Role of Proteinase-3 in Apoptosis and Drug Resistance

描述(由申请人提供)： 我们最近发现，丝氨酸蛋白酶-3(Protease-3，PR3)的表达降低与耐药有关。反义PR3寡核苷酸抑制细胞PR3的表达可显著减少细胞内ROS的产生和药物诱导的细胞凋亡，增加细胞对阿霉素(DOX)或柔红霉素(DNR)的耐药性。在这一应用中，我们建议探索Pr3介导药物诱导的细胞凋亡和增强抗癌药物疗效的分子机制(S)，以及它的缺失如何导致耐药。我们的长期目标是应用从这些研究中获得的知识来识别和/或开发新的和更有效的癌症治疗方案。其具体目的是(1)通过基因转移实验确定PR3表达增加是否增强了DOX和DNR的细胞毒作用；(2)研究PR3在介导活性氧自由基(ROS)增加中的作用，并揭示PR3介导药物诱导细胞凋亡的分子机制(S)；(3)通过鉴定具有凋亡功能的PR3结构域，确定PR3介导的药物诱导的细胞凋亡是否依赖于PR3的蛋白分解活性。具体目标1将(A)评估PR3的增加是否增强了DOX或DNR诱导的药物敏感和耐药细胞的凋亡，(B)确定PR3在药物诱导的ROS产生和凋亡中的作用，(C)研究PR3的亚细胞定位，以及(D)从生化角度确定PR3的表达如何调节DOX在触发细胞凋亡中的作用。具体目的2将(A)确定PR3介导的ROS增加是否通过线粒体依赖的机制和通过增加细胞肿瘤坏死因子-a(TNFct)的激活来发生，(B)研究抑制PR3的表达是否阻止中性鞘磷脂酶(N-sMase)的激活和神经酰胺的产生，以及PR3的表达增加是否激活这些过程，以及(C)评估PR3介导的药物诱导的ROS产生增加是否诱导神经酰胺的产生，并激活c-Jun末端激酶(JNK)和AP1转录因子。在具体目标3中，我们将(A)构建含有PR3区域的标记表达载体，该PR3区域在细胞中表达时产生含有或缺少PR3催化结构域的完整成分的PR3片段，(B)用这些表达载体转染缺少PR3的细胞，并鉴定可促进药物诱导的细胞凋亡的PR3片段，(C)评估该PR3片段在调节药物敏感和耐药细胞系中诱导的细胞凋亡的作用，(D)产生缺失突变体以及进行定点突变研究，以提炼具有凋亡功能的PR3区域，以及(E)设计该结构域的多肽，并检测它们是否能够调节DOX和DNR的细胞毒性。这些研究将有助于理解PR3如何能够介导药物诱导的细胞凋亡，并将有助于开发更有效的化疗或潜在的基因治疗策略。

DESCRIPTION (provided by applicant): We have recently discovered that decreased expression of proteinase-3 (PR3), a serine protease, is associated with drug resistance. Inhibition of cellular PR3 expression by an antisense PR3 oligodeoxynucleotide resulted in a significant reduction fin reactive oxygen species (ROS) generation and drug-induced apoptosis, and increased cellular resistance to doxorubicin (DOX) or daunorubicin (DNR). In this application, we propose to explore the molecular mechanism(s) by which PR3 mediates drug-induced apoptosis and increases the efficacy of anticancer agents, and how its loss causes drug resistance. Our long-term objective is to apply the knowledge obtained from these studies to identify and/or develop novel and more effective cancer therapeutic regimens. The Specific Aims are to (1) determine whether increased PR3 expression by gene transfer experiments enhances the cytotoxic effects of DOX and DNR, and (2) investigate the role of the PR3 in mediating increased reactive oxygen species (ROS) and delineate the molecular mechanism(s) of PR3-mediated drug-induced apoptosis, and (3) determine whether the PR3-mediated drug-induced apoptosis is dependent on the proteolytic activity of PR3 by identifying the domain of PR3 with apoptotic function. Specific Aim 1 will (a) evaluate whether the increase in PR3 enhances DOX- or DNR-induced apoptosis in drug sensitive and resistant cells, (b) determine the role of PR3 in drug-induced ROS production and apoptosis, (c) examine the subcellular localization of PR3, and (d) ascertain biochemically how PR3 expression modulates the effect of DOX on triggering apoptosis. Specific Aim 2 will (a) determine whether the PR3-mediated increase in ROS occurs through a mitochondrion-dependent mechanism, and by increased activation of cellular tumor necrosis factor-a (TNFct), (b) investigate whether inhibition of PR3 expression blocks neutral sphingomyelinase (N-SMase) activation and ceramide generation, and whether increased expression of PR3 activates these processes, and (c) assess whether the PR3-mediated drug induced increase in ROS production induces ceramide generation, and activates c-Jun-Terminal Kinase (JNK) and AP1 transcription factor. In Specific Aim 3, we will (a) construct FLAG-tagged expression vectors containing regions of PR3 that, upon expression in cells, produce PR3 fragments which either contain or lack the full components of catalytic domain of PR3, (b) transfect cells lacking PR3 with these expression vectors, and identify a fragment of PR3 which enhances drug-induced apoptosis, (c) assess the effect of this PR3 fragment in modulating drug-induced apoptosis in the drug sensitive and resistant cell lines, (d) generate deletion mutants as well as perform site-directed mutagenesis studies to refine the domain of PR3 with apoptotic function, and (e) design peptides from this domain and examine whether they are able to modulate the cytotoxicity of DOX and DNR. These studies will aid in understanding how PR3 is capable of mediating drug-induced apoptosis, and will be useful for the development of more effective chemotherapeutic or potential gene therapy strategies.