Antitumor Effect Of A Novel Inhibitor Of Protein Phosphatase â… , IPP2, And The Related Mechanisms

Reversible protein phosphorylation controls many processes in plant and animal cells, including cell proliferation, differentiation , development, the activity of neuron, metabolism, tumorigenesis etc. As physiological responses of cells to extracellular stimuli are defined by the phosphorylation state of key proteins, coordinating the opposing actions of protein kinases (PK) and protein phosphatases (PP) becomes important for cells to evoke an appropriate response.Protein phosphatases 1 (PP1) accounts for a significant amount of Ser/Thr protein phosphatase activity in mammalian cells. PP1 is composed of catalytic subunits and regulatory subunits, which modulates metabolism, cell differentiation and signal transduction by dephosphorylation. The regulatory subunits determine the substrate specificity, activity and subcellular localization of PP1. Now, several inhibitor proteins have been identified, which inhibit the activity of PP1 specially. Protein phosphatase inhibitor-1 (IPP-1) is the first endogenetic molecule to be found to regulate the activity of protein phosphatase. It is expressed abundantly in skeletal muscle, brain, fat tissue and kidney. When phosphorylated by protein kinase A (PKA) at Thr-35, IPP-1 inhibited PP1 activity. Thus IPP-1 provided a potential mechanism for cross-talk between a protein kinase and phosphatase. Human protein phosphatase inhibitor 2 (IPP2), a novel protein phosphatase inhibitor, was identified by us from human bone marrow stromal cell cDNA library by large-scale random sequencing. The 116-residue protein shares great homology with of human, mouse and rat protein phosphatase inhibitor 1. One of our previous studies had shown that IPP2 specifically inhibits PP1 activity following phosphorylation.It was reported that the expression and activity of PP1 were enhanced in liposarcoma, chondrosarcoma, malignancy of osteogenic tumor and fibrous histiocytoma, suggesting that PP1 is involved in the accelerated growth of these tumorcells, while the serine-threonine phosphatase inhibitor, okadaic acid (OKA) and calyculin (CLA), synergistically augment TNF-induced apoptosis in TNF-sensitive tumor cell lines. OKA also inhibits the growth of tumor cells and causes mitotic arrest that is characterized by chromosome overcondensation and chromatin degeneration, suggesting that PP1 and its inhibitors are essential for cell growth and mitotic process.In this study, we investigated the effects of IPP2 on the growth of HeLa cells and the related mechanisms. We constructed expression vectors for wild type of IPP2 (IPP2/WT) and an activated mutant that covers 8-60 residues of IPP2 with Thr40 mutated to Asp (IPP2/SHI-2). HeLa cells stably expressing IPP2/WT and IPP2/SHI-2 were established.First, we investigated the possible roles of IPP2 in regulation of cell proliferation. The [3H]-TdR incorporation assay and soft agar colony formation assay suggest that the proliferation ability of HeLa-IPP2/SHI-2 is impaired. In addition, the migration assay showed that the migration ability of HeLa-IPP2/SHI-2 is also weakened. The results suggested that overexpression of the activated mutant of IPP2 (IPP2/SHI-2) might inhibit in vitro growth and migration of human cerix carcinoma HeLa cells. In addition, we compared the tumorigenicity of HeLa-IPP2/SHI-2, HeLa-IPP2/WT, HeLa-mock or parental HeLa cells in nude mice. The results showed that tumorigenicity of HeLa-IPP2/SHI-2 was profoundly decreased compared to both control groups. The tumorigenicity of HeLa-IPP2/WT also was decreased, indicating stable expression of IPP2 may suppress HeLa cell growth in vivo. In order to determine whether overexpression of IPP2 and its activated mutant in human HeLa cells could mediate therapeutic effects in vivo, HeLa-bearing nude mice were used as a model. Purified IPP2/WT and IPP2/SHI-2 plasmids were delivered into the tumors by in vivo electroporation. The results showed that intratumoral gene transfer of IPP2/SHI-2 could prolong the survival of HeLa-bearing nude mice significantly.The catalytic subunit of PP1 changes...