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 survival of DNA damaged cells which would otherwise be eliminated. In this study, we investigated the effects of mutant K-ras on apoptosis in vitro following DNA damage. To avoid complications resulting from mutations in other cancer-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-ras was reconstructed by stable plasmid transfection. The cell lines were treated with etoposide, cisplatin and UV radiation and apoptosis measured flow cytometrically. Mutant K-ras potentiated the effect of etoposide-derived DNA damage by increasing apoptosis, whereas absence of K-ras had the opposite effect. This pattern was similar but less marked with cisplatin, whereas UV yielded no difference in apoptosis with regard to K-ras status, suggesting that the effect of K-ras on apoptosis is dependent on the nature of the DNA damage. To investigate possible mechanisms, we examined the expression of p19(ARF) mRNA by RT-PCR. Cells expressing mutant K-ras produced elevated levels of p19(ARF) mRNA, which could link K-ras status with p53 expression and hence susceptibility to DNA damage-induced apoptosis.
Brooks
D G
DG
Sir Alastair Currie CRC Laboratories, Molecular Medicine Centre, University of Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK.
James
R M
RM
Patek
C E
CE
Williamson
J
J
Arends
M J
MJ
D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D016466:CHO Cells; D002460:Cell Line; D002945:Cisplatin / Q000633:toxicity; D006224:Cricetinae; D004249:DNA Damage / Q000502:physiology*; D004622:Embryo, Mammalian; D005047:Etoposide / Q000633:toxicity; D018507:Gene Expression Regulation, Developmental; D011905:Genes, ras / Q000235:genetics / Q000502:physiology*; D051379:Mice; D009154:Mutation; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D014162:Transfection; D025803:Tumor Suppressor Protein p14ARF; D014466:Ultraviolet Rays
D000970:Antineoplastic Agents; D011506:Proteins; D012333:RNA, Messenger; D025803:Tumor Suppressor Protein p14ARF; D005047:Etoposide; D002945:Cisplatin