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Expression of an Ornithine Decarboxylase Dominant-Negative Mutant Reverses Eukaryotic Initiation Factor 4E-induced Cell Transformation¹

Departments of Cellular and Molecular Physiology and Pharmacology, The Milton S. Hershey Medical Center, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

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 shown previously that this induction appears to be related to the transformed phenotype of these cells (L. M. Shantz and A. E. Pegg, Cancer Res., 54: 2313–2316, 1994). To test whether increased ODC activity is responsible for the transformation of 4E-P2 cells, a dominant-negative mutant of ODC was used to reduce the intracellular ODC activity in 4E-P2 cells, and the resulting phenotypic changes were examined. The mutant K69A/C360A contains mutations to alanine of two key active site residues, lysine 69 and cysteine 360, and is truncated at 425 amino acids. Combination of purified K69A/C360A and purified wild-type ODC resulted in a dose-dependent decrease in specific activity compared with wild-type ODC alone, with a 71% reduction at equimolar concentrations. This mutant was transfected into 4E-P2 cells, and stable clones that expressed the truncated K69A/C360A were isolated. Several clones were tested 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, suggesting 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 associated with transformation in both in vitro and in vivo systems.

¹ This work was supported by Grant CA-18138 from the National Cancer Institute (to A. E. P.), Grant IRG-196A from the American Cancer Society (to L. M. S.), and the Pennsylvania State University Four Diamonds Fund (to L. M. S.). C. S. C. is the recipient of a Wellcome Trust Travel Grant.

² To whom requests for reprints should be addressed, at Departments of Cellular and Molecular Physiology and Pharmacology, The Milton S. Hershey Medical Center, The Pennsylvania State University College of Medicine, P. O. Box 850, Hershey, PA 17033.

Received 9/ 6/96. Accepted 10/ 2/96.

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