The Rat 3-3 is a secondary transformant of the rat fibroblast cell line (Rat-1) transfectedwith total DNA of a gastrocarcinoma cell line BGC-823. The cells over-express the c-Ha-rasoncogene which contains point mutati...The Rat 3-3 is a secondary transformant of the rat fibroblast cell line (Rat-1) transfectedwith total DNA of a gastrocarcinoma cell line BGC-823. The cells over-express the c-Ha-rasoncogene which contains point mutation at the 12th codon. In order to determine how theactivated c-Ha-ras oncogene expression governs the cell's transformation, two pendadecadeoxy-nucleotides AS-1 and AS-2 were synthesized. AS-1 was complementary to the single strandof the first three codons and the upstream sequence close to the ribosome binding site of c-Ha-ras mRNA. AS-2 was complementary to the 3' end of the first intron and the 5' end ofthe second exon of c-Ha-ras unripe RNA enclosed in the nucleus. The oligonucleotides couldblock either the translation of c-Ha-ras mRNA or the splicing of c-Ha-ras unripe RNA, thusinhibiting the expression of the activated c-Ha-ras oncogene and the proliferation of thetransformed cells Rat 3-3. The inhibitory effect increased with a growing concentration ofthe antisense oligodeoxynucleotide (from 4- 10 μmol/L) and reached its peak at 12 h afterRat 3-3 was treated with AS-1 and AS-2. This effect became weaker afterwards. The p21level, product of c-Ha-ras, in the Rat 3-3, was examined by ELISA method. The resultshows that the amount of p21 in the growth-inhibited cellsis about 30% of that of the controlcells.展开更多
文摘The Rat 3-3 is a secondary transformant of the rat fibroblast cell line (Rat-1) transfectedwith total DNA of a gastrocarcinoma cell line BGC-823. The cells over-express the c-Ha-rasoncogene which contains point mutation at the 12th codon. In order to determine how theactivated c-Ha-ras oncogene expression governs the cell's transformation, two pendadecadeoxy-nucleotides AS-1 and AS-2 were synthesized. AS-1 was complementary to the single strandof the first three codons and the upstream sequence close to the ribosome binding site of c-Ha-ras mRNA. AS-2 was complementary to the 3' end of the first intron and the 5' end ofthe second exon of c-Ha-ras unripe RNA enclosed in the nucleus. The oligonucleotides couldblock either the translation of c-Ha-ras mRNA or the splicing of c-Ha-ras unripe RNA, thusinhibiting the expression of the activated c-Ha-ras oncogene and the proliferation of thetransformed cells Rat 3-3. The inhibitory effect increased with a growing concentration ofthe antisense oligodeoxynucleotide (from 4- 10 μmol/L) and reached its peak at 12 h afterRat 3-3 was treated with AS-1 and AS-2. This effect became weaker afterwards. The p21level, product of c-Ha-ras, in the Rat 3-3, was examined by ELISA method. The resultshows that the amount of p21 in the growth-inhibited cellsis about 30% of that of the controlcells.
