April 2002
Volume 43, Issue 4
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Retinal Cell Biology  |   April 2002
Polyamine-Dependent Migration of Retinal Pigment Epithelial Cells
Author Affiliations
  • Dianna A. Johnson
    From the Departments of Ophthalmology,
  • Carolyn Fields
    From the Departments of Ophthalmology,
  • Amy Fallon
    From the Departments of Ophthalmology,
    Biology Department, Christian Brothers University, Memphis, Tennessee.
  • Malinda E. C. Fitzgerald
    Anatomy and Neurobiology, and
    Biology Department, Christian Brothers University, Memphis, Tennessee.
  • Mary Jane Viar
    Physiology, University of Tennessee Health Science Center, Memphis, Tennessee; and the
  • Leonard R. Johnson
    Physiology, University of Tennessee Health Science Center, Memphis, Tennessee; and the
Investigative Ophthalmology & Visual Science April 2002, Vol.43, 1228-1233. doi:
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      Dianna A. Johnson, Carolyn Fields, Amy Fallon, Malinda E. C. Fitzgerald, Mary Jane Viar, Leonard R. Johnson; Polyamine-Dependent Migration of Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2002;43(4):1228-1233.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Migration of retinal pigment epithelial (RPE) cells can be triggered by disruption of the RPE monolayer or injury to the neural retina. Migrating cells may re-establish a confluent monolayer, or they may invade the neural retina and disrupt visual function. The purpose of this study was to examine the role of endogenous polyamines in mechanisms of RPE migration.

methods. Endogenous polyamine levels were determined in an immortalized RPE cell line, D407, using HPLC. Activities of the two rate-limiting enzymes for polyamine synthesis, ornithine decarboxylase (ODC), and S-adenosylmethionine decarboxylase (SAMdc), were measured by liberation of (14CO2). Migration was assessed in confluent cultures by determining the number of cells migrating into a mechanically denuded area. All measurements were obtained both in control cultures and in cultures treated with synthesis inhibitors that deplete endogenous polyamines. Subcellular localization of endogenous polyamines was determined using a polyamine antibody.

results. The polyamines, spermidine and spermine, as well as their precursor, putrescine, were normal constituents of RPE cells. The two rate-limiting synthetic enzymes were also present, and their activities were stimulated dramatically by addition of serum to the culture medium. Cell migration was similarly stimulated by serum exposure. When endogenous polyamines were depleted, migration was blocked. When polyamines were replenished through uptake, migration was restored. Polyamine immunoreactivity was limited to membrane patches in quiescent cells. In actively migrating and dividing cells, immunoreactivity was enhanced throughout the cytoplasm.

conclusions. Polyamines are essential for RPE migration. Pharmacologic manipulation of the polyamine pathway could provide a therapeutic strategy for regulating anomalous migration.

Retinal pigment epithelium (RPE) normally exists as a mitotically quiescent monolayer of stationary cells that provides structural and metabolic support for visual function of the neural retina. Migration of RPE cells may be triggered by release of chemotactic agents or growth factors. Release of these factors from blood or vitreous is triggered in response to trauma, such as retinal detachment, 1 2 or certain pathologic conditions, such as subretinal neovascularization. 3 4 5 6 In some cases, migratory RPE cells may play a beneficial role in the healing process. They may aggregate along injured capillaries to slow leakage and thus impede further neovascular growth. 7 In contrast, exuberant migration and proliferation can also lead to accumulation of ectopic RPE cells in the form of epiretinal membranes that cause severe vision loss. 8 Thus, it is of considerable interest to examine the migratory behavior of RPE and to understand ways in which migration can be controlled clinically. 
To achieve movement, specific cellular mechanisms interact to produce coordinated cycles of four basic steps: protrusion of the leading edge, formation of attachment sites (focal adhesions), generation of contractile forces, and detachment of the trailing edge. 9 Cytoskeletal elements provide the intracellular framework necessary for each of these steps. Migration is initiated at cellular protrusion sites by polymerization of an actin network along the cell’s leading edge. Focal adhesions are composed of complexes of integrin receptors, actin filaments, and associated proteins that provide anchor points for actin stress fibers. Movement is initiated and maintained by contraction of myosin in association with stress fibers. 9  
A number of factors appear to regulate these cytoskeletal elements. In RPE cells, 10 as well as other epithelial cell types, 11 protein kinase C stimulates migration, perhaps through phosphorylation of integrin. 12 Mitogen-activated protein kinase has also been shown to be necessary for RPE migration. 13 Other regulatory factors have been identified in several epithelial cell types but have not been studied in RPE. Among these are members of the Rho family of small GTPases, Rho, Cdc42, and Rac, which regulate new protrusions and adhesions through their effects on polymerization and reorganization of actin. 14 Through regulation of p21 activated kinase, they also promote phosphorylation of myosin light chain and hence dimerization and interactions of myosin with actin to drive contraction. 15  
Additional regulatory factors recently identified include Src family kinases, 16 focal adhesion kinase (FAK), 17 and calpain. 18 FAK is activated by integrins and targeted to the focal adhesion complex. There, it undergoes additional phosphorylation to provide binding sites for other proteins, such as paxillin, within the focal adhesion complex. Paxillin, in turn, recruits actin to the complex and stimulates formation of stress fibers. 19  
Additional potent regulators of migration, the polyamines, have been described in intestinal epithelial cells. 20 21 22 The effect of polyamines on RPE migration has not been studied. Polyamines are composed of flexible carbon chains with amino groups that are positively charged at physiologic pH, and their synthesis is tightly regulated through the inducible enzyme, ornithine decarboxylase (ODC; for review, see Ref. 23 ). Although the exact mechanism of action of polyamines has not been determined, blockage of polyamine synthesis has been shown to cause reorganization of F-actin and tropomyosin and to disrupt the association of F-actin with activated epidermal growth factor (EGF) receptors. 24 25 In addition, polyamine depletion decreases phosphorylation of FAK, recruitment of paxillin, and focal adhesion complex formation. 26  
Endogenous polyamines are present in primary cultures of bovine RPE cells, and they are essential for proliferation. 27 Given the role of polyamines in intestinal epithelial cell migration, we wanted to determine whether polyamines also play a role in RPE cell migration. Using a human RPE cell line (D407), we found that polyamine deficiency disrupted migration and that the disruption was prevented by addition of exogenous polyamines. Biochemical analysis coupled with immunocytochemical localization studies suggest that polyamine content increases dramatically, and its subcellular localization changes in cells that migrate in response to disruption of the RPE monolayer. 
Methods
Cell Culture
The D407 line of human RPE cells was a gift from Alberta Davis (Alcon Laboratories, Fort Worth, TX). 28 Cells were maintained in T-25 tissue culture flasks (BD Biosciences, Franklin Lake, NJ) at 37° in an atmosphere of 95% air and 5% CO2. The culture medium contained Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY) with 5% neonatal calf serum, insulin (50 mg/mL), and an antibiotic–antimycotic mixture (10,000 U/mL penicillin G sodium, 10 mg/mL streptomycin sulfate, and 25 μg/mL amphotericin B). Cells were passaged weekly and fed every other day. 
Enzyme Activity of ODC and S-Adenosylmethionine Decarboxylase
ODC activity was measured by the liberation of [14CO2] from l- [1-14C] ornithine (52.6 mCi/mmol; New England Nuclear, Boston, MA) as previously described. 29 Cell supernatants were incubated with 200 μL labeled ornithine for 15 minutes at 37°C. The released [14CO2] was trapped on a piece of filter paper impregnated with 20 μL of 2 N NaOH suspended in a center well above the reaction mixture. The reaction was be stopped by the addition of 10% trichloroacetic acid and shaken for an additional 10 minutes to allow complete absorption of labeled CO2. The trapped [14CO2] was measured by liquid scintillation spectrophotometry at a counting efficiency of 97%. Enzyme activity is expressed as picomoles of CO2 per hour per milligram protein. 
S-adenosylmethionine decarboxylase (SAMdc) activity was measured similarly by the liberation of [14CO2] from S-adenosyl l-methionine (59.8 mCi/mmol) as previously described. 30 The assay mixture contained 20 μL labeled substrate and 100 μL sodium phosphate buffer (pH 7.40) containing putrescine dihydrochloride. Samples were incubated for 30 minutes at 37°C and enzyme activity expressed as above. Quantitative protein determination for all enzyme assays was by the Bradford method. 31  
Assay of Endogenous Polyamines
The levels of endogenous putrescine, spermidine, and spermine were analyzed by HPLC. Samples were placed in 0.5 mL of 0.5 M perchloric acid and immediately frozen at −80°C until all samples were ready for dansylation, extraction, and HPLC. Total protein content was determined using the Bradford method, 31 with polyamine content expressed as nanomoles per milligram protein. 
Migration Assay
This assay has been described previously. 20 22 Cells were plated at 2 × 105 cells/cm2 on uncoated plastic Petri dishes. Samples were first serum starved for 24 hours and then serum stimulated. This procedure is routinely used, first to help synchronize cultures so that the metabolic (serum-starved) state of the cells is more uniform, and second to provide a general stimulus (serum) that has been shown to trigger migration. 20 In our experiments, cells were fed on day 2 and serum removed on day 3. Migration was begun 24 hours later on day 4 with the addition of dialyzed serum. Experimental groups received 5 mM α-difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis (described later), with or without exogenous polyamines, throughout the experiment. Cell migration was initiated by clearing an area on the plate with a razor blade. The medium was changed to remove loose cells, cell fragments, and any endogenous secreted products that had collected during the previous 24 hours. The cells were allowed to migrate for 3 hours, after which they were fixed with 4% formaldehyde. The cells migrating across the scratch line were counted by computer imaging using an image analysis program (Metaview; Update Software, Chevy Chase, MD). All experiments were performed in triplicate and repeated (n = 6). Results are reported as the number of cells migrating per millimeter of scratch. Data are expressed as the mean ± SEM of six dishes. Significance of differences was determined by analysis of variance, and the level of significance was determined by Dunnett’s multiple range test. 32  
Immunocytochemistry
Cultures were fixed in 4% paraformaldehyde, 4% sucrose (pH 7.2) for 1 hour at room temperature. For antibody staining, samples were preincubated in blocking buffer (PBS with 1% Triton, 5% BSA and 5% normal serum to match the animal in which the primary antibody was made) for 1.5 hours at room temperature. An antibody against polyamines was obtained from Jean Guy Delcros (Institut de Rechereche Contre le Cancer, Rennes, France). The antibody has been well characterized. 33 34 35 Samples were incubated in primary antibody for 1 hour at room temperature and rinsed with PBS. For visualization with 3,3′-diaminobenzidine (DAB), cultures were rinsed several times in PBS and then incubated in biotinylated secondary antibody for 30 minutes at room temperature. Endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide solution for 15 minutes. Cultures were then incubated in avidin-biotin complex solution (Elite ABC; Vector Laboratories, Burlingame, CA) for 30 minutes followed by reaction with DAB substrate for 4 minutes. Cultures were allowed to air dry and were mounted (Cytoseal 60 mounting medium; Stephens Scientific, Kalamazoo, MI). 
Results
Two specific inhibitors of polyamine synthesis have been well characterized (Fig. 1) . One of these, DFMO, is a suicide substrate for ODC, and it specifically and irreversibly blocks synthesis of putrescine and, hence, the subsequent formation of spermidine and spermine. 36 37 The effect of DFMO on polyamine levels in RPE cultures is shown in Figure 2 . In control cells, levels of putrescine were higher than either spermidine or spermine. Exposure to 5 mM DFMO for 8 hours effectively depleted the cells of both putrescine and spermidine but had no significant effect on spermine levels. 
In addition to DFMO, another inhibitor of polyamine synthesis, diethylglyoxal bis-guanylhydrazone (DEGBG), has been described. 38 This compound inhibits the second rate-limiting enzyme, SAMdc, and leads to a depletion of spermine and spermidine (Fig. 1) . In RPE cells, DFMO and DEGBG were shown to be effective in inhibiting ODC and SAMdc, respectively (Fig. 3) . Basal activities of both enzymes were low in serum-free medium (t = 0) but were stimulated roughly sixfold within 4 to 8 hours after addition of serum to the medium. Enzyme activity decreased to basal levels within 12 hours after stimulation. The serum stimulated surge in activity of both enzymes was completely blocked by their respective inhibitors. 
Migration of confluent RPE cells in culture was triggered by mechanical removal of a portion of the cell monolayer. A significant number of the cells adjacent to the denuded area migrated into the cell-free area within 6 hours (Fig. 4) . Addition of 5 mM DFMO inhibited the migration. These observations were verified by obtaining counts of migrating cells (Fig. 4) . DFMO reduced the number of migrating cells by more than half. The addition of exogenous putrescine (100 μm), spermidine (10 μm), or spermine (5 μm) prevented the inhibition of migration by DFMO. 
Confluent cultures and those that had been partially denuded were fixed, labeled with the polyamine antibodies, and visualized with the avidin-biotin method. Areas of confluence showed light overall staining but specific labeling in small patches of the plasma membrane (Figs. 5A 5B) . The stained patches in one cell were often in register with stained areas of adjacent cells within the confluent monolayer. Stained structures appeared to have a filamentous nature (Fig. 5B) . A different pattern of staining was observed in two other types of cells: those that were not part of the monolayer and formed small aggregates on the outer surface of the monolayer (Figs. 5A 5C) and those along the edge of a denuded area (Fig. 5D) . In both cases, these cells were likely to be actively dividing and/or migrating. Staining was dark throughout the cytoplasm, except for aggregates of unstained vesicles. Lighter staining was present in the nucleus. 
Discussion
Our results demonstrate that the polyamines, spermidine and spermine, as well as their precursor, putrescine, are normal constituents of RPE cells. The observation that the activities of both rate-limiting enzymes for polyamine synthesis, ODC and SAMdc, appeared to be highly regulated is important. Low enzyme activities were observed in serum-free medium, whereas the presence of serum transiently stimulated both activities many fold within 2 hours. This suggests that pools of newly synthesized polyamines in RPE can fluctuate rapidly in response to changes in the extracellular environment. 
Blockage of polyamine synthesis by DFMO led to a complete depletion of putrescine and spermidine, while spermine levels remained near control levels. This suggests that putrescine and spermidine turn over within 8 hours or less. We were unable to establish the turnover rate for spermine from these data. It is possible that spermine, because it is the most highly charged of the polyamines, remains tightly bound and thus protected from catabolism. Alternatively, if all the putrescine and spermidine lost after DFMO treatment is converted to a stable pool of spermine that does not turn over, the total levels of spermine after DFMO treatment should be several fold higher than control values. The fact that spermine levels were lower than this predicted value suggests that at least some portion, perhaps the major portion, of the spermine pool turns over within 8 hours. 
Based on studies in other epithelial cell types, 20 21 22 24 25 changes in newly synthesized polyamine pools may be tied to a number of important functions in RPE cells. Our studies showed for the first time that serum-stimulated migration of RPE cells is dependent on synthesis of polyamines. Cells normally migrate when contact inhibition is lost. This can occur with trauma (e.g., retinal detachment or various surgical procedures), or retinal disease (e.g., epiretinal membranes), or as a natural consequence of aging when random RPE cells die. In our experimental model, contact inhibition was removed by physically scraping away portions of a confluent culture and counting the number of cells that migrate into the denuded area. Blockage of polyamine synthesis by DFMO was found to inhibit the migration that is normally seen 6 hours after serum stimulation. Thus, the migratory response occurs within the time frame we observed for the serum stimulated increase in polyamine synthesis in control cultures. When exogenous polyamines were added to replenish endogenous pools, DFMO had no effect on migration. We thus conclude that RPE migration is dependent on the availability of polyamines that are either endogenously synthesized or taken up from endogenous sources. This also demonstrates that the effects of DFMO are caused by the absence of polyamines and not by a direct action of DFMO itself. 
It is interesting to note that the addition of exogenous spermine (or other polyamines) to DFMO treated cells was needed to restore migration, even though the endogenous level of spermine was found to be near normal under these conditions. This suggests that spermine could exist in two pools: one that is sequestered and not readily available for support of migration and a second that is provided by stimulated synthesis (or uptake) and is essential for migration. 
Our immunocytochemical data provide the first description of the subcellular localization of polyamines in RPE cells, as well as changes in distribution that occur when cells are actively dividing and migrating. In quiescent cells, polyamine immunoreactivity is limited to discrete membrane patches at apparent points of attachment among adjacent cells. Recent reports have shown that polyamines regulate the structure of the cellular cytoskeleton, particularly the distribution of actin filaments and stress fibers. 22 We speculate that the polyamine-stained regions we observe could represent junctional complexes that serve to anchor the actin cytoskeleton to the membrane and, through integrin, to elements of the extracellular matrix and adjacent cells. If our conjecture is correct, binding of polyamines to junctional complexes could represent one of the sites at which this regulation is effected. 
Polyamine immunoreactivity was greatly enhanced in random groups of D407 cells that did not appear contact inhibited and that accumulated on the outer surface of an underlying, confluent monolayer of cells. A similar staining pattern was observed in cells within the monolayer that had lost contact inhibition after adjacent cells had been experimentally removed. Under both of these conditions, dark staining was observed throughout the cytoplasm. Light staining was observed in the nucleus. Numerous unstained vesicles were seen throughout the cytoplasm. We interpret this staining pattern to be a reflection of the stimulated synthesis of polyamines that would be expected to occur in cells undergoing cell division and/or migration. 
The discrete subcellular localization of polyamines at presumptive sites of attachment for actin filaments in quiescent cells suggests that polyamines may also serve a housekeeping function in maintaining the organization of the cytoskeleton. DFMO has been shown to cause disruption of actin stress fibers in intestinal epithelial cells in culture and thus to alter cell shape. 22 It is reasonable to suggest that there may be a similar interaction between polyamines and actin filaments during migration, when both polyamine levels and the number of stress fibers increase. 
In summary, polyamine function has been well studied in a number of epithelial cell types; however, little is known about the specific roles polyamines may play in RPE. In our findings polyamines were essential for RPE cell migration. Migration is required to establish a confluent monolayer during development and to maintain the monolayer during random loss of cells during aging. Of specific clinical importance is the abnormal RPE cell migration that leads to the formation of epiretinal membranes and vision loss after various forms of retinal trauma or disease. Our results provide a useful experimental model for further examination of RPE cell migration. One future goal will be to develop potential strategies for the pharmacologic regulation of migration that could be suitable for clinical use. This experimental model will also be useful in further defining the basic mechanisms of cell migration and how they are influenced by signaling molecules, such as polyamines. 
 
Figure 1.
 
Inhibitors of polyamine synthesis. DFMO covalently and irreversibly binds to activated ODC and inhibits it. 34 35 This leads to depletion of putrescine and subsequent loss of spermine and spermidine. DEGBG inhibits SAMdc, thus preventing synthesis of the polyamines, spermine and spermidine. 36
Figure 1.
 
Inhibitors of polyamine synthesis. DFMO covalently and irreversibly binds to activated ODC and inhibits it. 34 35 This leads to depletion of putrescine and subsequent loss of spermine and spermidine. DEGBG inhibits SAMdc, thus preventing synthesis of the polyamines, spermine and spermidine. 36
Figure 2.
 
Levels of endogenous polyamines in RPE cells. Samples of the D407 cell line of RPE cells were cultured to confluence in medium containing 10% serum and then exposed for 8 additional hours to control medium or dialyzed medium containing 5 mM DFMO. Harvested cells were analyzed by HPLC. In DFMO-treated samples, putrescine and spermidine were not detectable (*), but spermine levels were not significantly changed from control levels. Data are the mean ± SEM; n = 3.
Figure 2.
 
Levels of endogenous polyamines in RPE cells. Samples of the D407 cell line of RPE cells were cultured to confluence in medium containing 10% serum and then exposed for 8 additional hours to control medium or dialyzed medium containing 5 mM DFMO. Harvested cells were analyzed by HPLC. In DFMO-treated samples, putrescine and spermidine were not detectable (*), but spermine levels were not significantly changed from control levels. Data are the mean ± SEM; n = 3.
Figure 3.
 
Enzyme activity of ODC (left) and SAMdc (right) in cultured RPE cells. Cultures were grown to confluence in the presence of 10% serum and placed in serum-free medium for 24 hours. Dialyzed serum was reintroduced to the cultures (at t = 0) in the presence or absence of enzyme-specific inhibitors (DFMO or DEGBG), and samples were analyzed at 4-hour intervals thereafter. Serum-stimulated activities of both enzymes were blocked by their respective inhibitors. Data are the mean ± SEM; n = 3.
Figure 3.
 
Enzyme activity of ODC (left) and SAMdc (right) in cultured RPE cells. Cultures were grown to confluence in the presence of 10% serum and placed in serum-free medium for 24 hours. Dialyzed serum was reintroduced to the cultures (at t = 0) in the presence or absence of enzyme-specific inhibitors (DFMO or DEGBG), and samples were analyzed at 4-hour intervals thereafter. Serum-stimulated activities of both enzymes were blocked by their respective inhibitors. Data are the mean ± SEM; n = 3.
Figure 4.
 
Effect of DFMO on migration of RPE cells in culture. Top left: Cells in control cultures migrated into a mechanically denuded area within 6 hours. In the three micrographs shown, the edge of the original cell-free area is noted by a black line. Top right: Cell migration was inhibited by addition of 5 mM DFMO in the culture medium. Bottom left: Addition of spermine (5 μm) prevented the effects of DFMO. Bottom right: Quantitative analysis of RPE cell migration in DFMO-treated cultures in the presence and absence of exogenous polyamines. In cultures treated with 5 mM DFMO, the average number of migrating cells was inhibited by more than 50% compared with control levels. Addition of exogenous putrescine (100 μm), spermidine (10 μm), or spermine (5 μm) prevented the effect of DFMO. Data are the mean ± SEM; n = 3. Scale bars, 20 μm.
Figure 4.
 
Effect of DFMO on migration of RPE cells in culture. Top left: Cells in control cultures migrated into a mechanically denuded area within 6 hours. In the three micrographs shown, the edge of the original cell-free area is noted by a black line. Top right: Cell migration was inhibited by addition of 5 mM DFMO in the culture medium. Bottom left: Addition of spermine (5 μm) prevented the effects of DFMO. Bottom right: Quantitative analysis of RPE cell migration in DFMO-treated cultures in the presence and absence of exogenous polyamines. In cultures treated with 5 mM DFMO, the average number of migrating cells was inhibited by more than 50% compared with control levels. Addition of exogenous putrescine (100 μm), spermidine (10 μm), or spermine (5 μm) prevented the effect of DFMO. Data are the mean ± SEM; n = 3. Scale bars, 20 μm.
Figure 5.
 
Immunocytochemical localization of polyamines in RPE cells. Cells grown to confluence showed two patterns of polyamine staining. (A) In cells that constituted a confluent monolayer, staining was confined to membrane-associated patches (A, B; arrowheads). At high magnification (B), the staining was seen to be associated with filamentous structures (staining of nuclei was artificially enhanced to show their location). Aggregates of actively dividing cells (A, C; arrows) have darkly stained cytoplasm filled with unstained vesicles. (D) Dark staining was observed in all cells along a mechanically disrupted edge of the culture.
Figure 5.
 
Immunocytochemical localization of polyamines in RPE cells. Cells grown to confluence showed two patterns of polyamine staining. (A) In cells that constituted a confluent monolayer, staining was confined to membrane-associated patches (A, B; arrowheads). At high magnification (B), the staining was seen to be associated with filamentous structures (staining of nuclei was artificially enhanced to show their location). Aggregates of actively dividing cells (A, C; arrows) have darkly stained cytoplasm filled with unstained vesicles. (D) Dark staining was observed in all cells along a mechanically disrupted edge of the culture.
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Figure 1.
 
Inhibitors of polyamine synthesis. DFMO covalently and irreversibly binds to activated ODC and inhibits it. 34 35 This leads to depletion of putrescine and subsequent loss of spermine and spermidine. DEGBG inhibits SAMdc, thus preventing synthesis of the polyamines, spermine and spermidine. 36
Figure 1.
 
Inhibitors of polyamine synthesis. DFMO covalently and irreversibly binds to activated ODC and inhibits it. 34 35 This leads to depletion of putrescine and subsequent loss of spermine and spermidine. DEGBG inhibits SAMdc, thus preventing synthesis of the polyamines, spermine and spermidine. 36
Figure 2.
 
Levels of endogenous polyamines in RPE cells. Samples of the D407 cell line of RPE cells were cultured to confluence in medium containing 10% serum and then exposed for 8 additional hours to control medium or dialyzed medium containing 5 mM DFMO. Harvested cells were analyzed by HPLC. In DFMO-treated samples, putrescine and spermidine were not detectable (*), but spermine levels were not significantly changed from control levels. Data are the mean ± SEM; n = 3.
Figure 2.
 
Levels of endogenous polyamines in RPE cells. Samples of the D407 cell line of RPE cells were cultured to confluence in medium containing 10% serum and then exposed for 8 additional hours to control medium or dialyzed medium containing 5 mM DFMO. Harvested cells were analyzed by HPLC. In DFMO-treated samples, putrescine and spermidine were not detectable (*), but spermine levels were not significantly changed from control levels. Data are the mean ± SEM; n = 3.
Figure 3.
 
Enzyme activity of ODC (left) and SAMdc (right) in cultured RPE cells. Cultures were grown to confluence in the presence of 10% serum and placed in serum-free medium for 24 hours. Dialyzed serum was reintroduced to the cultures (at t = 0) in the presence or absence of enzyme-specific inhibitors (DFMO or DEGBG), and samples were analyzed at 4-hour intervals thereafter. Serum-stimulated activities of both enzymes were blocked by their respective inhibitors. Data are the mean ± SEM; n = 3.
Figure 3.
 
Enzyme activity of ODC (left) and SAMdc (right) in cultured RPE cells. Cultures were grown to confluence in the presence of 10% serum and placed in serum-free medium for 24 hours. Dialyzed serum was reintroduced to the cultures (at t = 0) in the presence or absence of enzyme-specific inhibitors (DFMO or DEGBG), and samples were analyzed at 4-hour intervals thereafter. Serum-stimulated activities of both enzymes were blocked by their respective inhibitors. Data are the mean ± SEM; n = 3.
Figure 4.
 
Effect of DFMO on migration of RPE cells in culture. Top left: Cells in control cultures migrated into a mechanically denuded area within 6 hours. In the three micrographs shown, the edge of the original cell-free area is noted by a black line. Top right: Cell migration was inhibited by addition of 5 mM DFMO in the culture medium. Bottom left: Addition of spermine (5 μm) prevented the effects of DFMO. Bottom right: Quantitative analysis of RPE cell migration in DFMO-treated cultures in the presence and absence of exogenous polyamines. In cultures treated with 5 mM DFMO, the average number of migrating cells was inhibited by more than 50% compared with control levels. Addition of exogenous putrescine (100 μm), spermidine (10 μm), or spermine (5 μm) prevented the effect of DFMO. Data are the mean ± SEM; n = 3. Scale bars, 20 μm.
Figure 4.
 
Effect of DFMO on migration of RPE cells in culture. Top left: Cells in control cultures migrated into a mechanically denuded area within 6 hours. In the three micrographs shown, the edge of the original cell-free area is noted by a black line. Top right: Cell migration was inhibited by addition of 5 mM DFMO in the culture medium. Bottom left: Addition of spermine (5 μm) prevented the effects of DFMO. Bottom right: Quantitative analysis of RPE cell migration in DFMO-treated cultures in the presence and absence of exogenous polyamines. In cultures treated with 5 mM DFMO, the average number of migrating cells was inhibited by more than 50% compared with control levels. Addition of exogenous putrescine (100 μm), spermidine (10 μm), or spermine (5 μm) prevented the effect of DFMO. Data are the mean ± SEM; n = 3. Scale bars, 20 μm.
Figure 5.
 
Immunocytochemical localization of polyamines in RPE cells. Cells grown to confluence showed two patterns of polyamine staining. (A) In cells that constituted a confluent monolayer, staining was confined to membrane-associated patches (A, B; arrowheads). At high magnification (B), the staining was seen to be associated with filamentous structures (staining of nuclei was artificially enhanced to show their location). Aggregates of actively dividing cells (A, C; arrows) have darkly stained cytoplasm filled with unstained vesicles. (D) Dark staining was observed in all cells along a mechanically disrupted edge of the culture.
Figure 5.
 
Immunocytochemical localization of polyamines in RPE cells. Cells grown to confluence showed two patterns of polyamine staining. (A) In cells that constituted a confluent monolayer, staining was confined to membrane-associated patches (A, B; arrowheads). At high magnification (B), the staining was seen to be associated with filamentous structures (staining of nuclei was artificially enhanced to show their location). Aggregates of actively dividing cells (A, C; arrows) have darkly stained cytoplasm filled with unstained vesicles. (D) Dark staining was observed in all cells along a mechanically disrupted edge of the culture.
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