TREATMENT OF MYELOID LEUKEMIC-CELLS WITH THE PHOSPHATASE INHIBITOR OKADAIC ACID INDUCES CELL-CYCLE ARREST AT EITHER G1/S OR G2/M DEPENDING ON DOSE

The phosphatase inhibitor okadaic acid was found to induce cell cycle arrest of human myeloid leukemic cell lines HL-60 and U937 in a concentration- and time-dependent manner. Exposure to low concentrations of okadaic acid (2-8nM) for 24-48 hr caused > 70% of cells to arrest at G2/M, with up to 40% of the cells arrested in early mitosis. Cell viability decreased rapidly after 48 hr of treatment, and morphological and DNA structure analysis indicated that this was primarily due to the induction of apoptosis. The cells arrested in mitosis by 8 nM okadaic acid could be highly enriched by density gradient centrifugation and underwent apoptosis when further cultured either with or without okadaic acid, indicating that the effects of okadaic acid were irreversible. In contrast to the effects of low concentrations of okadaic acid, high concentrations (500 nM), inhibited proliferation in less than 3 hr. Remarkably, the majority of cells also entered a mitosis-like state characterized by dissolution of the nuclear membrane and condensation and partial separation of chromosomes. However, these cells had a diploid content of DNA, indicating that the cell cycle arrest occurred at G1/S with premature chromosome condensation (PCC), rather than at G2/M. If cells were first blocked at G1/S with hydroxyurea and then treated with okadaic acid, > 90% developed PCC in less than 3 hr without replicating their DNA. Caffeine was not able to induce PCC in these cells, either with or without prior inhibition of DNA synthesis. These results provide further evidence that an interacting system of cell cycle associated phosphatases and kinases control the initiation of each phase of the cell cycle in higher eukaryotes and further suggest that okadaic acid will be useful in manipulating the cell cycle of leukemic cells.