Tumor promoters act by stimulating the clonal selection and outgrowth of initiated cell populations. This process involves the activation of cellular signalling pathways normally involved in the regulation of cell proliferation. Several distinct tumor promoters are known to exert specific-effects on these signaling pathpromoters such as 12-O-tetradecanoyl 13-phorbol acetate (TPA) can substitute for endogenous diacylglycerols in the activation of protein kinase C (pkC). Okadaic acid is a specific and potent inhibitor of the protein phosphatase 1 and 2A. Thapsigargin causes an elevation of intra-cellular Ca(2+) levels subsequent to inhibition of the Ca(2+)-ATPase responsible for sequestering Ca(2+) in intracellular stores. We have obtained data indicating that these disparate tumor promoters are capable of interacting both additively and synergistically to stimulate the expression of several genes, including VL30. VL30 is an endogenous murine retrovirus which is abundantly expressed in response to either transformation or proliferative stimuli. We have shown that a single VL30 element, transferred from the mouse genome to Rat-1 fibroblasts by pseudovirion-mediated transfection, is capable of responding transcriptionally to a number of agonists, including TPA and EGF. At least two independent signaling pathways, one involving pkC and the other increased intracellular Ca(2+), are involved in the regulation of VL30 expression. Deletional mutagenesis studies have implicated a triple repeat unit present in the VL30 LTR as an enhancer element capable of mediating responsiveness to TPA and EGF. The ability of thapsigargin and okadaic acid to interact positively with TPA in stimulating gene expression also maps to this repeat unit, and to an AP-1-like element present in this repeat. The initial goal of this proposal is to determine the role of other putative response elements, primarily a CArG element, in this regulation, and to identify and characterize the DNA binding proteins involved in transcriptional regulation from these elements. Once the functional response element has been full defined, we propose to produce transgenic mice containing the tumor promoter-responsive element linked to a lacZ reporter gene. A major goal of this proposal is to determine the effects of the three tumor promoters on gene expression in vivo, by monitoring lacZ expression in these transgenic mice in response to topical applications of TPA, thapsigargin, and okadaic-acid, alone and in combination. These experiments will indicate whether the observed interactions between the signaling pathways responding to these tumor promoters in vitro also function in vivo. We will determine whether cell-type- or tissue-specific changes in lacZ reporter gene expression are associated with the growth of tumors. We will use primary cell lines established from these transgenic mice to study the effect of targeted disruptions of specific signaling pathways and DNA-protein interactions on the ability of tumor promoters to modulate gene expression. These studies will be conducted both on populations of primary epidermal cells, and on individual cells microinjected with specific kinase inhibitors or inactivating antibodies. These experiments should contribute to our understanding of the mechanisms linking tumor promoter-induced signaling events to changes in gene transcription.
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