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OBJECTIVE To examine effects of a common mutation (2-base insertion in exon 5) of the TP53 gene on biological function of p53 protein in canine histiocytic sarcoma cells. SAMPLE Canine histiocytic tumor cell lines DH82 with deletion of TP53 and CHS-3 with the wild-type TP53 and canine wild-type and mutant TP53 fragments. PROCEDURES Wild-type or mutant TP53 with a polyprotein peptide tag at the N-terminus was transduced into DH82 and CHS-3 cells. Expression of p53 protein, changes in function as a transcription factor, and susceptibility to doxorubicin and nimustine were compared. RESULTS Transduced p53 protein was detected in wild-type TP53–transduced DH82 and CHS-3 cells, whereas expression was not detected in mutant TP53–transduced cells. There were significant increases in expression of target genes of p53 protein, including p21 and MDM2, in wild-type TP53–transduced cells, compared with results for native and mock-transfected cells, but not in mutant TP53–transduced cells. There was no significant difference in drug susceptibilities among native and derivative cells of CHS-3. However, cell viabilities of wild-type TP53–transduced DH82 cells incubated with doxorubicin were significantly lower than viabilities of native, mock-transfected, and AT insertion mutation–TP53–transduced DH82 cells; susceptibility to nimustine did not differ significantly among cells. CONCLUSIONS AND CLINICAL RELEVANCE Expression of p53 protein and its function as a transcription factor were lost after addition of a 2-base insertion in the TP53 gene in canine histiocytic tumor cells. Additional studies are needed to investigate the clinical relevance of this mutation in histiocytic sarcomas of dogs.

Canine histiocytic sarcoma (HS) is an aggressive tumor type originating from dendritic cells or macrophages. We previously reported high incidence of the two-base (AT) insertion mutation (insAT) in the tumor protein p53 (TP53) gene in dogs with HS, and the aim of this study was to investigate the clinical significance of insAT in canine HS. The present study established a sensitive digital PCR-based assay for detecting insAT and examined its associations with clinical variables and survival time. The mutation was detected in 26 of 64 dogs (41%), and the mean mutant allele frequency was 1.9% (range, 0.014-35%), indicating that not all tumor cells harbor insAT. The incidence of insAT was significantly higher in dogs with metastatic lesions than in those without metastatic lesions. However, the existence of insAT was not associated with survival time or response to chemotherapy with lomustine or nimustine. This study suggested that HS cells might acquire insAT in the TP53 gene during development of metastasis, but insAT was not a prognostic factor in canine HS. Further studies are needed to investigate the contribution of insAT to the development of metastatic lesions of canine HS.

The mutations of TP53 gene are frequently observed in canine histiocytic sarcoma (HS). The objective of this study was to examine the distribution of tumor cells with TP53 gene mutations. Tumor tissues were divided into three or four regions and TP53 gene mutations were examined. TP53 gene mutations were detected only in parts of the HS tissues from six of the eight dogs, and the frequency of the mutant allele varied (0-65%) among the tumor regions. This study suggests that canine HS can exhibit intratumor heterogeneity. Further studies are needed to examine the clinical significance of the intratumor heterogeneity of TP53 gene mutations.

To examine effects of a common mutation (2-base insertion in exon 5) of theCanine histiocytic tumor cell lines DH82 with deletion ofWild-type or mutantTransduced p53 protein was detected in wild-typeExpression of p53 protein and its function as a transcription factor were lost after addition of a 2-base insertion in the

Canine histiocytic sarcoma (HS) is a malignancy originating from the histiocytic cell lineage and characterized by poor response to chemotherapy and short survival time. Mutation of the TP53 gene and its association with poor prognosis has been reported in several canine tumors. However, the mutation of this gene has not been investigated in canine HS. The aim of this study was to examine a TP53 gene mutation in dogs with HS. Aberrations of the TP53 gene were examined by polymerase chain reaction-single strand conformational polymorphism analysis and DNA sequence analysis, revealing mutations of the TP53 gene in 12 (46%) of 26 dogs affected by HS. The incidence of the TP53 gene mutation was relatively high in canine HS compared with other canine tumors. Among these mutations, 10 of 12 dogs (83%) with a TP53 gene mutation harbored the same mutation: a 2-base (AT) insertion in exon 5, resulting in the introduction of a stop codon (c.446_447insAT, p.Tyr150SerfsX8). Further studies are needed to examine the functional change due to the mutation and its association with the pathogenesis of canine HS.