FUNCTION AND REGULATION OF TRESPIN, A NOVEL SERPIN

本研究的目的是阐明我们在NRP-152细胞中通过差异显示RT-PCR鉴定到的一种新的丝氨酸--Tresin的生物学功能和作用机制。我们还将研究转化生长因子-β和其他细胞凋亡诱导剂下调天冬氨酸氨基转移酶转录下调的机制，以及天冬氨酸氨基转移酶致癌的潜在作用。我们认为Tresin在转化生长因子-β等多种细胞凋亡诱导剂诱导的细胞凋亡中起负调控作用，其原因如下。Tresin的丢失与多种诱导剂诱导的细胞凋亡密切相关，重组Tresin可阻断HL60和Jurkat细胞胞浆中caspase 3的激活以及NRP-152细胞胞浆中ICE样半胱氨酸酶的激活。此外，转化生长因子-β在诱导细胞凋亡之前失去了Tresin的表达，并通过转录机制发生。我们计划通过在体内检测Tresin在体内的生物学功能来验证Tresin是一种细胞凋亡调节因子的假设，这些细胞被含有正义或反义Tresin cDNAs的表达载体转染或逆转录病毒感染。我们计划通过鉴定启动子元件和转录因子来研究Tresin的表达调控，这些启动子元件和转录因子负责通过凋亡诱导剂对其进行负调控。为此，我们将分离Tresin启动子，研究Tresin启动子的各种元件与荧光素酶报告构建体融合的转录调控，并通过与这些因子的表达构建体共转染鉴定所涉及的转录因子，以及利用核提取物进行迁移率改变分析。我们认为，从后一项研究中获得的数据将有助于识别一个新的转录因子和/或一个已知的转录因子在细胞凋亡调节中的作用及其作用机制。最后，我们将研究Tresin在癌症发生中的作用。这将通过将Tresin在各种恶性和癌前细胞和组织中的表达与其恶性表型相关联来完成。这也将通过确定在正义或反义Tresin基因转染/感染的细胞中Tresin基因的过度表达或低表达是否会改变它们的致瘤表型来实现这一点。

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.