anti -Benzo[a]pyrene-7,8-diol-9,10-epoxide Treatment Increases Levels of the Proteins p53 and p21 WAF1 in the Human Mammary Carcinoma Cell Line MCF-7

The tumor suppressor p53 is involved in the recognition of DNA damage induced by radiation and chemicals. The effect of polycyclic hydrocarbons on p53 was investigated by treating MCF-7 cells with anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), an ultimate carcinogenic metabolite of benzo[a]pyrene. Western blotting of lysates with antibody mAb1801 showed that B[a]PDE doses of 0.1 to 0.5 μM caused detectable increases in p53 protein. In cells treated with 0.3 μM B[a]PDE, p53 protein levels increased by 2 hours after treatment, reached a maximum between 8 and 24 hours and returned to control value by 120 hours after treatment. Levels of p21WAF1 protein increased by 8 hours after treatment, reached a maximum by 48 hours and returned to control value by 168 hours after treatment. B[a]PDE-DNA adducts were quantitated by [γ-33P]ATP postlabeling and separation on reverse-phase HPLC. Adduct levels dropped rapidly between 2 and 24 hrs after treatment (48 to 15 pmol/mg DNA), and subsequently decreased more slowly (12 pmol/mg DNA at 48 hrs to 3 at 168 hrs). By 96 hours after treatment, p53 protein levels were comparable to those from untreated controls, although a low level of B[a]PDE-DNA adducts remained. Cells treated with B[a]PDE also incorporated significantly less [3H]thymidine into DNA than did untreated cells for at least 24 hours after treatment. These results provide preliminary evidence that B[a]PDE-induced DNA damage is recognized by a p53-dependent signal transduction pathway and suggest that cell cycle arrest occurs during the repair of the majority of the B[a]PDE-DNA adducts.