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Direct ferritin immunoelectron microscopy was applied to visualize the distribution of the hepatocyte cell surface of the asialoglycoprotein receptor which is responsible for the rapid clearance of serum glycoproteins and lysosomal catabolism. For this purpose, rabbit antibody against the purified hepatic binding protein specific for asialoglycoproteins was prepared and coupled to ferritin by glutaraldehyde. The specific antibody conjugates were incubated with the hepatocytes, which were isolated from rat liver homogenate after fixation by glutaraldehyde perfusion. These cells preserved well the original polygonal shape and polarity, and it was easy to identify the sinusoidal, lateral, and bile canalicular faces. The surface density of the ferritin particles bound to the sinusoidal face was about four times higher than that of particles bound to the lateral face, while the bile canalicular face was hardly labeled and almost at the control level. Using the surface area of hepatocyte measured by morphometrical analyses, it was estimated that ~90% of bound ferritin particles were at the sinusoidal face, ~10% at the lateral face, and ~1% at the bile canalicular face. Nonhepatic cells such as endothelial and Kupffer cells had no receptor specific for asialoglycoproteins.Ashwell and co-workers found that the asialoglycoproteins (ASGPs) produced by removal of the terminal sialic acid from serum glycoproteins are recognized by a hepatic receptor, rapidly removed from the circulation, and taken up by the hepatocytes to be catabolized in lysosomes (2,23).Ultrastructural analyses of the carbohydrate recognition system in rat liver have been extensively carried out by Hubbard et al. (9,10,32) by using E2~I-ASGPs and two electron microscopic tracers, asialoorosomucoid (ASOR) covalently coupled to horseradish peroxidase (ASOR-HRP) and lactosaminated ferritin. According to them, the tracers injected intravenously into rat were rapidly internalized into the hepatocytes via coated pits and coated vesicles, began to accumulate in a complex arrangement of larger smooth-surfaced vesicles, and tubular structures at the sinusoidal periphery of the cells (30 sec-2 min), appeared in Golgi-lysosome regions either in small vesicles <200 nm in diameter, larger irregular vesicles or tubules (5 min), and finally, some of these vesicles fused with lysosomes (15 min). Very recently, it has been further shown by Wall and Hubbard (33) that, at low temperature or after formaldehyde prefixation, ASGP binding sites are present over much of the sinusoidal cell surface but are concentrated heavily over coated pits. Electron microscopic observations on the endocytosis of ASGP-enzyme conjugates by hepatocytes also have been reported by Stockert et al. (30).We are interested in the distribution of the ASGP receptor on the cell surface of rat hepatocytes in situ. We asked whether the receptor exists homogeneously on all the cell surfaces or exclusively on the sinusoidal face, and, if the latter is true, whether it exists homogeneously on the s...

SummaryMany colorectal cancer cells are resistant to the anti-proliferative effects of transforming growth factor-β (TGF-β). TGF-β also acts as paracrine factor from cancer cells on their mesenchymal cells. The aim of this study was to examine the expression of TGF-β and its receptors in human colorectal cancer tissue and determine any relationship with cancer growth. In situ hybridization and Northern blot hybridization detection of TGF-β 1 , type I and type II receptor mRNA and immunohistochemical staining of TGF-β 1 were performed using 11 human colorectal adenomas, 22 colorectal cancers and ten normal colorectal mucosas as control. TGF-β receptor mRNAs were expressed mainly by normal colorectal epithelial cells and adenoma. However, mRNAs for TGF-β receptors were only faintly, if at all, expressed in eight of 22 human colorectal cancers. In addition, intense signals of TGF-β 1 mRNA and the protein were detected in all colorectal cancers. TGF-β receptor mRNAs and TGF-β 1 protein were also distributed in fibroblasts and endothelial cells in the interstitium. Moreover, Smad 4 protein was translocated to nucleus in primarily cultured adenoma cells, but not in cancer cells after TGF-β stimulation. The escape of human colon cancer from TGF-β -mediated growth inhibition by down-regulation of TGF-β receptors as well as the effects of TGF-β on stroma formation and angiogenesis indicate a possible role for TGF-β in the progression of colon cancer in an intact host.Keywords: TGF-β; TGF-β receptor; Smad, colorectal cancer; colorectal adenoma 194British Journal of Cancer (1999) 80(1/2), 194-205 © 1999 Cancer Research Campaign Article no. bjoc.1998 Received 30 receptors, and alterations in post-receptor pathways. Several reports indicate that elevated levels of TGF-β mRNA and protein in colorectal cancer are associated with cancer progression (Tsushima et al, 1996). If TGF-β is indeed an inhibitor for epithelial cells, how then do colorectal cancer cells manage to proliferate despite elevated TGF-β production by tumour cells? Concerning the mechanisms of resistance to the anti-proliferative effects of TGF-β in colorectal cancer, Markowitz et al (1995) identified a specific TGF-β RII mutation that is associated with defective DNA mismatch repair in colon cancer cells. However, numerous reports also indicate that transcriptional regulation makes an important contribution to determination of the expression level of TGF-β receptors (Birchenall-Roberts et al, 1995;Kim et al, 1997). Accordingly, to clarify the mechanisms of resistance to the antiproliferative effects of TGF-β in vivo, we estimated the transcriptional level of TGF-β receptors in colorectal cancer using in situ hybridization and Northern blot hybridization as the standard for those in normal colorectal epithelial cells.In addition, no studies of human colorectal tumour to date have compared in situ levels of TGF-β 1 with those of its receptors as a presumptive target gene, and with the presence of ligand protein.To ascertain if TGF-β from colorectal cancer act...

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