Isolation of preneoplastic rat liver cells by centrifugal elutriation and binding to asialofetuin.
Putative preneoplastic hepatocytes were isolated from male Fischer 344 rats treated with a single dose of diethylnitrosamine, 2-acetylaminofluorene feeding, and partial hepatectomy (Solt-Farber model). The isolation procedure involved, after collagenase dispersion of the liver, separation of the hepatocytes into small- and large-cell fractions by centrifugal elutriation, and subsequent selection of cells deficient in asialoglycoprotein receptor(s) by plating onto asialofetuin (ASF)-coated plates. The number of cell surface binding sites for the asialoglycoprotein receptor was measured with both asialoorosomucoid and ASF as ligands. There was a 50% reduction of binding sites for both ligands in the original cell suspensions obtained from preneoplastic livers. The reduction in receptor binding sites was most pronounced in the large cell fraction (less than or equal to 30% of control value) after separating the original cell suspension by elutriation into small and large cell fractions. Immunohistochemical studies showed a lack of asialoglycoprotein receptor in preneoplastic (i.e., hyperplastic foci) areas. These areas were entirely super-imposable with glucose-6-phosphatase-deficient areas and partially overlapped the gamma-glutamyltranspeptidase-positive areas in serial liver sections. The attachment of preneoplastic hepatocytes to ASF-coated tissue culture dishes was greatly impaired, and the number of gamma-glutamyltranspeptidase-positive cells on the ASF dishes was reduced to less than 7% as compared to 45 to 70% on the collagen-coated plates. Thus, the lack of asialoglycoprotein (asialofetuin) surface receptors and the increased size of the early preneoplastic hepatocytes are characteristics that can be used to separate the preneoplastic cell population from normal liver cells.
D015073:2-Acetylaminofluorene / Q000633:toxicity; D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D002498:Centrifugation / Q000379:methods; D004052:Diethylnitrosamine / Q000633:toxicity; D060748:Fetuins; D008099:Liver / Q000473:pathology*; D008114:Liver Neoplasms, Experimental / Q000473:pathology*; D008297:Male; D009961:Orosomucoid / Q000031:analogs & derivatives / Q000032:analysis; D011230:Precancerous Conditions / Q000473:pathology*; D051381:Rats; D011916:Rats, Inbred F344; D011971:Receptors, Immunologic / Q000032:analysis; D014316:Tritium; D000509:alpha-Fetoproteins
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D060748:Fetuins; D009961:Orosomucoid; D011971:Receptors, Immunologic; D000509:alpha-Fetoproteins; C017029:asialofetuin; C017030:asialoorosomucoid; D014316:Tritium; D004052:Diethylnitrosamine; D015073:2-Acetylaminofluorene