Gaurnier-Hausser Anita, Mason Nicola J
Department of Professional Studies in the Health Sciences, Drexel University, Philadelphia, PA, 19102, USA.
Methods Mol Biol. 2015;1280:469-504. doi: 10.1007/978-1-4939-2422-6_29.
Companion dogs with spontaneous malignancies are clinically relevant models in which to study the corresponding human diseases and potential therapies. In both dogs and people, non-Hodgkin's lymphoma (NHL) is the most common hematopoietic malignancy. Diffuse large B-cell lymphoma (DLBCL) is the most common NHL subtype in dogs and people, sharing similar biologic, behavioral, genetic, and molecular characteristics in both species. One such molecular characteristic is the constitutive activation of the canonical NF-κB pathway, which in health regulates the expression of target genes that control cellular proliferation, survival, and immune and inflammatory responses as well as multidrug resistance. We found that canine and human DLBCL patients share similar NF-κB activity profiles. Using the cell-permeable NBD peptide, which blocks NF-κB signaling, we inhibited constitutive NF-κB activity and induced apoptosis of primary canine malignant B cells in vitro. In addition, we found that NBD peptide administration to dogs with relapsed B-cell lymphoma inhibited the expression of NF-κB target genes and reduced tumor burden. In this chapter, we describe our methods for processing canine malignant lymphoid tissue. We also describe our methods for treating the lymphocytes isolated from this tissue with NBD peptide and evaluating constitutive canonical NF-κB activity in these cells via immunoblot and electrophoretic mobility shift assay (EMSA). We highlight the nuances of working with canine primary cells.
患有自发性恶性肿瘤的伴侣犬是研究相应人类疾病和潜在治疗方法的临床相关模型。在犬类和人类中，非霍奇金淋巴瘤（NHL）是最常见的造血系统恶性肿瘤。弥漫性大B细胞淋巴瘤（DLBCL）是犬类和人类中最常见的NHL亚型，在这两个物种中具有相似的生物学、行为、遗传和分子特征。其中一个分子特征是经典NF-κB途径的组成性激活，在健康状态下，该途径调节控制细胞增殖、存活、免疫和炎症反应以及多药耐药性的靶基因的表达。我们发现犬类和人类DLBCL患者具有相似的NF-κB活性谱。使用可穿透细胞的NBD肽阻断NF-κB信号传导，我们在体外抑制了组成性NF-κB活性并诱导了原发性犬恶性B细胞的凋亡。此外，我们发现给复发B细胞淋巴瘤犬施用NBD肽可抑制NF-κB靶基因的表达并减轻肿瘤负担。在本章中，我们描述了处理犬恶性淋巴组织的方法。我们还描述了用NBD肽处理从该组织中分离的淋巴细胞并通过免疫印迹和电泳迁移率变动分析（EMSA）评估这些细胞中组成性经典NF-κB活性的方法。我们强调了处理犬原代细胞的细微差别。
