INCREASED EXPRESSION OF RAT RIBOSOMAL-PROTEIN L4 MESSENGER-RNA IN 5-AZACYTIDINE-TREATED PC12 CELLS PRIOR TO APOPTOSIS

5-Azacytidine (5AzC), a cytidine analogue, is thought to induce apopto sis in fetal neuronal cells and PC12 cells through DNA hypomethylation . However, apoptosis can be inhibited by adding protein synthesis inhi bitors, indicating de novo protein synthesis may be partially responsi ble for apoptosis. Therefore, genes expressed just before apoptosis fr om 5AzC-treated PC12 cells were cloned. cDNA Libraries were prepared f rom both 5AzC-treated and untreated PC12 cells and these libraries wer e subtracted. One clone overexpressed in 5AzC-treated PC12 cells was o btained, and was identified as the nearly full length (9 nt at 5' end and 1 nt at 3' end missing) rat ribosomal protein L4 (rpLA) gene. Time course study of Northern blot analysis in 5AzC-treated PC12 cells rev ealed that the peak of rat rpL4 gene expression preceded DNA fragmenta tion. COS-7 cells transfected with different amounts of cDNA from the subtracted clone expressed rat rpL4 dose-dependently. DNA fragmentatio n in the transfected COS-7 cells occurred proportional to the amount o f the cDNA used for transfection. The present study indicates that rat rpLA gene expression selectively increases in PC12 cells prior to 5Az C-induced apoptosis and that COS-7 cells transfected with and expressi ng the rat rpL4 gene also undergo apoptosis. (C) 1998 Academic Press.