J. Radiat. Res., 48, 77–85 (2007) Regular Paper Split Dose Recovery Studies using Homologous Recombination Deficient Gene Knockout Chicken B Lymphocyte Cells B. S. Satish RAO1,2, Kaori TANO1,3, Shunichi TAKEDA4 and Hiroshi UTSUMI1,5* Split-Dose Recovery/Sublethal Damage Repair/DNA Double-Strand Break Repair/Homologous Recombination/DT40 Chicken B-Cell Line. To understand the role of proteins involved in DSB repair modulating SLD recovery, chicken B lym- phoma (DT 40) cell lines either proficient or deficient in RAD52, XRCC2, XRCC3, RAD51C and RAD51D were subjected to fractionated irradiation and their survival curves charted. Survival curves of both WT DT40 and RAD52–/– cells had a big shoulder while all the other cells exhibited small shoulders. However, at the higher doses of radiation, RAD51C–/– cells displayed hypersensitivity comparable to the data obtained for the homologous recombination deficient RAD54–/– cells. Repair of SLD was measured as an increase in survival after a split dose irradiation with an interval of incubation between the radiation doses. All the cell lines (parental DT40 and genetic knockout cell lines viz., RAD52–/–, XRCC2–/–, XRCC3–/– RAD51C–/– and RAD51D–/–) used in this study demonstrated a typical split-dose recovery capacity with a specific peak, which varied depending on the cell type. The maximum survival of WT DT40 and RAD52–/– was reached at about 1–2 hours after the first dose of radiation and then decreased to a minimum thereafter (5h). The increase in the survival peaked once again by about 8 hours. The survival trends observed in XRCC2–/–, XRCC3–/–, RAD51C–/– and RAD51D–/– knockout cells were also similar, except for the difference in the initial delay of a peak survival for RAD51D–/– and lower survival ratios. The second phase of increase in the survival in these cell lines was much slower in XRCC2–/–, XRCC3–/–, RAD51C–/– and RAD51D–/– and further delayed when compared with that of RAD52–/– and parental DT40 cells suggesting a dependence on their cell cycle kinetics. This study demonstrates that the participation of RAD52, XRCC2, XRCC3, RAD51C and RAD51D in the DSB repair via homologous recombination is of less importance in compar- ison to RAD54, as RAD54 deficient cells demonstrated complete absence of SLD recovery. INTRODUCTION and nuclear proteins. Among these, the double strand break (DSB) is considered to be the critical lesion for radiation All normal mammalian cells exhibit innate capacities to induced cell death.1–5) Deficiency in DSB repair or inhibition recover from both endogenous and exogenous insults, of DSB repair results in higher rates of mammalian cell death, including ionizing radiation. Irradiation of mammalian cells as evidenced by a number of studies on radiosensitive DSB produces a variety of lesions in DNA, such as the base damage, repair-deficient mutant cells1,6,7) and cells from scid mice.8) strand breaks and cross links within the DNA or between DNA The ability of mammalian cells to repair sub-lethal dam- age (SLD) induced by ionizing radiation is very well estab- lished both experimentally and clinically.9–14) Several reports *Corresponding author: Phone: 075-702-1141, Fax: 075-702-2141, have documented the inability of repair of SLD in scid 15–17) E-mail: [email protected] cells. These cells are defective in the catalytic subunit 1Research Reactor Institute, Kyoto University, Kumatori-cho Noda, Sennan- of DNA dependent protein kinase (DNA-PKcs) or the DNA- 2 gun, Osaka 590-0494 Japan; Department of Radiobiology, MAHE Life binding sub unit of DNA-PK, which are involved in repair Sciences Center, Manipal Academy of Higher Education, Manipal 576 104, of DNA strand breaks. SLD repair has an important role in Karnataka State, Udupi District, India; 3Institute of Traditional Chinese Medicine, Suzuka University of Medical Science, Kishioka-cho, Suzuka 510- radiotherapy through fractionation, as radiation is generally 0293 Japan; 4Department of Radiation Genetics, Graduate School of Medicine, given in multiple fractions of low doses to allow the normal Kyoto University, Konoe-Yoshida, Sakyo-ku, Kyoto 606-8315 Japan; 5Health cells to recover from radiation damage, while attaining max- Research Foundation, Tanaka-Monzen-cho, Sakyo-ku, Kyoto 606-8225, Japan. imum damage to tumour cells. Rao, B.S.S., Tano, K., Takeda, S. and Utsumi, H. Split dose recovery studies It is clear from the earlier studies that, basically two path- using homologous recombination deficient gene knockout chicken B lymphocyte cells. ways namely, homologous recombination (HR) and non- doi:10.1269/jrr.06050 homologous end-joining (NHEJ) are operating in eukaryotic J. Radiat. Res., Vol. 48, No. 1 (2007); http://jrr.jstage.jst.go.jp 78 B. S. S Rao et al. cells for the repair of DSBs.18–20) Several regulatory proteins RAD51D–/–, RAD54–/– DT 40 cells have been explained else- involved in the DSB repair mechanism have also been iden- where.25–27) The above cell lines as well as the control DT tified. It is also known that the X-ray repair cross comple- 40 cells were maintained as suspension cultures in alpha- menting (XRCC) genes correct the phenotypes of certain MEM medium supplemented with mercaptoethanol (10 mutant rodent cell lines that are sensitive to DNA damaging µM), penicillin (100 µM/ml), streptomycin (100 µM/ml), agents including ionizing radiation. Recently, the sequence 10% fetal bovine serum (FBS, Hyclone, London, UK) and analysis of cloned human XRCC2 and XRCC3,21–23) have 1% chicken serum (Sigma, USA) in a humidified atmo- revealed its homology to the Rad51 of yeast and mam- sphere of 5% CO2 in air at 39.5°C. mals.22,23) Although, a few studies have demonstrated the involvement of RAD52, XRCC2, XRCC3, RAD51C and Irradiation and cell survival assay RAD51D in DSB repair through homologous recombination Exponentially growing cells in suspension were diluted to in mammalian cells, the knowledge accumulated is far from get appropriate cell counts (2 × 105 cells/ml), divided into clear understanding. The molecular mechanism of SLD various groups and exposed to X-rays. Radiation was admin- recovery is also not completely understood. However, our istered using a X-ray machine (Softex Co., Tokyo, Japan) at recent studies on SLD recovery, using chicken B lymphocyte a dose rate of 7.5 cGy/sec, with a 2.0 mm Al filter, operating cell line DT40 (wild type) cells and its RAD54, RAD54/KU70, at 150 kVp and 20 mA. For the split dose recovery studies, KU70 gene knockout cells after treatment with X-rays, a first dose which kills approximately 90% cells was select- showed that the repair of sublethal damage is due to DSB ed from the dose response curve of various cells used in this repair mediated by homologous recombination.24) The present study. X-irradiation was given as two equal fractions sepa- study using chicken DT40 cells proficient or deficient in the rated by repair intervals of 0–24 h at 39.5°C. above key proteins further broadens our understanding on the Immediately after the treatment, serially diluted cells role of RAD52, XRCC2, XRCC3, RAD51C and RAD51D in were plated in triplicate petri dishes with 6 ml of 1.5% (w/ DSB repair mechanism in influencing the SLD recovery. v) methylcellulose (Aldrich, Milwaukee, WI) containing D- MEM/F-12 (Gibco-BRL), 5% chicken serum and 10mM MATERIALS AND METHODS beta-mercaptoethanol. Colonies were counted 8–10 days after plating. Percentage survival was calculated relative to Cells and cell culture conditions the number of colonies from the control.27) All experiments Generation of RAD52–/–, XRCC2–/–, XRCC3–/–, RAD51C–/–, were repeated 2–3 times to check for consistency. Fig. 1. Survival curves of exponentially growing chicken B-cell lines, DT40, RAD54–/–, RAD52–/–, XRCC2–/–, XRCC3–/–, RAD51C–/– and RAD51D–/–. Two dotted lines are DT40 and RAD54–/– cells that already published (with permission, Utsumi et al. 2001). Cells were irradiated in suspension at a concentration of about 2 × 105 cells/ml of growth medium at room temperature. The cells growing as suspension cultures were then transferred to CO2 incubator at 39.5°C for the time interval indicated, diluted and plated in methylcellulose medium for colony formation. Plating efficiencies of DT40, RAD54–/–, RAD52–/–, XRCC2–/–, XRCC3–/–, RAD51C–/– and RAD51D–/–cells are about, 98,70, 95, 82, 76, 90 and 89% respectively. Each survival curve consists of data from three to four independent experiments and each point were Mean ± SE. J. Radiat. Res., Vol. 48, No. 1 (2007); http://jrr.jstage.jst.go.jp SLD Recovery and Homologous Recombination 79 Fig. 2. Surviving fractions of exponentially growing chicken B-cell lines, DT40, RAD52–/–, XRCC2–/–, XRCC3–/–, RAD51C–/– and RAD51D–/– cells after single-dose and two-dose X-irradiation. The first dose of the two-dose exposure was given together with the exposure for the single-dose experiment for the same cell suspensions. After an appropriate time interval, a part of the cell suspension was used for the single dose experiment and the rest of the samples were exposed to second dose of X-irradiation (doses same as the first). The first and the second doses of X-irradiation are indicated inside the each panel of the graphs. Other details as in Fig. 1. J. Radiat. Res., Vol. 48, No. 1 (2007); http://jrr.jstage.jst.go.jp 80 B. S. S Rao et al. Flow cytometry The secondary moderate increase in the cell survival in these Immediately after the second dose exposure to X-ray, cell lines was much slower and delayed until 10–12 h when cells were either used for clonogenic assay or fixed in cold compared with that of DT40 and RAD52–/– cells (8h), prob- 70% ethanol and kept overnight refrigerated at 4°C. Subse- ably an indication of delayed progression through the cell quently, cells were processed for univariate DNA analysis cycle (Fig.