ORNITHINE DECARBOXYLASE GENE-EXPRESSION IS ABERRANTLY REGULATED VIA THE CAMP SIGNAL-TRANSDUCTION PATHWAY IN MALIGNANT H-RAS TRANSFORMED-CELL LINES

We have tested the hypothesis that H-ras transformed cells contain alt erations in signal pathways important in controlling the expression of ornithine decarboxylase (ODC), the highly regulated rate-limiting act ivity in the biosynthesis of polyamines. Mouse 10T1/2 fibroblasts and a series of 10T1/2 H-ras transformed cell lines were treated with stim ulators of cAMP synthesis (forskolin and cholera toxin), a biologicall y stable analogue of cAMP (8-bromo-cAMP), and an inhibitor of cAMP deg radation (3-isobutyl-1-methylxanthine). Elevations in ODC gene express ion were noted in H-ras transformed cells that were not observed in pa rental 10T1/2 fibroblasts. The forskolin-mediated effects were not det ected with 1,9-dideoxyforskolin, a compound structurally related to fo rskolin, which does not activate adenyl cyclase. The effects observed with cholera toxin were not detected when cells were treated with the purified subunits of this compound, indicating that the toxin-induced effects were cAMP-specific. Actinomycin D treatment prior to forskolin exposure reduced the elevation observed in ODC gene expression indica ting the involvement of the transcriptional process. Furthermore, we o bserved that cycloheximide treatment of malignant but not benign H-ras transformed cells significantly elevated ODC message level. Treatment of malignant cells with both cycloheximide and forskolin together res ulted in a further additive elevation in ODC message, but a similar tr eatment of benign tumor cells reduced the forskolin-mediated increase in ODC message. In addition, treatment of H-ras transformed cells with the tumor promoter, 12-0-tetradecanoylphorbol-13-acetate (TPA) led to an elevation in ODC mRNA levels not observed in parental 10T1/2 fibro blasts. However, an increase in ODC message levels was observed in par ental 10T1/2 cells treated with a combination of TPA and forskolin. Ex posure of H-ras transformed cells to TPA and forskolin together led to further pronounced increases in ODC message when compared to treatmen t with TPA or forskolin alone. These results demonstrate aberrant regu lation of signal pathways involved in controlling ODC gene expression in H-ras transformed cells and provide further insight into the altere d growth regulatory program associated with malignant transformation. (C) 1994 Wiley-Liss, Inc.