Polyamine regulation of plasma membrane phospholipid flip-flop during apoptosis

During apoptosis, phosphatidylserine (PS) is moved from the plasma membrane inner leaflet to the outer leaflet where it triggers recognition and phago cytosis of the apoptotic cell. Although the mechanisms of PS appearance dur ing apoptosis are not well understood, it is thought that declining activit y of the aminophospholipid translocase and calcium-mediated, nonspecific fl ip-flop of phospholipids play a role, As previous studies in the erythrocyt e ghost have shown that polyamines can alter flip flop of phospholipids, we asked whether alterations in cellular polyamines in intact cells undergoin g apoptosis would affect PS appearance, either by altering aminophospholipi d translocase activity or phospholipid flip-flop. Cells of the human leukem ic cell line, HL-60, were incubated with or without the orni thine decarbox ylase inhibitor, difluoromethylornithine (DFMO), and induced to undergo apo ptosis by ultraviolet irradiation. Whereas DFMO treatment resulted in profo und depletion of putrescine and spermidine (but not spermine), it had no ef fect on caspase activity, DNA fragmentation, or plasma membrane vesiculatio n, typical characteristics of apoptosis, Notably, DFMO treatment prior to u ltraviolet irradiation did not alter the decline in PS inward movement by t he aminophospholipid translocase as measured by the uptake of 6-[(7-nitrobe nz-2-oxa-1,3-diazol-4-yl) aminocaproyl] (NBD)-labeled PS detected in the fl ow cytometer, Conversely, the appearance of endogenous PS in the plasma mem brane outer leaflet detected with fluorescein isothiocyanate-labeled annexi n V and enhanced phospholipid flip-flop detected by the uptake of 1-palmito yl-1-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)aminocaproyl]-sn-glycero-3-phosp hocholine (NBD-PC) seen during apoptosis were significantly inhibited by pr ior DFMO treatment, Importantly, replenishment of spermidine, by treatment with exogenous putrescine to bypass the metabolic blockade by DFMO, restore d both enhanced phospholipid flip-flop and appearance of PS during apoptosi s, Such restoration was seen even in the presence of cycloheximide but was not seen when polyamines were added externally just prior to assay, Taken t ogether, these data show that intracellular polyamines can modulate PS appe arance resulting from nonspecific flip-flop of phospholipids across the pla sma membrane during apoptosis.