[Frontiers in Bioscience 3, d1262-1273, December 15, 1998]

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Axel H. Schönthal

Department of Molecular Microbiology and Immunology, K. Norris Jr. Comprehensive Cancer Center, University of Southern California, 2011 Zonal Ave., HMR-405, Los Angeles, CA 90033

Received 8/25/98 Accepted 12/2/98


Originally, it was suspected that the tumor promoting activity of okadaic acid was due to its regulation of the same pathway that is affected by the well-established phorbol ester tumor promoters, namely the protein kinase C (PKC) pathway (50, 59, 60). The idea was that activation of PKC by phorbol esters initiated a kinase cascade that lead to the increased phosphorylation/activation of various downstream components of this signal transduction pathway. Similarly, okadaic acid, through the inhibition of phosphatases that dephosphorylate/inactivate the same components, would generate the same net effect, namely the increased activity of this pathway. Further support for this hypothesis was provided by the finding that several growth-regulatory genes that are activated by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) are also activated by okadaic acid (see detailed references in (12)). Most prominent among these genes are the proto-oncogenes c-fos and c-jun (61-64), whose protein products are able to interact and form the heterodimeric transcription factor AP-1 (65). And indeed, either agent, TPA or okadaic acid, causes increased AP-1 expression and activity (66-70). (For a detailed list of growth-regulatory genes that are regulated by okadaic acid, see ref. (71)). Activation of the c-fos gene is regulated through the reversible phosphorylation of ternary complex factor (TCF) by kinases of the mitogen-activated protein kinase (MAPK) family, and both agents, TPA and okadaic acid, stimulate TCF via the activation of the MAPK pathway (72). There are further examples of transcription factors that are activated by TPA and by okadaic acid. For example, the activity of nuclear factor kappa B (NF-kappaB) is stimulated by either agent (73, 74). Activation of NF-kappaB requires the degradation of protein inhibitors, IkappaB-alpha and IkappaB-beta, which is induced by the hyperphosphorylation of these proteins. It has been demonstrated that treatment of cells with okadaic acid or calyculin A results in the increased phosphorylation and subsequent degradation of these NF-kappaB inhibitors (75, 76). In the case of IkappaB-alpha, increased phosphorylation appears to be mediated through the (indirect) activation of ERK1, a member of the MAPK family, in response to okadaic acid treatment (76). Activation of the MAPK pathway by okadaic acid has been described by several groups (77-79).

However, while several cellular responses to drug treatment have been documented that are similar between okadaic acid and phorbol esters, there are also numerous differences. For example, using high definition two-dimensional gel electrophoresis, Guy et al. (80) identified 74 proteins that exhibited altered levels of phosphorylation in response to okadaic acid treatment of human fibroblasts. However, when the same cells were treated with TPA, a rather different pattern of protein phosphorylation was found. Moreover, in other studies physiological differences between okadaic acid-generated and TPA-generated transformed cells have been reported (61, 81).

In addition, depending on the experimental approach, TPA and okadaic acid are able to antagonize each otherís effects. For instance, it has been well documented that okadaic acid is able to induce apoptosis in various primary as well as transformed cells (82-85). In Balb/c 3T3 fibroblasts, this effect was found to be dependent on the presence of the p53 tumor suppressor protein (86), whereas in human breast carcinoma cells p53 function was not found to be required for this process (87). Further, in H-ras oncogene transformed cells okadaic acid-induced apoptosis appeared to involve the modulation of raf-1, PKC, and MAPK activities (88). Interestingly, the simultaneous treatment of certain breast cancer cell lines with okadaic acid and TPA has been shown to greatly diminish the induction of apoptosis by okadaic acid (89). Similarly, in THP-1 meyloid leukemia cells, cell death induced by okadaic acid is strongly reduced in the presence of TPA (90).

The various cellular responses to treatment with phosphatase inhibitors can differ enormously, and sometimes even yield contradicting results, which likely is due to variations in the length of treatment, the applied concentration, or the cell type used. In this regard, it was observed that okadaic acid, calyculin A, and cantharidin were able to prevent apoptosis in short term, but not in long term experiments (91). Similarly, the level of phosphorylation of the retinoblastoma (Rb) tumor suppressor protein, an important regulator of cell cycle progression, is affected differentially by okadaic acid, depending on the time of incubation and concentration of the drug that is used (92-95). Moreover, several groups have reported okadaic acid as an inhibitor of transformation in different in vitro transformation assays (96, 97), which is in contrast to those reports discussed further above that established this compound as a potent tumor promoter. Whereas the details of these discrepancies remain to be investigated, the above data argue against the view that TPA and okadaic acid exert their effects through two sides of the same coin, i.e. the phosphorylation versus the inhibition of dephosphorylation of the same substrates of signal transduction pathways.