FUNCTION AND REGULATION OF TRESPIN, A NOVEL SERPIN

本研究的目的是阐明一种名为Trespin的新型蛇形蛋白（用于抑制tgf - β的蛇形蛋白）的生物学功能和作用机制，我们已经通过差异显示RT-PCR在NRP-152细胞中发现了这种新型蛇形蛋白。我们还将研究tgf - β和其他凋亡诱导剂下调Trespin转录的机制，以及Trespin致癌的潜在作用。我们认为，由于以下原因，Trespin在tgf - β和许多其他诱导细胞凋亡的药物诱导的负调控细胞凋亡中发挥作用。许多药物诱导凋亡与Trespin的缺失密切相关，重组Trespin阻断了HL60和Jurkat细胞质中caspase 3的激活以及NRP-152细胞质中ice样caspase的激活。此外，tgf - β介导的Trespin表达缺失先于细胞凋亡的诱导，并通过转录机制发生。我们计划通过检测含有正义或反义Trespin cdna的表达载体转染或逆转录病毒感染的细胞中Trespin的生物学功能，来验证Trespin是细胞凋亡调节剂的假设。我们计划通过鉴定细胞凋亡诱导剂对Trespin负调控的启动子元件和转录因子来研究Trespin的表达调控。为此，我们将分离Trespin启动子，研究与荧光素酶报告基因构建融合的Trespin启动子的各种元件的转录调控，并通过与这些因子的表达构建体共转染和核提取物的迁移转移测定来鉴定涉及的转录因子。我们认为，从这些研究中获得的数据将有助于鉴定新的转录因子和/或已知转录因子在细胞凋亡调控中的作用及其作用机制。最后，我们将研究Trespin在癌变中的作用。这将通过在各种恶性和癌前细胞和组织中与它们的恶性表型相关的Trespin表达来实现。这也将通过确定转染/感染了正义或反义Trespin的细胞中Trespin cDNA的过表达或过表达是否会改变其致瘤表型来完成。

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.