The mechanisms by which tumor promoters contribute to carcinogenesis remain unclear. Palytoxin presents a unique tool for investigating molecular mechanisms underlying tumor promotion. This novel tumor promoter is as potent as the phorbol esters in the two-stage mouse skin model, but does not activate protein kinase C. Investigating the biochemical mechanisms of action of palytoxin may help reveal critical targets in tumor promotion. Preliminary studies show that palytoxin activates c-Jun N-terminal kinase (JNK) in Swiss 3T3 fibroblasts. JNK may be a significant mediator of palytoxin action. JNK phosphorylates the proto-oncogene product c-Jun, which is a transcription factor, on Ser-63 and Ser-73 in vitro and in whole cells. Phosphorylation of c-Jun on Ser-63 and Ser-73 results in transcriptional activation. This observation suggests that palytoxin may modulate gene expression by activating JNK, which in turn phosphorylates and activates c-Jun. Phorbol esters can modulate c-Jun activity through a different signaling pathway. Therefore, modulation of c-Jun function may play a significant role in tumor promotion. The focus of this study is to investigate the hypothesis that palytoxin modulates c-Jun function through the regulation of JNK.
The specific aims for this study are:
Specific Aim 1. Characterize the activation of JNK by palytoxin in Cos7 cells. These studies will extend the model for investigating the regulation of JNK by palytoxin to a transfectable cell line to be used in Specific Aims 2 and 3.
Specific Aim 2. Elucidate the signaling cascade that regulates activation of JNK by palytoxin. These studies will determine if palytoxin regulates JNK through activation of all or part of a protein kinase cascade that has recently been shown to regulate JNK activation (the Rac, Cdc42/Pak1/MEKK1/SEK1/JNK pathway).
Specific Aim 3. Determine if palytoxin stimulates c-Jun transcriptional activity via JNK. c-Jun activation will be measured using a luciferase reporter gene system that has been used by others to demonstrate the regulation of c-Jun by other JNK activators. The results from these studies will determine if c-Jun is a common target for diverse skin tumor promoters, which do or do not function through protein kinase C. These studies may reveal a biochemical mechanism for the transmission of palytoxin-induced signals, and may also suggest a role for JNK in carcinogenesis.
