title: Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase.
abstract: Thymidylate deprivation brings about "thymineless death" in prokaryotes and eukaryotes. Although the precise mechanism for thymineless death has remained elusive, inhibition of the enzyme thymidylate synthase (TS), which catalyzes the de novo synthesis of TMP, has served for many years as a basis for chemotherapeutic strategies. Numerous studies have identified a variety of cellular responses to thymidylate deprivation, including disruption of DNA replication and induction of DNA breaks. Since stalled or collapsed replication forks and strand breaks are generally viewed as being recombinogenic, it is not surprising that a link has been demonstrated between recombination induction and thymidylate deprivation in bacteria and lower eukaryotes. A similar connection between recombination and TS inhibition has been suggested by studies done in mammalian cells, but the relationship between recombination and TS inhibition in mammalian cells had not been demonstrated rigorously. To gain insight into the mechanism of thymineless death in mammalian cells, in this work we undertook a direct investigation of recombination in human cells treated with raltitrexed (RTX), a folate analog that is a specific inhibitor of TS. Using a model system to study intrachromosomal homologous recombination in cultured fibroblasts, we provide definitive evidence that treatment with RTX can stimulate accurate recombination events in human cells. Gene conversions not associated with crossovers were specifically enhanced several-fold by RTX. Additional experiments demonstrated that recombination events provoked by a double-strand break (DSB) were not impacted by treatment with RTX, nor was error-prone DSB repair via nonhomologous end-joining. Our work provides evidence that thymineless death in human cells is not mediated by corruption of DSB repair processes and suggests that an increase in chromosomal recombination may be an important element of cellular responses leading to thymineless death.
authors.affiliation: Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA. awaldman@biol.sc.edu
authors.forename: Barbara Criscuolo
authors.initials: BC
authors.lastname: Waldman
authors.forename: Yibin
authors.initials: Y
authors.lastname: Wang
authors.forename: Kasturi
authors.initials: K
authors.lastname: Kilaru
authors.forename: Zhengguan
authors.initials: Z
authors.lastname: Yang
authors.forename: Alaukik
authors.initials: A
authors.lastname: Bhasin
authors.forename: Michael D
authors.initials: MD
authors.lastname: Wyatt
authors.forename: Alan S
authors.initials: AS
authors.lastname: Waldman
mesh_terms: D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D002877:Chromosomes, Human / Q000201:enzymology / Q000378:metabolism*; D003434:Crossing Over, Genetic / Q000187:drug effects; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D005785:Gene Conversion / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011799:Quinazolines / Q000494:pharmacology*; D011995:Recombination, Genetic / Q000187:drug effects*; D013379:Substrate Specificity / Q000187:drug effects; D013876:Thiophenes / Q000494:pharmacology*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics
chemical_list: D005493:Folic Acid Antagonists; D011799:Quinazolines; D013876:Thiophenes; D013940:Thymidylate Synthase; C068874:raltitrexed