Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/81/010209-OsM)2.00/0 Experimental Cell Research 131 (1981) 209-216 DEPLETION OF 9L RAT BRAIN TUMOR CELL POLYAMINE CONTENT BY TREATMENT WITH D,L-a- DIFLUOROMETHYLORNITHINE INHIBITS PROLIFERATION AND THE Gl TO S TRANSITION JEROME SEIDENFELD,’ JOE W. GRAY2 and LAURENCE J. MARTON1,3. * ‘Brain Tumor Research Center, Department of Neurological Surgery, School of Medicine, University of Cahyornia, San Francisco, CA 94143, ZBiomedical Division, Lawrence Livermore Laboratory, University of California, Berkeley, at Livermore, CA 94550, USA, and 3Department of Laboratory Medicine, School of Medicine, University of California, San Francisco, CA 94143, USA SUMMARY o,L-cr-Difluoromethylornithine (DFMO), an irreversible inactivator of omithine decarboxvlase. inhibited 9L monolayer culture rat brain tumor cell proliferation at concentrations as low as 1 mM DFMO to about 25 % of control arowth when cells were seeded at an initial densitv of 5 x 1OVflask. DFMO reduced intracellular p&escine content to <5 % of control by 8 h and sp&midine content to <5 % of control by 48 h post-treatment. Cytostasis caused by 10 or 25 mM DFMO could both be reversed and blocked by addition of exogenous putrescine. Cells pretreated for 48 h with DFMO and then replated in its absence could not enter exponential growth until polyamine production resumed. Addition of exogenous putrescine at the time of replating allowed pretreated cells to resume exnonential growth at the same time as controls. Flow cvtometrv revealed that the fraction of cells inbl increased until polyamine accumulation resumed, implying the presence of a Gl-S block. Within 6 h of replating, there was a decrease in the fraction of control cells in Gl. These observations support the hypothesis that entry of 9L cells into S phase depends on an adequate intracellular pool of polyamines. D,L-cw-Difluoromethylornithine (DFMO) is an enzyme-activated, irreversible inhibitor of omithine decarboxylase (ODC, EC 4.1.1.17), the first enzyme in the polyamine biosynthetic pathway [l]. A number of studies have shown that the intracellular putrescine (Pu) and spermidine (Sd) content of cultured mammalian cells is depleted after treatment with DFMO [24]. Inhibi- tion of cellular proliferation [2, 31 or re- duced rates of intracellular DNA synthesis [4] occur subsequent to significant Sd de- pletion . Recently we reported that treatment of 9L rat brain tumor cells with 1 or 10 mM DFMO 24 h after seeding 1X lo6 cells/flask produced only minimal cytostasis despite significant Pu and Sd depletion [5]. These findings were in conflict with observations made on other cell lines [2, 3, 61. We have therefore extended our original studies to determine if 9L cells can maintain control rates of proliferation despite almost com- plete Pu and Sd depletion, or if our initial observations were a result of the culture conditions used in that study [5]. * To whom offprint requests should be addressed. Exp Cell Res 131 (1981)