Establishment of Immortalized Primate Epithelial Cells with Sub-genomic EBV DNA

The genetic information in a sub-fragment of EBV DNA, designated p31 (containing less than a quarter of the viral genome and derived from a recombinant DNA cosmid library) allows epithelial cells from primary monkey and human kidney cultures to escape senescence under standard tissue culture conditions. A number of epithelial cell lines, designated M1/31, 483/31, 199/31 and HK/31, have been established and characterized following transfection of primary cells with p31 DNA. They share many properties, although morphologically they are not all identical. The cultures are immortalized but not fully transformed or tumorigenic. They appear to be phenotypically stable, although DNA hybridization studies indicate that genotypic alterations, including amplification, occur subsequent to transfection with p31 DNA and the establishment of a continuously proliferating epithelium. All cell lines consistently express high levels of cytokeratin 18 and varying amounts of cytokeratin 7, demonstrating their epithelial origin. From a single marmoset kidney (designated 199) a series of related immortalized cells, with subtle phenotypic differences, have been generated by p31 or sub-fragments of it. Although hallmarks of a "hit-and-run" mechanism are apparent in all of our studies, 2 different techniques (in situ hybridization or selection for cell survival in semi-solid media, followed by nucleic acid hybridization) show that, in late-passaged cultures, a small proportion of the cells still contain some viral DNA. The studies focus on genetic information within the BamHI A and I regions as being relevant to immortalization. The role of the EBV DNA fragment in the genesis of epithelial cell lines is considered.