Analysis of Protein Phosphatase in Oral Colorectal Cancer

口腔结直肠癌蛋白磷酸酶分析

• 批准号: 07672159
• 负责人: FUJII Eiji
• 金额: $ 1.28万
• 依托单位: Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07672159/
• 关键词: pRb; TGF-beta; protein phosphatase (PP); G1 arrest; specific antibody; western blot; プロテイン・ホスファターゼ; 発がん物質(PhIP); TPR; GST; MBP; 融合蛋白質

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.

已知视网膜母细胞瘤蛋白(Rb)的磷酸化是细胞周期从G1向S转变所必需的。经转化生长因子-β处理后，来源于正常皮肤的HaCaT细胞生长停滞于G1期，同时pRb被去磷酸化。蛋白磷酸酶(PP)的激活分别是G2/M和G1/S转变所必需的。在本研究中，我们比较了HaCaT和其他口腔癌细胞系HSC4、HSC3和HOC313中蛋白磷酸酶(PP)的磷酸化状态和PPS的细胞内定位。经转化生长因子-β处理后，HSC4细胞生长停滞于G1期，pRb和HaCaT被去磷酸化，而HSC3和HOC313未被抑制在G1期，pRb被磷酸化。为了进一步研究PPS在细胞内的定位，在2 ng/mlTGF-β处理24小时后，提取每个细胞的核蛋白和胞浆蛋白，并用针对PP亚型的特异性抗体进行蛋白质印迹分析。HSC3、HOC313、HSC4和HaCaT细胞核中PP1Gamma1的表达水平为1：1：1：4。经转化生长因子-β处理后，HSC3、HOC313的表达水平仍为1：1，而HSC4、HaCaT的表达水平降至0：1。