Defective post-translational modification of collagen IV in a mutant F9 teratocarcinoma cell line is associated with delayed differentiation and growth arrest in response to retinoic acid.

We have selected a mutant F9 teratocarcinoma stem cell line, RA-5-1, which does not exhibit normal differentiation into parietal endoderm in the presence of retinoic acid, dibutyryl cyclic AMP, and theophylline (RACT). In this report, we demonstrate that the RA-5-1 mutant possesses a prolyl-4-hydroxylase enzyme with a higher Km for a synthetic collagen substrate and that this alteration results in a 6-7-fold reduction in the amount of collagen IV in the medium of RACT-treated mutant cells, as compared to wild type F9 cells. In addition, the collagen IV that is secreted by RACT-treated RA-5-1 cells has an abnormally low molecular weight and contains 6-9-fold less 4-hydroxyproline than the collagen IV secreted by RACT-treated wild type F9 cells. A brief ascorbate treatment can increase the hydroxyproline content of the collagen IV secreted by RACT-treated RA-5-1 cells. A large reduction in the amount of laminin in the medium of RACT-treated RA-5-1 mutant cells is also observed. Concomitant with the reduction in collagen IV and laminin polypeptides in the medium, the expression of several other differentiation-specific mRNAs is delayed in the RACT-treated RA-5-1 cells relative to wild type F9 cells. Moreover, the mutant cells do not exhibit the morphology or the complete growth arrest of wild type terminally differentiated parietal endoderm cells in the presence of RACT. These results suggest that a defect in the post-translational modification of collagen IV in the mutant RA-5-1 prevents the complete expression of the differentiation program in response to RACT. These experiments also demonstrate that the expression of certain differentiation-specific genes is compatible with continued proliferation in the mutant line.