Growth Regulatory Effects of Depletion Are Dissociable in Cells Cyclic AMP and Cultured Mouse Polyamine Lymphoma

Treatment of mouse lymphoma $49 cells with D,L-~x-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, depleted cellular polyamine levels and stopped cell growth. The cells were arrested predominantly in G1. Thus, polyamine depletion may lead to a regulatory growth arrest in $49 cells. We tested two hypotheses regarding the relationship of growth arrest mediated by polyamine limitation to that mediated by cyclic AMP (cAMP). The hypothesis that cAMP-induced arrest results from polyamine depletion is not tenable, because the arrest could not be reversed by addition of exogenous polyamines, and because cellular polyamine levels do not drop in dibuturyl cyclic AMP (Bt2cAMP)-arrested cells. The hypothesis that polyamine-mediated growth arrest is effected via modulation of cAMP levels or cAMP-dependent protein kinase activity was also shown to be incorrect, because a $49 variant deficient in cAMP-dependent protein kinase was arrested by DFMO. The activities of the polyamine-synthesizing enzymes ornithine decarboxylase (ODC) and S-adenosyl methionine decarboxylase (SAMD) are both reduced in Bt2cAMP-treated cells to about 10% of that in control populations, as shown previously. DFMO diminishes ODC activity and augments SAMD activity in both untreated and Bt2cAMP-treated cells, leading to polyamine depletion in both cases. Numerous studies indicate that the polyamines putrescine, spermidine, and spermine influence cell proliferation. Two sorts of evidence support this: first, polyamine levels and the activities of their synthesizing enzymes, ornithine decarboxylase (ODC) and S-adenosyl methionine decarboxylase (SAMD), vary with the proliferative state of the cell (1), and second, pharmacologically induced depletion of cellular polyamines leads to reduced cellular proliferation (2-11). Polyamines may simply be necessary constituents for proliferating cells or they may be regulatory molecules, signaling the cells to initiate or cease proliferation. We ask here whether polyamines are necessary for growth of mouse lymphoma $49 cells, whether they behave as regulatory molecules with respect to cell cycle specificity of arrest, and finally whether causal connections exist among regulatory systems controling cyclic AMP (cAMP), polyamines, and cell growth. ODC is the enzyme responsible for synthesis of putrescine from ornithine. Spermidine and spermine are generated from putrescine by addition of one and two aminopropyl groups, respectively. SAMD generates the aminopropyl donor molecules for this reaction and is the rate-limiting enzyme for the synthesis of these higher polyamines. We show here that treatment of $49 cells with a-difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ODC, depletes cells of polyamines and halts cell proliferation. Because regulators of cell growth act primarily to change the duration of the G1 phase of the cell cycle (12) we assume that a G1 arrest is suggestive of a regulatory growth arrest. S49 cells show a predominantly GI phase arrest with DFMO treatment. This indicates that in these cells polyamines are potentially regulatory molecules. Previous work has demonstrated that treatment of $49 cells with biologically active cAMP analogues such as dibutyryl cAMP (Bt2cAMP) arrests cells in the GE phase (13) and extinguishes the activities of ODC and of SAMD (14, 15). Both responses are mediated via the cAMP-dependent protein kinase, as both are abolished in $49 mutant cells devoid of that kinase (13-15). THE JOURNAL OF CELL BIOLOGY -VOLUME 96 MARCH 1983 762-767 762 © The Rockefeller University Press • 0021-9525/83/03/0762/06 $1.00 on A ril 3, 2017 D ow nladed fom Published March 1, 1983