title: Short hairpin RNA suppression of thymidylate synthase produces DNA mismatches and results in excellent radiosensitization.
abstract: OBJECTIVE
  To determine the effect of short hairpin ribonucleic acid (shRNA)-mediated suppression of thymidylate synthase (TS) on cytotoxicity and radiosensitization and the mechanism by which these events occur.
  
  
  METHODS
  shRNA suppression of TS was compared with 5-fluoro-2'-deoxyuridine (FdUrd) inactivation of TS with or without ionizing radiation in HCT116 and HT29 colon cancer cells. Cytotoxicity and radiosensitization were measured by clonogenic assay. Cell cycle effects were measured by flow cytometry. The effects of FdUrd or shRNA suppression of TS on dNTP deoxynucleotide triphosphate imbalances and consequent nucleotide misincorporations into deoxyribonucleic acid (DNA) were analyzed by high-pressure liquid chromatography and as pSP189 plasmid mutations, respectively.
  
  
  RESULTS
  TS shRNA produced profound (≥ 90%) and prolonged (≥ 8 days) suppression of TS in HCT116 and HT29 cells, whereas FdUrd increased TS expression. TS shRNA also produced more specific and prolonged effects on dNTPs deoxynucleotide triphosphates compared with FdUrd. TS shRNA suppression allowed accumulation of cells in S-phase, although its effects were not as long-lasting as those of FdUrd. Both treatments resulted in phosphorylation of Chk1. TS shRNA alone was less cytotoxic than FdUrd but was equally effective as FdUrd in eliciting radiosensitization (radiation enhancement ratio: TS shRNA, 1.5-1.7; FdUrd, 1.4-1.6). TS shRNA and FdUrd produced a similar increase in the number and type of pSP189 mutations.
  
  
  CONCLUSIONS
  TS shRNA produced less cytotoxicity than FdUrd but was equally effective at radiosensitizing tumor cells. Thus, the inhibitory effect of FdUrd on TS alone is sufficient to elicit radiosensitization with FdUrd, but it only partially explains FdUrd-mediated cytotoxicity and cell cycle inhibition. The increase in DNA mismatches after TS shRNA or FdUrd supports a causal and sufficient role for the depletion of dTTP thymidine triphosphate and consequent DNA mismatches underlying radiosensitization. Importantly, shRNA suppression of TS avoids FP-mediated TS elevation and its negative prognostic role. These studies support the further exploration of TS suppression as a novel radiosensitizing strategy.
authors.affiliation: Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan, USA. sflan@umich.edu
authors.forename: Sheryl A
authors.initials: SA
authors.lastname: Flanagan
authors.forename: Kristin S
authors.initials: KS
authors.lastname: Cooper
authors.forename: Sudha
authors.initials: S
authors.lastname: Mannava
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Nikiforov
authors.forename: Donna S
authors.initials: DS
authors.lastname: Shewach
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D003570:Cytidine Triphosphate / Q000378:metabolism; D053843:DNA Mismatch Repair; D004789:Enzyme Activation / Q000187:drug effects; D005467:Floxuridine / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D011836:Radiation Tolerance / Q000235:genetics*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D014410:Tumor Stem Cell Assay / Q000379:methods
chemical_list: D000964:Antimetabolites, Antineoplastic; D034741:RNA, Small Interfering; D005467:Floxuridine; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D013940:Thymidylate Synthase; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1