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  • Author or Editor: Takayuki Nakagawa x
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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)

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in American Journal of Veterinary Research


Objective—To perform molecular cloning of the canine telomerase reverse transcriptase (TERT) gene and determine its expression in neoplastic and nonneoplastic cells.

Sample Population—9 canine tumor cell lines derived from various neoplasms, 16 primary canine tumors, and tissues from 15 normal canine organs.

Procedure—Tumor cell lines were derived from canine tumors that included osteosarcoma, mammary gland adenocarcinoma, melanoma, acute lymphoblastic leukemia, lymphoma, and mastocytoma and a canine primary fibroblast culture. Canine TERT complementary DNA (cDNA) was amplified by use of polymerase chain reaction (PCR) and sequenced. Expression of TERT mRNA was examined by reverse transcription (RT)-PCR assay. Telomerase activity was measured by use of the telomeric repeat amplification protocol assay.

Results—The canine TERT cDNA clone was 237 base pairs in length and contained a central region encoding the reverse transcriptase motif 2. Expression of TERT mRNA was detected in canine tumor cell lines that had telomerase activity but not in telomerasenegative canine primary fibroblasts. The TERT mRNA was detected in 13 of 16 canine tumor tissues and several normal tissues such as liver, ovary, lymph node, and thymus. A significant correlation between TERT expression level and telomerase activity was noted.

Conclusions and Clinical Relevance—Expression of TERT mRNA was closely associated with telomerase activity in neoplastic cells as well as some non-neoplastic cells from dogs. In addition to telomerase activity, expression of TERT mRNA can be used as a marker of tumor cells. (Am J Vet Res 2003;64:1395–1400)

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in American Journal of Veterinary Research