ANTI APOPTOTIC SIGNALING IN MYELOMA--BCL 2 AND NF KAPPA

骨髓瘤中的抗凋亡信号传导——BCL 2 和 NF KAPPA

• 批准号: 6377791
• 负责人: RENA R FEINMAN
• 金额: $ 21.2万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6377791
• 关键词: BCL2 gene /protein; antisense nucleic acid; apoptosis; biological signal transduction; cell line; clinical research; dexamethasone; drug resistance; human subject; longitudinal human study; multiple myeloma; mutant; neoplasm /cancer chemotherapy; neoplasm /cancer pharmacology; nuclear factor kappa beta; oligonucleotides; posttranslational modifications

Understanding the molecular mechanisms by which myeloma cells evade signals leading to chemotherapy-induced apoptosis is the long term goal of this project. Glucocorticoids such as dexamethasone (Dex], all potent inducers of apoptosis are frequently used to treat multiple myeloma [MM], a clonal B cell-malignancy. Using standard doses of these agents, complete remissions are rare [5% of patients]. Drug resistance increases with prolonged treatment, suggesting primary resistance of tumor cells as a major obstacle to cure. This resistance maybe due to MM cell expression of moderate to high levels of the anti-apoptotic protein bcl-2. There is compelling evidence that bcl-2 imparts its anti-apoptotic signal by ducking and modulating the expression of genes critical for apoptosis, including transcription factors such as NF-kappaB. We hypothesize that chemoresistance in MM by bcl-2 requires activation of NF-kappaB. To investigate this hypothesis we will address the following specific aims: 1. Test whether persistent NF-kappaB activation observed in MM cells confers resistance in chemotherapy-induced apoptosis in MM cell lines. P50:p65 NF-kappaB subunits will be over-expressed in drug-sensitive MM cell lines to inactivated NF-kB. Antisense phosphorothiorate oligonucleotides for p50 and p65 will also be used to inhibit NF-kappaB. 2. Determine whether bcl-2 contributes to maintaining NF-kappaB activity through modulation of drug-mediated post-translational regulation of IkappaB proteins and/or p65 transactivation. Different modes of IkBalpha, Ikbeta and Ikepsilon protein regulation will be investigated: 1] basal and signal-induced IkappaB phosphorylation and degradation 2) IKK-alpha and -beta functions and 3) inhibitory properties of IkappaB proteins. For p65 transactivation: 1] a Gal4-BASED hybrid system will be used to test whether clinically relevant drugs and enforced bcl-2 interfere with the transactivation potential of p65 and 2] a bcl-2 deletion mutant that lacks the transmembrane domain will be expressed in MM cell lines to determine whether membrane-anchored bcl-2 augments p65 transactivation. 3. Investigate whether the inhibitory effects of Dex on NF-kappaB activity predict response to chemotherapy. Longitudinal studies will be conducted to determine whether patient responsiveness to glucocorticoid based therapy can predicted from their in vitro response to Dex in terms of NF-kappaB DNA binding, p65 immunostaining. There studies will hopefully provide deeper insight into the molecular basis of drug resistance in MM and should result in the identification of potential targets for therapeutic intervention.

了解骨髓瘤细胞逃避信号导致化疗诱导细胞凋亡的分子机制是该项目的长期目标。糖皮质激素，如地塞米松 (Dex)，都是有效的细胞凋亡诱导剂，常用于治疗多发性骨髓瘤 [MM]，一种克隆性 B 细胞恶性肿瘤。使用这些药物的标准剂量，完全缓解的情况很少见 [5% 的患者]。随着治疗时间的延长，耐药性增加，表明肿瘤细胞的原发性耐药性是治愈的主要障碍，这种耐药性可能是由于 MM 细胞表达中度至高水平的抗凋亡蛋白所致。 bcl-2. 有令人信服的证据表明，bcl-2 通过回避和调节对细胞凋亡至关重要的基因（包括 NF-kappaB 等转录因子）的表达来传递其抗细胞凋亡信号，我们假设 bcl-2 需要在 MM 中产生化疗耐药性。为了研究这一假设，我们将解决以下具体目标： 1. 测试在 MM 细胞中观察到的持续 NF-kappaB 激活是否会对化疗诱导的细胞凋亡产生抵抗力。在 MM 细胞系中，P50:p65 NF-kappaB 亚基将在药物敏感的 MM 细胞系中过度表达，导致 NF-kB 失活。 p50 和 p65 的反义硫代磷酸寡核苷酸也将用于抑制 NF-κB。 2. 确定 bcl-2 是否通过调节药物介导的 IkappaB 蛋白翻译后调节和/或 p65 反式激活来维持 NF-kappaB 活性。将研究 IkBalpha、Ikbeta 和 Ikepsilon 蛋白调节的不同模式：1] 基础和信号诱导的 IkappaB 磷酸化和降解 2) IKK-alpha 和 -beta 功能以及 3) IkappaB 蛋白的抑制特性。对于 p65 反式激活：1] 基于 Gal4 的混合系统将用于测试临床相关药物和强制 bcl-2 是否干扰 p65 的反式激活潜力，2] 将表达缺乏跨膜结构域的 bcl-2 缺失突变体在 MM 细胞系中确定膜锚定的 bcl-2 是否增强 p65 反式激活。 3. 研究 Dex 对 NF-kappaB 活性的抑制作用是否可以预测化疗反应。将进行纵向研究，以确定是否可以根据 NF-kappaB DNA 结合、p65 免疫染色等方面对 Dex 的体外反应来预测患者对糖皮质激素治疗的反应。这些研究有望更深入地了解多发性骨髓瘤耐药性的分子基础，并确定治疗干预的潜在靶点。