To address this issue, we examined the effect of an 18-base pair oligodeoxynucleotide complementary to a sequence starting of ATG initiation codon of mRNA of c-sis upon glioma cell growth. First, we investigated the expression of c-sis within cultured human glioma cell lines and also fresh glioma specimens by using polymerase chain reaction. We could detect messenger ribonucleic acid transcripts of c-sis in three of four glioma cell lines and two of five glioblastoma multiforme specimens. This finding was confirmed by dot-blot hybridization with a specific oligonucleotide probe of c-sis. The antisense oligonucleotides complementary to c-sis messenger ribonucleic acid were efficiently incorporated into A172 cells in vitro, and kinetic studies showed that maximum uptake was seen after 48 hours incubation with antisense oligomers. Exposure of human glioma cell lines to antisense oligodeoxynucleotides targeted against the first initiation codon of c-sis inhibited cell proliferation in a time- and dose-dependent fashion. From flow cytometric analysis with anti-c-sis sera, it was demonstrated that the antisense oligomers specifically block the de novo synthesis of intracellular c-sis protein by glioma cells. In contrast, the corresponding sense oligomers did not inhibit either synthesis of c-sis protein or glioma cell growth. These results clearly support a role of c-sis protein in the proliferation of these human neuroglial tumors and show that inducible protein expression can be blocked by means of synthetic oligonucleotides complementary to a coding exon.

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