Regulation of alphafetoprotein synthesis in mouse hepatoma cells

Partially purified AFP mRNA was obtained from specifically immunoprecipitated polysomes from an AFP-producing tumor, Hepatoma 75-35775. This mRNA translates AFP and albumin in vitro and was used as a template to prepare complementary DNA (cDNA) of 5.3X10⁵ daltons. Purification of AFP-cDNA was achieved by preparative hybridization of the cDNA to adult liver poly(A)-RNA, and subsequent hydroxylapatite chromatography to separate cDNA-RNA hybrids. The single stranded cDNA isolated by this procedure showed no albumin sequences by hybridization to albumin mRNA, and was found to consist of a major component of unique sequence complexity.

AFP comprises 1.9-2.0% of the total protein synthesized by Hepa-2 cells. The rate of synthesis is equal to the rate of secretion indicating that all the protein made is eventually secreted. Treatment of these cells with 10^-6 hydrocortisone (HC) results in a slight increase (1.5-2.0 fold) in AFP synthesis; treatment with 10^-3 Dibutyryl-cAMP or 8Br-cAMP results in a 3-6 fold increase in AFP synthesis. There is no synergistic effect, such as that seen with albumin synthesis, when the cells are treated with a combination of glycocorticoid and cAMP analogs.

During the course of our studies it was observed that Hepa-2 cells underwent significant changes in AFP secretion during a long period of in vitro cultivation. These cells are referred to as "high passage" cultures. In these cells, the rate of AFP synthesis is significantly higher than the rate of secretion. Treatment with hydrocortisone and analogs of cAMp had a greater effect on the rate of secretion than on the rate of synthesis, and a combined treatment resulted in a state in which the rate of synthesis and secretion become equal. We interpret these data to indicate that these cells have a defect in their ability to secrete AFP and that the glucocorticoid and cyclic nucleotide treatment corrects this defect. Transferrin, another glycoprotein, showed characteristics similar to AFP whereas albumin synthesis and secretion responded as expected from previous studies. We conclude that these cells may have a general defect in the secretion of glycoproteins.

AFP-mRNA levels were measured by cDNA-RNA reassociation kinetics to determine whether the increase in synthesis by treatment of HC and analogs of cAMP is due to an increase in mRNA levels. It was demonstrated that the increases in AFP synthesis correspond very closely to the increases in mRNA levels. We conclude that the expression of the AFP gene in low passage cells can be modulated by HC and analogs of cAMP. On the other hand, preliminary studies indicate that mRNA levels are not increased in high passage Hepa-2 cells. This would be expected if the main effect of these substances is in secretion rather than in synthesis.