Microscopic Evidence of Ferritin Uptake by Ehrlich Ascites Tumor Cells

Ferritin, added to the incubation medium of ascites tumor cells, was used as an electron microscopic marker to study the uptake of large protein molecules by morphologically intact cells. A definite uptake could be detected after I hour of incubation in Tyrode bicarbonate solution containing 0.04 to 13.3 mg ferritin/ml. Ferritin was found in a variety of membrane-surrounded structures, suggesting that pinocytcsis and related membrane movements are occurring under physiological conditions and can account for the penetration of intact macromolecules into isolated tumor cells. Supplementation of the medium with serum albumin (33 mg/ml) increased the average amount of ferritin per cell and per pinocytotic structure. Ferritin was strongly adsorbed by fragments of lysed cells, which were readily taken up by intact cells. Besides its role as carrier, this debris appeared to stimulate membrane movements. Only rare examples were found to suggest the release of ferritin from the pinocytotic structures into the cytoplasm. Thus, the disintegration of such structures cannot be considered an obvious step towards a rapid metabolic utilization of protein by the cell. Particles of colloidal gold presented to the cell under the same conditions were not taken up to any significant extent, thus providing good evidence for a selective ingestion of particles of comparable sizes. I N T R O D U C T I O N It has been known for some decades that unicellular organisms and the phagocytic elements of higher species share the property of ingesting intact macromolecules and snpramolecular aggregates. Morphological studies of this uptake, inspired by Lewis (24), taken up by Holter and Marshall (17), and pursued by ChapmanAndresen (6-8), Schumaker (35), Brandt (3), and Holtzer and Holtzer (21) have emphasized the importance of membrane invaginations and vesiculations in this process of ingestion. The term pinocytosis, used originally by Lewis (24) and then by Mast and Doyle (26) to describe membrane movements leading to cell drinking, was extended by Holter (18) to include movements leading to macromolecular ingestion. Bennett, furthermore, proposed the hypothesis that pinocytosis might be a transport mechanism of general importance for mammalian cells (1). The demonstration of an uptake of unaltered 255 on D ecem er 3, 2017 jcb.rress. org D ow nladed fom protein molecules by mammal ian cells other than the well recognized phagocytes came from experiments of Coons et al., using fluorescent antibody techniques (10). These authors studied the tissues of rats after the injection of various proteins into the whole animal. By exposing frozen tissue sections to specific fluorescent antibodies they could detect antigen-antibody complexes localized intracellularly, indicating a penetration of the cells by proteins. Simple labeling of albumin with fluorescein was used by Holter and Holtzer (19. 21) to compare the albumin uptake by various mammal ian tissues in vitro. By analogy to observations made on unicellular organisms (35) and in spite of the relative inadequacy of fluorescent microscopy for localization studies on small cells, these authors postulated that albumin was taken up by pinocytotic processes. In these comparative studies, ascites tumor cells appeared to display a moderate pinocytotic activity. The uptake of specific proteins has been subsequently documented both by histochemical (38) and electron microscopic (28) observations on the tubular cells of the rat kidney. Likewise: tumor cells in culture have been shown to ingest gold particles when grown for several days in the presence of colloidal gold (16). There are no electron microscopic observations, however, on the uptake of protein by tumor cells, nor any electron microscopic demonstration of sizeable ingestion of macromolecules or supramolecular particles by tumor cells after short periods of incubation. Such data would be valuable to assess the importance of a direct protein uptake in the protein metabolism of tumor cells. Data on the nitrogen metabolism of tissue culture cells indicate that proteins of the medium are utilized to an important extent by the growing normal (15, 27) and tumor cell (22). I t has been suggested also by Busch and Greene (4) that tumor tissue might display a greater uptake of whole proteins than most normal tissues. These authors, on the basis of protein-C 14 fixation by the tissue of tumor-bearing rats, have postulated that the protein uptake could account for doubling the weight of the tumor within 24 hours. These studies, however, do not provide conclusive evidence that proteins are taken up as whole macromolecules. Furthermore, they stand in contradiction to the relatively low degree of pinocytosis described for ascites tumor cells (21) and HeLa cells in culture (16). It appeared desirable, therefore, to clarify this point, in particular to re-examine the optical evidence of protein uptake using an iron-containing and electron-opaque protein, ferritin, as a marker. The adequacy of ferritin for studying the cellular ingestion of proteins has been well documented by the work of Farquhar and Palade (12, 13). I t was felt that an electron microscopic study of ascites tumor cells, incubated in the presence of ferritin, would provide conclusive information on the occurrence, the mechanism, and the order of magnitude of this penetration process. This paper will also compare briefly the uptake of ferritin and colloidal gold. Its main purpose, however, is to serve as a basis for further studies on the quantitative aspect of protein uptake by tumor cells (33). M A T E R I A L S A N D M E T H O D S A hyperdiploid strain of Ehrlich ascites tumor cells maintained in A strain mice was used exclusively. The tumor ceils were harvested 7 to 8 days after intraperitoneal inoculation of 0.1 ml of ascites fluid. The cells were gently spun, the ascites fluid decanted, and the resulting pellet was resuspended with cold Tyrode bicarbonate solution. This suspension was counted and in all experiments 5 X 107 cells were incubated in a total of 1.5 ml. The basic incubating medium contained a bicarbonate buffer which was adjusted to pH 7.3-7.4 before use. The incubation was performed in a metabolic shaking incubator, model Dubnoff Precision, in an atmosphere of 95 per cent 02 and 5 per cent CO2. The pH at the termination of the incubation was determined. The Dubnoff