The ability of tumor promoters to stimulate clonal selection and outgrowth of initiated cell populations is generally attributed to their ability to induce a coordinated change in the expression of various immediate early genes associated with cell proliferation. We have shown that both the phorbol ester tumor promoter 12-0-tetradecanoyl-phorbol acetate and the sesquiterpene tumor promoter thapsigargin can effect the coordinate regulation of immediate early genes including c-fos and the endogenous murine retrovirus VL30. Although proximal targets have been identified for several major classes of tumor promoters, the precise pathways by which disparate tumor promoters act to inhibit the Ca2 plus -ATPase of the endoplasmic reticulum. However, it is not known how this aberration of calcium homoestasis is transduced into induction of c-fos and VL30. In our progress report, we present data indicating that thapsigargin treatment results in an inhibition of protein synthesis, which is associated with decreased MAP kinase phosphatase (MKP-1) activity and increased activation of the stress-activated protein kinases SAPK/JNK and p38/HOG1. Based on these observations, we hypothesize that treatment with thapsigargin results in the activation of protein kinase R (PKR), which phosphorylates eIF2 alpha. Phosphorylation of elF2 alpha results in a block to translation initiation, and decreased synthesis of MKP-1. Loss of MKP-1 results in increased activation of MAP kinases, and this leads to increased phosphorylation of the transcription factor Elk-1 and induction of c-fos mRNA. We propose to test the applicability of this model to calcium-dependent induction of VL30 and c-fos. We plan to identify the transcription factors responsible for induction of VL30. We will also characterize the role of CBF/A, a putative negative regulator of VL30 expression which acts via the VL30 CArG element. Since the c-fos promoter bears significant sequence similarity to the VL30 enhancer, including the presence of a CArG motif, we will determine whether CBF/A and other modulators of VL30 enhancer, including the presence of a CArG motif, we will determine whether CBF/A and other modulators of VL30 expression can also modulate c-fos expression. The role of a PKR-dependent pathway in mediating the response to thapsigargin will be determined for both VL30 and c-fos. These experiments will include determination of possible PKR-dependent changes in the phosphorylation and activation of the transcription factor(s) operating on the promoters for these two genes. The research described in this proposal will provide new insight into the molecular mechanisms by which tumor promoters cause coordinated changes in the expression of proliferation-associated genes.
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