Abstract
During apoptosis, phosphatidylserine (PS) is moved from the plasma membrane inner leaflet to the outer leaflet where it triggers recognition and phagocytosis of the apoptotic cell. Although the mechanisms of PS appearance during apoptosis are not well understood, it is thought that declining activity of the aminophospholipid translocase and calcium-mediated, nonspecific flip-flop of phospholipids play a role. As previous studies in the erythrocyte ghost have shown that polyamines can alter flip-flop of phospholipids, we asked whether alterations in cellular polyamines in intact cells undergoing apoptosis would affect PS appearance, either by altering aminophospholipid translocase activity or phospholipid flip-flop. Cells of the human leukemic cell line, HL-60, were incubated with or without the ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and induced to undergo apoptosis by ultraviolet irradiation. Whereas DFMO treatment resulted in profound depletion of putrescine and spermidine (but not spermine), it had no effect on caspase activity, DNA fragmentation, or plasma membrane vesiculation, typical characteristics of apoptosis. Notably, DFMO treatment prior to ultraviolet irradiation did not alter the decline in PS inward movement by the aminophospholipid translocase as measured by the uptake of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS detected in the flow cytometer. Conversely, the appearance of endogenous PS in the plasma membrane outer leaflet detected with fluorescein isothiocyanate-labeled annexin V and enhanced phospholipid flip-flop detected by the uptake of 1-palmitoyl-1-[6-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)aminocaproyl]-sn-glycero-3-phosphocholine (NBD-PC) seen during apoptosis were significantly inhibited by prior DFMO treatment. Importantly, replenishment of spermidine, by treatment with exogenous putrescine to bypass the metabolic blockade by DFMO, restored both enhanced phospholipid flip-flop and appearance of PS during apoptosis. Such restoration was seen even in the presence of cycloheximide but was not seen when polyamines were added externally just prior to assay. Taken together, these data show that intracellular polyamines can modulate PS appearance resulting from nonspecific flip-flop of phospholipids across the plasma membrane during apoptosis.
Highlights
Under normal conditions, plasma membrane phospholipids of cells appear to be asymmetrically distributed across the bilayer with phosphatidylserine found almost entirely in the inner leaflet and sphingomyelin in the outer leaflet of the bilayer [1,2,3]
These observations prompted us to ask whether alteration of polyamine levels in intact cells during the process of apoptosis would alter the appearance of PS in the outer leaflet of the plasma membrane [8, 14]
In earlier work from this laboratory, we have shown that enhanced phospholipid flip-flop and PS appearance seen during apoptosis are entirely dependent on the presence of extracellular calcium [8]
Summary
The human leukemia cell line HL-60 was obtained from ATCC (Rockville, MD) and cultured in RPMI 1640 (Mediatech, Herndon, VA) supplemented with 20% heat-inactivated fetal bovine serum (Gemini Biological Products, Inc., Calabasas, CA) and maintained in the undifferentiated state at 37 °C in a 5% CO2, humidified atmosphere. HL-60 cells were plated at a cell density of 1.25 ϫ 105 cells/ml in RPMI with 20% fetal calf serum and treated with 1 mM DFMO ((2-(difluoromethyl)-DL-ornithine monohydrochloride monohydrate), a generous gift of Dr Ekkehard Bohme, Hoechst-Roussel, Cincinnati, OH) and incubated 120 h at 37 °C in a 5% CO2 humidified atmosphere. Reconstitution of polyamines was accomplished by the addition of putrescine in RPMI at a final concentration of 10 M at 72 and 96 h post-seeding. In experiments in which protein synthesis was inhibited by cycloheximide, the inhibitor was added at a final concentration of 0.5 g/ml concurrently with the initial addition of putrescine. This concentration and incubation period was shown to be sufficient to stop most of protein synthesis as determined by a 75% reduction in [35S]Cys incorporation but which resulted in less than 10% cell death as determined by trypan blue staining
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
