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

Abstract

Alpha-fetoprotein (AFP) is a major globulin of embryonic plasma and a physiological carrier of unesterified fatty acids. In the present work, we have characterized the interaction of AFP and albumin, a major serum protein of adult mammals which presents numerous biochemical analogies with AFP, with the plasma membrane of the rat Morris 7777 hepatoma cells. Time course analysis of the uptake of AFP and albumin by these cells showed a saturable profile at 4°C and 37°C. Saturable binding of ¹²⁵I-AFP or ¹²⁵I-albumin were observed when the concentration of these proteins increased (ranging from 0.3 to 4.5 µM). The Hill and Scatchard analysis revealed the existence of binding sites in the surface of hepatoma cells, with a k′d = 2.2 × 10^–6 M (2.9 × 10⁶ sites/cell) in the case of AFP and a k′d = 4.5 × 10^–6 M (3.9 × 10⁶ sites/cell) in the case of albumin. ¹²⁵I-AFP and ¹²⁵I-albumin bound to the cells were completely displaced in the presence of a 200-fold excess of unlabeled AFP or albumin, respectively, suggesting that these interactions were specific. We have observed crossed competition between AFP and albumin for their respective binding sites; no such crossed competition was observed when an excess of unlabeled transferrin was added. Pulse-chase experiments showed that about 50% of the AFP and 75% of the albumin taken up by the cells were released undegraded into the medium after 1 h. Cytochemical studies performed with covalent conjugates of AFP, albumin and transferrin with horseradish peroxidase have shown that AFP and albumin entered the cells via a vesicular system. This intracellular pathway is different from that of transferrin, a plasma protein whose internalization mediated by specific receptors via coated pits has been reported in other cells. The results presented here suggest that AFP and albumin interact with sites in the membrane of hepatoma cells, probably physically related, and then they are transported inside the cells by a mechanism different from that described for transferrin.

Review of the putative cell-surface receptors for alpha-fetoprotein: identification of a candidate receptor protein family

“…Following uptake, AFP was observed in coated pits of the cell membrane and was then tracked to vesicles, multivesicular bodies, and the trans-Golgi network surrounding the nucleus (Table 1) [18,22]. A series of studies subsequently showed that the property of AFP uptake was present in neoplastic cells such as breast/mammary carcinomas, hepatomas, neuroblastomas, lymphomas, and T and B cell malignancies [17,19,22,33,34] (Table 2). Those studies and others strongly supported the presence of an AFP cellsurface receptor as an integral cell membrane protein.…”

Alpha‐fetoprotein‐specific transfer factors downregulate alpha‐fetoprotein expression and specifically induce apoptosis in Bel7402 alpha‐fetoprotein‐positive hepatocarcinoma cells

“…Many hepatocarcinoma cell lines, such as Bel7402, HepG2 and SMMC‐7721, can express AFP mRNA and protein and AFP receptors exist on membranes of these cells. So AFP and its receptors on the membranes form autocrine loops that play an important role in maintaining the growth of hepatocarcinoma cells 4,5 . The importance of AFP to hepatocarcinoma cells also means that suppression or influence of AFP expression and its biological activity may be a new method to inhibit the growth of hepatocarcinoma cells.…”

“…So AFP and its receptors on the membranes form autocrine loops that play an important role in maintaining the growth of hepatocarcinoma cells. 4,5 The importance of AFP to hepatocarcinoma cells also means that suppression or influence of AFP expression and its biological activity may be a new method to inhibit the growth of hepatocarcinoma cells. Many results indicate that downregulation of AFP expression is able to induce the growth inhibition of hepatocarcinoma cells.…”

“…Therefore, AFP has been used as a serological marker for HCC in clinical diagnosis 3 . Researchers have found that there are specific membrane AFP receptors or AFP‐binding sites in many malignant cells and low concentrations of AFP can stimulate the proliferation of hepatocarcinoma cells 4,5 . Besides, AFP can function as a kind of suppressive factor on the immune system and help hepatocarcinoma cells escape from immune surveillance 6,7 .…”

“…3 Researchers have found that there are specific membrane AFP receptors or AFP-binding sites in many malignant cells and low concentrations of AFP can stimulate the proliferation of hepatocarcinoma cells. 4,5 Besides, AFP can function as a kind of suppressive factor on the immune system and help hepatocarcinoma cells escape from immune surveillance. 6,7 So when HCC produces, with the secretion of AFP, AFP can not only enhance the growth of HCC, but also help HCC to escape the watch of the immune system.…”