Imbalance of deoxyribonucleoside triphosphates and DNA double-strand breaks in mouse mammary tumor FM3A cells treated in vitro with an antineoplastic tropolone derivative

Abstract

The mechanism by which alpha,alpha-bis(2-hydroxy-6-isopropyltropon-3-yl)-4-methoxytolu ene (JCI-3661) kills mouse mammary tumor FM3A (F28-7) cells was studied. When the cells were exposed to the drug at 3.7 microM, the intracellular dNTP pool became imbalanced because of decreases in dGTP and dATP and an increase in dTTP. The pattern of the dNTP imbalance was the same as that caused by hydroxyurea. When JCI-3661 was added to the culture medium, mature DNA strands broke, giving fragments of 100-200 kilobase pairs long as found by orthogonal-field-alternation gel electrophoresis. DNA strand breaks, detected by this technique, were observed in the cells at 12 h after the addition. The beginning of cell death was observed at about 14 h (trypan blue staining) or at about 12 h (colony-forming ability) after cultivation Breaks in the single and double strands of DNA, as measured by alkaline and neutral filter elution assay, became evident 24 h after treatment with 3.7 microM JCI-3661. Comparison of the ratio of single- and double-strand breaks caused by JCI-3661 to that following radiation suggested that JCI-3661 broke only double strands. Cycloheximide inhibited both the breakage of double strands and the cell death caused by JCI-3661. JCI-3661 decreased DNA synthesis more than RNA or protein synthesis. The breaks in double strands of DNA were probably important in the cell death caused by JCI-3661.