Analysis of Protein Phosphatase in Oral Colorectal Cancer

已知视网膜母细胞瘤蛋白（retinoblastoma protein，pRb）的磷酸化是G1期向S期转变所必需的。通过转化生长因子（transforming growth factor，TGF）-β处理，来源于正常人上皮的HaCaT细胞显示生长停滞在G1期，并且pRb在该时间被去磷酸化。另外，我们还研究了HaCaT细胞中2A和1型蛋白磷酸酶（PP）的激活对G2/M和G1/S转换的影响，并与其他口腔癌细胞系HSC 4、HSC 3和HOC 313进行了比较。用2ng/ml的TGF-β处理HSC 4细胞24小时后，提取细胞核和胞浆蛋白，用流式细胞仪检测细胞周期，发现细胞周期中PPs的表达与细胞周期的变化有关，而HSC 3和HOC 313细胞则相反。并使用针对单个PP同种型的特异性抗体进行蛋白质印迹分析。HSC 3、HOC 313、HSC 4和HaCaT细胞核中PP 1 γ 1的表达水平为1：1：1：4。经TGF-β处理后，HSC 3、HOC 313仍为1：1，而HSC 4、HaCaT降至0：1; PP 1 α为1：1：0：2。经TGF-β处理后，HSC 3、HOC 313和HSC 4的pRb磷酸化程度仍为1：1：0，而HaCaT降至1。

It is known that phosphorylation of retinoblastoma protein (pRb) is necessary for G1 to S phase transition. By the treatment of transforming growth factor (TGF)-beta, HaCaT cell which is derived from normal human epithelia shows growth arrest at G1 phase and pRb is dephosphorylated at that time. It is also known that activation of protein phosphatase (PP) type 2A and 1 are necessary for G2/M and G1/S transition, respectively.In this study, we investigated the phospholyration status of pRb and intracellular localization of PPs in HaCaT compared with other oral cancer cell lines, HSC4, HSC3 and HOC313. By TGF-beta treatment, HSC4 showed growth arrest at G1 phase and dephosphorylation of pRb as well as HaCaT.In contrast, HSC3 and HOC313 did not arrest at G1 and pRb was phospholyrated.To further characterize the intra-cellular localization of PPs, nuclear and cytoplasmic protein from each cell were extracted 24 hours after the treatment of 2ng/ml TGF-beta, and western blot analysis were performed using specific antibodies against individual PP isotypes. The expression levels of PP1gamma1 in nucleus of HSC3, HOC313, HSC4 and HaCaT were rated 1 : 1 : 1 : 4. With TGF-beta treatment, those of HSC3, HOC313 were still 1 : 1, but HSC4, HaCaT were reduced to 0 : 1.For PP1alpha, the levels were 1 : 1 : 0 : 2. With TGF-beta treatment, those of HSC3, HOC313 and HSC4 were still 1 : 1 : 0, but HaCaT was reduced to 1.These results demonstrated that the extents of phosphorylated pRb were related inversely to the expression levels of PP1gamma1 and PP1alpha in nucleus.