Role of Proteinase-3 in Apoptosis and Drug Resistance

描述（由申请人提供）： 我们最近发现，蛋白酶-3（PR 3）（一种丝氨酸蛋白酶）表达的降低与耐药性有关。反义PR 3寡核苷酸抑制细胞PR 3表达可显著减少活性氧（ROS）的产生和药物诱导的细胞凋亡，并增加细胞对阿霉素（DOX）或柔红霉素（DNR）的耐药性。在本申请中，我们提出探索PR 3介导药物诱导的细胞凋亡并增加抗癌剂功效的分子机制，以及其损失如何导致耐药性。我们的长期目标是应用从这些研究中获得的知识，以确定和/或开发新的和更有效的癌症治疗方案。具体目的是（1）确定通过基因转移实验增加的PR 3表达是否增强DOX和DNR的细胞毒性作用，和（2）研究PR 3在介导活性氧（ROS）增加中的作用，并描述PR 3介导的药物诱导细胞凋亡的分子机制，以及（3）通过鉴定具有凋亡功能的PR 3结构域来确定PR 3介导的药物诱导的凋亡是否依赖于PR 3的蛋白水解活性。具体目标1将（a）评估PR 3的增加是否增强药物敏感性和抗性细胞中DOX或DNR诱导的细胞凋亡，（B）确定PR 3在药物诱导的ROS产生和细胞凋亡中的作用，（c）检查PR 3的亚细胞定位，和（d）确定PR 3表达如何以生物化学方式调节DOX对触发细胞凋亡的作用。具体目标2将（a）确定PR 3介导的ROS增加是否通过依赖于TNF α的机制以及通过增加细胞肿瘤坏死因子-α（TNF α）的活化而发生，（B）研究PR 3表达的抑制是否阻断中性鞘磷脂酶（N-SM酶）活化和神经酰胺生成，以及PR 3表达的增加是否激活这些过程，和（c）评估PR 3介导的药物诱导的ROS产生的增加是否诱导神经酰胺产生，并激活c-Jun末端激酶（JNK）和AP 1转录因子。在具体目标3中，我们将（a）构建含有PR 3区域的FLAG标记的表达载体，其在细胞中表达时产生含有或缺乏PR 3催化结构域的完整组分的PR 3片段，（B）用这些表达载体转染缺乏PR 3的细胞，并鉴定增强药物诱导的细胞凋亡的PR 3片段，（c）评估该PR 3片段在药物敏感性和抗性细胞系中调节药物诱导的细胞凋亡的作用，（d）产生缺失突变体以及进行定点诱变研究以改进具有细胞凋亡功能的PR 3结构域，和（e）设计来自该结构域的肽并检查它们是否能够调节DOX和DNR的细胞毒性。这些研究将有助于了解PR 3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.