Inhibition of cellular protein phosphatases results in the disruption of p53-regulated homeostasis

Two important serine/threonine protein phosphatases (PP), PP1 and PP2A, are involved in many cellular processes including cell cycle regulation, cell signaling, and programmed cell death. A number of natural product toxins have been identified which selectively and potently inhibit PP1 and 2A. Two of these are the cyanobacteria-derived cyclic heptapeptide microcystin-LR, and the polyether fatty acid okadaic acid obtained from black sponge. Inhibition of protein phosphatases can lead to hyperphosphorylation of many cellular proteins. This hyperphosphorylation results in a variety of cellular alterations including cell cycle arrest and cell death/apoptosis. Studies in many different cell lines have shown that okadaic acid and microcystin cause mitotic arrest and induce apoptosis. Paradoxically, both okadaic acid and microcystin are also tumor promoters. The cellular and molecular mechanisms underlying their tumor promoting activities are not completely understood, although it is theorized that p53 may play a role. With okadaic acid-induced mitotic arrest and apoptosis, the role of p53 has only been briefly studied and the results are not consistent. In microcystin induced-mitotic arrest and apoptosis, p53 has only recently been examined and again the results have not been definitive. The primary goal of our research is to further examine the involvement of p53 in protein phosphatase inhibitor-induced apoptosis/tumor promotion. In vitro, it was found that RKO-P cells (wild type p53 function) were more sensitive to okadaic acid-mediated cytotoxicity, induction of apoptosis and G2 growth arrest than RKO-E6 cells (a lack of appreciable functional p53). Conversely in vivo, the results showed that p53 homozygous knockout mice were more sensitive to the hepatic proliferative effects associated with chronic, sublethal microcystin exposure. The increase in hepatic proliferation was associated with an increase in the expression of Ki-67, the mitotic cyclins, cyclin A and cyclin B, the mitotic cyclin dependent kinase, cdc2a. No significant changes were seen in the expression of genes associated with apoptosis or oxidative damage. These data indicate that p53 is important in regulating the cytotoxic and proliferative responses associated with protein phosphatase inhibition by okadaic acid and microcystin, and that pre-neoplastic hyperplasia may be associated with an increase in cellular proliferation than a decrease in apoptosis