In spite of the number of studies that have been conducted, the role of elevated cyclooxygenase (COX)-2 (and its prostaglandin (PG) products) in skin tumor development remains elusive. Several observations suggest a contributory role, including a reduction in skin tumors following an initiation-promotion protocol in either COX-1 or COX-2 knockout mice, and that application of COX inhibitory non-steroidal anti-inflammatory drugs (NSAIDs) usually reduce skin carcinogenesis. We and others, however, have shown that for several cell types NSAIDs induce apoptosis through PG-independent mechanisms, suggesting that this pharmacological approach is not informative with regard to understanding the involvement of PGs in carcinogenesis. To more directly assess the role of COX-2, we took the genetic approach of generating transgenic mice in which COX-2 was overexpressed through the use of a keratin-14 (K14) promoter. These mice are almost completely resistant to phorbol ester promotion. In earlier studies, the topical application of PGE2, but not PGF2alpha, inhibited phorbol ester promotion. Collectively these studies indicate the contribution of COX-2 to skin carcinogenesis is incompletely understood. It is hypothesized that upregulated COX- 2 represses the development of skin tumors through the generation of high levels of PGE2. This hypothesis will be tested by answering the following questions: (1) Is the inhibitory effect of COX-2 overexpression stage-specific or tumor-promoter-specific? Inducible COX-2 transgenic mice will be generated and COX-2 turned on or off at specified times during tumor development. (2) Do specific PGs have inhibitory effects on promotion? Does cAMP mimic this effect? First, PGE2, PGF2? or PGD2 will be applied topically at the time of phorbol ester treatment during promotion of wild-type mice. Second, the role of cAMP will be assessed with regard to its ability to interfere with signaling from PKC and its ability to inhibit tumor promotion. (3) What is the basis for the resistance to tumor promotion in the K14.COX-2 transgenics? 2-D gel electrophoresis and mass spec will to be used to identify changes in protein expression in K14.COX-2 transgenic skin. The function of one of the proteins found to be upregulated in K14.COX-2 mice, galectin-7, which has been implicated in apoptosis, and other candidate proteins will be determined using transient transfection and the significance of candidate proteins assessed with regard to markers for tumor development, including proliferation, apoptosis and invasion.
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