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Effect of an adenoviral vector that expresses the canine p53 gene on cell growth of canine osteosarcoma and mammary adenocarcinoma cell lines

Mitsuhiro Yazawa DVM1, Asuka Setoguchi DVM, PhD2, Sung-Hyeok Hong DVM, PhD3, Rina Uyama DVM, PhD4, Takayuki Nakagawa DVM5, Noriko Kanaya DVM6, Ryohei Nishimura DVM, PhD7, Nobuo Sasaki DVM, PhD8, Kenichi Masuda DVM9, Koichi Ohno DVM, PhD10, and Hajime Tsujimoto DVM, PhD11
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  • 1 Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
  • | 2 Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
  • | 3 Department of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
  • | 4 Department of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
  • | 5 Department of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
  • | 6 Department of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
  • | 7 Department of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
  • | 8 Department of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
  • | 9 Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
  • | 10 Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
  • | 11 Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.

Abstract

Objective—To generate an adenoviral vector that expressed the canine p53 gene and investigate its growth-inhibiting effect on canine osteosarcoma and mammary adenocarcinoma cell lines.

Sample Population—2 canine osteosarcoma cell lines (HOS, OOS) and 3 canine mammary adenocarcinoma cell lines (CHMp, CIPm, and CNMm).

Procedure—An adenoviral vector that expressed the canine p53 gene (AxCA-cp53) was generated. p53 gene expression was examined by use of reverse transcription (RT)-polymerase chain reaction (PCR) assay and immunohistochemistry. Susceptibility of cell lines to the adenoviral vector was determined by infection with an adenoviral vector that expresses β-galactosidase (AxCA-LacZ) and 3-indolyl-β-D-galactopyranoside staining. Growth inhibitory effects were examined by monitoring the numbers of cells after infection with mock (PBS) solution, AxCA-LacZ, or AxCA-cp53. The DNA contents per cell were measured by flow cytometry analysis. Apoptotic DNA fragmentation was detected by use of a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay.

Results—AxCA-cp53-derived p53 gene mRNA and P53 protein were detected by RT-PCR analysis and immunohistochemistry, respectively. Multiplicity of infection at which 50% of cells had positive 3-indolyl- β-D-galactopyranoside staining results ranged from 10 to 50. AxCA-cp53 induced growth inhibition in a dosedependent manner. Arrest of the G1-phase population and apoptotic DNA fragmentation were observed in cells infected with AxCA-cp53.

Conclusions and Clinical Relevance—AxCA-cp53 inhibits cell growth via induction of cell cycle arrest and apoptosis in canine osteosarcoma and mammary adenocarcinoma cell lines that lack a functional p53 gene. AxCA-cp53 may be useful to target the p53 gene in the treatment of dogs with tumors. (Am J Vet Res 2003;64:880–888)

Abstract

Objective—To generate an adenoviral vector that expressed the canine p53 gene and investigate its growth-inhibiting effect on canine osteosarcoma and mammary adenocarcinoma cell lines.

Sample Population—2 canine osteosarcoma cell lines (HOS, OOS) and 3 canine mammary adenocarcinoma cell lines (CHMp, CIPm, and CNMm).

Procedure—An adenoviral vector that expressed the canine p53 gene (AxCA-cp53) was generated. p53 gene expression was examined by use of reverse transcription (RT)-polymerase chain reaction (PCR) assay and immunohistochemistry. Susceptibility of cell lines to the adenoviral vector was determined by infection with an adenoviral vector that expresses β-galactosidase (AxCA-LacZ) and 3-indolyl-β-D-galactopyranoside staining. Growth inhibitory effects were examined by monitoring the numbers of cells after infection with mock (PBS) solution, AxCA-LacZ, or AxCA-cp53. The DNA contents per cell were measured by flow cytometry analysis. Apoptotic DNA fragmentation was detected by use of a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay.

Results—AxCA-cp53-derived p53 gene mRNA and P53 protein were detected by RT-PCR analysis and immunohistochemistry, respectively. Multiplicity of infection at which 50% of cells had positive 3-indolyl- β-D-galactopyranoside staining results ranged from 10 to 50. AxCA-cp53 induced growth inhibition in a dosedependent manner. Arrest of the G1-phase population and apoptotic DNA fragmentation were observed in cells infected with AxCA-cp53.

Conclusions and Clinical Relevance—AxCA-cp53 inhibits cell growth via induction of cell cycle arrest and apoptosis in canine osteosarcoma and mammary adenocarcinoma cell lines that lack a functional p53 gene. AxCA-cp53 may be useful to target the p53 gene in the treatment of dogs with tumors. (Am J Vet Res 2003;64:880–888)