In order to provide a valuable cell model for studying the mechanism of DNA damage to kill tumor cells and enhance the curative effect, an antisense cDNA, which effectively expressed ATM gene encoding carboxyl terminal 100 kD domain fragment, was constructed, and a cell line U937-ASPI3K, which stably expressed the antisense ATM/PI3K cDNA was established through transfection and Zeocin selection. The results showed the constructed ATM/PI3K cDNA is in the opposite orientation in expression vector pZeoSV2(+) through examination of gene sequence. The cell line U937-ASPI3K was total loss of constitutive ATM protein, while the ATM abundance was not influenced in both the control cell lines U937-pZeoSV(-) and U937. The present experiment provided a practical cell model to elusidate mechanism of cell cycle checkpoints in DNA damage signal transduction pathway and search a new approach for tumor therapy.