Previous studies have shown that structurally diverse tumor promoters can modulate protein kinases involved in signal transduction. In this study, we show that palytoxin, a potent non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type skin tumor promoter, induces a signaling pathway leading to the activation of the stress-activated protein kinases/c-Jun N-terminal kinases (JNK) in Swiss 3T3 fibroblasts. Treatment of cells with doses as low as 0.1 nm palytoxin results in significant activation of JNK. In contrast to epidermal growth factor, which induces a transient activation of JNK in Swiss 3T3 cells, palytoxin causes prolonged enzyme activation. Since stimulation of ion flux appears to play an important role in the mechanism of action of palytoxin in other systems, we investigated the role of sodium and calcium in the activation of JNK: (a) our results show that incubation of Swiss 3T3 cells in a sodium-free medium dramatically reduced the magnitude of JNK activation by palytoxin; and (b) we found that the sodium ionophore gramicidin activates JNK. Together, these results suggest that sodium influx, which is a hallmark of palytoxin action, may play a key role in the activation of JNK by palytoxin. Our results indicate that calcium influx is not necessary or sufficient for palytoxin-induced activation of JNK. In contrast to palytoxin, the TPA-type tumor promoter phorbol 12,13-dibutyrate and the non-TPA-type tumor promoters thapsigargin and okadaic acid do not appear to activate JNK in this system. In contrast to phorbol 12,13-dibutyrate, palytoxin does not activate the p42/p44 mitogen-activated protein kinases. Our results demonstrate that in Swiss 3T3 fibroblasts, palytoxin can activate a protein kinase signaling pathway that is distinct from that activated by the prototypical phorbol ester tumor promoters and other potent skin tumor promoters.

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This work was supported in part with the assistance of the American Cancer Society Institutional Research Grant IN-13-35-17 and by funds provided through the Grant-in-Aid Program of the Office of the Vice President for Research at the University of Minnesota.

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