Inhibition of Crandell-Rees Feline Kidney cell proliferation by X-ray-induced senescence

Abstract

Radioresistance and radiotoxicity have been reported following cancer treatments in felines. Optimizing radiation doses to induce cytotoxic effects to only cancer cells and not normal cells is critical in achieving effective radiation therapy; however, the mechanisms of radiation resistance, radiotoxicity, and DNA damage response (DDR) in feline cells have not yet been elucidated. A DNA double-strand break (DSB) is the most toxic type of DNA damage induced by X-rays and heavy ion beams used in treating cancers. Crandell-Rees Feline Kidney (CRFK) cells is one of the most widely used cat cells in life science research. Here, we report that DSB-triggered senescence induced by X-rays is important in inhibiting the proliferation of CRFK cells. We demonstrated through cell proliferation assay that X-rays at doses 2 Gy and 10 Gy are toxic to CRFK cells that irradiating CRFK cells inhibits their proliferation. In X-irradiated CRFK cells, a dose-dependent increase in DSB-triggered senescence was detected according to morphological changes and using senescence-associated β galactosidase staining assay. Moreover, our data indicated that in CRFK cells, the major DDR pathway, which involves the phosphorylation of H2AX at Ser139, was normally activated by ATM kinases. Our findings are useful in the understanding of X-rays-induced cellular senescence and in elucidating biological effects of radiation, e.g. , toxicity, in feline cells. Furthermore, our findings suggest that the CRFK cell line is an excellent matrix for elucidating radioresistance and radiotoxicity in cat cells.

Combined ataxia telangiectasia mutated and DNA-dependent protein kinase inhibition radiosensitizes Madin–Darby canine kidney cells

“…Trypan blue exclusion test was used to determine the number of viable cells in accordance with our previously described methods [ 23 ] with the following modifications. For the radiation toxicity assay, cells were seeded at a density of 5.0 × 10 4 cells per 60-mm dish.…”

“…Nuclei were stained with 0.025 μg/mL of 4,6-diamino-2-phenylindole (DAPI) fluorescent dye (Boehringer Mannheim, Mannheim, Germany), in accordance with our previously described methods [ 15 , 23 ]. Images of cells were obtained using an Olympus Fluorescence Microscope BX51 equipped with a digital camera Olympus DP50.…”

“…The mean values and standard deviations of each assay were calculated from the mean of three independent experiments. Statistical analysis was performed using the analysis of variance (ANOVA) and Ryan’s multiple comparison tests (ANOVA4 on the web, https://www.hju.ac.jp/~kiriki/anova4/ ) as previously described [ 23 ]. Statistical significance was set at a P -value of less than 0.05.…”

The potential for senotherapy as a novel approach to extend life quality in veterinary medicine

“…With similar pathologic findings to humans ( 216 , 217 ), they represent a naturally occurring model for study of senotherapeutics ( 215 ). In vitro studies utilizing feline renal cells have shown a dose dependent correlation with radiation and SC ( 218 ). Further studies can utilize this data to understand the etiopathology of radiotoxicity induced renal senescence and investigation of senotherapeutics in vivo in this context ( Figure 4 ).…”

Protective Effect of Natural Antioxidant Compounds on Methimazole Induced Oxidative Stress in a Feline Kidney Epithelial Cell Line (CRFK)

“…As the kidney is a target organ of MMI-induced toxicity, an immortalized cell line derived from the renal cortex of a normal cat (CRFK) was used [ 25 ]. Although uncertainty has been suggested about the epithelial vs. mesenchymal phenotype of CRFK cells [ 26 ], they have been originally described as epithelial [ 25 ], and they are widely used accordingly [ 27 , 28 ].…”