Analysis of Protein Phosphatase in Oral Colorectal Cancer

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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.