Two-dimensional gel analysis of apoptosis-specific p53 isoforms induced by 2-methoxyestradiol in human lung cancer cells

The natural metabolic byproduct of estradiol, 2-methoxyestradiol (2-MeOE₂), induces apoptosis in human lung cancer cells by a p53-dependent mechanism. The expression of wild-type p53 isoforms was investigated in H1299 non-small cell lung carcinoma cells induced into apoptosis by 2-MeOE₂. H1299 cells lack endogenous p53 and undergo predominantly a G₁ arrest when infected with a recombinant wild-type p53 adenovirus. However, when H1299 cells transfected with p53 were treated with 2-MeOE₂, they underwent rapid and extensive apoptosis. H1299 cells expressing mutant his273 p53 were unaffected by 2-MeOE₂, indicating a dependence of 2-MeOE₂-mediated apoptosis on the presence of a functional p53. Analysis of wild-type p53 phosphoisoforms in H1299 cells by two-dimensional gel electrophoresis revealed that 2-MeOE₂ induced a unique group of acidic p53 isoforms. Although most of the wild-type p53 in untreated H1299 cells migrated as at least five diffuse species with isoelectric points from pH 5.5-6.3, as many as nine additional forms migrating toward the acidic region with pl values from 4.4-5.3 were detected in 2-MeOE₂-treated apoptotic cells. Two other agents known to induce apoptosis, vinblastine and actinomycin D, induced a similar pattern of acidic p53 species as that observed for 2-MeOE₂. The results indicated that the induction of apoptosis in H1299 cells by 2-MeOE₂ is dependent on the upregulation of specific p53 isoforms. Identification of the specific p53 phosphoisoforms induced by MeOE₂ will be an important step in understanding the regulation and function of p53 in apoptosis.