CYCLOSPORINE AND GENE EXPRESSION BY HUMAN THYMOCYTES

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The objective of this proposal is the elucidation of the mechanism of action of cyclosporine (CsA). Cyclosporine acts by inhibiting the activation of tissue specific genes which can be induced in vitro by activating thymocytes and T-cells with agents which initiate a partially known cascade of transducing signals. The mechanism of action of a drug such as cyclosporine which specifically inhibits the expression of genes which are coordinately expressed during the activation of thymocytes or T- cells can be analysed by using approaches aimed at identifying and characterizing cis-acting DNA sequences (recognition elements) required for eukaryotic gene regulation. The known transcription factors of several inducible tissue-specific genes are preexisting and are modified during activation by a posttranscriptional mechanism which does not require protein synthesis; whereas others require newly synthesized proteins to induce their expression. Positive and negative regulatory elements which function in response to extracellular agents have been identified. It is my working hypothesis, that CsA can inhibit the induction of genes coding for lymphokines either directly, by binding to DNA in concert with a regulatory protein, or indirectly by affecting the modification of one or more putative, regulatory proteins (this modification can be either covalent or allosteric), or by influencing the binding of regulatory proteins to "CsA regulatory sequences" on the gene. My goal is to identify subsets of thymocytes resistant to the effects of CsA since it has been proposed that a specific subset of T-cells is relatively resistant to CsA. The comparison of the effects of CsA on uninduced and induced thymocytes will be useful for the understanding of the molecular events which are elicited during induction and inhibited by CsA. The effect of CsA on the cascade of transducing signals elicited by extracellular inducers and its effect on Ca+2 will also be studied.