Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Rome, Italy.
Res Vet Sci. 2012 Aug;93(1):240-5. doi: 10.1016/j.rvsc.2011.07.030. Epub 2011 Sep 3.
We have investigated SIRT1, p53 and cell cycle-checkpoint kinase 2 (CHK2) gene dysfunction in a dog with a multicancer syndrome-like in order to evaluate their potential role in the determinism of the disease and to establish a possible correlation between SIRT1 transcript level and p53 expression status.
Blood sample and tumour samples from a pure breed English Setter dog with different tumours were used for this study. Nucleotide sequence analysis was performed with a DNA autosequencer in order to examine p53 and CHK2 mutations. In addition, the expression level of SIRT1 was quantified by Southern Blot analysis of Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR).
Cytological examination revealed five different tumours: a cutaneous sebaceous epithelioma, a cutaneous mast cell tumour, a testicular Sertoli cell tumour, an oral malignant melanoma, and a cutaneous squamous cell carcinoma. Sequencing analysis revealed the presence of a nucleotide substitution, (CGG>CAG) exon 7 of the p53 gene in DNA from peripheral blood mononuclear cells (PBMCs) as well as in the melanoma; whereas the other four cancers showed the loss of the wild-type allele. Furthermore, CHK2 mutation at codon 311 has been identified in the melanoma and sebaceous epithelioma. In addition, SIRT1 cDNA expression decreased in all tumour samples compared to cDNA SIRT1expression level in peripheral blood mononuclear cells (PBMCs) in the same dog.
These results suggest that the germ line mutation of the p53 gene at codon 248 might be, at least, one cause of the multicancer syndrome-like in our dog; furthermore, we show a possible correlation between SIRT1 transcript level and p53 mutations status. The regulatory role of SIRT1 in tumour suppressor pathways suggests that the net effect seen may represent both direct and indirect downstream regulation and it is likely to depend on the presence or absence of functional p53.
我们研究了一只具有多种癌症综合征样病症的犬的 SIRT1、p53 和细胞周期检查点激酶 2（CHK2）基因功能障碍，以评估它们在疾病决定中的潜在作用，并确定 SIRT1 转录本水平与 p53 表达状态之间的可能相关性。
本研究使用了一只不同肿瘤的纯种英国塞特犬的血液样本和肿瘤样本。通过 DNA 自动测序仪对 p53 和 CHK2 突变进行了核苷酸序列分析。此外，通过逆转录聚合酶链反应（RT-PCR）的 Southern Blot 分析来定量 SIRT1 的表达水平。
细胞学检查显示了五种不同的肿瘤：皮肤皮脂腺上皮瘤、皮肤肥大细胞瘤、睾丸支持细胞瘤、口腔恶性黑色素瘤和皮肤鳞状细胞癌。测序分析显示，在来自外周血单核细胞（PBMCs）的 DNA 以及黑色素瘤中，p53 基因第 7 外显子的核苷酸取代（CGG>CAG）存在，而其他四种癌症则显示出野生型等位基因的缺失。此外，黑色素瘤和皮脂腺上皮瘤中已鉴定出 CHK2 密码子 311 的突变。此外，与同一犬的外周血单核细胞（PBMCs）中的 SIRT1 cDNA 表达水平相比，所有肿瘤样本中的 SIRT1 cDNA 表达均降低。
这些结果表明，p53 基因第 248 密码子的种系突变至少是我们犬的多种癌症综合征样的原因之一；此外，我们显示了 SIRT1 转录本水平与 p53 突变状态之间的可能相关性。SIRT1 在肿瘤抑制途径中的调节作用表明，所观察到的净效应可能代表直接和间接的下游调节，并且可能取决于功能性 p53 的存在与否。
