ORNITHINE DECARBOXYLASE OVER-EXPRESSION STIMULATES MITOGEN-ACTIVATED PROTEIN-KINASE AND ANCHORAGE-INDEPENDENT GROWTH OF HUMAN BREAST EPITHELIAL-CELLS

In these experiments we tested the hypothesis that constitutive activa tion of polyamine(PA) biosynthesis may contribute to mammary carcinoge nesis. Spontaneously immortalized normal human MCF-10A breast epitheli al cells were infected with the retroviral vector pLOSN containing a c DNA which codes for a truncated and more stable ornithine decarboxylas e (ODC), the rate-limiting enzyme in PA synthesis. Upon chronic select ive pressure with alpha-difluoromethylornithine (DFMO) (an irreversibl e inhibitor of ODC), infected MCF-10A cells exhibited an approximately 250-fold increase in ODC activity, which persisted despite discontinu ation of DFMO. ODC over-expressing MCF-10A cells showed a modest decre ase in S-adenosylmethionine decarboxylase and an increase in spermidin e/spermineN(1)-acetyltransferase. Analysis of cellular PA profile reve aled a selective accumulation of putrescine without alterations in spe rmidine and spermine contents. Lesser degrees of increased ODC activit y were obtained reproducibly by re-exposing the cells to incremental s mall doses of DFMO. We observed a bell-shaped dose-related positive ef fect of ODC activity on clonogenicity in soft agar of MCF-10A cells. S ince anchorage dependent growth was actually reduced, such positive in fluence on this feature of transformation was not a non-specific conse quence of a growth advantage provided by ODC over-expression. In addit ion, we observed a close parallelism between the dose-dependent effect s of ODC expression on clonogenicity and activity of the ERK-2 kinase, a central element of the MAPK cascade. Our data demonstrate an intera ction between PA and the MAPK signalling pathway and suggest that the latter may be involved in ODC-induced transformation of mammary epithe lial cells. (C) 1997 Wiley-Liss, Inc.