Polyamine Depletion Inhibits Apoptosis

Ray et al. (21) showed for the first time that polyamine depletion activates signal transduction pathways leading to cell-cycle arrest. Incubating IEC-6 cells with DFMO for 4 d inhibited proliferation, and arrested cells in the G1 phase of the cell cycle. Cell-cycle arrest was accompanied by an increase in the level of p53 protein and the cell-cycle inhibitors, p21Waf1/Cip1 and p27Kip1. Unlike findings in many other cell lines, the induction of p53 did not induce apoptosis. p53 is activated by a variety of stimuli in

Dna Damage Cell Cycle Sphase Increase

different cell types. These stimuli include DNA damage, withdrawal of growth factors, and the expression of Myc (22). Whether apoptosis occurs depends on the severity of damage and other existing factors. The mechanisms involved in determining whether cell-cycle arrest or apoptosis occurs aren't entirely known, but the deletion of p21Waf1/Cip1 can cause cells to undergo apoptosis that would otherwise have entered into cell-cycle arrest and repair (22). Because damage-induced apoptosis of intestinal cells is p53-dependent (23), we predicted that polyamine-deficient IEC-6 cells might be protected from apoptosis.

We examined the involvement of polyamines in the induction of apoptosis by the DNA topoisomerase I inhibitor, camptothecin (24). In IEC-6 cells, camptothecin-induced apoptosis occurred within 6 h, accompanied by detachment of cells and an increase in caspase 3 activity. DFMO decreased the number of floating cells by 80% and inhibited the increase in caspase 3 activity by 75%. The addition of 10 p,M putrescine to the incubate along with DFMO prevented all the effects of polyamine depletion. Similar results were obtained when apoptosis was stimulated by TNF-a and cycloheximide (CHX) (24). The inhibition of S-adenosylmethione decarboxylase with 1 mM diethylglyoxal ¿tfs-(guanyl-hydrozone) doubled the rate of apoptosis in response to camptothecin, while significantly increasing cell putrescine levels and depleting spermidine and spermine. Thus in this system, putrescine itself appears sufficient to

Fig. 4. Apoptotic cells in mice subjected to 15 Gy y-irradiation and killed 4 h later. Mice received vehicle (control) or 2% DFMO in their drinking water for 4 d before irradiation. (A) Apoptotic cells per crypt-villus unit. (B) Position of apoptotic cells along the crypt villus axis. (Reproduced with permission from ref. 25.)

Fig. 4. Apoptotic cells in mice subjected to 15 Gy y-irradiation and killed 4 h later. Mice received vehicle (control) or 2% DFMO in their drinking water for 4 d before irradiation. (A) Apoptotic cells per crypt-villus unit. (B) Position of apoptotic cells along the crypt villus axis. (Reproduced with permission from ref. 25.)

support normal levels of apoptosis. This study also demonstrated that polyamine depletion was able to inhibit apoptosis, regardless of whether it was initiated by the intrinsic (camptothecin) or the extrinsic (TNF-a) pathway.

Polyamine depletion has the same effect on apoptosis in vivo as it does in vitro (25). Pretreatment of IEC-6 cells with DFMO for 4 d significantly reduced radiation-induced apoptosis, as determined by DNA fragmentation and caspase 3 activity. Radiation exposure in mice resulted in a high frequency of apoptosis over cell positions 4 through 7 in the crypt (Fig. 4A). Pretreatment of mice with 2% DFMO in drinking water significantly reduced apoptotic cells from approx 2.75 to 1.61 per crypt-villus unit (Fig. 4B), accompanied by significant decreases in caspase 3 levels. Pretreatment with DFMO also inhibited the radiation-induced increase in the proapoptotic protein, Bax. Moreover, DFMO significantly enhanced the intestinal crypt survival rate by 2.1 times as determined 4 d after irradiation (25). Thus these experiments indicate that the IEC-6 cell line reflects the responses to apoptosis and polyamines that occur in the intestine of the whole animal.

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