EXPRESSION OF AN ORNITHINE DECARBOXYLASE DOMINANT-NEGATIVE MUTANT REVERSES EUKARYOTIC INITIATION-FACTOR 4E-INDUCED CELL-TRANSFORMATION

pMV7-4E cells (4E-P2), which overexpress translation initiation factor eIF-4E, contain elevated levels of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine biosynthesis. We have sho wn previously that this induction appears to be related to the transfo rmed phenotype of these cells (L. M. Shantz and A. E. Pegg, Cancer Res ., 54: 2313-2316, 1994). To test whether increased ODC activity is res ponsible for the transformation of 4E-P2 cells, a dominant-negative mu tant of ODC was used to reduce the intracellular ODC activity in 4E-P2 , cells, and the resulting phenotypic changes were examined. The mutan t K69A/C360A contains mutations to alanine of two key active site resi dues, lysine 69 and cysteine 360, and is truncated at 425 amino acids. Combination of purified K69A/C360A and purified wild-type ODC resulte d in a dose-dependent decrease in specific activity compared with wild -type ODC alone, with a 71% reduction at equimolar concentrations. Thi s mutant was transfected into 4E-P2 cells, and stable clones that expr essed the truncated K69A/C360A were isolated. Several clones were test ed for their ability to form transformed foci on a monolayer, grow in soft agar, and form tumors in nude mice. When ODC activity was reduced by 60%, the transformed phenotype of 4E-P2 cells was abolished, sugge sting strongly that high ODC levels are critical to the transformation of these cells. In addition, K69A/C360A can be used to determine the ODC activity associate with transformation in both in vitro and in viv o systems.