CELLULAR PHARMACOLOGY OF N(1)-AZIRIDINYL AND N(8)-AZIRIDINYL ANALOGS OF SPERMIDINE

We have previously described the synthesis and cytotoxic properties of 2 polyamine analogues in which either the N1- or N8-amino group of sp ermidine was replaced by the alkylating moiety, aziridine. However, th e mechanisms by which these aziridinyl analogues of spermidine inhibit cell growth remain unknown. As a result, we have studied: (a) the eff ect of pretreatment with difluoromethyl ornithine (DFMO) and coincubat ion with exogenous spermidine on cytotoxicity induced by the aziridiny l spermidines; (b) the reversibility of the cytotoxicity induced by th e aziridinyl spermidines; (c) the accumulation of N1- and N8-aziridiny l spermidine by cells and the effects of DFMO on this process; and (d) the impact of N1- and N8-aziridinyl spermidine on cellular polyamine pools and on cellular accumulation of spermidine. The cytotoxicity ind uced by these 2 aziridinyl derivatives of spermidine [concentration re quired to inhibit cell growth or incorporation of radiolabeled precurs or into trichloroacetic acid-precipitable material by 50% (IC50) N1 = 0.2 muM, IC50 N8 = 0.4 muM)] was potentiated by pretreatment of L1210 cells for 24 h with 100 muM DFMO (IC50 N1 = 0.05 muM, IC50 N8 = 0.15 m uM) and was prevented by coincubation with 3.7 muM spermidine (IC50 N1 = 1.1 muM, IC50 N8 = 2.4 muM). In contrast, similar pretreatment with DFMO or coincubation with spermidine had no effect on the cytotoxicit y induced by the aziridine-containing alkylating agent, N,N',N''-triet hylenethiophosphoramide (thiotepa) (IC50 = 2.4 muM). The cytotoxicity induced by 24-h incubation with either N1- or N8-aziridinyl spermidine was not altered by removal of those compounds and incubating treated cells in medium augmented with 3.7 Am spermidine. However, and as expe cted, similar maneuvers did not reverse the cell growth-inhibitory eff ect induced by 24-h incubation with 100 muM DFMO. Cellular accumulatio n of both N1- and N8-aziridinyl spermidine increased with increasing e xtracellular concentrations. N1-Aziridinyl spermidine was accumulated to a greater degree than was the N8-analogue, achieving up to 6-fold h igher intracellular concentrations at the same extracellular concentra tion. Cellular accumulation of both aziridinyl compounds was greatly e nhanced by 24-h pretreatment with DFMO. Both N1- and N8-aziridinyl spe rmidine inhibited the uptake of spermidine in a dose-dependent manner. The perturbation of polyamine biochemistry by the test compounds was characterized b their ability to deplete cellular putrescine, as well as spermidine and spermine. These results imply that the cytotoxic mec hanism of the aziridinyl spermidine analogues is. to a great extent, d ependent on their polyamine nature and may imply selectivity for rapid ly growing and neoplastic cells.