Determination of connexin gene contributing to suppression of canine mammary carcinogenesis and investigation of the mechanism

缝隙连接蛋白(Cx)基因是缝隙连接中的一员，其表达模式具有组织和细胞特异性，并且在致癌过程中表达大多下调，因此这些基因足以监测肿瘤的发展。应用逆转录偶联聚合酶链式反应(RT-PCR)和RT-Real-Time PCR方法检测了5例正常乳腺和乳腺肿瘤组织中Cx基因的表达。我们用Cx26和Cx43基因的特异性引物进行RT-PCR，观察了Cx26和Cx43基因在所有正常组织中的表达。此外，我们还利用RT-PCR分析了Cx26和Cx43基因在几种类型的犬乳腺肿瘤中的表达模式，我们观察到Cx43基因的下调与犬乳腺肿瘤的恶性程度有关。RT-Real-time聚合酶链式反应证实了上述RT-PCR结果。这些结果提示Cx43在犬乳腺肿瘤的发生发展中起着重要的作用。为了确定通过Cx43基因的诱导对肿瘤细胞具有抑制作用的化学预防药物，我们试图通过建立癌细胞培养系统和异种移植模型来筛选候选药物。因此，我们从食物中确定了一个有希望的成分来诱导Cx43基因在癌细胞中的诱导。事实上，该组分对Cx43基因的诱导在异种移植模型的有效肿瘤抑制中起到了关键作用。总体而言，诱导Cx43基因高表达的药物似乎对犬乳腺肿瘤具有预防和治疗作用。

The expression patterns of connexin (Cx) genes, a member of gap junctions, are tissue- and cell-specific, and the expressions are mostly down-regulated during carcinogenic processes, so the genes were adequate to monitor the development of the tumors. We examined the expression of Cx mRNAs in five canine normal mammary glands and mammary gland tumor samples by reverse transcriptase-coupled polymerase chain reaction (RT-PCR) and RT-realtime PCR. We observed the expression of Cx26 and Cx43 genes in all of the normal tissues by RT-PCR using specific primers from Cx26 and Cx43 genes. Also, we analyzed the expression patterns of Cx26 and Cx43 genes in several types of canine mammary gland tumors using the RT-PCR, and we observed that the down-regulation of Cx43 was associated with malignancy of canine mammary gland tumors. These RT-PCR results were confirmed by RT-realtime PCR. These results suggest that Cx43 plays an important role in progression of canine mammary gland tumors.In order to determine chemopreventive agents which have suppressive effects on cancer cells via the induction of Cx43 gene, we tried to pick up candidate agents, using a cancer cell culture system and a xenograft model. As a result, we determined a promising component from foods to induce the induction of Cx43 gene in cancer cells. Actually, the induction of Cx43 gene by the component played a critical role in causing effective tumor suppression of the xenograft model. Overall, it seems that agents inducing high expression of Cx43 gene acts as a preventive and therapeutic agent against canine mammary tumors.