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Comparison of DNA-Protein Cross-Links Induced by 4'-(9-Acridinylamino)-methanesulfon-m-anisidide and by -Radiation¹

Division of Biochemical Oncology, Department of Radiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

The antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) inhibits topoisomerase II activity through the formation of a complex of DNA and covalently bound enzyme which, upon protein denaturation, yields DNA breaks (single strand breaks). In the present study, this complex served as a standard for analysis of radiation-induced DNA-protein cross-links (DPC). Following the treatment of exponentially growing mouse L929 cells with 0–100 ng/ml of m-AMSA for 1 h, a linear dose-dependent increase was found in the amount of DNA retained on nitrocellulose filters during subsequent analysis. This result indicates that the assay can detect DPC that have a single protein bound to each DNA fragment. The results of fractionation of nuclear DNA show that m-AMSA induces 20- to 45-fold more DPC in nuclear matrix-associated DNA than in the majority distal loop DNA, supporting the notion that topoisomerase II is located at the nuclear matrix. The frequency of single strand breaks induced by m-AMSA, which should be equal to the frequency of DPC, was determined by alkaline elution. Results of the alkaline elution assay could be correlated with the percentage of DNA retained on nitrocellulose filters; i.e., 1% DNA retention corresponded to 2560 DPC per log-phase L929 cell, which has been determined to have a DNA content of 22.25 pg. Using this standard curve, DPC induced by -irradiation in air were estimated to be formed at a frequency of 133 DPC/cell/Gy, a frequency approximately 3% that of -ray-induced single strand breaks. The radiation dose response for DPC production was unaffected by the high levels of DPC present in cells previously treated with m-AMSA. In addition, DPC induced by m-AMSA were rapidly reversed after the removal of the drug, in contrast to a slower removal of DPC induced by -radiation. These observations suggest that although m-AMSA and -radiation both preferentially induce DPC with matrix-attached DNA, they produce independent types of DPC.

¹ Supported by USPHS Research Grant CA-15378 and Cancer Core Support Grant P30 CA-43703 from the National Cancer Institute, DHHS.

² To whom requests for reprints should be addressed, at Wearn Building B-21, Division of Biochemical Oncology, Department of Radiology, University Hospitals, Cleveland, Ohio 44106.

Received 5/16/88. Revised 9/18/88. Accepted 11/11/88.