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Time Dependence of DNA Lesions and Growth Inhibition by ICI D1694, a New Quinazoline Antifolate Thymidylate Synthase Inhibitor¹

Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263

DNA single-strand breaks and associated growth inhibition induced by the thymidylate synthase inhibitor N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazoline-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid (ICI D1694) were quantitated using the human ileocecal adenocarcinoma cell line, HCT-8. The effects of different concentrations and schedules of [6R,S]-5-formyltetrahydrofolate ([6RS]LV) and 2'-deoxythymidine (dThd) on drug growth inhibition and DNA damage were also evaluated. The drug concentrations for 50% inhibition of cell growth in culture following 2-h and 72-h exposures were 0.073 and 0.003 µM, respectively. After a 2-h drug exposure, the occurrence of DNA single-strand breaks (SSBs) was time dependent. It was detectable at 8 h and reached a maximum at about 24 h, 34 ± 3 (SD) and 305 ± 34 rad equivalents with 0.1 µM (50% inhibition concentration) and 1.0 µM (90% inhibition concentration) ICI D1694, respectively. A significant level of DNA SSBs (101 ± 13 rad equivalents) was still detectable at 72 h after the 2-h treatment with 1 µM ICI D1694. No significant level of DNA SSBs was detected when cells were exposed simultaneously to ICI D1694 and 20 µM [6RS]LV. Complete rescue of drug-induced DNA SSBs could be achieved when cells were exposed to 10 µM dThd starting no later than 4 h after drug treatment.

The growth inhibition of ICI D1694 was abrogated by [6RS]LV in a concentration-dependent manner. Complete protection was achieved when cells were exposed simultaneously to 1 µM ICI D1694 and 5 µM [6RS]LV or to 3 µM dThd immediately after drug treatment. The results demonstrate that: (a) the growth inhibition of ICI D1694 is a function of time and schedule; (b) the growth inhibition is accompanied by extensive DNA single-strand breaks and slow repair; (c) at 1 µM ICI D1694, 3 µM dThd and 5 µM [6RS]LV can completely rescue cells from drug effects when dThd is added up to 4 h following drug treatment or when [6RS]LV is given in combination with the drug; (d) interference of [6RS]LV with ICI D1694 action may be occurring at the level of drug uptake and at intracellular targets, while dThd interferes with the drug action at intracellular targets.

¹ Supported in part by Program Project Grant CA21071 and Surgical Oncology Training Grant CA09581 (M. A. A.) from the National Cancer Institute and American Cancer Society Clinical Oncology Fellowship 90-130 (M. A. A.).

² To whom requests for reprints should be addressed, at Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263.

Received 2/ 3/92. Accepted 8/26/92.

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