The hypothesis to be tested is that reactive oxygen species (ROS) mediate skin tumor malignant progression in part through altered activities of certain transcription factors via Mitogen Activated Protein (MAP) kinase signal transduction pathways. It is proposed that modulation of antioxidant defense systems during skin tumor malignant progression plays a functional role by leading to elevated ROS. We have developed an in vitro model to study molecular mechanisms of tumor progression. Using repeated treatments with ionizing radiation or N-methyI-N'-nitro-N-nitrosoguanidine (MNNG), we malignantly progressed a papilloma cell line, 308 cells. These cells have an activated Harvey-ras gene. We have demonstrated that both radiation and MNNG malignant variants (6R90 and 6M90, respectively) showed elevated ROS levels that contribute to their proliferative capacity in vitro as well as in vivo. We have also found increased Erk-l/2 and p38 MAP kinase activities to be important components of ROS-mediated signaling. The pro-oxidant state also contributed to constitutive elevation ofAP-1, NF-KB and cAMP response element transactivation in the malignant phenotype. We have shown a reduction in several antioxidant defense mechanisms in the malignant variants including a significant reduction in catalase protein and activity. These data are the rationale for a novel mechanistic model in which an activated Harvey-ras oncogene causes through Rac 1 signaling an increase in ROS that is attenuated in benign papilloma cells by catalase activity. This attenuation is lost in malignant progression through a down-regulation in catalase expression. Through ROS mediated signaling there is enhanced activities of Erkl/2 and p38. The increase in these kinase activities in turn contributes to constitutive elevation of certain transcription factor activities, AP-1, NF-kappaB and CRE that are involved in the maintenance of the malignant phenotype.
Specific Aim 1 : to test the hypothesis that the increase in ROS in the malignant variants is due in part to activated Ha-ras signaling downstream through Rac 1 to increase ROS and also due to a decrease in the expression of the catalase gene.
Specific Aim 2 : to test the hypothesis that the decrease in catalase expression is due to alterations in the transcription of the catalase gene.
Specific Aim 3 : to test the hypothesis that activation of Erkl/2 and p38 kinases in malignant variant cells leads to activation of AP-1 and CRE cis elements.
Specific Aim 4 : to test the hypothesis that activation of AP-1, NF-kappaB and CRE play functional roles in the maintenance of the malignant phenotype. An understanding of both gene alterations and signal transduction pathways in the progression of mouse skin carcinomas can lead to new strategies for intervention including chemoprevention and treatment of human squamous carcinomas.
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