Retinoids, a class of compounds comprising natural and synthetic analogs of retinol (vitamin A), have been identified as inhibitors of malignant transformation. Retinoids are thought to inhibit carcinogenesis through their profound effects on cell differentiation and cell proliferation, but the mechanism(s) by which retinoids regulate these processes are not understood.
The aims of this grant were to characterize the earliest actions of retinoic acid (RA) in the F9 murine teratocarcinoma stem cell line, which differentiates into endoderm, a type of epithelial cell, in the presence of RA. We have now identified at least two genes, ERA-1 (Early Retinoic Acid-1) and ERA-2, which are expressed rapidly (within 2 hr.) after RA addition. The increases in the ERA-1 and ERA-2 mRNAs in response to RA are insensitive to protein synthesis inhibitors, but are prevented by RNA synthesis inhibitors (eg. actinomycin D) suggesting that the ERA-1 and ERA-2 mRNAs may represent a primary, direct response to RA. We've demonstrated by nuclear transcriptional run-off assays that the ERA-1 gene is regulated by RA at the level of transcription, and we also have preliminary evidence that the half-life of ERA-1 mRNA doesn't change upon RA addition. To our knowledge, this is the first discovery of such rapid regulation of genes by RA in any differentiation system. Therefore, analysis of these ERA genes should provide much new information about the mechanism of RA action. We propose to identify additional genes which exhibit rapid mRNA induction in response to RA. We also propose to sequence the ERA- 1, and ERA-2 genes, to determine their genomic structures, including their 5' flanking regions, and to delineate the DNA sequences and nuclear proteins required for their transcriptional regulation by RA. We want to produce antibodies specific for the ERA-1, and ERA-2 gene products as we now have data which suggests that the ERA-1 gene product is a DNA binding protein. To analyze the functions of their gene products, these ERA genes, under the control of a heterologous promoter (in the sense, and antisense orientations), will be transfected into F9 stem cells, and these transfected lines will be assessed for their ability to differentiate. These studies should result in major advances in the understanding of RA action in differentiation, the regulation of cell proliferation, and the inhibition of malignant transformation.
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