Effect of the VP3 gene of chicken anemia virus on canine mammary tumor cells

Jih-Jong Lee Department and Graduate Institute of Veterinary Medicine, College of Bio-Resources and Agriculture, National Taiwan University, No. 1, Section 4, Roosevelt Rd, Taipei 106, Taiwan.

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Poshen B. Chen Genomics Research Center, Academia Sinica, No. 128, Section 2, Academia Rd, Taipei 115, Taiwan.

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Su-Hua Yang Department and Graduate Institute of Veterinary Medicine, College of Bio-Resources and Agriculture, National Taiwan University, No. 1, Section 4, Roosevelt Rd, Taipei 106, Taiwan.

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Chiung-Hsiang Cheng Department and Graduate Institute of Veterinary Medicine, College of Bio-Resources and Agriculture, National Taiwan University, No. 1, Section 4, Roosevelt Rd, Taipei 106, Taiwan.

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Ling-Ling Chueh Department and Graduate Institute of Veterinary Medicine, College of Bio-Resources and Agriculture, National Taiwan University, No. 1, Section 4, Roosevelt Rd, Taipei 106, Taiwan.

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Victor F. Pang Department and Graduate Institute of Veterinary Medicine, College of Bio-Resources and Agriculture, National Taiwan University, No. 1, Section 4, Roosevelt Rd, Taipei 106, Taiwan.

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Michael Hsiao Genomics Research Center, Academia Sinica, No. 128, Section 2, Academia Rd, Taipei 115, Taiwan.

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Chung-Tien Lin Department and Graduate Institute of Veterinary Medicine, College of Bio-Resources and Agriculture, National Taiwan University, No. 1, Section 4, Roosevelt Rd, Taipei 106, Taiwan.

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Abstract

Objective—To investigate the antitumor effect of the chicken anemia virus (CAV) VP3 gene in canine mammary tumor (CMT) cells.

Sample Populations—Established primary canine cell lines that originated from epithelial cells of resected CMTs and nonneoplastic mammary gland epithelial (MGE) cells.

Procedures—Expression vectors and lentiviral vectors encoding the VP3 gene from a Taiwan-Ilan isolate of CAV were used to deliver the VP3 gene into CMT cells and nonneoplastic MGE cells. Ectopic gene expression and the pro-apoptotic effect of the VP3 gene on CMT and nonneoplastic MGE cells by either transfection or viral infection were evaluated via immunofluorescence microscopy, western blot analysis, and terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling analysis.

Results—Overexpression of the enhanced green fluorescent protein–VP3 fusion protein was detected predominantly in the nuclei of CMT cells. In contrast, the VP3 protein was localized to the cytoplasm of nonneoplastic MGE cells. Among the fusion protein–expressing CMT cells, most underwent characteristic changes of apoptosis, whereas apoptosis was not detected in fusion protein–expressing, nonneoplastic MGE cells. Induction of apoptosis by VP3 gene overexpression in CMT cells was associated with the caspase-9–, but not the caspase-8–, mediated apoptosis pathway.

Conclusions and Clinical Relevance—These data indicate that the VP3 gene of the CAV induces apoptosis in malignant CMT cells, but not in nonneoplastic canine MGE cells. On the basis of such tumor cell–specific killing, the VP3 gene may be a promising agent for the treatment of malignant mammary gland tumors in dogs.

Abstract

Objective—To investigate the antitumor effect of the chicken anemia virus (CAV) VP3 gene in canine mammary tumor (CMT) cells.

Sample Populations—Established primary canine cell lines that originated from epithelial cells of resected CMTs and nonneoplastic mammary gland epithelial (MGE) cells.

Procedures—Expression vectors and lentiviral vectors encoding the VP3 gene from a Taiwan-Ilan isolate of CAV were used to deliver the VP3 gene into CMT cells and nonneoplastic MGE cells. Ectopic gene expression and the pro-apoptotic effect of the VP3 gene on CMT and nonneoplastic MGE cells by either transfection or viral infection were evaluated via immunofluorescence microscopy, western blot analysis, and terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling analysis.

Results—Overexpression of the enhanced green fluorescent protein–VP3 fusion protein was detected predominantly in the nuclei of CMT cells. In contrast, the VP3 protein was localized to the cytoplasm of nonneoplastic MGE cells. Among the fusion protein–expressing CMT cells, most underwent characteristic changes of apoptosis, whereas apoptosis was not detected in fusion protein–expressing, nonneoplastic MGE cells. Induction of apoptosis by VP3 gene overexpression in CMT cells was associated with the caspase-9–, but not the caspase-8–, mediated apoptosis pathway.

Conclusions and Clinical Relevance—These data indicate that the VP3 gene of the CAV induces apoptosis in malignant CMT cells, but not in nonneoplastic canine MGE cells. On the basis of such tumor cell–specific killing, the VP3 gene may be a promising agent for the treatment of malignant mammary gland tumors in dogs.

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