Induced Apoptosis of Cancer Cells by Antisense ATM Gene Therapy

Yi, H K; Chang, S H; Lee, D Y; Kim, J S; Hwang, R H

Abstract

PURPOSE: Recently cloned AT gene mutated (ATM) in ataxia telangiectasia is involved in DNA damage response at different cell cycle checkpoints and also appears to have a wide role in signal transduction. ATM phenotype cells increased sensitivity to ionizing radiation and chemother- apeutic agents, and are defective Gl/S checkpoints in response to DNA damage. We have trying to focus on the possible role of hypersensitivity to DNA damage-induced apoptosis in the ATM cells. We hypothesized that cancers could be made more sensitive to therapeutic x-rays and agents by disabling the AT gene in tumor cells using ATM antisense strategy. Therefore, we investigated whether antisense ATM transduced cancer cells show ATM phenotype cells and undergo apoptotic death after exposure to low concentration of chemotherapeutic agents or irradiation doses that do not induce appreciable apoptosis in control cells.
MATERIALS AND METHODS
NH2-terminal portion of AT gene was recloned and ligated in reverse orientation in pcDNA3. This antisense ATM expression vector transfected to MCF-7 cells, and Ve3 cells by calcium phosphate method. The transformed cells were selected in the presence of G418 and confirmed AT protein expression by immunohistochemistry. We have analyzed the induction of apoptosis of antisense ATM transduced MCF-7 and Ve3 cells after treatment of chemotherapeutic agents or irradiation using DNA fragmentation, MTT assay and FACScan.
RESULTS
Antisense ATM transduced Ve3 and MCF-7 cells were showed the inhibition of ATM protein expression. Its expression was predominantly detected in the nucleus. The antisense ATM transduced Ve3 and MCF-7 cells showed increased progress of DNA fragmentaion induced by etoposide treatment, increased radiosensitization and induced apototic cells (Ao phase) of FACScan after irradiation, and increased susceptibility of apoptosis induced by treatment with low concentration of chemotherapeutic agents.
CONCLUSIONS
ATM playing in cell cycle progression and cell death in response to DNA damage may help identify potential targets for improved cancer therapies, and ATM antisense gene therapy can be a useful therapeutic tool to combat various cancers.