Feline XLF accumulates at DNA damage sites in a Ku‐dependent manner

Abstract

Resistance to radiotherapy and chemotherapy is a common problem in the treatment of cancer in humans and companion animals, including cats. There is thus an urgent need to develop new treatments. Molecularly targeted therapies hold the promise of high specificity and significant cancer‐killing effects. Accumulating evidence shows that DNA double‐strand break ( DSB ) repair proteins, which function in Ku‐dependent non‐homologous DNA ‐end joining ( NHEJ ), are potential target molecules for next‐generation cancer therapies. Although cancer radioresistance in cats has been previously described, there are no reports on feline Ku‐dependent NHEJ . Here, we cloned and sequenced feline XLF cDNA and characterized X‐ray repair cross‐complementing protein 4‐like factor ( XLF ), which is one of the core NHEJ proteins. We demonstrated that feline XLF localizes to the nuclei of feline cells and that feline XLF immediately accumulates at laser‐induced DSB sites in a Ku‐dependent manner. Amino acid sequence alignment analysis showed that feline XLF has only 80.9% identity with human XLF protein, while the predicted nuclear localization signal and putative 14‐3‐3‐binding motif are perfectly conserved among human, cat, dog, chimpanzee, and mouse. These findings are consistent with the hypothesis that regulation of subcellular localization is important for the function of XLF . Furthermore, these findings may be useful in clarifying the mechanisms underlying feline Ku‐dependent DSB repair and feline cell radioresistance, and possibly facilitate the development of new molecularly targeted therapies that target common proteins in human and feline cancers.

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

“…γH2AX is a gold standard biomarker for DSBs [ 8 , 20 , 28 ]. Previously, using western blotting, we determined the change in γH2AX expression in CRFK cells between from 1 hr to 24 hr post-irradiation with X-rays at a single dose of 10 Gy [ 17 ]. As a result, the expression level of γH2AX was the highest after 1 hr [ 17 ].…”

“…CRFK cells (HSRRB, Osaka, Japan) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum as previously described [ 17 ]. Adherent cells were irradiated with X-rays at 1, 2, or 10 Gy and at a dose rate of 0.71–0.77 Gy/min (200 kVp/20 mA with 0.5-mm Al and 0.5-mm Cu filters) using Pantak HF320S (Shimadzu, Kyoto, Japan) at room temperature, as described in previous studies [ 17 ]. KU-55933, an ATM kinase inhibitor, was purchased from Wako Pure Chemical (Osaka, Japan).…”

“…Total protein extraction and western blot analysis were performed according to our previously described methods [ 11 , 17 ] with the following modifications. The total proteins were electrophoresed on Extra PAGE One Precast Gel 5–20% (Nacalai Tesque, Kyoto, Japan) or Super Sep ACE Gel 5–20% (Wako Pure Chemical).…”

“…Immunostaining was performed as previously described [ 12 , 17 ] with the following modifications. Briefly, cultured cells were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 in PBS for 5 min, blocked using a blocking solution, and incubated with mouse anti-γH2AX monoclonal antibody (JBW301) (Upstate Biotechnology Inc.) for 60 min at room temperature.…”

“…As a result, the cells recover from radiation injury, and the affected DNA is accurately and efficiently repaired [ 8 , 25 ]. In dog and cat cells, DSBs may mainly be repaired by non-homologous end joining (NHEJ) repair mechanism, which induces the binding of DSB ends of the Ku heterodimer, consisting of Ku70 and Ku80 [ 14 , 15 , 16 , 17 ]; however, further studies are needed to clarify this. Additionally, in the early repair stage, kinases, such as DNA-PK and ATM kinases, are activated, and these phosphorylate Ser139 of the histone H2AX near DSB sites [ 5 , 6 ].…”