Tousled kinase activator, gallic acid, promotes homologous recombinational repair and suppresses radiation cytotoxicity in salivary gland cells

Abstract

Accidental or medical radiation exposure of the salivary glands can gravely impact oral health. Previous studies have shown the importance of Tousled-like kinase 1 (TLK1) and its alternate start variant TLK1B in cell survival against genotoxic stresses. Through a high-throughput library screening of natural compounds, the phenolic phytochemical, gallic acid (GA), was identified as a modulator of TLK1/1B. This small molecule possesses anti-oxidant and free radical scavenging properties, but in this study, we report that in vitro it promotes survival of human salivary acinar cells, NS-SV-AC, through repair of ionizing radiation damage. Irradiated cells treated with GA show improved clonogenic survival compared to untreated controls. And, analyses of DNA repair kinetics by alkaline single-cell gel electrophoresis and γ-H2AX foci immunofluorescence indicate rapid resolution of DNA breaks in drug-treated cells. Study of DR-GFP transgene repair indicates GA facilitates homologous recombinational repair to establish a functional GFP gene. In contrast, inactivation of TLK1 or its shRNA knockdown suppressed resolution of radiation-induced DNA tails in NS-SV-AC, and homology directed repair in DR-GFP cells. Consistent with our results in culture, animals treated with GA after exposure to fractionated radiation showed better preservation of salivary function compared to saline-treated animals. Our results suggest that GA-mediated transient modulation of TLK1 activity promotes DNA repair and suppresses radiation cytoxicity in salivary gland cells.

Keywords: Acinar; DNA repair; Gallic acid; Homologous recombination; Radiation; Salivary; Tousled; Xerostomia.

Figure 1. Modulators of TLK1

A. Chemical structures of gallic acid (GA) and thiorizadine (THD). B. In vitro kinase reaction with recombinant TLK1B and histone H3 in the presence of GA or THD. Immunoblotted proteins were reacted with antibody to phospho-histone H3 (Ser10).

Figure 2. Effect of GA on NS-SV-AC viability after radiation

A. Experimental design, B. MTS assay, and C. Colony formation assay. NS-SV-AC were irradiated and treated with GA for 16 h. Cells were switched to drug-free medium thereafter, and viability assessed at day 3 and day 10 by MTS assay and colony formation assay, respectively.

Figure 3. Effect of GA on radiation-induced checkpoint delay

A, B. NS-SV-AC or stably-transfected shRNA TLK1 cells were exposed to 8 Gy, and treated, or not, with GA for indicated times before they were analyzed by propidium iodide flow cytometry. Inset: Immunoblotting of cell lysates with antiserum to TLK1. C, D. Immunoblotting of irradiated NS-SV-AC cells with antibodies to phospho-histone H3 (Ser10), phospho-Chk1 (Ser317), and β-actin. UT: untreated; GA: gallic acid; IR: irradiation.

Figure 4. Resolution of radiation (IR)- induced DNA breaks in NS-SV-AC and shRNA TLK1 cells

Single cells alkaline gel electrophoresis of A. NS-SV-AC cells and C. shRNA TLK1 cells. Cells were irradiated, and treated, or not, with GA. At indicated times, cells were electrophoresed, and cellular DNA analyzed by propidium iodide staining. B. The quantification of tail moment (mean ± SEM) of irradiated NS-SV-AC cells is graphed. D. Immuno-localization of phospho- S139 H2AX in NS-SV-AC cells 24 h after irradiation (IR). NS-SV-AC cells were irradiated prior to treatment with GA for 16 h. Cells were placed in drug-free medium for 8 h thereafter. Immuno-labeling with antiserum to gamma-H2AX (S139) is shown. E. Quantification of phospho-H2AX foci/ cell is shown. UT: untreated, IR: irradiation.

Figure 5. Assessment of homologous recombinational repair in MCF-7 DR-GFP cells

A. Schematic of HR assay, B. Evaluation of I-SceI double strand break repair after treatment with GA and, or, THD in NS-SV-AC and shRNA TLK1 transfected cells. UT: untreated, GA: gallic acid, THD: thioridazine.

Figure 6. Salivary glands post irradiation

A. The head and neck region of the animal was exposed to 2 Gy × 5 days. Animals were injected with saline or GA (50 mg/kg) each day after radiation. Stimulated saliva was collected at 5 weeks after the onset of radiation. Data shown is mean ± SEM. B–E. Histopathologic images of salivary glands; non-irradiated (B), irradiated (C), gallic acid-treated (D), irradiated and treated with gallic acid (E). Inset: enlargement of select area showing nucleo- and cytomegaly.