Mouse teratocarcinoma (TC) stem cells are pluripotent cells which are similar in some respects to the cells of the inner cell mass of the mouse embryo. The teratocarcinoma stem cells are tumorigenic; these TC stem cells exhibit malignant properties such as progressive growth and transplantability. However, after treatment with low (less than 10 to the-6 concentrations of retinoic acid (RA), these TC stem cells are induced to differentiate into nonmalignant differentiated cell types such as fibroblasts or parietal endoderm cells. This proposal addresses the question of how retinoic acid converts the tumorigenic TC stem cells into benign differentiated cells with limited growth potential. Although the mechanism by which RA causes differentiation is not understood, it is clear from our previous work (PNAS 80: 5880-5884) and the work of others that RA can regulate gene expression. In this grant, we propose to use molecular biological, and biochemical techniques to isolated specific TC cell genes which are rapidly induced by RA during the differentiation process. We will employ both the F9 and P19 TC stem cell lines for the isolation of these """"""""early"""""""" RA-inducible genes. We then plan to use the cDNA clones for these genes which are expressed at very early times, (1-6 hrs.) after RA addition to determine whether RA directly regulates these genes at the level of transcription, to determine whether dibutyryl cyclic AMP regulates these genes at the transcriptional level, to assay for the proteins encoded by these genes, and to determine if the expression of these """"""""early"""""""" RA-inducible genes in transient or stable. We will determine if a functional cellular RA binding protein is required for expression of these """"""""early"""""""" RA-inducible genes. We will also examined the expression of these """"""""early"""""""" RA-inducible genes in several different teratocarinoma lines. Further characterization of these RA-inducible genes will include the isolation of genomic clones, in order to determine the sequences of these genes, including the conding, noncoding, and flanking sequences. Since retinoic acid can profoundly influence developmental processes in vivo, and RA inhibits carcinogenesis in several experimental systems, the studies outlined in this proposal will provide important and relevant information concerning the mechanism by which RA causes differentiation in this experimental system. The studies in this grant proposal will also lead to future studies in which we will determine whether expression of these genes, (without RA addition, under control of a different promotor sequence), will promote differentiation and loss of tumorigenicity.
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