Porcine epidemic diarrhoea virus (PEDV) infection causes watery diarrhoea, vomiting, anorexia, and weight loss, especially among neonatal piglets, inflicting on them morbidity and mortality potentially reaching 90%–100%. Despite it being known that certain mammalian cell phases are arrested by PEDV, the mechanisms have not been elucidated, and PEDV pathogenesis is poorly understood. This study determined the effect of an epidemic PEDV strain on cell cycle progression. We observed the effect of the PEDV SHpd/2012 strain on an infected Vero cell cycle through flow cytometry and Western blot, investigating the interrelationships of cell-cycle arrest, the DNA damage–signalling pathway caused by PEDV and the phosphorylation levels of the key molecules Chk.2 and H2A.X involved upstream and downstream in this pathway. PEDV induced Vero cell-cycle arrest at the G1/G0 phase. The phosphorylation levels of Chk.2 and H2A.X increased with the prolongation of PEDV infection, and no significant cell-cycle arrest was observed after treatment with ATM or Chk.2 inhibitors. The proliferation of PEDV was also inhibited by treatment with ATM or Chk.2 inhibitors. PEDV-induced cell-cycle arrest is associated with activation of DNA damage–signalling pathways. Our findings elucidate the molecular basis of PEDV replication and provide evidence to support further evaluation of PEDV pathogenesis.

Objective: To evaluate in vitro effects of gemcitabine alone and in combination with carboplatin on canine transitional cell carcinoma (TCC) cell lines. Sample: In vitro cultures of 5 canine TCC cell lines. Procedures: Cells were treated with gemcitabine, carboplatin, or a combination of both at various concentrations. Cell proliferation was assessed via a fluorescence-based microplate cell proliferation assay. Cell cycle was evaluated via propidium iodide staining, and apoptosis was assessed by measurement of caspase 3 and 7 enzymatic activity. Synergy between gemcitabine and carboplatin was quantified via combination index analyses. Results: Treatment of 5 canine TCC cell lines with gemcitabine or carboplatin decreased cell proliferation, increased apoptosis, and induced cell cycle arrest. Cell cycle arrest and apoptosis were markedly increased when cell lines were treated with both gemcitabine and carboplatin simultaneously or sequentially. Order of administration during sequential treatment did not consistently affect cell proliferation results in TCC cell lines. When TCC cell lines were treated with gemcitabine and carboplatin in combination at therapeutically relevant concentrations (gemcitabine concentration, < 10μM; carboplatin concentration, < 250μM), a significant decrease in cell proliferation was observed, compared with cell proliferation following treatment with gemcitabine or carboplatin alone. In combination, the effects of gemcitabine and carboplatin were synergistic in 3 of 5 cell lines and additive in the other 2. Conclusions and Clinical Relevance: Gemcitabine had antitumor effects on canine TCC cells in vitro, and the combination of gemcitabine and carboplatin had synergistic activity at biologically achievable concentrations.

Objective-To determine cellular changes associated with secondary epidermal laminae (SEL) in forefeet and hind feet of ponies with insulin-induced laminitis. Animals-8 ponies. Procedures-Laminitis was induced in 4 ponies by IV administration of insulin and glucose; 4 control ponies received saline (0.9% NaCl) solution IV. Laminar tissue samples obtained from the dorsal aspects of the hooves were histologically evaluated. Primary epidermal lamina (PEL) length and width and SEL length, width, and angle were determined. Numbers of epidermal cell nuclei per micrometer and per total length of SEL and numbers of apoptotic and proliferative cells in axial, middle, and abaxial laminar regions were determined. Results-SEL in treatment group ponies were significantly longer, were significantly narrower, and had a smaller angle relative to PEL in all laminar regions versus control ponies. In treatment group ponies, the number of epidermal cell nuclei per SEL was typically higher and the number of cells per micrometer of SEL was lower in laminar regions, apoptotic cell numbers were higher in abaxial and middle regions in forefeet and hind feet, and proliferating cell numbers were higher in axial laminar regions in forefeet and all laminar regions in hind feet, versus control ponies. Conclusions and Clinical Relevance-Results indicated SEL elongation, narrowing, and alteration in orientation developed in all feet of ponies with insulin-induced laminitis. This was primarily attributable to cell stretching that developed at the same time as an accelerated cell death-proliferation cycle; differences in cell cycle responses among laminar regions between forefeet and hind feet may have been attributable to differences in load bearing.

The LEC rat is reported to exhibit hypersensitivity to X-irradiation, deficiency in DNA double-strand break repair, and radio-resistant DNA synthesis. This character of the LEC rat has been thought to be due to abnormal G1 arrest in cells after X-irradiation. In this report, we re-investigated the effect of X-irradiation on the cell cycle in primary-cultured fibroblasts. Primary-cultured fibroblasts derived from LEC and BN rats were exposed to 4 Gy of X-ray and their cell cycle analysis was performed with a flow cytometer. Fibroblasts derived from both rats showed normal response of the cell cycle, indicating the arrest at both G1- and G2/M-phase and no difference in the cell cycle population between fibroblasts derived from both rats. In contrast, when the same analysis was performed using the cell line, L7 and W8, which had been established from the lung fibroblasts of LEC and control WKAH rats, respectively, by immortalizing with SV40 T-antigen, L7 cells but not W8 cells showed impaired G1 arrest and abnormal cell cycle. These results suggest that fibroblasts derived from LEC rats possess the normal cell cycle response after X-irradiation, if they are kept naive as not immortalized with SV40 T-antigen.