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The inhibitory effects of horse antiserum against rat alpha-fetoprotein (AFP) on the uptake of 2-deoxy-D-glucose (2dG) by the AFP-producing rat ascites hepatoma AH66 cells was studied. AH66 cells cultured in medium containing 20% heat-inactivated antiserum had a 1.5-fold lower rate of sugar uptake than did AH66 cells which were cultured in medium containing 20% heat inactivated normal horse serum. The inhibition of 2dG uptake by antiserum was dependent on both the concentration and the exposure time of antiserum. Preincubation of AH66 tumor cells for 2 and 6 h with antiserum prior to the measurement of 2dG uptake resulted in a 70.1% and 58.2% decrease in 2dG uptake compared to control cells. Antiserum did not inhibit the rate of phosphorylation of 2dG by tumor cells. Kinetic constants for the uptake of 2dG in both AH66 cells treated with antiserum to AFP and in control cells were calculated from Lineweaver-Burk plots. The Km remained constant at approximately 1.2 mM, but the Vmax was twice as small for the cells treated with antiserum as for the control cells (571 vs 923 nanomoles/2 X 10(5) cells/min). These studies suggest that the inhibition of 2dG uptake by treatment with antiserum was the result of a decrease in the number of transport sites, or a decrease in the amount of carrier protein for the sugar which was present on the surface of the plasma membrane of the AH66 cells.

Five rat yolk sac tumor cell lines were cloned from a yolk sac tumor line which originally arose following fetectomy. The doubling time of each of the cloned tumor lines was about 50 h. All of the cloned tumor cell lines synthesized and secreted AFP and albumin but there was a gradual decrease in the synthesis of these proteins during serial passage. The cells formed clusters which looked like vitelline ducts or the parietal yolk sac and they also had basement membranes which closely resembled Reichert's membrane. When the cloned cell lines were cultivated in the presence of 1 mM dibutyryl cyclic-AMP, myotube-like and neuron-like cells appeared. Acetylcholine esterase and creatine phosphokinase activity were present when myotube-like cells were present whereas acetylcholine esterase activity predominated when neuron-like cells were present.

A daunorubicin (DNR)-resistant variant (AH66DR) of al-pha-fetoprotein (AFP)-producing rat ascites hepatoma AH66 was established. AH66DR was 169 times more resistant to DNR than AH66. A growth-inhibitory effect due to the decrease in sugar uptake following treatment of the cell lines with specific antibody against highly purified rat AFP was also observed. Pre-incubation of both lines with antibody prior to the measurement of 2-deoxy-D-glucose (2dG) uptake resulted in a 45-55% decrease in 2dG uptake compared to control cells with a 2-fold reduction of Vmax value while the Km remained unchanged. Upon pre-incubating AH66DR with antibody, an increased intracellular level of DNR concentration with reduction of efflux rate was observed. A significantly superior cytotoxic effect against AH66DR, which reached almost the same level as the cytotoxic effect of DNR against AH66, was found, as compared to various other controls when tumor cells were cultured with a mixture of antibody and DNR. Our in vitro results suggest that the DNR-resistance of the AFP-producing tumor cells may be overcome if the cells are treated with specific antibody.