Rapid induction of apoptosis by deregulated uptake of polyamine analogues.

Treatment of Chinese hamster ovary cells with alpha-difluoromethylornithine for 3 days, followed by exposure to cycloheximide, led to an unregulated, rapid and massive accumulation of polyamine analogues. This accumulation led to cell death by apoptosis within a few hours. Clear evidence of DNA fragmentation was seen in response to both N-terminally ethylated polyamines and to polyamines containing methyl groups on the terminal carbon atoms. Programmed cell death was induced within 2-4 h of exposure to 1 microM or higher concentrations of N1,N11-bis(ethyl)norspermine. The presence of cycloheximide increased the uptake of the polyamine analogues and therefore led to cell death at lower analogue concentrations, but it was not essential for the induction of apoptosis, since similar effects were seen when the protein synthesis inhibitor was omitted and the concentration of N1, N11-bis(ethyl)norspermine was increased to 5 microM or more. The induction of apoptosis was blocked both by the addition of the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, or by the addition of the polyamine oxidase inhibitor N1-methyl-N2-(2,3-butadienyl)butane-1,4-diamine (MDL 72,527). These experiments provide evidence to support the concepts that: (1) polyamines or their oxidation products may be initiators of programmed cell death; (2) regulation of polyamine biosynthesis and uptake prevents the accumulation of toxic levels of polyamines; and (3) the anti-neoplastic effects of bis(ethyl) polyamine analogues may be due to the induction of apoptosis in sensitive tumour cells.