Tumour-associated antigens reacting with cytotoxic antibodies in serum of hepatoma-bearing rats

Abstract

Summary.-3M-KC1 extracts of the hepatoma D23 contain antigens that inhibit the complement-dependent cytotoxicity for D23 hepatoma cells of serum from D23 tumour-bearing rats (D23 TBS). Inhibition was not due to a general anticomplementary activity of the extracts. Although a minor part (25%) of the protein of D23-KC1 extract was insoluble in PBS, this part contained most of the inhibitory activity. Fractionation of the PBS-soluble material of the extract on Concanavalin A-Sepharose showed that the inhibitory activity did not bind to the lectin. Analysis of D23-KC1 extracts on a Sepharose CL-4B column showed that the antigens involved in the cytotoxicity were heterogeneously distributed in the high -mol. wt region ( > 200,000). Precipitation with 10% trichloroacetic acid (TCA) of D23 KC1 extracts revealed that most of the antigenicity was insoluble in TCA. Heating of D23 KC1 extracts at 100°C did not affect the antigenicity. Enzyme treatment of D23 extra nuclear membranes (D23 ENP) revealed that the inhibitory activity was not sensitive to proteolytic digestion, while treatment with phospholipase A2, C or D abrogated partly the inhibitory activity. The lipid nature of the antigenicity was indicated by its solubility in organic solvents as chloroform or n-butanol.

The Lipid Nature of a Tumour‐Associated Autoantigen from a Chemically Induced Rat Hepatoma

“…In previous reports [13,14,16], wehaveshown that tbe target antigen(s) for the cytotoxic antibodies in D23 TBS arc present in plasma membranes, ENP and 3 M KC! extracts of bepatomas, and some normal organs.…”

“…We have previously reported thai the inhibition of cytotoxicity of D23 ENP was abrogated by phospholipase digestion [13]. It was, however, not clear whether this effect was due to soiubilizalion of the antigenic activity or whether the enzymes acted directly on the aniigen(s).…”

“…16] we have shown that the larget antigen(s) for the cytotoxic anlibodies in D23 TBS arc present in ENP, plasma membranes, and 3 M KCl extracts of the D23 hepatoma. A lipid nature of the antigents) was suggested by the solubility of the antigen(s) in //-bulanol and in chloroform/ methanol [13].…”

“…Furthermore, the antigen(s) was found in extranuclear membranes and 3 M KCl extracts of several tumours and normal tissues. Several lines of evidence suggest that the antigen(s) are ofa lipid nature [13], and the present report deals witb tbe further characterization of these hydrophobic autoantigens.…”

Cytotoxicity of Tumour‐Bearer Serum to D23 Hepatoma Cells Derived from Solid Tumours, Ascites or in Vitro Cultures

“…hepatoma develop a complement-dependent cytotoxicity against the tumour [13,14]. The cytotoxic antibodies, which are of the IgM class [15], react with hydrophobic antigens [11] present on several lumours and in certain normal tissues and arc thus of an autoimmune nature [13. 14].…”