Ornithine decarboxylase induction in transformation by H-Ras and RhoA

Abstract

The objective of these studies has been to develop a better understanding of the regulation of ornithine decarboxylase (ODC) during the neoplastic process, and to determine whether induction of ODC is a necessary component in the action of the ras oncogene. Specifically, we have studied the role of ODC overexpression in signaling pathways mediated by Raf or RhoA. Cells transformed by ras are known to have constitutively high levels of ODC activity that correlate with oncogenic transformation. To determine which pathways downstream of Ras contribute to the regulation of ODC activity, NIH-3T3 cells were transfected with plasmids coding for activated mutants of either H-Ras or RhoA, or oncogenic nu-Raf. There was a good correlation between increasing ODC specific activity and change in morphology from normal to transformed in the nu-Raf, HRas(61L), and RhoA(63L) clones. Increasing ODC activity also correlated positively with the ability to grow in soft agar in both the H-Ras- and RhoA-expressing cells. In stable transfections, coexpression of the ODC dominant negative mutant K69A/C360A with either HRas(61L) or RhoA(63L) both inhibited intracellular ODC activity and caused a reversion of the transformed phenotype, as measured by a dramatic reduction in the ability of these cells to grow in soft agar and form foci on a monolayer. These results suggest strongly that ODC induction is necessary for transformation by oncogenic Ras. In contrast, expression of K69A/ C360A had no effect on the ability of nu-Raf-transformed cells to grow in soft agar, although intracellular ODC levels were inhibited. When grown on a monolayer, these cells also maintained their transformed appearance. Furthermore, expression of the ODC dominant negative mutant did not affect the phosphorylation of mitogen-activated protein kinase in nu-Raf-transformed cells. These experiments provide strong support for the concept that transformation by activated ras is accompanied by an induction of ODC. The results using RhoA(63L) and nu-Raf suggest that this increase in ODC activity is mediated at least in part through a Raf/ mitogen-activated protein kinase independent pathway.