A subset of cells expressing SV40 large T antigen contain elevated p53 levels and have an altered cell cycle phenotype

Cells transformed by the simian virus 40 (SV40) large T antigen (Tag) conta in elevated levels of cellular p53 protein. To quantify this relationship, levels of p53 were measured in NIH 3T3 cells that expressed different conce ntrations of Tag. Using immunoblotting, average p53 levels were shown to in crease linearly with Tag concentrations in these cell lines. Single-cell me asurements were also performed using flow cytometry to measure p53 immunofl uorescence. Surprisingly, the flow cytometry experiments showed that two di stinct cell populations, based on p53 content, were present in cells expres sing high levels of Tag. One cell population contained elevated p53 levels. A second population did not contain elevated p53, even though high concent rations of Tag were present in the cells. This latter cell population did n ot appear to arise because of mutations in either Tag or p53. The two cell populations also had phenotypic differences. In exponentially growing cells , Tag alters the cell cycle distribution (decreases the percentage of G(1) phase cells and increases the percentages of S and G(2) + M phase cells). T his phenotype was maximum in the cell population containing elevated p53. A lesser phenotype was found in the cell population that did not contain ele vated p53. These data show, firstly, that cells can express significant lev els of Tag and not contain elevated levels of p53 and, secondly, that eleva ted p53 correlates with the altered cell cycle distribution produced by Tag in growing cells.