Simultaneous Analysis of the p16 Gene and Protein in Canine Lymphoma Cells and Their Correlation with pRb Phosphorylation

SIMPLE SUMMARY: Lymphoma is one of the most frequently diagnosed malignancies in dogs. The most common epigenetic alteration is gene methylation. Methylation of the p16 gene leads to decreased expression of its protein. The p16 protein inhibits the activity of cyclin-dependent kinase, as a negative control of the cell cycle to prevent phosphorylation of the retinoblastoma (pRb) protein. The methylation of the p16 gene has been reported in canine lymphomas, however, p16 protein expression has not been examined in previous studies. In this study, the gene and protein expression of p16, and phosphorylation of pRb, were examined simultaneously in canine lymphoma/leukemia cell lines treated with or without a demethylation drug in vitro. We identified the hypermethylation of the p16 gene, the decreased expression of p16 protein and the hyperphosphorylation of pRb in four out of eight cell lines. Furthermore, we revealed that the expression of the p16 protein was more stable than that of the p16 gene and more closely related to the phosphorylation of pRb. In conclusion, the p16 protein expression is suggested as a promising biomarker for canine lymphoma cells, and the p16–pRb pathway could be a target for the better treatment of canine lymphomas. ABSTRACT: Cyclin-dependent kinase inhibitor p16 (CDKN2A) primarily functions as a negative regulator of the retinoblastoma protein (pRb) pathway to prevent pRb phosphorylation, thus playing a critical role in cell cycle arrest. In canine lymphoma cells, methylation due to inactivation of the p16 gene has been reported. However, its protein expression has not been examined in previous studies. In our in vitro study, the gene and protein expression of p16 and phosphorylated pRb were examined simultaneously in eight canine lymphoma and leukemia cell lines (17-71, CLBL-1, GL-1, CLC, CLGL-90, Ema, Nody-1, and UL-1). Methylation of the p16 gene was also explored using the demethylation drug 5-Aza-2′-deoxycytidine (5-Aza). After 5-Aza treatment, p16 gene and protein expression increased and pRb phosphorylation decreased, suggesting that both hypermethylation of the p16 gene and pRb hyperphosphorylation occurred in four out of eight cell lines (CLBL-1, CLC, Nody-1, and UL-1). Moreover, the estimation of p16’s protein expression was better than that of p16’s mRNA expression because the expression of the protein was more stable than those of the gene, and highly related to the phosphorylation of pRb. These results revealed that p16’s protein expression could be a promising biomarker for canine lymphoma cells.