title: Late DNA Damage Mediated by Homologous Recombination Repair Results in Radiosensitization with Gemcitabine.
abstract: Gemcitabine (dFdCyd) shows broad antitumor activity in solid tumors in chemotherapeutic regimens or when combined with ionizing radiation (radiosensitization). While it is known that mismatches in DNA are necessary for dFdCyd radiosensitization, the critical event resulting in radiosensitization has not been identified. Here we hypothesized that late DNA damage (≥24 h after drug washout/irradiation) is a causal event in radiosensitization by dFdCyd, and that homologous recombination repair (HRR) is required for this late DNA damage. Using γ-H2AX as a measurement of DNA damage in MCF-7 breast cancer cells, we demonstrate that 10 or 80 nM dFdCyd alone produced significantly more late DNA damage compared to that observed within 4 h after treatment. The combination of dFdCyd treatment followed by irradiation did not produce a consistent increase in DNA damage in the first 4 h after treatment, however, there was a synergistic increase 24-48 h later relative to treatment with dFdCyd or radiation alone. RNAi suppression of the essential HRR protein, XRCC3, significantly decreased both radiosensitization and late DNA damage. Furthermore, inhibition of HRR with the Rad51 inhibitor B02 prevented radiosensitization when added after, but not during, treatment with dFdCyd and radiation. To our knowledge, this is the first published study to show that radiosensitization with dFdCyd results from a synergistic increase in DNA damage at 24-48 h after drug and radiation treatment, and that this damage and radiosensitization require HRR. These results suggest that tumors that overexpress HRR will be more vulnerable to chemoradiotherapy, and treatments that increase HRR and/or mismatches in DNA will enhance dFdCyd radiosensitization.
authors.affiliation: Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan 48109.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Im
authors.affiliation: Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan 48109.
authors.forename: Sheryl A
authors.initials: SA
authors.lastname: Flanagan
authors.affiliation: Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan 48109.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Ackroyd
authors.affiliation: Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan 48109.
authors.forename: Brendan
authors.initials: B
authors.lastname: Knapp
authors.affiliation: Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan 48109.
authors.forename: Aaron
authors.initials: A
authors.lastname: Kramer
authors.affiliation: Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan 48109.
authors.forename: Donna S
authors.initials: DS
authors.lastname: Shewach
mesh_terms: D053903:DNA Breaks, Double-Stranded / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005720:Gamma Rays; D006657:Histones / Q000378:metabolism; D006801:Humans; D061986:MCF-7 Cells; D051135:Rad51 Recombinase / Q000037:antagonists & inhibitors; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D059767:Recombinational DNA Repair / Q000187:drug effects* / Q000528:radiation effects*; D013997:Time Factors; D000093542:Gemcitabine
chemical_list: D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D011838:Radiation-Sensitizing Agents; C110901:X-ray repair cross complementing protein 3; D003841:Deoxycytidine; D051135:Rad51 Recombinase; D000093542:Gemcitabine