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

D G Brooks, R M James, C E Patek, J Williamson, M J Arends

Research output: Contribution to journalArticlepeer-review

Abstract

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.
Original languageEnglish
Pages (from-to)2144-52
Number of pages9
JournalOncogene
Volume20
Issue number17
DOIs
Publication statusPublished - 19 Apr 2001

Keywords

  • Animals
  • Antineoplastic Agents
  • Apoptosis
  • CHO Cells
  • Cell Line
  • Cisplatin
  • Cricetinae
  • DNA Damage
  • Embryo, Mammalian
  • Etoposide
  • Gene Expression Regulation, Developmental
  • Genes, ras
  • Mice
  • Mutation
  • Protein Biosynthesis
  • Proteins
  • RNA, Messenger
  • Stem Cells
  • Transfection
  • Tumor Suppressor Protein p14ARF
  • Ultraviolet Rays

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