Alphafoetoprotein uptake by cloned cell lines derived from a nickel-induced rat rhabdomyosarcoma

Abstract

Summary Rat, mouse, pig and chicken alphafoetoproteins (AFP), rat serum albumin and egg albumin, or their fluoresceinated conjugates were added to cultures of several cloned cell lines isolated from a nickelinduced rat rhabdomyosarcoma. The intracellular uptake of assayed proteins was revealed by the indirect immunoperoxidase technique and/or by direct fluorescence microscopy. All (Trojan & Uriel, 1979;Uriel et al., 1982) and birds (Moro & Uriel, 1981). We have subsequently demonstrated that neuron-like elements in primary cultures of dissociated cells from foetal mouse brain hemispheres can incorporate exogenous AFP . This supports the conclusion that the wide distribution of intracellular AFP through the immature nervous system results from protein uptake, as opposed to an eventual in situ AFP synthesis. The same conclusion can probably be extended to other foetal tissues of ecto-and mesodermal origin where intracellular AFP has also been demonstrated during normal ontogenic development (Basteris, 1979; Dziadek & Adamson, 1978; (ABE et al., 1976).AFP and other proteins Rat, mouse, pig and chicken AFP were isolated as previously described (De Nechaud & Uriel, 1971;Hassoux et al., 1977; Lampreave et al., 1980;Moro & Uriel, 1981 Kummer et al. (1981) for tritiation of monoclonal antibodies. Briefly 2mg of lyophilized AFP dissolved in 1 ml of 0.1 M Na borate buffer pH 8.5, were added to 1 mCi of dried [3H]NSP. The mixture was kept for 36 min at 4°C with stirring. Labelled AFP was separated from unincorporated reactants by Sephadex G-25 chromatography using PBS, pH 7.2, as the eluant. Fractions containing AFP were pooled. Aliquots, 50pl each, were put in small vials and stored at -18°C until use. The specific activity of the preparation was of 29uCi mg-' AFP. High-resolution autoradiography Cloned cell lines were incubated in 1 ml fresh MEM medium to whom either 0.45 or 0.90 pCi of [3H]-AFP were added. After 3 h at 37°C, the cells were washed 3 times with PBS and fixed for 1 h at 4°C with 1.6% glutaraldehyde in 0.1 Sorensen phosphate buffer, pH 7. After several washes in buffer, including overnight, the cells were postfixed in 2% OS04 in the same buffer, for 1 h, at room temperature, scraped from the dishes and pelleted. The pellets were dehydrated in alcohol and embedded in Epon. Ultrathin sections were harvested on forward-coated copper grids and covered with a monogranular layer of Ilford L4 emulsion, using a loop. Autoradiograms were developed in a phenidon-containing developer, after gold latensification (Bouteille, 1976). The sections were finally stained with uranyl acetate and lead citrate. AFP incubation of cultured cellsImmunochemical and immunocytochemical reactions Specific rabbit antisera to rat and mouse AFP were obtained as previously described (De Nechaud & Uriel, 1971;Hassoux et al., 1977). Pure antibodies were isolated from their respective antisera by affinity chromatography on AFP-immunoabsorbents prepared by the procedure of Avrameas ALPHAFOETOPROTEIN UPTAKE BY RAT RHABDOMYOSARCOMA CLONES ...

Specific Uptake of Alpha-Fetoprotein and Albumin by Rat Morris 7777 Hepatoma Cells

“…AFP and albumin were mainly localized in uncoated vesicles near the cell surface. Previous work centered on the study of AFP binding and uptake has been performed in highly specialized cells, including different lymphoid and leukemia cell lines [27-29, 31, 36, 38-40], the MCF-7 breast cancer cell line [18], skeletal muscle cells [14] or rhabdomyosarcoma cell lines [17,19,30]. This is the first study using hepatoma cells.…”

“…Previous studies have shown that the ability to internalize AFP, which is characteristic of developing cells [14][15][16], may reappear in neoplastic cells [17][18][19]. The existence of an albumin receptor has been postulated in different cell types [20][21][22].…”

Cell-type-specific receptors for alpha-fetoprotein in a mouse T-lymphoma cell line.

“…Fluorescein conjugates were prepared by incubating proteins with fluorescein isothiocyanate (FITC; Sigma) as reported elsewhere (9). The FITC/protein molar ratio ranged from 1.5 to 3 for all proteins except a2M, which was -10.…”

“…The intracellular presence of AFP in fetal tissues other than the liver and the yolk sac is due to active uptake of the protein (5,6) and not local synthesis (3,7). It has also been shown that the ability to internalize AFP may be shared by neoplastic cells of different origin, such as spontaneous mammary carcinomas growing in the mouse (8), as well as in two cultured cell lines derived from a rat rhabdomyosarcoma (9) and a human breast carcinoma (10). Preliminary morphological observations of AFP uptake by these cultured cells suggested the existence of a receptor-mediated mechanism.…”

Increased Alpha-Fetoprotein Receptor in the Serum of Patients with Early-Stage Breast Cancer

“…In vitro and in vivo studies showed that malignant cells regain the ability to take up afp via a receptor that would be present in undifferentiated cells of either embryonic or tumour origin [10][11][12][13][14][15] , but mostly absent in normal adult cells. The existence of such a receptor for afp (recaf) was then demonstrated and functionally characterized in several cell lines [16][17][18] .…”