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. 2008 Jan;175(1):89-95.
doi: 10.1016/j.tvjl.2006.12.001. Epub 2007 Feb 1.

Molecular characterisation of canine nonsteroidal anti-inflammatory drug-activated gene (NAG-1)

Kiyoshi Yamaguchi  1 , Nichelle C Whitlock, Jason L Liggett, Alfred M Legendre, Michael M Fry, Seung Joon Baek
Affiliations

Molecular characterisation of canine nonsteroidal anti-inflammatory drug-activated gene (NAG-1)

Kiyoshi Yamaguchi et al. Vet J. 2008 Jan.

Abstract

Nonsteroidal anti-inflammatory drug (NSAID)-activated gene (NAG-1), a divergent member of the transforming growth factor beta superfamily, was previously identified as a gene induced by several anti-tumorigenic compounds, including NSAIDs and peroxisome proliferator-activated receptor gamma (PPARgamma) ligands in humans. In this study, canine NAG-1 was characterised from a canine genomic database. Gene induction by some NSAIDs and PPARgamma ligands was demonstrated in canine osteosarcoma cell lines. Phylogenetic analysis indicates that canine NAG-1 is more homologous with the corresponding mouse and rat genes than with human NAG-1. Expression of canine NAG-1 was increased by treatment with piroxicam and SC-560 (NSAIDs) and the PPARgamma ligand rosiglitazone. This study demonstrates that canine NAG-1 is up-regulated by some anti-tumorigenic compounds in osteosarcoma cell lines and may provide an important target of chemotherapy in canine cancer.

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Figures

Fig. 1
Fig. 1
Predicted nucleotide sequence (top) and deduced amino acid sequence (bottom) of canine NAG-1 (GenBank XM_541938).
Fig. 2
Fig. 2
Alignment of NAG-1 amino acid sequence from the dog, human, mouse, rat, and chimpanzee (GenBank: human, AAH08962; mouse, NP_035949; rat, CAA09891; chimpanzee, XP_524157). (a) Amino acid sequence alignment of the C-terminal region begins with the first conserved cysteine residue. Conserved cysteine residues are marked with an asterisk (*). Bold letter indicates RXXR motifs. (b) Phylogenetic tree based on NAG-1 amino acid sequence homology among the dog, human, mouse, rat, and chimpanzee.
Fig. 2
Fig. 2
Alignment of NAG-1 amino acid sequence from the dog, human, mouse, rat, and chimpanzee (GenBank: human, AAH08962; mouse, NP_035949; rat, CAA09891; chimpanzee, XP_524157). (a) Amino acid sequence alignment of the C-terminal region begins with the first conserved cysteine residue. Conserved cysteine residues are marked with an asterisk (*). Bold letter indicates RXXR motifs. (b) Phylogenetic tree based on NAG-1 amino acid sequence homology among the dog, human, mouse, rat, and chimpanzee.
Fig. 3
Fig. 3
Expression profiles of NAG-1 protein in canine tissues demonstrated by Western blot analysis using 30 μg of cell lysate from each tissue separated by SDS-PAGE. (a) Polyclonal anti-human NAG-1 antibody. Canine NAG-1 appeared as a band ~ 35 kDa. (b) Result from incubating NAG-1 antibody with human NAG-1 peptide.
Fig. 4
Fig. 4
Effects of NSAID and PPARγ ligands on NAG-1 expression in canine osteosarcoma cancer cells determined by RT-PCR. (a) CCL-183 cells were treated for 24 h with DMSO vehicle (Veh) or NSAIDs: DFU: 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2 (5H)-furanonel, 100 μM; Dic: diclofenac, 100 μM; Pir: piroxicam, 100 μM; SC: SC-560, 50 μM; SS: sulindac sulphide, 30 μM. Plasmid containing canine NAG-1 cDNA was used as a positive control (Cont). (b) CCL-183 cells were treated for 24 h with vehicle (Veh) or PPARγ ligands: MCC: MCC-555, 5 μM; RGZ: rosiglitazone, 5 μM. RT-PCR was performed using total RNA isolated from cells. Equal loading was demonstrated by GAPDH RT-PCR.
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