AN ANTISENSE CONSTRUCT OF FULL-LENGTH ATM CDNA IMPOSES A RADIOSENSITIVE PHENOTYPE ON NORMAL-CELLS

The cloning of a full-length cDNA for the gene (ATM) mutated in the hu man genetic disorder ataxia-telangiectasia (A-T) has been described re cently. This cDNA, as well as a fragment representing a functional reg ion from ATM, are capable of rescuing various aspects of the radiosens itive phenotype in A-T cells. We have subcloned full-length ATM cDNA i n the opposite orientation in an EBV-based vector under the control of an inducible promoter to determine whether this anti-sense construct might sensitize control lymphoblastoid cells to ionizing radiation, Th e effectiveness of expression of this construct in control cells was m onitored by loss of ATM protein which was evident over a period 6-12 h after induction. Under these conditions radiosensitivity was enhanced approximately threefold in control cells, approaching the degree of r adiosensitivity observed in A-T cells. Expression of the anti-sense co nstruct also increased the number of radiation-induced chromosomal bre aks and led to the appearance of radioresistant DNA synthesis in these cells. Abrogation of the G1/S checkpoint was evident from the loss of the p53 response and that of its downstream effector, p21/WAF1, post- irradiation. The extent of accumulation of transfected cells in G2/M p hase at 24 h post-irradiation was similar to that observed in A-T cell s and the induction of stress-activated protein kinase by ionizing rad iation was prevented by antisense ATM cDNA expression. These data demo nstrate that full-length ATM anti-sense cDNA, by reducing the amount o f ATM protein, is effective in imposing a series of known defects char acteristic of the A-T phenotype, This inducible system provides an exp erimental model to further investigate mechanisms underlying radiosens itivity and cell cycle control.