TRANSFORMED-CELLS REQUIRE CONTINUOUS ACTIVITY OF RNA-POLYMERASE-II TORESIST ONCOGENE-INDUCED APOPTOSIS

Studies have indicated that deregulated oncogene expression can result in either programmed cell death or proliferation, depending on the ce llular microenvironment, However, little is known about whether oncoge nic signals in themselves are able to activate a cellular apoptotic pr ogram. We have tested the hypothesis that oncogenic signals in the abs ence of gene expression are sufficient to induce cell death, which wou ld indicate that constitutive expression of antiapoptotic genes is nec essary for maintenance of the transformed state, Using two highly spec ific RNA polymerase (RNAP) II inhibitors, 5,6-dichloro-1-beta-D-ribofu ranosylbenzimidazole (DRB) and alpha-amanitin, which inhibit RNAP II f unction by two distinct mechanisms, we found that inhibition of gene e xpression substantially increased apoptosis in a time-and dose-depende nt manner in p53(+/+)- and p53(-/-)-transformed mouse embryonic fibrob lasts and ha HeLa cells, demonstrating that this type of apoptosis doe s not require wild-type p53. Engineered expression of an alpha-amaniti n resistance RNAP II gene rendered cells resistant to induction of apo ptosis by alpha-amanitin without affecting their sensitivity to DPB, i ndicating that alpha-amanitin induces apoptosis solely by inhibiting R NAP II function and not by a nonspecific mechanism, DRB-induced apopto sis was independent of the cell cycle or ongoing DNA replication, sinc e DRB induced similar levels of apoptosis in asynchronous cells and ce lls synchronized by collection at mitosis. Inhibition of RNAP II in un transformed cells like Rat-1 or human AG1522 fibroblasts resulted not in apoptosis but in growth arrest, In contrast, deregulated expression of c-Myc in Rat-1 cells dramatically increased their sensitivity to D RB, directly demonstrating that apoptosis following inhibition of RNAP II function is greatly enhanced by oncogenic expression. The requirem ent for RNAP II function to prevent oncogene-induced apoptosis implies the need for the constitutive expression of an antiapoptotic gene(s) to maintain the transformed state, The differential sensitivities of u ntransformed and transformed cells to induction of apoptosis by transc riptional inhibition, coupled with the finding that this type of apopt osis is independent of p53 status, suggest that inhibition of RNAP II may be exploited therapeutically for the design of successful antitumo r agents.