Yonemaru Kayoko, Sakai Hiroki, Murakami Mami, Kodama Atsushi, Mori Takashi, Yanai Tokuma, Maruo Kohji, Masegi Toshiaki
Laboratoriy of Veterinary Pathology, Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, Japan.
J Vet Med Sci. 2007 Mar;69(3):271-8. doi: 10.1292/jvms.69.271.
To investigate whether inactivation of the p53 and retinoblastoma (Rb) protein pathways contributes to the development of canine hemangiosarcoma (HSA), we examined immunohistochemically the expression of p53, Rb, phosphorylated Rb (phospho-Rb), p16, and cyclin D1 in 39 spontaneous canine HSAs and 10 hemangiomas. In addition, mutations in the p53 gene were analyzed by polymerase chain reaction (PCR)-single-stranded conformation polymorphism and PCR direct sequencing; furthermore, we quantified cyclin D1 mRNA by semiquantitative real-time reverse transcription-PCR. Positive immunoreactivity for p53 was observed in 17.9% of HSAs. However, mutations were not detected in these cases. The labeling indices for Rb, phospho-Rb, and cyclin D1 were markedly higher in all HSAs than in hemangiomas. Of the 7 cases with cyclin D1-positive immunoreactivity, 4 overexpressed cyclin D1 mRNA (to a level more than 10-fold higher than that of GAPDH mRNA). The p16 protein was clearly detected in all hemangiomas; however, 82% of the neoplastic cells in HSA showed a loss of or low immunoreactivity. These results suggest that alteration of the p16-cyclin D1-Rb pathway, rather than the p53 pathway, may be associated with the pathogenesis of canine HSA.
为了研究p53和视网膜母细胞瘤（Rb）蛋白通路失活是否与犬血管肉瘤（HSA）的发生有关，我们采用免疫组织化学方法检测了39例自发性犬HSA和10例血管瘤中p53、Rb、磷酸化Rb（phospho-Rb）、p16和细胞周期蛋白D1的表达。此外，通过聚合酶链反应（PCR）-单链构象多态性和PCR直接测序分析p53基因的突变；此外，我们通过半定量实时逆转录PCR对细胞周期蛋白D1 mRNA进行定量。在17.9%的HSA中观察到p53阳性免疫反应。然而，在这些病例中未检测到突变。所有HSA中Rb、phospho-Rb和细胞周期蛋白D1的标记指数均明显高于血管瘤。在7例细胞周期蛋白D1阳性免疫反应的病例中，4例细胞周期蛋白D1 mRNA过表达（水平比甘油醛-3-磷酸脱氢酶mRNA高10倍以上）。在所有血管瘤中均清晰检测到p16蛋白；然而，82%的HSA肿瘤细胞显示p16蛋白缺失或免疫反应性低。这些结果表明，p16-细胞周期蛋白D1-Rb通路而非p53通路的改变可能与犬HSA的发病机制有关。
