Absence of DNA damage‐mediated induction of human methyltransferase specific for precarcinogenic O⁶‐methylguanine

Abstract

Abstract The ability of cultured normal human fetal liver and kidney epithelial cells to repair the premutagenic and precarcinogenic O 6 ‐methylguanine (O 6 ‐MeGua) DNA adduct was determined by directly monitoring its loss in cellular DNA and quantitating the number of O 6 ‐MeGua‐DNA‐methyltransferase (O 6 ‐MT) molecules per cell. Following treatment of the epithelial cells with the direct acting carcinogen N‐methyl‐N‐nitrosourea (MNU), the loss of the O 6 ‐MeGua adduct was biphasic, exhibiting a half‐life of 2.0 and 1.5 h in the liver and kidney cells, respectively. The activity of O 6 ‐MT in the liver and kidney epithelial cells in culture was 0.19 pmol/mg protein or 18,500 molecules/cell. The activity of O 6 ‐MT was maintained throughout the life of the cultures, i.e., 20 subpassages or 50 cumulative population doublings for the liver and kidney. In order to ascertain whether human fetal epithelial cells exhibit an induction of O 6 ‐MT, the cell cultures were treated with single and multiple conditioning doses of N‐methyl‐N‐nitro‐N‐nitroso‐guanidine (MNNG) or γ‐irradiated and assayed for the amount of O 6 ‐MT. A 1 h exposure of cells to 2, 4, and 8 μM MNNG resulted in an 80–100% decrease of the initial O 6 ‐MT activity which was restored to the constitutive levels within 48 and 72 h post‐treatment. Rat hepatoma cells, used as a positive control, increased their levels of O 6 ‐MT to 2.8‐fold the constitutive levels following treatment with MNNG. Treatment of the human liver and kidney epithelial cells with chronic low doses of MNNG exhibited O 6 ‐MT levels identical to untreated cells. The O 6 ‐MT activity in epithelial cells remained unaffected upon pre‐irradiation with 1.2 or 2.5 Gy of γ‐irradiation. These result indicate that (i) the normal human fetal liver and kideny epithelial cells in culture actively repair O 6 MeGua; (ii) resynthesis of O 6 ‐MT in cultured cell and recovery to the basal constitutive levels occurs followinggonotoxin exposure; and (iii) O 6 ‐MT is not induced following the chllenge does of physical and chemical agents.