Mutant K-ras enhances apoptosis in embryonic stem cells in combination with DNA damage and is associated with increased levels of p19(ARF)

The roles of K-ras in neoplasia are not entirely understood, although there is evidence that K-ras affects susceptibility to apoptosis, modulating sur vival of DNA damaged cells which would otherwise be eliminated. In this stu dy, we investigated the effects of mutant K-ras on apoptosis in vitro follo wing DNA damage. To avoid complications resulting from mutations in other c ancer-related genes and from the presence of endogenous K-ras, we derived K -ras null embryonic stem cells. Expression of either wild-type or mutant K- vas was reconstructed by stable plasmid transfection, The cell lines were t reated with etoposide, cisplatin and UV radiation and apoptosis measured fl ow cytometrically. Mutant K-ras potentiated the effect of etoposide-derived DNA damage by increasing apoptosis, whereas absence of K-ras had the oppos ite effect. This pattern was similar but less marked with cisplatin, wherea s UV yielded no difference in apoptosis with regard to K-ras status, sugges ting that the effect of K-ras on apoptosis is dependent on the nature of th e DNA damage, To investigate possible mechanisms, we examined the expressio n of p19(ARF) mRNA by RT-PCR, Cells expressing mutant K-ras produced elevat ed levels of p19(ARF) mRNA, which could link K-ras status with p53 expressi on and hence susceptibility to DNA damage-induced apoptosis.