We examined the expression of p53 in three lines of pluripotent embryonal carcinoma (EC) and ES cells. p53 mRNA and protein levels were constitutively high in two lines but absent from one. In the P19 line of EC cells neither p53 protein nor mRNA was detected. The ®rst intron of the p53 gene in these cells had been invaded by a murine leukemia virus and there was extensive hypermethylation of the p53 gene accompanying its inactivation. In all three cell lines, irradiation resulted in arrest of the cells in the G2 but not in the G1 phase of the cell cycle despite the induction of p21 cip1 in the cell lines expressing p53. Thus, the chromosomal stability of EC and ES cells appears to be not dependent on the p53 protein and we interpret our results to suggest that these cells may require the deletion of p53 dependent cell cycle regulation in order to become immortalized.

Embryonal carcinoma (EC) cells can be efficiently transfected with cloned DNAs but there is a strong tendency for expression from transfected genes to be lost from stably transformed cells. To investigate the mechanism responsible for this loss of expression, we transfected P19 EC cells with a gene encoding the E. coli beta-galactosidase and examined expression of this gene in clonal populations of cells. Cells that carry and express the beta-galactosidase gene give rise to cells that do not express at a rate of about 0.02 events per cell per cell division. These non-expressing cells were of two types, some had lost the transfected genes while others had inactivated them. In those cells that retained but inactivated the transfected genes, the inactive state was stable and suppression was at the level of transcription initiation but not associated with increased DNA methylation. Because transfected DNAs integrate into the genome as tandem arrays, the gene loss and inactivation seen in EC cells may be analogous to the repeat-induced gene inactivation seen in lower eukaryotes.

Plasmid DNA can be efficiently transfected into embryonal carcinoma cells but it is difficult to isolate clones of cells stably expressing genes present on the transfected plasmids. Even in clonal populations derived from transfected cells, the introduced genes are expressed in some but not all cells. Cotransfection with a region of the Pgk-1 gene results in more efficient, stable cotransformation due to increased numbers of copies of the transfected plasmids integrated into the genomic DNA. The PgK-1 genomic sequences did not allow the plasmid DNA to replicate autonomously but seemed to enhance the ligation of transfected plasmids before their integration into the host genome. Our results suggest a model in which the plasmid DNAs are able to integrate and subsequently excise from the host genome by recombination events enhanced by transcription through the tandemly repeated sequences of the transfected plasmids.