Internalization of cationized ferritin receptors by rat hepatoma ascites cells

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Experimental

INTERNALIZATION

Cell Research 114 (1978) 39-45

OF CATIONIZED RAT HEPATOMA

FERRITIN

ASCITES

RECEPTORS

BY

CELLS

PETER C. MOLLER and JEFFREY P. CHANG Division of Cell Biology, Department of Human Biological Chemistry and Genetics, Graduate School of Biomedical Sciences, The University of Texas Medical Branch, Galveston, TX 77550, USA

SUMMARY Cationized ferritin (CF) was used to label the cell surface anionic sites of Chang rat hepatoma ascites cells. If the hepatoma cells were fixed with glutaraldehyde and treated with CF, the label was distributed evenly over the external surface of the plasma membrane. Treatment of unfixed ascites cells with CF yielded clusters of ferritin particles separated by label-free areas of the plasma membrane. Some unfixed ascites cells were treated firstly with CF, then incubated in veronal buffered saline at 37°C for 10, 20, 30 and 45 min, subsequently fixed in glutaraldehyde and re-exposed to CF. After 10 min of incubation, the label was arranged into large clusters with the remaining areas of the plasma membrane lightly labelled with CF. At 20 min, only clusters of ferritin were present on the plasma membrane; the remaining area of the cell surface was totally free of label. The ability of the plasma membrane to bind additional CF was completely restored after 45 min of incubation. These results suggest that for some period of time after internalization of CF label on cell surface the plasma membrane is devoid of any detectable negative charge.

Changes in the surface properties of cells occurring as a result of carcinogenic transformation have been well documented [l]. These alterations are expressed, at least in part, as changes in the carbohydrate portion of plasma membrane glycoproteins [2]. The presence of N-acetyl-neuraminic acid (NANA) in cell surface coat glycoproteins contributes to the cell surface charge. Recently a polycationic derivative offerritin, cationized ferritin (CF), was developed to visualize negative charges on the surface of plasma membranes [3]. With this technique, the surface of unfixed cells can be labelled with CF at a physiologic pH and the distribution of ferritin particles can be observed with an electron microscope. Thus CF has been used to label the surfaces of normal [4, 51 and transformed [6, 71 cells.

However, labeling of anionic sites by CF in hepatoma cells has not been previously reported. In the present study we have labeled the surface of Chang hepatoma ascites cells [8, 93 to study the time course of CF-induced rearrangement and the internalization of cell surface CF receptors. MATERIALS

AND METHODS

Under ether anesthesia, the ascites fluid containing the ascites tumor cells was withdrawn from rats and centrifuged at 250 g for 10 min [lo]. The loosely nacked cells were resuspended 3 times in Verona1 buffered saline (VBS) (O.g5% sodium barbital in 0.9% NaCl pH 7.2) to remove blood corpuscles. Approx. 95 % of the ascites cells excluded trypan blue. Some cells were fixed for 30 mitt in 2% glutaraldehyde in VBS at room temperature (RT) and-washed 3 times in VBS. The material was then exposed to CF solution (0.33 mg CF/ml VBS) for 5 min at RT, rinsed 3 times in VBS and post-fixed in 1% 0~0, for 45 min [ 111.The cells were dehydrated in a graded series of alcohols and embedded in Spurr low viscosity embedding media [ 121in the usual manner. Exp Cell Res 114 (1978)

Figs I-IO.

x60000

Fig. I. Chang hepatoma ascites cell fixed before expo-

sure to CF. The ferritin label is distributed over the cell surface in an even and continuous layer. Lead citrate stain.

Other cells were washed 3 times in VBS at 37°C. The washed cells were treated with CF solution at 37°C for 5 min. After washing with VFJS at RT, the cells were fixed with glutaraldehyde, post-fixed in OsO,, dehydrated and embedded for electron microscopy. Additionally, live ascites cells were washed in warm VBS and treated with CF for 5 min at 37°C. The cells were then washed with warm VBS and reincubated in CF-free VBS at 37°C for 10, 20, 30 or 45 min. After reincubation, the cells were fixed in alutaraldehyde for 30 min. at RT, rinsed 3 times with VBS and re-exposed to CF for 5 min at RT. After being rinsed 3 times in VBS, the cells were postfixed in 0~0,. Dehydration and embedding were carried out as above. Controls consisted of treatment of fixed ascites cells for 20 min at 25°C before CF exnosure with either poly+lysine (Miles-Yeda, Kankakde, Ill.) (10 pg/ml VBS) or with 0.05 % cetyl nyridinium chloride (Sigma, - rSt Louis, MO) in 0.05 Tns-maleate buffer at pa 7.4 to which had been added 8.72% sucrose. As an additional control unfixed ascites cells were treated for 60 Exp Cell Res II4 (1978)

Fig. 2. Chang hepatoma ascites cell after 5 min of

CF treatment at 37°C before fixation. Fenitin label aggregated into clusters presumably as a result of lateral migration of CF receptors. Lead citrate stain.

min at 37°C with neuraminidase (Behrina Diagnostics. Somerville, N.J.) (83 U/ml VBS) before &taraldehyde fixation and CF labeling. The cells were subsequently processed for electron microscopy as above.

RESULTS Distribution of CF on fixed and unfixed cells

When fixed Chang hepatoma ascites cells are treated with CF, the label is distributed over the entire cell surface in a uniform and continuous layer (fig. 1). This is in contrast with clustering of CF receptors on plasma membrane of unfixed hepatoma cells which were treated with CF for 5 min

Fig. 3. Chang hepatoma ascites cell treated with polyL-lysine before CF labeling. CF particles on cell surface in an even and continuous layer. No apparent reduction of ferritin particles. Lead citrate stain. Fig. 4. Chang hepatoma ascites cell fixed before exposure to cetyl pyridinium chloride and CF labeling.

Greatly reduced CF binding with very little CF on cell surface. Lead citrate stain. Fig. 5. Chang hepatoma ascites cell treated with neuraminidase, fixed and labeled with CF. No apparent reduction of CF binding. Lead citrate stain.

at 37°C before fixation. The CF particles were arranged into patch-like areas on the external plasma membrane surface of the hepatoma cells. Typically, CF clusters

were randomly distributed over the cell surface and separated by narrow areas of unlabeled plasma membrane (fig. 2). Occasional detachment of the CF clusters from the Exp Ceil Res 114 (I 978)

42

Moller and Chang

surface of the plasma membrane could be seen in some cells. Incubation of fixed ascites cells with poly-L-lysine before CF treatment resulted in no detectable reduction in ferritin binding (fig. 3). There was a smooth and even layer of CF particles on the cell surface with no apparent discontinuities present. If, however, cetyl pyridinum chloride was used, CF binding on the surface of the hepatoma cells was greatly reduced (fig. 4). The reduction in CF binding was not localized, but rather general on all parts of the cell surface with individual particles and clusters of CF separated by ferritin-free regions of varying widths. When ascites cells were treated with neuraminidase before fixation and CF labeling, there was no apparent diminution in the amount of ferritin bound to the cell surface (fig. 5). The CF labeling presented the same appearance as cells treated with poly+lysine before exposure to CF: a continuous and uniform layer of CF on the surface of the plasma membrane. CF distribution in living cells reincubated in warm VBS If ascites cells were treated with CF for 5 min and reincubated in CF-free VBS at 37°C for up to 60 min, and then re-exposed to CF for 5 min, the following effects were noted. After 10 min of reincubation in warm VBS, the CF particles were arranged into a clustered configuration. The areas of the plasma membrane between CF clusters were covered with CF particles, but reduced from levels usually seen on the surface of hepatoma cells fixed before CF treatment (fig. 6). At 20 min the CF particles were also arranged into clusters, but there was an apparent lack of any CF binding on many portions of the cell surface (fig. 7). In this Eq Cell Res I14 (1978)

respect, the surface of cells incubated for 20 min resembles the surface of cells treated with CF before fixation (fig. 2): clustered CF particles with most of the plasma membrane free of ferritin label. The ability of the cell surface to bind CF began to recover after 30 min of reincubation, with CF particles re-appearing on the plasma membrane (fig. 8). While the CF binding capacity has started to regenerate, the CF particles seen in fig. 8 are arranged into an uneven and occasionally discontinuous layer. Fig. 6 shows the surface of a hepatoma ascites cells after 45 min of reincubation. The CF-binding ability appears to be almost completely restored with CF distributed evenly around the periphery of the cell and with no gaps in the layer of ferritin (fig. 9). One final point, CF-labeled vesicles were present within the cytoplasm of many unfixed and reincubated ascites cells (figs 7, 10). This would suggest that internalization of CF-labeled surface material probably contributes to the removal of CF receptors and NANA from the cell surface. DISCUSSION The results of this study have demonstrated the presence of anionic sites on the surface of Chang hepatoma ascites cells. The distribution of CF on the surface of the ascites cells prefixed before labeling was continuFigs 6-9. Chang hepatoma ascites cells treated with

CF for 5 mitt, incubated in CF-free VBS at 37°C for 10 (fig. 6), 20 (fig. 7), 30 (fig. 8), 45 (fig. 9) mitt, fixed ;n giutara~d~hyde, then re-exposed m CF. All sections stained with lead citrate. Fig. 6, Ferritin particles arranged into clusters with light labeling around periphery of cell;flg. 7, aggregates of ferritin particles with large areas of the plasma membrane surface totally of label; jig. 8, CF binding beginning to reappear on areas of the plasma membrane between CF aggregateqfig. 9, CF binding ability increased with heavier CF binding present over entire surface of cell.

CF uptake by hepatoma cells

43

44

Moller and Chang

Fig. IO. Chang hepatoma ascites cell incubated as in fig. 7. Ferritin containing vesicles are present in cytoplasm directly beneath an unlabeled portion of the plasma membrane. Lead citrate stain.

ous and uniform. This pattern of CF labeling is similar to that observed on Ehrlich ascites cells [7] and Moloney-virus-transformed lymphoma cells and SV40-transformed fibroblasts [6]. Distribution of CF on the surface of ascites cells labelled before fixation was also similar to that reported for various cell types [4, 61: aggregation of CF, areas of the plasma membrane surface without label and some internalization of CF via endocytosis. The clustering of the CF receptors on the surface of hepatoma cells occurs presumably as result of cross-linking of negatively charged groups [5]. These results suggest that CF, like other multivalent ligands such as lectins and antibodies, can Exp Cdl Res I 14 ( 1978)

induce clustering of plasma membrane components [4]. The absence of ferritin particles from those portions of the cell surface where internalization has occurred suggests that for a period of time following endocytosis the cell membrane may be devoid of charge. The reduction in CF-binding ability and subsequent recovery by the ascites cells reincubated in warm VBS supports this observation. The temporary redistribution of anionic sites may be the result of CFinduced lateral migration of receptors for this ligand and subsequent internalization of labeled plasma membrane. This could account for the unlabeled areas of the plasma membrane in the ascites cells labeled be-

CF uptake by hepatoma cells

45

fore fixation and for the reduced CF-bind- with these compounds yielded no noticeing capacity of the reincubated cells. In the able reduction of ferritin particles on the latter example, this would mean that some surface of the hepatoma ascites cells. By period of time is required before the tem- contrast, cetyl pyridinium chloride is an exporarily depleted anionic surface material, cellent blocking agent and can almost comNANA, could be regenerated. pletely eliminate CF binding on the surface The time required for the regeneration of of the plasma membrane. Cetyl pyridinium the CF-binding capacity of Chang hepatoma chloride is a long chain aliphatic ammonium ascites cells was on the order of 20-30 min, salt that is sometimes used to block ruthewith a more or less completely restored nium red staining [14]. Because it can preCF-binding ability present after 45 min of cipitate cell surface polyanions [15], cetyl reincubation. This is in contrast to the 3 h pyridinium chloride can reduce the number required by mouse peritoneal macrophages of receptor sites available to CF. [5] and 1 h required by guinea pig aortic endothelium [4] for complete regeneration This work was supported by USPHS Grant CA 16663 from the NCI. It is a pleasure to acknowledge the of CF-binding activities. technical assistance of D. H. Hodges and L. R. The presence of detached clusters of CF Partridge. particles suggests two additional points of interest. (1) The retention of the clustered REFERENCES configuration by the detached CF aggreEmmelot, P, Eurj cancer 9 (1973) 319. gates is probably due to surface anionic Nicolson, G L, Int rev cytol39 (1974) 89. Danon, D, Goldstein, L, Marikovsky, Y & groups bound to the CF; (2) the detached Skutelsky, E, J ultrastr res 38 (1972) 500. CR clusters could originate, also via exoSkutelsky, E & Danon, D, J cell biol 71 (1976) 232. cytosis, from vesicles bounded by newly Skutelsky, E & Hardy, B, Exp cell res 101 (1976) synthesized plasma membrane or may 337. simply represent CF clusters that have be- 6. Marikovsky, Y, Inbar, M, Danon, D & Sachs, L, Exp cell res 89 (1974) 359. come detached during the course of EM 7. Subjeck, J R & Weiss, L, J cell physiol 85 (1975) 529. preparation. In any event, the origin and 8. Chang, J P, Gibley, Jr, C W & Ichinoe, K, Cancer composition of the detached CF clusters reres 27 ( 1967)2065. 9. Smith, D F, Walborg, Jr, E F & Chang, J P, mains to be established. res 30 (1970) 2306. Neuraminidase and poly-L-lysine have IO. Cancer Moller, P C, Yokoyama, M & Chang, J P, J natl frequently been utilized as controls for CFcancer inst 58 (1977) 1401. II. Caulfield, J B, J biophys biochem cytol 3 (1957) binding studies [4, 51. Neuraminidase, by 827. virtue of its ability to cleave sialic acid re- 12. Spurr, A R, J ultrastr res 26 (1969) 3 I. 13. Roth, J. Exn oath 11 (1975) 291. sidues from cell surface coat glycoproteins 14. Gustavson,‘G T & Pihl, E, Acta path et microb Stand 69 (1%7) 393. should also reduce CF binding to the cell 15. Martin, B J & Philpott, C W, Cell tissue res 150 surface. Similarly, poly-L-lysine, because it (1974) 193. is a polycationic molecule, can interact with July 7, 1977 cell surface anions and prevent their bind- Received Revised version received October 3 1, 1977 ing with CF. Attempts to block CF binding Accepted January 18, 1978

E.~I Cell

Re., 114 fIY78)