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  • Author or Editor: Mitsuhiro Yazawa x
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Abstract

Objective—To measure telomere length and telomerase activity in naturally occurring canine mammary gland tumors.

Sample Population—27 mammary gland tumor specimens obtained during resection or necropsy and 12 mammary gland tissue specimens obtained from healthy (control) dogs.

Procedure—Telomere length in tissue specimens was measured by use of restriction endonuclease digestion and Southern blot analysis. Telomerase activity was measured by use of a telomeric repeat amplification protocol assay.

Results—Telomere length in mammary gland tumors ranged from 11.0 to 21.6 kilobase pairs (kbp; mean ± SEM, 14.5 ± 0.5 kbp) but did not differ among tumor types. Telomeres in mammary gland tumors were slightly shorter than in normal tissue specimens, but telomere length could not be directly compared between groups, because mean age of dogs was significantly different between groups. Age was negatively correlated with telomere length in control dogs but was not significantly correlated with length in affected dogs. Telomerase activity was detected in 26 of 27 mammary gland tumors and in 4 of 12 normal tissue specimens. However, telomerase activity and telomere length were not correlated in tumor specimens.

Conclusion and Clinical Relevance—Telomere length is maintained in canine mammary gland tumors regardless of the age of the affected dog. Measurement of telomere length may be a useful tool for monitoring the in vivo effects of telomerase inhibitors in dogs with tumors. (Am J Vet Res 2001; 62:1539–1543)

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in American Journal of Veterinary Research

Abstract

Objective—To evaluate aberrations of the p53 tumor suppressor gene in naturally developing tumors in dogs.

Sample Population—Tumor specimens from 15 dogs with various tumors, including malignant lymphoma (7 dogs), monocytic leukemia (1), mammary gland adenoma (1), mammary gland benign mixed tumor (1), rhabdomyosarcoma (1), colon cancer (1), and osteosarcoma (3).

Procedure—Aberrations of the p53 gene in these tumor tissues were examined by reverse transcriptase- polymerase chain reaction and single-strand conformation polymorphism analysis, using 3 fragments that covered the entire open reading frame of the canine p53 gene, followed by nucleotide sequencing of the abnormal bands.

Results—Point mutations, deletions, and insertions resulting in a number of amino acid substitutions of wild-type p53 were detected in 7 of the 15 tumor specimens from dogs with malignant lymphoma, monocytic leukemia, rhabdomyosarcoma, colon cancer, and osteosarcoma. Of these 7 dogs, 2 had aberrations of the p53 gene on both alleles, whereas 5 had aberrations of the p53 gene on 1 allele and concurrently lacked the wild-type p53 transcript. Many of the aberrations of the p53 gene detected in these tumors were located in the transactivation, DNA binding, and oligomerization domains.

Conclusions and Clinical Relevance—Various naturally developing tumors in dogs often have inactivation of the p53 tumor suppressor gene, which may be 1 of the multiple step-wise genetic changes during tumorigenesis. This study indicates that p53 gene can be a target for gene therapy for tumors in dogs. (Am J Vet Res 2001;62:433–439)

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in American Journal of Veterinary Research

Abstract

Objective—To evaluate the mechanism of multidrug resistance in feline lymphoma cell lines.

Sample Population—A feline lymphoma cell line (FT-1) and its adriamycin (ADM)-resistant subline (FT-1/ADM).

Procedures—The FT-1 cell line was cultivated in the presence of a gradually increasing concentration of ADM to generate its ADM-resistant subline (FT-1/ADM). Susceptibility of cells from the parental FT-1 cell line and the FT-1/ADM subline to antineoplastic drugs was determined. From the complementary DNA (cDNA) template of FT-1/ADM cells, feline MDR1 cDNA was amplified by use of polymerase chain reaction (PCR) and sequenced. Reverse transcription (RT)-PCR and Western blot analyses were performed to assess expression of the MDR1 gene and P-glycoprotein (P-gp) in FT-1/ADM cells, compared with that in FT-1 cells.

Results—A drug sensitivity assay revealed that FT-1/ADM cells were much more resistant to ADM and vincristine than the parental FT-1 cells. The feline MDR1 cDNA amplified by use of PCR was 3,489 base pairs long, corresponding to approximately 90% of the whole open reading frame of human MDR1 cDNA; its amino acid sequence was 91.5, 87.0, and 79.4% identical to that of human MDR1, mouse mdr1a, and mdr1b cDNA, respectively. By RT-PCR analysis, expression of MDR1 messenger RNA was clearly detected in FT-1/ADM cells but not in the parental FT-1 cells. Western blot analysis also revealed the expression of P-gp encoded by the MDR1 gene in FT-1/ADM cells but not in FT-1 cells.

Conclusions—The basic structure of the feline MDR1 gene was essentially the same as that of multidrug- resistance genes of other species. Expression of P-gp appeared to be one of the mechanisms responsible for the development of multidrug resistance in feline lymphoma cell lines in vitro. (Am J Vet Res 2000;61:1122–1127)

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in American Journal of Veterinary Research

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)

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in American Journal of Veterinary Research

Abstract

Objective—To evaluate results of centrosome hyperamplification in naturally developing tumors of dogs.

Sample Population—Tumor specimens from 9 dogs with tumors (rhabdomyosarcoma, osteosarcoma, chondrosarcoma, myxosarcoma, and mammary gland tumor) and 2 canine osteosarcoma cell lines.

Procedure—3 antibodies for centrosome proteins (ie, anti-γ-tubulin, anti-BRCA1, and anti-pericentrin) were used for immunohistochemical analysis. Double immunostaining for centrosomes was used to confirm the specificity of these antibodies for centrosomes. Mutational analysis of the canine p53 gene was carried out by polymerase chain reaction–singlestrand conformation polymorphism analysis, and expression of canine MDM2 protein was evaluated by use of immunohistochemical analysis, using anti- MDM2 antibody.

Results—Immunohistochemical analysis of dog osteosarcoma cell lines with apparent aneuploidy revealed frequent hyperamplification of centrosomes in the osteosarcoma cell lines. Similar hyperamplified centrosomes were detected in the tumor tissues from all of the 9 tumors. The frequency of cells with hyperamplified centrosomes (3 to 20/cell) in each tumor tissue ranged from 9.50 to 48.1%, whereas centrosome hyperamplification was not observed in normal lymph nodes from these dogs. In 8 of the 9 tumors, mutation of p53 gene or overexpression of MDM2, or both, was detected.

Conclusions and Clinical Relevance—Various types of naturally developing tumors in dogs often have hyperamplification of centrosomes associated with chromosome instability. Hyperamplification of centrosomes is a novel tumor marker for use in cytologic and histologic examinations of clinical specimens obtained from dogs. (Am J Vet Res 2001;62:1134–1141)

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in American Journal of Veterinary Research

Abstract

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)

Full access
in American Journal of Veterinary Research