title: Time dependence of DNA lesions and growth inhibition by ICI D1694, a new quinazoline antifolate thymidylate synthase inhibitor.
abstract: 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'-deoxy-thymidine (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 microM, 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 microM (50% inhibition concentration) and 1.0 microM (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 microM ICI D1694. No significant level of DNA SSBs was detected when cells were exposed simultaneously to ICI D1694 and 20 microM [6RS]LV. Complete rescue of drug-induced DNA SSBs could be achieved when cells were exposed to 10 microM 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 microM ICI D1694 and 5 microMs [6RS]LV or to 3 microMs 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 microM ICI D1694, 3 microMs dThd and 5 microMs [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.
authors.affiliation: Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263.
authors.forename: M B
authors.initials: MB
authors.lastname: Yin
authors.forename: M A
authors.initials: MA
authors.lastname: Guimaraes
authors.forename: Z G
authors.initials: ZG
authors.lastname: Zhang
authors.forename: M A
authors.initials: MA
authors.lastname: Arredondo
authors.forename: Y M
authors.initials: YM
authors.lastname: Rustum
mesh_terms: D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000187:drug effects; D005472:Fluorouracil / Q000494:pharmacology; D005575:Formyltetrahydrofolates / Q000494:pharmacology; D008727:Methotrexate / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology*; D012235:Rhodamines / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology*; D013936:Thymidine / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D013997:Time Factors
chemical_list: D004277:DNA, Single-Stranded; D005575:Formyltetrahydrofolates; D011799:Quinazolines; D012235:Rhodamines; D013876:Thiophenes; D004247:DNA; D013940:Thymidylate Synthase; C068874:raltitrexed; D005472:Fluorouracil; D013936:Thymidine; D008727:Methotrexate