The goal of this proposal is to elucidate the biological function, and mechanism of action of a novel serpin named Trespin (for TGF-beta- repressible serpin) that we have identified in NRP-152 cells by differential display RT-PCR. We will also study the mechanism of transcriptional down-regulation of Trespin by TGF-beta and other apoptosis inducers, and the potential role of Trespin carcinogenesis. We believe that Trespin plays a role as a negative-regulator apoptosis induced by TGF-beta and many other apoptosis inducing agents for the following reasons. Loss of Trespin correlates well with the induction of apoptosis by many agents, and recombinant Trespin blocks the activation of caspase 3 in HL60 and Jurkat cell cytosols and an ICE-like caspase in NRP-152 cell cytosol. Moreover, loss of Trespin expression by TGF-beta precedes the induction of apoptosis and occurs through a transcriptional mechanism. We plan to test the hypothesis that Trespin is a regulator of apoptosis by examining the biological function of Trespin in vivo in cells transfected or retrovirally infected with expression vectors containing sense or anti-sense Trespin cDNAs. We plan to study the regulation of Trespin expression by characterizing the promoter elements and transcription factors responsible for its negative regulation by apoptosis-inducing agents. For this we will isolate the Trespin promoter, study transcriptional regulation of various elements of the Trespin promoter fused to a luciferase reporter construct and identify the transcription factors involved by co-transfection with expression constructs for such factors and mobility shift assays with nuclear extracts. We feel that the data obtained from these latter studies will help the identification of a novel transcriptional factor and/or the role of a known transcription factor in the regulation of apoptosis and its mechanism of action. Lastly, we will study the role of Trespin in carcinogenesis. This will be done by correlating expression Trespin in a variety of malignant and pre-malignant cells and tissues with their malignant phenotype. This will also be done by determining whether over- expression or under-expression of Trespin cDNA in cells transfected/infected with sense or anti-sense Trespin will alter their tumorigenic phenotype.
